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Posts Tagged ‘Epigenetics’


Effect of mitochondrial stress on epigenetic modifiers

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Early Mitochondrial Stress Alters Epigenetics, Secures Lifelong Health Benefits

GEN 5/3/2016  http://www.genengnews.com/gen-news-highlights/early-mitochondrial-stress-alters-epigenetics-secures-lifelong-health-benefits/81252685/

A little adversity builds character, or so the saying goes. True or not, the saying does seem an apt description of a developmental phenomenon that shapes gene expression. While it knows nothing of character, the gene expression apparatus appears to respond well to short-term mitochondrial stress that occurs early in development. In fact, transient stress seems to result in lasting benefits. These benefits, which include improved metabolic function and increased longevity, have been observed in both worms and mice, and may even occur—or be made to occur—in humans.

Gene expression is known to be subject to reprogramming by epigenetic modifiers, but such modifiers generally affect metabolism or lifespan, not both. A new set of epigenetic modifiers, however, has been found to trigger changes that do just that—both improve metabolism and extend lifespan.

Scientists based at the University of California, Berkeley, and the École Polytechnique Fédérale de Lausanne (EPFL) have discovered enzymes that are ramped up after mild stress during early development and continue to affect the expression of genes throughout the animal’s life. When the scientists looked at strains of inbred mice that have radically different lifespans, those with the longest lifespans had significantly higher expression of these enzymes than did the short-lived mice.

“Two of the enzymes we discovered are highly, highly correlated with lifespan; it is the biggest genetic correlation that has ever been found for lifespan in mice, and they’re both naturally occurring variants,” said Andrew Dillin, a UC Berkeley professor of molecular and cell biology. “Based on what we see in worms, boosting these enzymes could reprogram your metabolism to create better health, with a possible side effect of altering lifespan.”

Details of the work, which appeared online April 29 in the journal Cell, are presented in a pair of papers. One paper (“Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity”) resulted from an effort led by Dillin and the EPFL’s Johan Auwerx. The other paper (“Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt”) resulted from an effort led by Dillin and his UC Berkeley colleague Barbara Meyer.

According to these papers, mitochondrial stress activates enzymes in the brain that affect DNA folding, exposing a segment of DNA that contains the 1500 genes involved in the work of the mitochondria. A second set of enzymes then tags these genes, affecting their activation for much or all of the lifetime of the animal and causing permanent changes in how the mitochondria generates energy.

The first set of enzymes—methylases, in particular LIN-65—add methyl groups to the DNA, which can silence promoters and thus suppress gene expression. By also opening up the mitochondrial genes, these methylases set the stage for the second set of enzymes—demethylases, in this case jmjd-1.2 and jmjd-3.1—to ramp up transcription of the mitochondrial genes. When the researchers artificially increased production of the demethylases in worms, all the worms lived longer, a result identical to what is observed after mitochondrial stress.

“By changing the epigenetic state, these enzymes are able to switch genes on and off,” Dillin noted. This happens only in the brain of the worm, however, in areas that sense hunger or satiety. “These genes are expressed in neurons that are sensing the nutritional status of the animal, and these signals emanate out to the periphery to change peripheral metabolism,” he continued.

When the scientists profiled enzymes in short- and long-lived mice, they found upregulation of these genes in the brains of long-lived mice, but not in other tissues or in the brains of short-lived mice. “These genes are expressed in the hypothalamus, exactly where, when you eat, the signals are generated that tell you that you are full. And when you are hungry, signals in that region tell you to go and eat,” Dillin explained said. “These genes are all involved in peripheral feedback.”

Among the mitochondrial genes activated by these enzymes are those involved in the body’s response to proteins that unfold, which is a sign of stress. Increased activity of the proteins that refold other proteins is another hallmark of longer life.

These observations suggest that the reversal of aging by epigenetic enzymes could also take place in humans.

“It seems that, while extreme metabolic stress can lead to problems later in life, mild stress early in development says to the body, ‘Whoa, things are a little bit off-kilter here, let’s try to repair this and make it better.’ These epigenetic switches keep this up for the rest of the animal’s life,” Dillin stated.

 

Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity

Carsten Merkwirth6, Virginija Jovaisaite6, Jenni Durieux,…., Reuben J. Shaw, Johan Auwerx, Andrew Dillin

Highlights
  • H3K27 demethylases jmjd-1.2 and jmjd-3.1 are required for ETC-mediated longevity
  • jmjd-1.2 and jmjd-3.1 extend lifespan and are sufficient for UPRmt activation
  • UPRmt is required for increased lifespan due to jmjd-1.2 or jmjd-3.1 overexpression
  • JMJD expression is correlated with UPRmt and murine lifespan in inbred BXD lines

Across eukaryotic species, mild mitochondrial stress can have beneficial effects on the lifespan of organisms. Mitochondrial dysfunction activates an unfolded protein response (UPRmt), a stress signaling mechanism designed to ensure mitochondrial homeostasis. Perturbation of mitochondria during larval development in C. elegans not only delays aging but also maintains UPRmt signaling, suggesting an epigenetic mechanism that modulates both longevity and mitochondrial proteostasis throughout life. We identify the conserved histone lysine demethylases jmjd-1.2/PHF8 and jmjd-3.1/JMJD3 as positive regulators of lifespan in response to mitochondrial dysfunction across species. Reduction of function of the demethylases potently suppresses longevity and UPRmt induction, while gain of function is sufficient to extend lifespan in a UPRmt-dependent manner. A systems genetics approach in the BXD mouse reference population further indicates conserved roles of the mammalian orthologs in longevity and UPRmt signaling. These findings illustrate an evolutionary conserved epigenetic mechanism that determines the rate of aging downstream of mitochondrial perturbations.

Figure thumbnail fx1

 

Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt
Ye Tian, Gilberto Garcia, Qian Bian, Kristan K. Steffen, Larry Joe, Suzanne Wolff, Barbara J. Meyer, Andrew Dillincorrespondence
http://dx.doi.org/10.1016/j.cell.2016.04.011             Publication stage: In Press Corrected Proof
Highlights
  • LIN-65 accumulates in the nucleus in response to mitochondrial stress
  • Mitochondrial stress-induced chromatin changes depend on MET-2 and LIN-65
  • LIN-65 and DVE-1 exhibit interdependence in nuclear accumulation
  • met-2 and atfs-1 act in parallel to affect mitochondrial stress-induced longevity

Organisms respond to mitochondrial stress through the upregulation of an array of protective genes, often perpetuating an early response to metabolic dysfunction across a lifetime. We find that mitochondrial stress causes widespread changes in chromatin structure through histone H3K9 di-methylation marks traditionally associated with gene silencing. Mitochondrial stress response activation requires the di-methylation of histone H3K9 through the activity of the histone methyltransferase met-2 and the nuclear co-factor lin-65. While globally the chromatin becomes silenced by these marks, remaining portions of the chromatin open up, at which point the binding of canonical stress responsive factors such as DVE-1 occurs. Thus, a metabolic stress response is established and propagated into adulthood of animals through specific epigenetic modifications that allow for selective gene expression and lifespan extension

 Siddharta Mukherjee’s Writing Career Just Got Dealt a Sucker Punch
Author: Theral Timpson

Siddharha Mukherjee won the 2011 Pulitzer Prize in non-fiction for his book, The Emperor of All Maladies.  The book has received widespread acclaim among lay audience, physicians, and scientists alike.  Last year the book was turned into a special PBS series.  But, according to a slew of scientists, we should all be skeptical of his next book scheduled to hit book shelves this month, The Gene, An Intimate History.

Publishing an article on epigenetics in the New Yorker this week–perhaps a selection from his new book–Mukherjee has waltzed into one of the most active scientific debates in all of biology: that of gene regulation, or epigenetics.

Jerry Coyne, the evolutionary biologist known for keeping journalists honest, has published a two part critique of Mukherjee’s New Yorker piece.  The first part–wildly tweeted yesterday–is a list of quotes from Coyne’s colleagues and those who have written in to the New Yorker, including two Nobel prize winners, Wally Gilbert and Sidney Altman, offering some very unfriendly sentences.

Wally Gilbert: “The New Yorker article is so wildly wrong that it defies rational analysis.”

Sidney Altman:  “I am not aware that there is such a thing as an epigenetic code.  It is unfortunate to inflict this article, without proper scientific review, on the audience of the New Yorker.”

The second part is a thorough scientific rebuttal of the Mukherjee piece.  It all serves as a great drama about one of the most contested ideas in biology and also as a cautionary tale to journalists, even experienced writers such as Mukherjee, about the dangers of wading into scientific arguments.  Readers may remember that a few years ago, science writer, David Dobbs, similarly skated into the same topic with his piece, Die, Selfish Gene, Die, and which raised a similar shitstorm, much of it from Coyne.

Mukherjee’s mistake is in giving credence to only one side of a very fierce debate–that the environment causes changes in the genome which can be passed on; another kind of evolution–as though it were settled science.   Either Mukherjee, a physicisan coming off from a successful book and PBS miniseries on cancer, is setting himself up as a scientist, or he has been a truly naive science reporter.   If he got this chapter so wrong, what does it mean about an entire book on the gene?

Coyne quotes one of his colleagues who raised some questions about the New Yorker’s science reporting, one particular question we’ve been asking here at Mendelspod.  How do we know what we know?  Does science now have an edge on any other discipline for being able to create knowledge?

Coyne’s colleague is troubled by science coverage in the New Yorker, and goes so far as to write that the New Yorker has been waging a “war on behalf of cultural critics and literary intellectuals against scientists and technologists.”

From my experience, it’s not quite that tidy.  First of all, the New Yorker is the best writing I read each week.  Period.  Second, I haven’t found their science writing to have the slant claimed in the quote above.  For example, most other mainstream outlets–including the New York Times with the Amy Harmon pieces–have given the anti-GMO crowd an equal say in the mistaken search for a “balance” on whether GMOs are harmful.  (Remember John Stewart’s criticism of Fox News?  That they give a false equivalent between two sides even when there is no equivalent on the other side?)

But the New Yorker has not fallen into this trap on GMOs and most of their pieces on the topic–mainly by Michael Specter–have been decidedly pro science and therefore decided pro GMO.

So what led Mukherjee to play scientist as well as journalist?  There’s no question about whether I enjoy his prose.  His writing beautifully whisks me away so that I don’t feel that I’m really working to understand.  There is a poetic complexity that constantly brings different threads effortlessly together, weaving them into the same light.  At one point he uses the metaphor of a web for the genome, with the epigenome being the stuff that sticks to the web.  He borrows the metaphor from the Hindu notion of “being”, or jaal.

“Genes form the threads of the web; the detritus that adheres to it transforms every web into a singular being.”

There have been a few writers on Twitter defending Mukherjee’s piece.  Tech Review’s Antonio Regalado called Coyne and his colleagues “tedious literalists” who have an “issue with epigenetic poetry.”

At his best, Mukherjee can take us down the sweet alleys of his metaphors and family stories with a new curiosity for the scientific truth.  He can hold a mirror up to scientists, or put the spotlight on their work.   At their worst, Coyne and his scientific colleagues can reek of a fear of language and therefore metaphor.  The always outspoken scientist and author, Richard Dawkins, who made his name by personifying the gene, was quick to personify epigentics in a tweet:   “It’s high time the 15 minutes of underserved fame for “epigenetics” came to an overdue end.”  Dawkins is that rare scientist who has consistently been as comfortable with rhetoric and language as he is with data.

Hats off to Coyne who reminds us that a metaphor–however lovely–does not some science make. If Mukherjee wants to play scientist, let him create and gather data. If it’s the role of science journalist he wants, let him collect all the science he can before he begins to pour it into his poetry.

 

Same but Different  

How epigenetics can blur the line between nature and nurture.

Annals of Science MAY 2, 2016 ISSUE     BY

The author’s mother (right) and her twin are a study in difference and identity. CREDIT: PHOTOGRAPH BY DAYANITA SINGH FOR THE NEW YORKER

October 6, 1942, my mother was born twice in Delhi. Bulu, her identical twin, came first, placid and beautiful. My mother, Tulu, emerged several minutes later, squirming and squalling. The midwife must have known enough about infants to recognize that the beautiful are often the damned: the quiet twin, on the edge of listlessness, was severely undernourished and had to be swaddled in blankets and revived.

The first few days of my aunt’s life were the most tenuous. She could not suckle at the breast, the story runs, and there were no infant bottles to be found in Delhi in the forties, so she was fed through a cotton wick dipped in milk, and then from a cowrie shell shaped like a spoon. When the breast milk began to run dry, at seven months, my mother was quickly weaned so that her sister could have the last remnants.
Tulu and Bulu grew up looking strikingly similar: they had the same freckled skin, almond-shaped face, and high cheekbones, unusual among Bengalis, and a slight downward tilt of the outer edge of the eye, something that Italian painters used to make Madonnas exude a mysterious empathy. They shared an inner language, as so often happens with twins; they had jokes that only the other twin understood. They even smelled the same: when I was four or five and Bulu came to visit us, my mother, in a bait-and-switch trick that amused her endlessly, would send her sister to put me to bed; eventually, searching in the half-light for identity and difference—for the precise map of freckles on her face—I would realize that I had been fooled.

But the differences were striking, too. My mother was boisterous. She had a mercurial temper that rose fast and died suddenly, like a gust of wind in a tunnel. Bulu was physically timid yet intellectually more adventurous. Her mind was more agile, her tongue sharper, her wit more lancing. Tulu was gregarious. She made friends easily. She was impervious to insults. Bulu was reserved, quieter, and more brittle. Tulu liked theatre and dancing. Bulu was a poet, a writer, a dreamer.

….. more

Why are identical twins alike? In the late nineteen-seventies, a team of scientists in Minnesota set out to determine how much these similarities arose from genes, rather than environments—from “nature,” rather than “nurture.” Scouring thousands of adoption records and news clips, the researchers gleaned a rare cohort of fifty-six identical twins who had been separated at birth. Reared in different families and different cities, often in vastly dissimilar circumstances, these twins shared only their genomes. Yet on tests designed to measure personality, attitudes, temperaments, and anxieties, they converged astonishingly. Social and political attitudes were powerfully correlated: liberals clustered with liberals, and orthodoxy was twinned with orthodoxy. The same went for religiosity (or its absence), even for the ability to be transported by an aesthetic experience. Two brothers, separated by geographic and economic continents, might be brought to tears by the same Chopin nocturne, as if responding to some subtle, common chord struck by their genomes.

One pair of twins both suffered crippling migraines, owned dogs that they had named Toy, married women named Linda, and had sons named James Allan (although one spelled the middle name with a single “l”). Another pair—one brought up Jewish, in Trinidad, and the other Catholic, in Nazi Germany, where he joined the Hitler Youth—wore blue shirts with epaulets and four pockets, and shared peculiar obsessive behaviors, such as flushing the toilet before using it. Both had invented fake sneezes to diffuse tense moments. Two sisters—separated long before the development of language—had invented the same word to describe the way they scrunched up their noses: “squidging.” Another pair confessed that they had been haunted by nightmares of being suffocated by various metallic objects—doorknobs, fishhooks, and the like.

The Minnesota twin study raised questions about the depth and pervasiveness of qualities specified by genes: Where in the genome, exactly, might one find the locus of recurrent nightmares or of fake sneezes? Yet it provoked an equally puzzling converse question: Why are identical twins different? Because, you might answer, fate impinges differently on their bodies. One twin falls down the crumbling stairs of her Calcutta house and breaks her ankle; the other scalds her thigh on a tipped cup of coffee in a European station. Each acquires the wounds, calluses, and memories of chance and fate. But how are these changes recorded, so that they persist over the years? We know that the genome can manufacture identity; the trickier question is how it gives rise to difference.

….. more

But what turns those genes on and off, and keeps them turned on or off? Why doesn’t a liver cell wake up one morning and find itself transformed into a neuron? Allis unpacked the problem further: suppose he could find an organism with two distinct sets of genes—an active set and an inactive set—between which it regularly toggled. If he could identify the molecular switches that maintain one state, or toggle between the two states, he might be able to identify the mechanism responsible for cellular memory. “What I really needed, then, was a cell with these properties,” he recalled when we spoke at his office a few weeks ago. “Two sets of genes, turned ‘on’ or ‘off’ by some signal.”

more…

“Histones had been known as part of the inner scaffold for DNA for decades,” Allis went on. “But most biologists thought of these proteins merely as packaging, or stuffing, for genes.” When Allis gave scientific seminars in the early nineties, he recalled, skeptics asked him why he was so obsessed with the packing material, the stuff in between the DNA.  …. A skein of silk tangled into a ball has very different properties from that same skein extended; might the coiling or uncoiling of DNA change the activity of genes?

In 1996, Allis and his research group deepened this theory with a seminal discovery. “We became interested in the process of histone modification,” he said. “What is the signal that changes the structure of the histone so that DNA can be packed into such radically different states? We finally found a protein that makes a specific chemical change in the histone, possibly forcing the DNA coil to open. And when we studied the properties of this protein it became quite clear that it was also changing the activity of genes.” The coils of DNA seemed to open and close in response to histone modifications—inhaling, exhaling, inhaling, like life.

Allis walked me to his lab, a fluorescent-lit space overlooking the East River, divided by wide, polished-stone benches. A mechanical stirrer, whirring in a corner, clinked on the edge of a glass beaker. “Two features of histone modifications are notable,” Allis said. “First, changing histones can change the activity of a gene without affecting the sequence of the DNA.” It is, in short, formally epi-genetic, just as Waddington had imagined. “And, second, the histone modifications are passed from a parent cell to its daughter cells when cells divide. A cell can thus record ‘memory,’ and not just for itself but for all its daughter cells.”

…..

 

 

The New Yorker screws up big time with science: researchers criticize the Mukherjee piece on epigenetics

Jerry Coyne
https://whyevolutionistrue.wordpress.com/2016/05/05/the-new-yorker-screws-up-big-time-with-science-researchers-criticize-the-mukherjee-piece-on-epigenetics/

Abstract: This is a two part-post about a science piece on gene regulation that just appeared in the New Yorker. Today I give quotes from scientists criticizing that piece; tomorrow I’ll present a semi-formal critique of the piece by two experts in the field.

esterday I gave readers an assignment: read the new New Yorkerpiece by Siddhartha Mukherjee about epigenetics. The piece, called “Same but different” (subtitle: “How epigenetics can blur the line between nature and nurture”) was brought to my attention by two readers, both of whom praised it.  Mukherjee, a physician, is well known for writing the Pulitzer-Prize-winning book (2011) The Emperor of All Maladies: A Biography of Cancer. (I haven’t read it yet, but it’s on my list.)  Mukherjee has a new book that will be published in May: The Gene: An Intimate History. As I haven’t seen it, the New Yorker piece may be an excerpt from this book.

Everyone I know who has read The Emperor of All Maladies gives it high praise. I wish I could say the same for Mukherjee’s New Yorker piece. When I read it at the behest of the two readers, I found his analysis of gene regulation incomplete and superficial. Although I’m not an expert in that area, I knew that there was a lot of evidence that regulatory proteins called “transcription factors”, and not “epigenetic markers” (see discussion of this term tomorrow) or modified histones—the factors emphasized by Mukherjee—played hugely important roles in gene regulation. The speculations at the end of the piece about “Lamarckian evolution” via environmentally induced epigenetic changes in the genome were also unfounded, for we have no evidence for that kind of adaptive evolution. Mukherjee does, however, mention that lack of evidence, though I wish he’d done so more strongly given that environmental modification of DNA bases is constantly touted as an important and neglected factor in evolution.

Unbeknownst to me, there was a bit of a kerfuffle going on in the community of scientists who study gene regulation, with many of them finding serious mistakes and omissions in Mukherjee’s piece.  There appears to have been some back-and-forth emailing among them, and several wrote letters to the New Yorker, urging them to correct the misconceptions, omissions, and scientific errors in “Same but different.” As I understand it, both Mukherjee and the New Yorker simply batted these criticisms away, and, as far as I know, will not publish any corrections.  So today and tomorrow I’ll present the criticisms here, just so they’ll be on the record.

Because Mukherjee writes very well, and because even educated laypeople won’t know the story of gene regulation revealed over the last few decades,  they may not see the big lacunae in his piece. It is, then,  important to set matters straight, for at least we should know what science has told us about how genes are turned on and off. The criticism of Mukherjee’s piece, coming from scientists who really are experts in gene regulation, shows a lack of care on the part of Mukherjee and theNew Yorker: both a superficial and misleading treatment of the state of the science, and a failure of the magazine to properly vet this piece (I have no idea whether they had it “refereed” not just by editors but by scientists not mentioned in the piece).

Let me add one thing about science and the New Yorker. I believe I’ve said this before, but the way the New Yorker treats science is symptomatic of the “two cultures” problem. This is summarized in an email sent me a while back by a colleague, which I quote with permission:

The New Yorker is fine with science that either serves a literary purpose (doctors’ portraits of interesting patients) or a political purpose (environmental writing with its implicit critique of modern technology and capitalism). But the subtext of most of its coverage (there are exceptions) is that scientists are just a self-interested tribe with their own narrative and no claim to finding the truth, and that science must concede the supremacy of literary culture when it comes to anything human, and never try to submit human affairs to quantification or consilience with biology. Because the magazine is undoubtedly sophisticated in its writing and editing they don’t flaunt their postmodernism or their literary-intellectual proprietariness, but once you notice it you can make sense of a lot of their material.

. . . Obviously there are exceptions – Atul Gawande is consistently superb – but as soon as you notice it, their guild war on behalf of cultural critics and literary intellectuals against scientists, technologists, and analytic scholars becomes apparent.

…. more

Researchers criticize the Mukherjee piece on epigenetics: Part 2

Trigger warning: Long science post!

Yesterday I provided a bunch of scientists’ reactions—and these were big names in the field of gene regulation—to Siddhartha Mukherjee’s ill-informed piece in The New Yorker, “Same but different” (subtitle: “How epigenetics can blur the line between nature and nurture”). Today, in part 2, I provide a sentence-by-sentence analysis and reaction by two renowned researchers in that area. We’ll start with a set of definitions (provided by the authors) that we need to understand the debate, and then proceed to the critique.

Let me add one thing to avoid confusion: everything below the line, including the definition (except for my one comment at the end) was written by Ptashne and Greally.

by Mark Ptashne and John Greally

Introduction

Ptashne is The Ludwig Professor of Molecular Biology at the Memorial Sloan Kettering Cancer Center in New York. He wrote A Genetic Switch, now in its third edition, which describes the principles of gene regulation and the workings of a ‘switch’; and, with Alex Gann, Genes and Signals, which extends these principles and ideas to higher organisms and to other cellular processes as well.  John Greally is the Director of the Center for Epigenomics at the Albert Einstein College of Medicine in New York.

 

The New Yorker  (May 2, 2016) published an article entitled “Same But Different” written by Siddhartha Mukherjee.  As readers will have gathered from the letters posted yesterday, there is a concern that the article is misleading, especially for a non-scientific audience. The issue concerns our current understanding of “gene regulation” and how that understanding has been arrived at.

First some definitions/concepts:

Gene regulation refers to the “turning on and off of genes”.  The primary event in turning a gene “on” is to transcribe (copy) it into messenger RNA (mRNA). That mRNA is then decoded, usually, into a specific protein.  Genes are transcribed by the enzyme called RNA polymerase.

Development:  the process in which a fertilized egg (e.g., a human egg) divides many times and eventually forms an organism.  During this process, many of the roughly 23,000 genes of a human are turned “on” or “off” in different combinations, at different times and places in the developing organism. The process produces many different cell types in different organs (e.g. liver and brain), but all retain the original set of genes.

Transcription factors: proteins that bind to specific DNA sequences near specific genes and turn transcription of those genes on and off. A transcriptional ‘activator’, for example, bears two surfaces: one binds a specific sequence in DNA, and the other binds to, and thereby recruits to the gene, protein complexes that include RNA polymerase. It is widely acknowledged that the identity of a cell in the body depends on the array of transcription factors present in the cell, and the cell’s history.  RNA molecules can also recognize specific genomic sequences, and they too sometimes work as regulators.  Neither transcription factors nor these kinds of RNA molecules – the fundamental regulators of gene expression and development – are mentioned in the New Yorker article.

Signals:  these come in many forms (small molecules like estrogen, larger molecules (often proteins such as cytokines) that determine the ability of transcription factors to work.  For example, estrogen binds directly to a transcription factor (the estrogen receptor) and, by changing its shape, permits it to bind DNA and activate transcription.

Memory”:  a dividing cell can (often does) produce daughters that are identical, and that express identical genes as does the mother cell.  This occurs because the transcription factors present in the mother cell are passively transmitted to the daughters as the cell divides, and they go to work in their new contexts as before.  To make two different daughters, the cell must distribute its transcription factors asymmetrically.

Positive Feedback: An activator can maintain its own expression by  positive feedback.  This requires, simply, that a copy of the DNA sequence to which the activator binds is  present  near its own gene. Expression of the activator  then becomes self-perpetuating.  The activator (of which there now are many copies in the cell) activates  other target genes as it maintains its own expression. This kind of ‘memory circuit’, first described  in  bacteria, is found in higher organisms as well.  Positive feedback can explain how a fully differentiated cell (that is, a cell that has reached its developmental endpoint) maintains its identity.

Nucleosomes:  DNA in higher organisms (eukaryotes) is wrapped, like beads on a string, around certain proteins (called histones), to form nucleosomes.  The histones are subject to enzymatic modifications: e.g., acetyl, methyl, phosphate, etc. groups can be added to these structures. In bacteria there are no nucleosomes, and the DNA is more or less ‘naked’.

“Epigenetic modifications: please don’t worry about the word ”epigenetic”; it is misused in any case. What Mukherjee refers to by this term are the histone modifications mentioned above, and a modification to DNA itself: the addition of methyl groups. Keep in mind that the organisms that have taught us the most about development – flies (Drosophila) and worms (C. elegans)—do not have the enzymes required for DNA methylation. That does not mean that DNA methylation cannot do interesting things in humans, for example, but it is obviously not at the heart of gene regulation.

Specificity Development requires the highly specific sequential turning on and off of sets of genes.  Transcription factors and RNA supply this specificity, but   enzymes that impart modifications to histones  cannot: every nucleosome (and hence every gene) appears the same to the enzyme.  Thus such enzymes cannot pick out particular nucleosomes associated with particular genes to modify.  Histone modifications might be imagined to convey ‘memory’ as cells divide – but there are no convincing indications that this happens, nor are there molecular models that might explain why they would have the imputed effects.

Analysis and critique of Mukherjee’s article

The picture we have just sketched has taken the combined efforts of many scientists over 50 years to develop.  So what, then, is the problem with the New Yorker article?

There are two: first, the picture we have just sketched, emphasizing the primary role of transcription factors and RNA, is absent.  Second, that picture is replaced by highly dubious speculations, some of which don’t make sense, and none of which has been shown to work as imagined in the article.

(Quotes from the Mukherjee article are indented and in plain text; they are followed by comments, flush left and in bold, by Ptashne and Greally.)

In 1978, having obtained a Ph.D. in biology at Indiana University, Allis began to tackle a problem that had long troubled geneticists and cell biologists: if all the cells in the body have the same genome, how does one become a nerve cell, say, and another a blood cell, which looks and functions very differently?

The problems referred to were recognized long before 1978.  In fact, these were exactly the problems that the great French scientists François Jacob and Jacques Monod took on in the 1950s-60s.  In a series of brilliant experiments, Jacob and Monod showed that in bacteria, certain genes encode products that regulate (turn on and off) specific other genes.  Those regulatory molecules turned out to be proteins, some of which respond to signals from the environment.  Much of the story of modern biology has been figuring out how these proteins – in bacteria and in higher organisms  – bind to and regulate specific genes.  Of note is that in higher organisms, the regulatory proteins look and act like those in bacteria, despite the fact that eukaryotic DNA is wrapped in nucleosomes  whereas bacterial DNA is not.   We have also learned that certain RNA molecules can play a regulatory role, a phenomenon made possible by the fact that RNA molecules, like regulatory proteins, can recognize specific genomic sequences.

In the nineteen-forties, Conrad Waddington, an English embryologist, had proposed an ingenious answer: cells acquired their identities just as humans do—by letting nurture (environmental signals) modify nature (genes). For that to happen, Waddington concluded, an additional layer of information must exist within a cell—a layer that hovered, ghostlike, above the genome. This layer would carry the “memory” of the cell, recording its past and establishing its future, marking its identity and its destiny but permitting that identity to be changed, if needed. He termed the phenomenon “epigenetics”—“above genetics.”

This description greatly misrepresents the original concept.  Waddington argued that development proceeds not by the loss (or gain) of genes, which would be a “genetic” process, but rather that some genes would be selectively expressed in specific and complex cellular patterns as development proceeds.  He referred to this intersection of embryology (then called “epigenesis”) and genetics as “epigenetic”.We now understand that regulatory proteins work in combinations to turn on and off genes, including their own genes, and that sometimes the regulatory proteins respond to signals sent by other cells.  It should be emphasized that Waddington never proposed any “ghost-like” layer of additional information hovering above the gene.  This is a later misinterpretation of a literal translation of the term epigenetics, with “epi-“ meaning “above/upon” the genetic information encoded in DNA sequence.  Unfortunately, this new and pervasive definition encompasses all of transcriptional regulation and is of no practical value.

…..more

By 2000, Allis and his colleagues around the world had identified a gamut of proteins that could modify histones, and so modulate the activity of genes. Other systems, too, that could scratch different kinds of code on the genome were identified (some of these discoveries predating the identification of histone modifications). One involved the addition of a chemical side chain, called a methyl group, to DNA. The methyl groups hang off the DNA string like Christmas ornaments, and specific proteins add and remove the ornaments, in effect “decorating” the genome. The most heavily methylated parts of the genome tend to be dampened in their activity.

It is true that enzymes that modify histones have been found—lots of them.  A striking problem is that, after all this time, it is not at all clear what the vast majority of these modifications do.  When these enzymatic activities are eliminated by mutation of their active sites (a task substantially easier to accomplish in yeast than in higher organisms) they mostly have little or no effect on transcription.  It is not even clear that histones are the biologically relevant substrates of most of these enzymes.  

 In the ensuing decade, Allis wrote enormous, magisterial papers in which a rich cast of histone-modifying proteins appear and reappear through various roles, mapping out a hatchwork of complexity. . . These protein systems, overlaying information on the genome, interacted with one another, reinforcing or attenuating their signals. Together, they generated the bewildering intricacy necessary for a cell to build a constellation of other cells out of the same genes, and for the cells to add “memories” to their genomes and transmit these memories to their progeny. “There’s an epigenetic code, just like there’s a genetic code,” Allis said. “There are codes to make parts of the genome more active, and codes to make them inactive.”

By ‘epigenetic code’ the author seems to mean specific arrays of nucleosome modifications, imparted over time and cell divisions, marking genes for expression.  This idea has been tested in many experiments and has been found not to hold.

….. and more

 

Larry H. Bernstein, MD, FCAP

I hope that this piece brings greater clarity to the discussion.  I have heard the use of the term “epigenetics” for over a decade.  The term was never so clear.  I think that the New Yorker article was a reasonable article for the intended audience.  It was not intended to clarify debates about a mechanism for epigenetic based changes in evolutionary science.  I think it actually punctures the “classic model” of the cell depending only on double stranded DNA and transcription, which deflates our concept of the living cell.  The concept of epigenetics was never really formulated as far as I have seen, and I have done serious work in enzymology and proteins at a time that we did not have the technology that exists today.  I have considered with the critics that protein folding, protein misfolding, protein interactions with proximity of polar and nonpolar groups, and the regulatory role of microRNAs that are not involved in translation, and the evolving concept of what is “dark (noncoding) DNA” lend credence to the complexity of this discussion.  Even more interesting is the fact that enzymes (and isoforms of enzymes) have a huge role in cellular metabolic differences and in the function of metabolic pathways.  What is less understood is the extremely fast reactions involved in these cellular reactions.  These reactions are in my view critical drivers.  This is brought out by Erwin Schroedinger in the book What is Life? which infers that there can be no mathematical expression of life processes.

 

 

 

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Metabolic insight into cancer cell survival

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Revised 4/20/2016

AACR 2016: Novel Epigenetic Drug Therapeutics Revealed

http://www.genengnews.com/gen-news-highlights/aacr-2016-epigenetic-drug-therapeutics/81252634/

As the 2016 American Association for Cancer Research meeting begins to downshift toward a close, the presentation sessions certainly did not suffer from a lack of enthusiasm from attendees or high-quality research from presenters. Of particular note was a major symposium that discussed next-generation epigenetic therapeutics.

In the past several years, there have been a variety of epigenetic targets exploited by newly developed drug compounds, many of which have already progressed into clinical trials. Often these compounds will target specific classes of epigenetic regulators like acetylases and histone demethylases, for instance, the small-molecule inhibitors of protein interacting bromodomains—implicated in a diverse range of cancers—and methyltransferase inhibitors, such as lysyl demethylases (KDM).

However, for all of their recently achieved success, researchers are continually searching for increasingly rapid methods to validate epigenetic drug targets. Session Chairperson Udo Oppermann, Ph.D., principal investigator at the University of Oxford, stressed that open access research and continued investigator cooperation were key factors for driving rapid development of novel therapeutics in the field. Anecdotally, Dr. Oppermann noted that if biologists were a bit more like the international cooperative teams of physicists that discovered the Higgs boson or gravitational waves, many biological endeavors would advance rather quickly.

After providing the audience with a brief introduction to the symposium’s topic, Dr. Oppermann described his current research on histone demethylase inhibitors in multiple myeloma and the connection to metabolic pathways. He surmised that tricarboxylic acid (TCA) cycle-derived metabolites can link cellular metabolism to cancer—impacting epigenetic landscapes. Specifically, the TCA intermediates are inhibitors of KDMs, ultimately controlling epigenetic and metabolic regulation.

Furthermore, Dr. Oppermann’s group was able to show that treatment of myeloma cell lines with the potent and specific histone demethylase inhibitor GSK-J4 was able to reverse the Myc-driven metabolic dependency, forcing a selected amino acid depletion. This deficiency led to the integrated stress response and the activation of proapoptotic genes. This work helps to solidify further the potent nature of GSK-J4 in cancer while simultaneously uncovering the metabolic mechanisms that cancer cells employ to keep their overproliferative phenotypes progressing forward.

Next, Tomasz Cierpicki, Ph.D., assistant professor at the University of Michigan, described his work on targeting leukemic stem cells with small-molecule inhibitors of the protein regulator of cytokinesis 1 (PRC1). Dr. Cierpicki took the audience through his research design, which was to target BMI1, an oncogene that determines the proliferative capacity and self-renewal potential of normal and leukemic stem cells. BMI1 has been implicated in a variety of tumors and is essential for the Polycomb Repressive Complex 1 (PRC1). Moreover, BMI1 interacts with the RING1B protein to form an active E3 ubiquitin ligase that targets histone H2A, modifying epigenetic regulation mechanisms.

Dr. Cierpicki’s laboratory looked at inhibitors of the RING1B–BMI1 E3 ligase complex as potential therapeutic agents targeting cancer stem cells. Using an array of techniques from fragment screening to medicinal chemistry, the researchers were able to identify potent compounds that bound to RING1B–BMI1 and inhibit its E3 ubiquitin ligase activity with low micromolar affinities. When testing in vitro, the inhibitors revealed robust downregulation of H2A ubiquitination. Dr. Cierpicki and his colleagues found that the RING1–BMI1 inhibitor blocked the self-renewal capacity of the stem cells and induced cellular differentiation—validating RING ligases as a novel epigenetic drug target.

Finishing up the session was William Sellers, M.D., vice president and global head of oncology for the Novartis Institutes for BioMedical Research. Dr. Sellers’ research is focused on what genes are necessary for epigenetic regulation of cancer and how they are linked to essential metabolic processes. He and his colleagues accomplished their studies through the use of large-scale shRNA screening across a diverse set of 390 cancer cell lines.

Utilizing deep small hairpin RNA (shRNA) screening libraries, at 20 shRNAs per genome, provided the investigators with highly robust gene-level data, which resulted in the emergence of several distinct classes of cancer-dependent genes. For example, Dr. Sellers’ group found that several known oncogenes fell into the genetic dependence class, whereas other genes were sorted into lineage, paralog, and collateral synthetic lethality dependent classes.

An interesting example from Dr. Sellers’ work was the link his laboratory discovered between polyamine metabolism and salvage and the protein arginine methyltransferase 5 (PRMT5). In particular, the loss of methylthioadenosine phosphorylase (MTAP)—which has been observed in many solid tumors and hematologic malignancies—resulted in the accumulation of S-methyl-5′-thioadenosine (MTA), which specifically inhibited the epigenetic mechanisms of PRMT5. The culmination Dr. Sellers’ analysis led to the finding that PRMT5 is a novel target for therapeutic development in MTAP deleted cancers.

These three presentations represented some of the excellent, cutting-edge research that is not only looking to develop novel drug therapeutics but also trying to uncover the underlying molecular mechanisms of epigenetic regulation and cancer.

 

 

 

A Metabolic Twist that Drives Cancer Survival

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=190262

A novel metabolic pathway that helps cancer cells thrive in conditions that are lethal to normal cells has been identified.

 

“It’s long been thought that if we could target tumor-specific metabolic pathways, it could lead to effective ways to treat cancer,” said senior author Dr. Ralph DeBerardinis, Associate Professor of CRI and Pediatrics, Director of CRI’s Genetic and Metabolic Disease Program, and Chief of the Division of Pediatric Genetics and Metabolism at UT Southwestern. “This study finds that two very different metabolic processes are linked in a way that is specifically required for cells to adapt to the stress associated with cancer progression.”

The study, available online today in the journal Nature, reveals that cancer cells use an alternate version of two well-known metabolic pathways called the pentose phosphate pathway (PPP) and the Krebs cycle to defend against toxins. The toxins are reactive oxygen species (ROS) that kill cells via oxidative stress.

This work builds on earlier studies by Dr. DeBerardinis’ laboratory that found the Krebs cycle, a series of chemical reactions that cells use to generate energy, could reverse itself under certain conditions to nourish cancer cells.

Dr. DeBerardinis said most normal cells and tumor cells grow by attaching to nutrient-rich tissue called a matrix. “They are dependent on matrix attachment to receive growth-promoting signals and to regulate their metabolism in a way that supports cell growth, proliferation, and survival,” he said.

Detachment from the matrix results in a sudden increase in ROS that is lethal to normal cells, he added. Cancer cells seem to have a workaround.

The destruction of healthy cells when detached from the matrix was reported in a landmark 2009 Nature study by Harvard Medical School cell biologist Dr. Joan Brugge. Intriguingly, that same study found that inserting an oncogene – a gene with the potential to cause cancer – into a normal cell caused it to behave like a cancer cell and survive detachment, said Dr. DeBerardinis, who also is affiliated with the Eugene McDermott Center for Human Growth & Development, holds the Joel B. Steinberg, M.D. Chair in Pediatrics, and is a Sowell Family Scholar in Medical Research at UT Southwestern.

“Another Nature study, this one from CRI Director Dr. Sean Morrison’s laboratory in November 2015, found that the rare skin cancer cells that were able to detach from the primary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis said. Dr. Morrison, also a CPRIT Scholar in Cancer Research and a Howard Hughes Medical Institute Investigator, holds the Mary McDermott Cook Chair in Pediatrics Genetics at UT Southwestern.

Working under the premise that the two findings were pieces of the same puzzle, a crucial part of the picture seemed to be missing, he said.

It was known for decades that the PPP was a major source of NADPH, which provides a source of reducing equivalents (that is, electrons) to scavenge ROS; however, the PPP produces NADPH in a part of the cell called the cytosol, whereas the reactive oxygen species are generated primarily in another subcellular structure called the mitochondria.

“If you think of ROS as fire, then NADPH is like the water used by cancer cells to douse the flames,” Dr. DeBerardinis said. But how could NADPH from the PPP help deal with the stress of ROS produced in a completely different part of the cell? “What we did was to discover how this happens,” Dr. DeBerardinis said.

The current study in Nature demonstrates that cancer cells use a “piggybacking” system to carry reducing equivalents from the PPP into the mitochondria. This movement involves an unusual reaction in the cytosol that transfers reducing equivalents from NADPH to a molecule called citrate, similar to a reversed reaction of the Krebs cycle, he said. The citrate then enters the mitochondria and stimulates another pathway that results in the release of reducing equivalents to produce NADPH right at the location of ROS creation, allowing the cancer cells to survive and grow without the benefit of matrix attachment.

“We knew that both the PPP and Krebs cycle provide metabolic benefits to cancer cells.  But we had no idea that they were linked in this unusual fashion,” he said. “Strikingly, normal cells were unable to transport NADPH by this mechanism, and died as a result of the high ROS levels.”

Dr. DeBerardinis stressed that the findings were based on cultured cell models, and more research will be necessary to test the role of the pathway in living organisms. “We are particularly excited to test whether this pathway is required for metastasis, because cancer cells need to survive in a matrix-detached state in the circulation in order to metastasize,” he said.

CRI scientists find novel metabolic twist that drives cancer survival
http://www.utsouthwestern.edu/newsroom/news-releases/year-2016/april/cancer-metabolism.html

https://pharmaceuticalintelligence.com/2016/04/09/programmed-cell-death-and-cancer-therapy/5 days ago Cancer Cell Survival Driven by Novel Metabolic Pathway … This new study describes an alternate version of two wellknown metabolic pathways, the pentose phosphate pathway (PPP) and the Krebs cycle,…

http://www.nature.com/nature/journal/v481/n7381/abs/nature10642.html Jan 19, 2012 Nature | Letter …. DeBerardinis, R. J. et al. Beyond aerobic …. Andrew R. Mullen,; Pei-Hsuan Chen,; Tzuling Cheng &; Ralph J. DeBerardinis ..   

Haematopoietic stem cells require a highly regulated protein – Nature
http://www.nature.com/nature/journal/v509/n7498/abs/nature13035.html May 1, 2014 Nature | Article. Print; Share/ ….. synthesis. Nature Methods 6, 275–277 (2009) …. Robert A. J. Signer,; Jeffrey A. Magee &; Sean J. Morrison …       
http://www.nature.com/nature/journal/v527/n7577/abs/nature15726.html Nov 12, 2015 Nature | Article ….. Multistep nature of metastatic inefficiency: dormancy of solitary cells after successful extravasation and ….. Sean J. Morrison …     
Deep imaging of bone marrow shows non-dividing stem Nature
http://www.nature.com/nature/journal/v526/n7571/abs/nature15250.html   Oct 1, 2015 Nature | Letter. Print; Share/ …… Morrison, S. J. & Scadden, D. T. The bone marrow niche for ….. Kiranmai S. Kocherlakota &; Sean J. Morrison …
https://pharmaceuticalintelligence.com/category/cancer-biology-innovations-in-cancer-therapy/genomic-expression/Glutamine and cancer: cell biology, physiology, and clinical opportunities …. Metabolism of glutamine-derived α-ketoglutarate in the TCA cycle serves … known as hexosamines, that are used to glycosylate growth factor receptors and …… of two wellknown metabolic pathways, the pentose phosphate pathway (PPP )
The Mitochondrial Warburg Effect: A Cancer Enigma – IBC Journal
http://www.ibc7.org/article/file_down.php?pid=48&mode=article This feature of cancer cells is known as the Warburg effect, named … new paradigm of collaboration and a well-designed systemic approach will supply … Krebs cycle. … The pentose phosphate pathway uses glucose to produce ribose, which is used … glucose is taken up into cells, it is used in two main metabolic pathways

 

New paper offers intriguing insights into tumor metabolism     William G. Gilroy    August 19, 2009

Posted In: Research

A paper appearing in this week’s edition of the journal Nature by a team of researchers that includes University of Notre Dame biologist Zachary T. Schafer has important new implications for understanding the metabolism of tumors.

Schafer, an assistant professor of biological sciences and Coleman Junior Chair of Cancer Biology, points out that in the early stages of tumor formation some cells become detached from their normal cellular matrix. These “homeless” cells tend to develop certain defects that stop them from becoming cancerous. In a process known as apoptosis, these precancerous cells essentially kill themselves, allowing them to be destroyed by immune system cells.

The prevailing wisdom among researchers has been that apoptosis was the only way that cells could die.

In studies conducted prior to the research described in the Nature paper, it was found that even when apoptosis was inhibited in detached, precancerous cells, they still eventually died. Intrigued by these results, a team of researchers led by Joan S. Brugge, Louise Foote Pfieffer Professor of Cell Biology at Harvard Medical School, and Schafer decided to take a closer look.

They report in this week’s Nature paper that they found that even when apoptosis was inhibited in detached cells endowed with a cancer-causing gene, they still were incapable of absorbing glucose, their primary energy source. Additionally, the cells displayed signs of oxidative stress, which is a harmful accumulation of oxygen-derived molecules called reactive oxygen species (ROS). The research also revealed decreased ATP production, a key factor in energy transport in the cells.

Schafer notes that this combination of loss of glucose transport, decreased ATP production and heightened oxidative stress reveal a manner of cell death that hadn’t been previously demonstrated to play a role in this context.

In the next phase of the study, Schafer engineered the cells to express a high level of HER2, a gene known to be hyperactive in many breast cancer tumors. He also treated the cells with antioxidants to relieve oxidative stress.

Both approaches helped the cells survive. The HER2-treated cells regained glucose transport, avoided oxidative stress and recovered ATP levels.
Most surprisingly, the antioxidants restored metabolic activity in the cells by allowing fatty acids to be effectively used instead of glucose as an energy source, providing them with a chance to survive.

“Our results raise the possibility that antioxidant activity might allow early stage tumor cells to survive where they would otherwise die from these metabolic defects,” Schafer said.

He also cautions that while the antioxidant findings were surprising, their research was done solely in cell cultures and more research needs to be done before there are clear implications for individuals and their diets.

The paper does, however, offer important new clues about the metabolism of tumor cells and important information that may lead to drugs that can developed to target them.

 

Antioxidant and Oncogene Rescue of Metabolic Defects Caused by
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931797/Aug 19, 2009
Nature. Author manuscript; available in PMC 2010 Sep 2. Published in … Y. Irie,1 Sizhen Gao,1 Pere Puigserver,1,2 and Joan S. Brugge1,*.

http://www.nature.com/cdd/journal/v10/n8/full/4401251a.html
Proteasome inhibitors have been shown to be effective in cancer treatment, an ability … a specific inhibitor of 26S proteasome, also reduced cell viability ( 80% with 10 mu …
be a consequence of the increased generation of ROS caused by MG132. …. vectors endowed with the wild type forms of RB or p53 genes (Figure 1f).

 

The Metastasis-Promoting Roles of Tumor-Associated Immune Cells

Tumor metastasis is driven not only by the accumulation of intrinsic alterations in malignant cells, but also by the interactions of cancer cells with various stromal cell components of the tumor microenvironment. In particular, inflammation and infiltration of the tumor tissue by host immune cells, such as tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells have been shown to support tumor growth in addition to invasion and metastasis. Each step of tumor development, from initiation through metastatic spread, is promoted by communication between tumor and immune cells via the secretion of cytokines, growth factors and proteases that remodel the tumor microenvironment. Invasion and metastasis requires neovascularization, breakdown of the basement membrane, and remodeling of the extracellular matrix for tumor cell invasion and extravasation into the blood and lymphatic vessels. The subsequent dissemination of tumor cells to distant organ sites necessitates a treacherous journey through the vasculature, which is fostered by close association with platelets and macrophages. Additionally, the establishment of the pre-metastatic niche and specific metastasis organ tropism is fostered by neutrophils and bone marrow-derived hematopoietic immune progenitor cells and other inflammatory cytokines derived from tumor and immune cells, which alter the local environment of the tissue to promote adhesion of circulating tumor cells. This review focuses on the interactions between tumor cells and immune cells recruited to the tumor microenvironment, and examines the factors allowing these cells to promote each stage of metastasis.

 

Once established, tumors are quite adept at preventing anti-tumor immune responses, and several defense mechanisms to circumvent immune detection have been described including antigen loss, down-regulation of major histocompatibility molecules (MHC), deregulation or loss of components of the endogenous antigen presentation pathway, and tumor-induced immune suppression mediated through cytokine secretion or direct interactions between tumor ligands and immune cell receptors [2]. These mechanisms contribute to the process of immunoediting in which tumor cell subpopulations susceptible to immune recognition are lysed and eliminated, while resistant tumor cells proliferate and increase their frequency in the developing neoplasia [3]. However, tumors not only effectively escape immune recognition, they also actively subvert the normal anti-tumor activity of immune cells to promote further tumor growth and metastasis.

During early stages of cancer development, infiltrating immune cell populations are primarily tumor suppressive, but depending on the presence of accessory stromal cells, the local cytokine milieu, and tumor-specific interactions, these immune cells can undergo phenotypic changes to enhance tumor cell dissemination and metastasis. For instance, CD4+ T cells, macrophages, and neutrophils have all been shown to possess opposing properties depending on the inflammatory state of the tumor environment, the tissue context, and other cellular stimuli intrinsic to the altered tumor cells [4, 5]. These features are dependent upon the inherent plasticity of immune cells in response to stimulatory or suppressive cytokines [6]. Notably, the switch from a Th1 tumor-suppressive phenotype such as CD4+ “helper” T cells, which aid cytotoxic CD8+ T cells in tumor rejection, to a Th2 tumor-promoting “regulatory” phenotype, which blocks CD8+ T-cell activity, is a characteristic outcome in the inflammatory, immune-suppressive tumor microenvironment [5, 7]. Likewise, M1 macrophages and N1 neutrophils are known to have pronounced anti-tumor activity; however, these immune cells are often subverted to a tumor-promoting M2 and N2 phenotype, respectively, in response to immune-suppressive cytokines secreted by tumor tissue [8].

 

The crosstalk that occurs between tumor and immune cells within the tumor microenvironment, the circulation, or at distant metastatic sites has been clearly shown to foster metastatic dissemination. Immune cells as well as the suppressive factors that they secrete represent potential targets for therapeutic intervention. Regardless of their source, cytokines, chemokines, proteases, and growth factors are some of the main factors contributing to immunosuppression and immune-mediated tumor progression. These molecules can be produced by immune, stromal, or malignant cells and can act in paracrine and autocrine fashion to promote each stage of tumor cell invasion and metastasis by enhancing inflammation, angiogenesis, tumor proliferation, and recruitment of additional immunosuppressive and tumor-promoting immune cells. These secreted factors provide the malignant cells with an abundant source of growth and survival signals that perpetuate a supportive microenvironment for tumor metastasis and represent some of the most attractive targets for directed anti-tumor therapy. Immune pathways provide numerous soluble targets for cancer treatment, and indeed, many drugs to target immune-suppressive molecules are moving forward in clinical trials. For instance, the anti-RANKL (Denosumab) antibody has been shown to effectively inhibit bone metastasis in prostate cancer patients [201], while a variety of neutralizing antibodies to IL-1β and IL-1 receptor have been shown to have efficacy in treating metastasis in pre-clinical animal models [202]. Several agents that target IL-1 or other immune-suppressive cytokines are already approved for the treatment of some inflammatory diseases and are prime candidates for human trails [202]. Additionally, other proteins involved in tumor progression that are induced directly or indirectly by immune cell populations, such as EMT-associated transcription factors, adhesion molecules, and tumor receptors and ligands which mediate immune suppression, could also be targeted with small molecules or blocking antibodies. Antibodies against two surface molecules expressed by suppressive lymphoid cells, anti-CTLA-4 (ipiliumimab) [203, 204] and anti-PD-1 have been recently gaining increasing support from clinical trials for their effective treatment for many forms of cancer including advanced melanoma and prostate cancer [205, 206]. Specifically, anti-CTLA-4 has been shown to be particularly efficacious in metastatic melanoma, while anti-PD-1 has only just begun a comprehensive evaluation in clinical trials [204, 207]. Likewise, non-steroidal anti-inflammatory drugs (NSAIDS) to prevent or treat chronic inflammation and lymphangiogenesis [208210], and anti-coagulants to prevent platelet aggregation on circulating tumor cells [211] are just two examples of a multitude of therapeutic agents that could be utilized to prevent immune-mediated tumor progression at unique stages of metastasis. Of course, new methods or biomarkers for the detection of patients at risk of tumor progression or metastasis are also desperately needed to tailor personalized therapy for patients to obtain the best possible clinical outcome.

 

  1. https://pharmaceuticalintelligence.com/category/cancer-and-therapeutics/Mar 26, 2016 This turns your immune systems ability to attack and kill cancer cells back on” …. the rare skin cancer cells that were able to detach from theprimary tumor and successfully metastasize to other parts of the body had the ability to keep ROS levels from getting dangerously high,” Dr. DeBerardinis remarked.

  2. https://pharmaceuticalintelligence.com/tag/histone-deacetylase-inhibitors-hdac/The HDAC-inhibiting agent romidepsin significantly increased T-cell tumor … skin cancer cells that were able to detach from the primary tumor and successfully … of the body had the ability to keep ROS levels from getting dangerously high,” Dr. …. Sensitivity for EGFR or KRAS was higher in patients with multiplemetastatic …

  3. https://pharmaceuticalintelligence.com/category/cancer-biology-innovations-in-cancer-therapy/genomic-expression/In a study involving 320 patients, the researchers were able to infer cell death in …. Glutamine and cancer: cell biology, physiology, and clinical opportunities … On the other hand, GLS2 expression is enhanced in some neuroblastomas, …… of the body had the ability to keep ROS levels from getting dangerously high,” Dr.

 

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Genomics and epigenetics link to DNA structure

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Sequence and Epigenetic Factors Determine Overall DNA Structure

http://www.genengnews.com/gen-news-highlights/sequence-and-epigenetic-factors-determine-overall-dna-structure/81252592/

http://www.genengnews.com/Media/images/GENHighlight/Atomiclevelsimulationsshowingelectrostaticforcesbetweeneachatom1259202113.jpg

Atomic-level simulations show electrostatic forces between each atom. [Alek Aksimentiev, University of Illinois at Urbana-Champaign]

 

The traditionally held hypothesis about the highly ordered organization of DNA describes the interaction of various proteins with DNA sequences to mediate the dynamic structure of the molecule. However, recent evidence has emerged that stretches of homologous DNA sequences can associate preferentially with one another, even in the absence of proteins.

Researchers at the University of Illinois Center for the Physics of Living Cells, Johns Hopkins University, and Ulsan National Institute of Science and Technology (UNIST) in South Korea found that DNA molecules interact directly with one another in ways that are dependent on the sequence of the DNA and epigenetic factors, such as methylation.

The researchers described evidence they found for sequence-dependent attractive interactions between double-stranded DNA molecules that neither involve intermolecular strand exchange nor are mediated by DNA-binding proteins.

“DNA molecules tend to repel each other in water, but in the presence of special types of cations, they can attract each other just like nuclei pulling each other by sharing electrons in between,” explained lead study author Hajin Kim, Ph.D., assistant professor of biophysics at UNIST. “Our study suggests that the attractive force strongly depends on the nucleic acid sequence and also the epigenetic modifications.”

The investigators used atomic-level supercomputer simulations to measure the forces between a pair of double-stranded DNA helices and proposed that the distribution of methyl groups on the DNA was the key to regulating this sequence-dependent attraction. To verify their findings experimentally, the scientists were able to observe a single pair of DNA molecules within nanoscale bubbles.

“Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation,” the authors wrote. “We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine act as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction.”

The findings from this study were published recently in Nature Communications in an article entitled “Direct Evidence for Sequence-Dependent Attraction Between Double-Stranded DNA Controlled by Methylation.”

After conducting numerous further simulations, the research team concluded that direct DNA–DNA interactions could play a central role in how chromosomes are organized in the cell and which ones are expanded or folded up compactly, ultimately determining functions of different cell types or regulating the cell cycle.

“Biophysics is a fascinating subject that explores the fundamental principles behind a variety of biological processes and life phenomena,” Dr. Kim noted. “Our study requires cross-disciplinary efforts from physicists, biologists, chemists, and engineering scientists and we pursue the diversity of scientific disciplines within the group.”

Dr. Kim concluded by stating that “in our lab, we try to unravel the mysteries within human cells based on the principles of physics and the mechanisms of biology. In the long run, we are seeking for ways to prevent chronic illnesses and diseases associated with aging.”

 

Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

Jejoong Yoo, Hajin Kim, Aleksei Aksimentiev, and Taekjip Ha
Nature Communications 7 11045 (2016)    DOI:10.1038/ncomms11045BibTex

http://bionano.physics.illinois.edu/sites/default/files/styles/large/public/telepathy_figures_0.png?itok=VUJIHX2_

Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA–DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

Formation of a DNA double helix occurs through Watson–Crick pairing mediated by the complementary hydrogen bond patterns of the two DNA strands and base stacking. Interactions between double-stranded (ds)DNA molecules in typical experimental conditions containing mono- and divalent cations are repulsive1, but can turn attractive in the presence of high-valence cations2. Theoretical studies have identified the ion–ion correlation effect as a possible microscopic mechanism of the DNA condensation phenomena3, 4, 5. Theoretical investigations have also suggested that sequence-specific attractive forces might exist between two homologous fragments of dsDNA6, and this ‘homology recognition’ hypothesis was supported by in vitro atomic force microscopy7 and in vivo point mutation assays8. However, the systems used in these measurements were too complex to rule out other possible causes such as Watson–Crick strand exchange between partially melted DNA or protein-mediated association of DNA.

Here we present direct evidence for sequence-dependent attractive interactions between dsDNA molecules that neither involve intermolecular strand exchange nor are mediated by proteins. Further, we find that the sequence-dependent attraction is controlled not by homology—contradictory to the ‘homology recognition’ hypothesis6—but by a methylation pattern. Unlike the previous in vitro study that used monovalent (Na+) or divalent (Mg2+) cations7, we presumed that for the sequence-dependent attractive interactions to operate polyamines would have to be present. Polyamine is a biological polycation present at a millimolar concentration in most eukaryotic cells and essential for cell growth and proliferation9, 10. Polyamines are also known to condense DNA in a concentration-dependent manner2, 11. In this study, we use spermine4+(Sm4+) that contains four positively charged amine groups per molecule.

Sequence dependence of DNA–DNA forces

To characterize the molecular mechanisms of DNA–DNA attraction mediated by polyamines, we performed molecular dynamics (MD) simulations where two effectively infinite parallel dsDNA molecules, 20 base pairs (bp) each in a periodic unit cell, were restrained to maintain a prescribed inter-DNA distance; the DNA molecules were free to rotate about their axes. The two DNA molecules were submerged in 100mM aqueous solution of NaCl that also contained 20 Sm4+molecules; thus, the total charge of Sm4+, 80 e, was equal in magnitude to the total charge of DNA (2 × 2 × 20 e, two unit charges per base pair; Fig. 1a). Repeating such simulations at various inter-DNA distances and applying weighted histogram analysis12 yielded the change in the interaction free energy (ΔG) as a function of the DNA–DNA distance (Fig. 1b,c). In a broad agreement with previous experimental findings13, ΔG had a minimum, ΔGmin, at the inter-DNA distance of 25−30Å for all sequences examined, indeed showing that two duplex DNA molecules can attract each other. The free energy of inter-duplex attraction was at least an order of magnitude smaller than the Watson–Crick interaction free energy of the same length DNA duplex. A minimum of ΔG was not observed in the absence of polyamines, for example, when divalent or monovalent ions were used instead14, 15.

Figure 1: Polyamine-mediated DNA sequence recognition observed in MD simulations and smFRET experiments.
Polyamine-mediated DNA sequence recognition observed in MD simulations and smFRET experiments.

(a) Set-up of MD simulations. A pair of parallel 20-bp dsDNA duplexes is surrounded by aqueous solution (semi-transparent surface) containing 20 Sm4+ molecules (which compensates exactly the charge of DNA) and 100mM NaCl. Under periodic boundary conditions, the DNA molecules are effectively infinite. A harmonic potential (not shown) is applied to maintain the prescribed distance between the dsDNA molecules. (b,c) Interaction free energy of the two DNA helices as a function of the DNA–DNA distance for repeat-sequence DNA fragments (b) and DNA homopolymers (c). (d) Schematic of experimental design. A pair of 120-bp dsDNA labelled with a Cy3/Cy5 FRET pair was encapsulated in a ~200-nm diameter lipid vesicle; the vesicles were immobilized on a quartz slide through biotin–neutravidin binding. Sm4+ molecules added after immobilization penetrated into the porous vesicles. The fluorescence signals were measured using a total internal reflection microscope. (e) Typical fluorescence signals indicative of DNA–DNA binding. Brief jumps in the FRET signal indicate binding events. (f) The fraction of traces exhibiting binding events at different Sm4+ concentrations for AT-rich, GC-rich, AT nonhomologous and CpG-methylated DNA pairs. The sequence of the CpG-methylated DNA specifies the methylation sites (CG sequence, orange), restriction sites (BstUI, triangle) and primer region (underlined). The degree of attractive interaction for the AT nonhomologous and CpG-methylated DNA pairs was similar to that of the AT-rich pair. All measurements were done at [NaCl]=50mM and T=25°C. (g) Design of the hybrid DNA constructs: 40-bp AT-rich and 40-bp GC-rich regions were flanked by 20-bp common primers. The two labelling configurations permit distinguishing parallel from anti-parallel orientation of the DNA. (h) The fraction of traces exhibiting binding events as a function of NaCl concentration at fixed concentration of Sm4+ (1mM). The fraction is significantly higher for parallel orientation of the DNA fragments.

Unexpectedly, we found that DNA sequence has a profound impact on the strength of attractive interaction. The absolute value of ΔG at minimum relative to the value at maximum separation, |ΔGmin|, showed a clearly rank-ordered dependence on the DNA sequence: |ΔGmin| of (A)20>|ΔGmin| of (AT)10>|ΔGmin| of (GC)10>|ΔGmin| of (G)20. Two trends can be noted. First, AT-rich sequences attract each other more strongly than GC-rich sequences16. For example, |ΔGmin| of (AT)10 (1.5kcalmol−1 per turn) is about twice |ΔGmin| of (GC)10 (0.8kcalmol−1 per turn) (Fig. 1b). Second, duplexes having identical AT content but different partitioning of the nucleotides between the strands (that is, (A)20 versus (AT)10 or (G)20 versus (GC)10) exhibit statistically significant differences (~0.3kcalmol−1 per turn) in the value of |ΔGmin|.

To validate the findings of MD simulations, we performed single-molecule fluorescence resonance energy transfer (smFRET)17 experiments of vesicle-encapsulated DNA molecules. Equimolar mixture of donor- and acceptor-labelled 120-bp dsDNA molecules was encapsulated in sub-micron size, porous lipid vesicles18 so that we could observe and quantitate rare binding events between a pair of dsDNA molecules without triggering large-scale DNA condensation2. Our DNA constructs were long enough to ensure dsDNA–dsDNA binding that is stable on the timescale of an smFRET measurement, but shorter than the DNA’s persistence length (~150bp (ref. 19)) to avoid intramolecular condensation20. The vesicles were immobilized on a polymer-passivated surface, and fluorescence signals from individual vesicles containing one donor and one acceptor were selectively analysed (Fig. 1d). Binding of two dsDNA molecules brings their fluorescent labels in close proximity, increasing the FRET efficiency (Fig. 1e).

FRET signals from individual vesicles were diverse. Sporadic binding events were observed in some vesicles, while others exhibited stable binding; traces indicative of frequent conformational transitions were also observed (Supplementary Fig. 1A). Such diverse behaviours could be expected from non-specific interactions of two large biomolecules having structural degrees of freedom. No binding events were observed in the absence of Sm4+ (Supplementary Fig. 1B) or when no DNA molecules were present. To quantitatively assess the propensity of forming a bound state, we chose to use the fraction of single-molecule traces that showed any binding events within the observation time of 2min (Methods). This binding fraction for the pair of AT-rich dsDNAs (AT1, 100% AT in the middle 80-bp section of the 120-bp construct) reached a maximum at ~2mM Sm4+(Fig. 1f), which is consistent with the results of previous experimental studies2, 3. In accordance with the prediction of our MD simulations, GC-rich dsDNAs (GC1, 75% GC in the middle 80bp) showed much lower binding fraction at all Sm4+ concentrations (Fig. 1b,c). Regardless of the DNA sequence, the binding fraction reduced back to zero at high Sm4+ concentrations, likely due to the resolubilization of now positively charged DNA–Sm4+ complexes2, 3, 13.

Because the donor and acceptor fluorophores were attached to the same sequence of DNA, it remained possible that the sequence homology between the donor-labelled DNA and the acceptor-labelled DNA was necessary for their interaction6. To test this possibility, we designed another AT-rich DNA construct AT2 by scrambling the central 80-bp section of AT1 to remove the sequence homology (Supplementary Table 1). The fraction of binding traces for this nonhomologous pair of donor-labelled AT1 and acceptor-labelled AT2 was comparable to that for the homologous AT-rich pair (donor-labelled AT1 and acceptor-labelled AT1) at all Sm4+ concentrations tested (Fig. 1f). Furthermore, this data set rules out the possibility that the higher binding fraction observed experimentally for the AT-rich constructs was caused by inter-duplex Watson–Crick base pairing of the partially melted constructs.

Next, we designed a DNA construct named ATGC, containing, in its middle section, a 40-bp AT-rich segment followed by a 40-bp GC-rich segment (Fig. 1g). By attaching the acceptor to the end of either the AT-rich or GC-rich segments, we could compare the likelihood of observing the parallel binding mode that brings the two AT-rich segments together and the anti-parallel binding mode. Measurements at 1mM Sm4+ and 25 or 50mM NaCl indicated a preference for the parallel binding mode by ~30% (Fig. 1h). Therefore, AT content can modulate DNA–DNA interactions even in a complex sequence context. Note that increasing the concentration of NaCl while keeping the concentration of Sm4+ constant enhances competition between Na+ and Sm4+ counterions, which reduces the concentration of Sm4+ near DNA and hence the frequency of dsDNA–dsDNA binding events (Supplementary Fig. 2).

Methylation determines the strength of DNA–DNA attraction

Analysis of the MD simulations revealed the molecular mechanism of the polyamine-mediated sequence-dependent attraction (Fig. 2). In the case of the AT-rich fragments, the bulky methyl group of thymine base blocks Sm4+ binding to the N7 nitrogen atom of adenine, which is the cation-binding hotspot21, 22. As a result, Sm4+ is not found in the major grooves of the AT-rich duplexes and resides mostly near the DNA backbone (Fig. 2a,d). Such relocated Sm4+ molecules bridge the two DNA duplexes better, accounting for the stronger attraction16, 23, 24, 25. In contrast, significant amount of Sm4+ is adsorbed to the major groove of the GC-rich helices that lacks cation-blocking methyl group (Fig. 2b,e).

Figure 2: Molecular mechanism of polyamine-mediated DNA sequence recognition.
Molecular mechanism of polyamine-mediated DNA sequence recognition.

(ac) Representative configurations of Sm4+ molecules at the DNA–DNA distance of 28Å for the (AT)10–(AT)10 (a), (GC)10–(GC)10 (b) and (GmC)10–(GmC)10 (c) DNA pairs. The backbone and bases of DNA are shown as ribbon and molecular bond, respectively; Sm4+ molecules are shown as molecular bonds. Spheres indicate the location of the N7 atoms and the methyl groups. (df) The average distributions of cations for the three sequence pairs featured in ac. Top: density of Sm4+ nitrogen atoms (d=28Å) averaged over the corresponding MD trajectory and the z axis. White circles (20Å in diameter) indicate the location of the DNA helices. Bottom: the average density of Sm4+ nitrogen (blue), DNA phosphate (black) and sodium (red) atoms projected onto the DNA–DNA distance axis (x axis). The plot was obtained by averaging the corresponding heat map data over y=[−10, 10] Å. See Supplementary Figs 4 and 5 for the cation distributions at d=30, 32, 34 and 36Å.

If indeed the extra methyl group in thymine, which is not found in cytosine, is responsible for stronger DNA–DNA interactions, we can predict that cytosine methylation, which occurs naturally in many eukaryotic organisms and is an essential epigenetic regulation mechanism26, would also increase the strength of DNA–DNA attraction. MD simulations showed that the GC-rich helices containing methylated cytosines (mC) lose the adsorbed Sm4+ (Fig. 2c,f) and that |ΔGmin| of (GC)10 increases on methylation of cytosines to become similar to |ΔGmin| of (AT)10 (Fig. 1b).

To experimentally assess the effect of cytosine methylation, we designed another GC-rich construct GC2 that had the same GC content as GC1 but a higher density of CpG sites (Supplementary Table 1). The CpG sites were then fully methylated using M. SssI methyltransferase (Supplementary Fig. 3; Methods). As predicted from the MD simulations, methylation of the GC-rich constructs increased the binding fraction to the level of the AT-rich constructs (Fig. 1f).

The sequence dependence of |ΔGmin| and its relation to the Sm4+ adsorption patterns can be rationalized by examining the number of Sm4+ molecules shared by the dsDNA molecules (Fig. 3a). An Sm4+ cation adsorbed to the major groove of one dsDNA is separated from the other dsDNA by at least 10Å, contributing much less to the effective DNA–DNA attractive force than a cation positioned between the helices, that is, the ‘bridging’ Sm4+ (ref. 23). An adsorbed Sm4+ also repels other Sm4+ molecules due to like-charge repulsion, lowering the concentration of bridging Sm4+. To demonstrate that the concentration of bridging Sm4+ controls the strength of DNA–DNA attraction, we computed the number of bridging Sm4+ molecules, Nspm (Fig. 3b). Indeed, the number of bridging Sm4+ molecules ranks in the same order as |ΔGmin|: Nspm of (A)20>Nspm of (AT)10Nspm of (GmC)10>Nspm of (GC)10>Nspm of (G)20. Thus, the number density of nucleotides carrying a methyl group (T and mC) is the primary determinant of the strength of attractive interaction between two dsDNA molecules. At the same time, the spatial arrangement of the methyl group carrying nucleotides can affect the interaction strength as well (Fig. 3c). The number of methyl groups and their distribution in the (AT)10 and (GmC)10 duplex DNA are identical, and so are their interaction free energies, |ΔGmin| of (AT)10Gmin| of (GmC)10. For AT-rich DNA sequences, clustering of the methyl groups repels Sm4+ from the major groove more efficiently than when the same number of methyl groups is distributed along the DNA (Fig. 3b). Hence, |ΔGmin| of (A)20>|ΔGmin| of (AT)10. For GC-rich DNA sequences, clustering of the cation-binding sites (N7 nitrogen) attracts more Sm4+ than when such sites are distributed along the DNA (Fig. 3b), hence |ΔGmin| is larger for (GC)10 than for (G)20.

Figure 3: Methylation modulates the interaction free energy of two dsDNA molecules by altering the number of bridging Sm4+.
Methylation modulates the interaction free energy of two dsDNA molecules by altering the number of bridging Sm4+.

(a) Typical spatial arrangement of Sm4+ molecules around a pair of DNA helices. The phosphates groups of DNA and the amine groups of Sm4+ are shown as red and blue spheres, respectively. ‘Bridging’ Sm4+molecules reside between the DNA helices. Orange rectangles illustrate the volume used for counting the number of bridging Sm4+ molecules. (b) The number of bridging amine groups as a function of the inter-DNA distance. The total number of Sm4+ nitrogen atoms was computed by averaging over the corresponding MD trajectory and the 10Å (x axis) by 20Å (y axis) rectangle prism volume (a) centred between the DNA molecules. (c) Schematic representation of the dependence of the interaction free energy of two DNA molecules on their nucleotide sequence. The number and spatial arrangement of nucleotides carrying a methyl group (T or mC) determine the interaction free energy of two dsDNA molecules.

Genome-wide investigations of chromosome conformations using the Hi–C technique revealed that AT-rich loci form tight clusters in human nucleus27, 28. Gene or chromosome inactivation is often accompanied by increased methylation of DNA29 and compaction of facultative heterochromatin regions30. The consistency between those phenomena and our findings suggest the possibility that the polyamine-mediated sequence-dependent DNA–DNA interaction might play a role in chromosome folding and epigenetic regulation of gene expression.

  1. Rau, D. C., Lee, B. & Parsegian, V. A. Measurement of the repulsive force between polyelectrolyte molecules in ionic solution: hydration forces between parallel DNA double helices. Proc. Natl Acad. Sci. USA 81, 26212625 (1984).
  2. Raspaud, E., Olvera de la Cruz, M., Sikorav, J. L. & Livolant, F. Precipitation of DNA by polyamines: a polyelectrolyte behavior. Biophys. J. 74, 381393 (1998).
  3. Besteman, K., Van Eijk, K. & Lemay, S. G. Charge inversion accompanies DNA condensation by multivalent ions. Nat. Phys. 3, 641644 (2007).
  4. Lipfert, J., Doniach, S., Das, R. & Herschlag, D. Understanding nucleic acid-ion interactions.Annu. Rev. Biochem. 83, 813841 (2014).
  5. Grosberg, A. Y., Nguyen, T. T. & Shklovskii, B. I. The physics of charge inversion in chemical and biological systems. Rev. Mod. Phys. 74, 329345 (2002).
  6. Kornyshev, A. A. & Leikin, S. Sequence recognition in the pairing of DNA duplexes. Phys. Rev. Lett. 86, 36663669 (2001).
  7. Danilowicz, C. et al. Single molecule detection of direct, homologous, DNA/DNA pairing.Proc. Natl Acad. Sci. USA 106, 1982419829 (2009).
  8. Gladyshev, E. & Kleckner, N. Direct recognition of homology between double helices of DNA in Neurospora crassa. Nat. Commun. 5, 3509 (2014).
  9. Tabor, C. W. & Tabor, H. Polyamines. Annu. Rev. Biochem. 53, 749790 (1984).
  10. Thomas, T. & Thomas, T. J. Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell. Mol. Life Sci. 58, 244258 (2001).

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Epigenetics, Environment and Cancer: Articles of Note @PharmaceuticalIntelligence.com

Author and Curator: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

 

Introduction

Author: Larry H. Bernstein, MD, FCAP

The following discussions are presented in two series. The first set of discussions is mainly concerned with the role of genomics in the rapidly emerging research domain of genomics and medicine. The recent advances in genomic research at the end of the 20th century brought into the new millennium a seminal accomplishment because of the mapping of the human genome. This development required advances in technology that touches on biochemistry, organic chemistry, physical chemistry, mathematics and computational sciences that have been followed by a surge of innovation for the last 15 years. This was an accomplishment of basic science research that can be ascribed to substantial leadership from the National Institutes of Health, and to a diversity of research centers within the United States, England, France, and Germany, and Israel among others.

In looking back at this development, it might appear to be weighted heavily in a concentrated work on the genetic code. This was predated by the discovery of genetic inborn errors of metabolism that was at least a half century precedent. Thus a model was constructed for the accounting for many human conditions that are expressed in-utero, perinatal, postnatal, and at critical life stages.   However, even allowing for over-simplification of a model of life reduced to the expression of a genetic code, this has led to the genesis of a concept of genetic clarification of life “maladies”, diagnostic, therapeutic, and prognostic implications. The concept of a “personalized medicine” emerges from such a construct.

I have already ceded considerable ground in an argument of what occurs in life, illness, and death at the cellular, organ, and organ system level. There are indeed gene amplifications and downregulation of genes that are expressed or have an “on-off” nature in transcription, which becomes a major driver of metabolic control. In this respect, the classic model of gene-RNA-protein has been superseded by a much more complicated model, but still in the realm of personalized medicine. The classic model of metabolism is tied to anabolic and catabolic pathways, glycolytic and mitochondrial substrates, amino acids, proteins and 3D-protein aggregates that have functional roles, and that is controlled by allosteric interactions, ion transport, membrane affinity, signaling pathways, and hydrophilic and hydrophobic effects. This leads to the second part of the discussion about epigenetics and environmental impacts on cellular function. It is by no means irrelevant because the evolution of organisms from sea to land, and the existence of living forms in mountainous and desert regions imposed restrictions that required adaptation. A full understanding of these factors is required in the immersion in personalized medicine.

Genetics Impact on Physiology

 

A Perspective on Personalized Medicine

Curator: Larry H. Bernstein, MD, FCAP

 

Precision Medicine for Future of Genomics Medicine is The New Era

Demet Sag, PhD, CRA, GCP

 

Epistemology of the Origin of Cancer: a New Paradigm – New Cancer Theory by two US Scientists in peer-reviewed Cancer Journal

Reporter: Aviva Lev-Ari, PhD, RN

 

A Reconstructed View of Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP

 

Signaling and Signaling Pathways

Curator: Larry H. Bernstein, MD, FCAP

 

Gene Amplification and Activation of the Hedgehog Pathway

Curator: Larry H Bernstein, MD, FCAP

 

Pancreatic Cancer and Crossing Roads of Metabolism

Curator: Demet Sag, PhD

 

Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

Reviewer and Curator: Larry H. Bernsteag, MD, FCAP

 

Acetylation and Deacetylation of non-Histone Proteins

Author and Curator: Larry H Bernstein, MD, FCAP

 

Pull at Cancer’s Levers

Author and Curator, Larry H. Bernstein, MD, FCAP

 

Epilogue: Envisioning New Insights in Cancer Translational Biology

Author and Curator: Larry H Bernstein, MD, FCAP

 

Directions for Genomics in Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP

 

What is the Future for Genomics in Clinical Medicine?

Author and Curator: Larry H Bernstein, MD, FCAP

 

Environmental Factors Impacting Genetic Mutations

 

Deciphering the Epigenome

Curator: Larry H. Bernstein, MD, FCAP

 

The Underappreciated EpiGenome

Author:  Demet Sag, PhD

 

Introduction to Metabolomics

Curator: Larry H Bernstein, MD, FCAP

 

The Metabolic View of Epigenetic Expression

Writer and Curator: Larry H Bernstein, MD, FCAP

 

Somatic, germ-cell, and whole sequence DNA in cell lineage and disease profiling

Curator: Larry H Bernstein, MD, FCAP

 

RNA and the transcription the genetic code

Curator: Larry H. Bernstein, MD, FCAP

 

Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H Bernstein, MD, FCAP

 

Genomics and Epigenetics: Genetic Errors and Methodologies – Cancer and Other Diseases

Writer and Curator: Larry H Bernstein, MD, FCAP

 

BET Proteins Connect Diabetes and Cancer

Author and Curator: Larry H. Bernstein, MD, FCAP

Cancer Metastasis

Author: Tilda Barliya PhD

 

Issues in Personalized Medicine: Discussions of Intratumor Heterogeneity from the Oncology Pharma forum on LinkedIn

Curator and Writer: Stephen J. Williams, Ph.D.

 

Immuno therapy at MassBio, 3/31 and 4/1/2016.

2016 MassBio Annual Meeting 03/31/2016 8:00 AM – 04/01/2016 3:00 PM Royal Sonesta Hotel, Cambridge, MA

Reporter: Aviva Lev-Ari, PhD, RN

Plenary Session: Immunotherapy in Combination, 2016 MassBio Annual Meeting 03/31/2016 8:00 AM – 04/01/2016 3:00 PM Royal Sonesta Hotel, Cambridge, MA

Plenary Session: Innovative Pricing Pricing Models: The Future is Now, 2016 MassBio Annual Meeting 03/31/2016 8:00 AM – 04/01/2016 3:00 PM Royal Sonesta Hotel, Cambridge, MA

2-Plenary Session: Advanced Manufacturing, 2016 MassBio Annual Meeting 03/31/2016 8:00 AM – 04/01/2016 3:00 PM Royal Sonesta Hotel, Cambridge, MA

Plenary Session: The 2016 National Landscape, 2016 MassBio Annual Meeting 03/31/2016 8:00 AM – 04/01/2016 3:00 PM Royal Sonesta Hotel, Cambridge, MA – 4/1 @11AM

LIVE @ Congressman Richard Neal – D-MA, Dean of MA Delegation 2016 MassBio Annual Meeting 04/01/2016 11AM Royal Sonesta Hotel, Cambridge, MA

LIVE Remarks by Rachel Kaprielian, Tony Chat and Mike Huckman @ 2016 MassBio Annual Meeting 04/01/2016 12:45 PM Royal Sonesta Hotel, Cambridge, MA

 

Summary

Larry H. Bernstein, MD, FCAP

 

The preceding chapters have provided a substantial insight into the growth and acceleration of work related to translational medicine and personalized medicine. I make note of the fact that a substantial knowledge has been from basic research using animal models, including C. Eligans. The amount of knowledge is quite impressive. Let me review some major points gained from these presentations.

  1. Non-coding areas of our DNA are far from being without function. But the ensuing work with RNAs is captivating. Whether regulating gene expression and transcription, or providing protein attachment sites, this once-dismissed part of the genome is vital for all life.

There are two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), each with two fused rings, and the pyrimidines (cytosine [C], thymine [T], and uracil [U]), each with a single ring. Furthermore, it is now widely accepted that RNA contains only A, G, C, and U (no T), whereas DNA contains only A, G, C, and T (no U).

There is no uncertainty about the importance of “Junk DNA”.  It is both an evolutionary remnant, and it has a role in cell regulation.  Further, the role of histones in their relationship the oligonucleotide sequences is not understood.  We now have a large output of research on noncoding RNA, including siRNA, miRNA, and others with roles other than transcription. This requires major revision of our model of cell regulatory processes.  The classic model is solely transcriptional.

  • DNA-> RNA-> Amino Acid in a protein.

Redrawn we have

  • DNA-> RNA-> DNA and
  • DNA->RNA-> protein-> DNA.

DNA is involved mainly with genetic information storage, while RNA molecules—mRNA, rRNA, tRNA, miRNA, and others—are engaged in diverse structural, catalytic, and regulatory activities, in addition to translating genes into proteins. RNA’s multitasking prowess, at the heart of the RNA World hypothesis implicating RNA as the first molecule of life, likely spurred the evolution of numerous modified nucleotides. This enabled the diversified complementarity and secondary structures that allow RNA species to specifically interact with other components of the cellular machinery such as DNA and proteins. The alphabet of RNA consists of at least 140 alternative nucleotide forms.

Among the 140 modified RNA nucleotide variants identified, methylation of adenosine at the N6 position (m6A) is the most prevalent epigenetic mark in eukaryotic mRNA. Identified in bacterial rRNAs and tRNAs as early as the 1950s, this type of methylation was subsequently found in other RNA molecules, including mRNA, in animal and plant cells as well. In 1984, researchers identified a site that was specifically methylated—the 3′ untranslated region (UTR) of bovine prolactin mRNA.1 As more sites of m6A modification were identified, a consistent pattern emerged: the methylated A is preceded by A or G and followed by C (A/G—methylated A—C).

Although the identification of m6A in RNA is 40 years old, until recently researchers lacked efficient molecular mapping and quantification methods to fully understand the functional implications of the modification. In 2012, we (D.D. and G.R.) combined the power of next-generation sequencing (NGS) with traditional antibody-mediated capture techniques to perform high-resolution transcriptome-wide mapping of m6A, an approach we termed m6A-seq.2 Briefly, the transcriptome is randomly fragmented and an anti-m6A antibody is used to fish out the methylated RNA fragments; the m6A-containing fragments are then sequenced and aligned to the genome, thus allowing us to locate the positions of methylation marks.

  1. The work of Warburg and Meyerhoff, followed by that of Krebs, Kaplan, Chance, and others built a solid foundation in the knowledge of enzymes, coenzymes, adenine and pyridine nucleotides, and metabolic pathways, not to mention the importance of Fe3+, Cu2+, Zn2+, and other metal cofactors.

Of huge importance was the work of Jacob, Monod and Changeux, and the effects of cooperativity in allosteric systems and of repulsion in tertiary structure of proteins related to hydrophobic and hydrophilic interactions, which involves the effect of one ligand on the binding or catalysis of another, demonstrated by the end-product inhibition of the enzyme, L-threonine deaminase (Changeux 1961), L-isoleucine, which differs sterically from the reactant, L-threonine whereby the former could inhibit the enzyme without competing with the latter. The current view based on a variety of measurements (e.g., NMR, FRET, and single molecule studies) is a ‘‘dynamic’’ proposal by Cooper and Dryden (1984) that the distribution around the average structure changes in allostery affects the subsequent (binding) affinity at a distant site.

Present day applications of computational methods to biomolecular systems, combined with      structural, thermodynamic, and kinetic studies, make possible an approach to that question, so as to provide a deeper understanding of the requirements for allostery. The current view is that a variety of measurements (e.g., NMR, FRET, and single molecule studies) are providing additional data beyond that available previously from structural, thermodynamic, and kinetic results. These should serve to continue to improve our understanding of the molecular mechanism of allostery

  1. Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. The measurement of free radicals has increased awareness of radical-induced impairment of the oxidative/antioxidative balance, essential for an understanding of disease progression. Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Various studies have confirmed that metals activate signaling pathways and the carcinogenic effect of metals has been related to activation of mainly redox sensitive transcription factors, involving NF-kappaB, AP-1 and p53.
  1. There is heterogeneity in the immediate interstices between cancer cells, which may seem surprising, but it should not be.  This refers to the complexity of the cells arranged as tissues and to their immediate environment, which I shall elaborate on. Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups.

IDH1 mutations have been identified at the Arg132 codon. Mutations in IDH2 have been identified at the Arg140 codon, as well as at Arg172, which is aligned with IDH1 Arg132. IDH1 and IDH2 mutations are heterozygous in cancer, and they catalyze the production of α-2-hydroxyglutarate. The study found human IDH1 transitions between an inactive open, an inactive semi-open, and a catalytically active closed conformation. In the inactive open conformation, Asp279 occupies the position where the isocitrate substrate normally forms hydrogen bonds with Ser94. This steric hindrance by Asp279 to isocitrate binding is relieved in the active closed conformation.

There are allelic variations that underlie common diseases and complete genome sequencing for many individuals with and without disease is required. However, there are advantages and disadvantages as we can carry out partial surveys of the genome by genotyping large numbers of common SNPs in genome-wide association studies but there are problems such as computing the data efficiently and sharing the information without tempering privacy.

Since the first report of p53 as a non-histone target of a histone acetyltransferase (HAT), there has been a rapid proliferation in the description of new non-histone targets of HATs. Of these,

  • transcription factors comprise the largest class of new targets.

The substrates for HATs extend to

  1. cytoskeletal proteins,
  2. molecular chaperones and
  3. nuclear import factors.
  • Deacetylation of these non-histone proteins by histone deacetylases (HDACs) opens yet another exciting new field of discovery in
  • the role of the dynamic acetylation and deacetylation on cellular function.

We capture the dynamic interactions between the systems under stress that are elicited by cytokine-driven hormonal responses, long thought to be circulatory and multisystem, that affect the major compartments of fat and lean body mass, and are as much the drivers of metabolic pathway changes that emerge as epigenetics, without disregarding primary genetic diseases.

The greatest difficulty in organizing such a work is in whether it is to be merely a compilation of cancer expression organized by organ systems, or whether it is to capture developing concepts of underlying stem cell expressed changes that were once referred to as “dedifferentiation”. In proceeding through the stages of neoplastic transformation, there occur adaptive local changes in cellular utilization of anabolic and catabolic pathways, and a retention or partial retention of functional specificities.

This effectively results in the same cancer types not all fitting into the same “shoe”. There is a sequential loss of identity associated with cell migration, cell-cell interactions with underlying stroma, and metastasis., but cells may still retain identifying “signatures” in microRNA combinatorial patterns. The story is still incomplete, with gaps in our knowledge that challenge the imagination.

What we have laid out is a map with substructural ordered concepts forming subsets within the structural maps. There are the traditional energy pathways with terms aerobic and anaerobic glycolysis, gluconeogenesis, triose phosphate branch chains, pentose shunt, and TCA cycle vs the Lynen cycle, the Cori cycle, glycogenolysis, lipid peroxidation, oxidative stress, autosomy and mitosomy, and genetic transcription, cell degradation and repair, muscle contraction, nerve transmission, and their involved anatomic structures (cytoskeleton, cytoplasm, mitochondria, liposomes and phagosomes, contractile apparatus, synapse.

We are a magnificent “magical” experience in evolutionary time, functioning in a bioenvironment, put rogether like a truly complex machine, and with interacting parts. What are those parts – organelles, a genetic message that may be constrained and it may be modified based on chemical structure, feedback, crosstalk, and signaling pathways. This brings in diet as a source of essential nutrients, exercise as a method for delay of structural loss (not in excess), stress oxidation, repair mechanisms, and an entirely unexpected impact of this knowledge on pharmacotherapy.

Despite what we have learned, the strength of inter-molecular interactions, strong and weak chemical bonds, essential for 3-D folding, we know little about the importance of trace metals that have key roles in catalysis and because of their orbital structures, are essential for organic-inorganic interplay. This will not be coming soon because we know almost nothing about the intracellular, interstitial, and intravesicular distributions and how they affect the metabolic – truly metabolic events.

  1. We must translate the sequence information from genomics locus of the genes to function with related polymorphism of these genes so that possible patterns of the gene expression and disease traits can be matched. Then, we may develop precision technologies for:
  1. Diagnostics
  2. Targeted Drugs and Treatments
  3. Biomarkers to modulate cells for correct functions

With the knowledge of:

  1. gene expression variations
  2. insight in the genetic contribution to clinical endpoints ofcomplex disease and
  3. their biological risk factors,
  4. share etiologic pathways

which requires an understanding of both:

  • the structure and
  • the biology of the genome.
  1. A new paradigm is summarized in a sequence of six steps:

“(1) A pathogenic stimulus (biological or chemical) leads at first to a normal reaction seen in wound healing, namely, inflammation. When the inflammatory stimulus is too great or too prolonged, the healing process is unsuccessful, and that results in

(2) chronic inflammation.

“That’s just the beginning. When chronic inflammation persists,

(3) fibrosis [thickening and scarring of the connective tissue,] develops. The fibrosis, with its ongoing alteration of the cellular microenvironment is different and creates

(4) a precancerous niche, resulting in a chronically stressed cellular matrix. In such a situation, the organism deploys

(5) a chronic stress escape strategy. But if this attempt fails to resolve the precancerous state, then

(6) a normal cell is transformed into a cancerous cell.”

Keep in mind:

  1. Nutritional resources that have been available and made plentiful over generations are not abundant in some climates.
  2. Despite the huge impact that genomics has had on biological progress over the last century, there is a huge contribution not to be overlooked in epigenetics, metabolomics, and pathways analysis.

I have provided mechanisms explanatory for regulation of the cell that go beyond the classic model of metabolic pathways associated with the cytoplasm, mitochondria, endoplasmic reticulum, and lysosome, such as, the cell death pathways, expressed in apoptosis and repair.  Nevertheless, there is still a missing part of this discussion that considers the time and space interactions of the cell, cellular cytoskeleton and extracellular and intracellular substrate interactions in the immediate environment.

  1. Signal transduction occurs when an extracellular signaling[1] molecule activates a specific receptor located on the cell surface or inside the cell. In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.[2] Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.[3] The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.[4]

In 1970, Martin Rodbell examined the effects of glucagon on a rat’s liver cell membrane receptor. He noted that guanosine triphosphate disassociated glucagon from this receptor and stimulated the G-protein, which strongly influenced the cell’s metabolism. Thus, he deduced that the G-protein is a transducer that accepts glucagon molecules and affects the cell.[5] For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.

Signal transduction involves the binding of extracellular signaling molecules and ligands to cell-surface receptors that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation of the receptor, known as receptor activation. This activation is always the initial step (the cause) leading to the cell’s ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.[13] Most steroid hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of steroid-responsive genes.[14] Examples of signaling molecules include the hormone melatonin,[15] the neurotransmitter acetylcholine[16] and the cytokine interferon γ.[17]

Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye,[20] and odorants binding to odorant receptors in the nasal epithelium.[21] Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune system response against invading pathogens mediated by signal transduction processes. This may occur independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling.

Unraveling the multitude of

  • nutrigenomic,
  • proteomic, and
  • metabolomic patterns

that arise from the ingestion of foods or their

  • bioactive food components

will not be simple but is likely to provide insights into a tailored approach to diet and health. The use of new and innovative technologies, such as

  • microarrays,
  • RNA interference, and
  • nanotechnologies,

will provide needed insights into molecular targets for specific bioactive food components and

  • how they harmonize to influence individual phenotypes(1).
  1. Oct4 has a critical role in committing pluripotent cells into the somatic cellular pathway. When embryonic stem cells overexpress Oct4, they undergo rapid differentiation and then lose their ability for pluripotency. Other studies have shown that Oct4 expression in somatic cells reprograms them for transformation into a particular germ cell layer and also gives rise to induced pluripotent stem cells (iPSCs) under specific culture conditions.

Oct4 is the gatekeeper into and out of the reprogramming expressway. By modifying experimental conditions, Oct4 plus additional factors can induce formation of iPSCs, epiblast stem cells, neural cells, or cardiac cells. Dr. Schöler suggests that Oct4 a potentially key factor not only for inducing iPSCs but also for transdifferention.  “Therapeutic applications might eventually focus less on pluripotency and more on multipotency,

  1. Epigenetics is getting a big attention recently to understand genomics and provide better results. However, this field is studied for many years under functional genomics and developmental biology for cellular and molecular biology. Stem cells have a free drive that we have not figured out yet. So genomics must be studied essentially with people training in developmental biology and comparative molecular genetics knowledge to make heads and tail for translational medicine.

There are three main routes of epigenetic modifications one

  • histone modifications via acetylation and methylation and the other is
  • DNA methylation, which are two classical mechanisms in epigenetics.

The third factor is

  • non-coding RNAs that are usually underestimated even not included.

In 1993, Kavai group showed brain development assays of mice showed that only 0.7% genome has tissue and cellular specificity, and 1.7% of genome was able to turn on and off. This conclusion is relevant to genome sequencing data. Also, previous studies in genome and RNA biology presented that RNA directed DNA modifications lead into splicing and transcriptional silencing for gene regulation in Arapsidosis, mice, and Drosophila. (Borge, F. and. Martiensse, R.A. 2013; Di Croce L, Raker VA, Corsaro M, et al. 2002; Piferrer, F, 2013; Jun Kawai1 et al. 1993)

The environment creates the epigenerators including temperature, differentiation signals and metabolites that trigger the cell membrane proteins for development of signal transduction within the cell to activate gene(s) and to create cellular response.  These changes can be modulated but they are not necessary for modulation. The second step involves epigenetic initiators that require precise coordination to recognize specific sequences on a chromatin in response to epigenerator signals. These molecules are

  • DNA binding proteins and
  • non coding RNAs.

After they are involved they are on for life and controlled by autoregulatory mechanisms, like Sxl (sex lethal) RNA binding protein in somatic sex determination and ovo DNA binding protein in germline sex determination of fruit fly. Both have autoregulation mechanisms, cross talks, differential signals and cross reacting genes since after the final update made the soma has to maintain the decision to stay healthy and develop correctly.  Then, this brings the third level mechanism called epigenetic maintainers that are DNA methylating enzymes, histone modifying enzymes and histone variants.  The good news is they can be reversed. As a result the phonotype establishes either a

  • short term phenotype, transient for transcription,
  • DNA replication and repair or
  • long term phenotype outcomes that are chromatin conformation and heritable markers.

Early in development things are short term and stop after the development seized but be able to maintain the short term phenotype during wound healing, coagulation, trauma, disease and immune responses.

The metabolome for each organism is unique, but from an evolutionary perspective has metabolic pathways in common, and expressed in concert with the environment that these living creatures exist. The metabolome of each has adaptive accommodation with suppression and activation of pathways that are functional and necessary in balance, for its existence.

Most interesting is a recent report from Johns Hopkins in Mar 28, PNAS on breast cancer and stem cell physiology. “Aggressive cancers contain regions where the cancer cells are starved for oxygen and die off, yet patients with these tumors generally have the worst outcome,” Semenza said in a release. “Our new findings tell us that low oxygen conditions actually encourage certain cancer stem cells to multiply through the same mechanism used by embryonic stem cells.”

One of the genes responsible for initiating a stem cell fate under low oxygen conditions is called NANOG. This gene is one of many turned on in oxygen-poor conditions by proteins called hypoxia-inducible factors, or HIFs. NANOG in turn instructs cells to become stem cells to resist the poor conditions and help survival.

NANOG levels can be artificially lowered in embryonic stem cells by experimentally methylating the respective mRNA transcript at the sixth position of its adenine nucleotide. Since this methylation is otherwise thought to stabilize the transcript from degradation, this may help NANOG abandon its proposed stem cell fate for the cell.

In addition to the basic essential nutrients and their metabolic utilization, they are under cellular metabolic regulation that is tied to signaling pathways.  In addition, the genetic expression of the organism is under regulatory control by the interaction of RNAs that interact with the chromatin genetic framework, with exosomes, and with protein modulators. This is referred to as epigenetics, but there are also drivers of metabolism that are shaped by the interactions between enzymes and substrates, and are related to the tertiary structure of a protein.  The framework for diseases in and Pharmaceutical interventions that are designed to modulate specific metabolic targets are addressed as the pathways are unfolded.

Personalized Medicine is here now

Two years ago AJP was found to have a positive test for BRCA1, carrying an 87 percent risk for breast cancer and a 50 percent risk for ovarian cancer. At that time she had a preventive mastectomy. The decision was not easy, but it also brought into consideration that her mother and grandmother both died of breast cancer. She did not have an oophorectomy at that time because on considering the advice of medical experts, she would have been left with no estrogen support. She wanted to delay her early vegetative senescence. She has reached the age of 39 years and on the advice of medical expert opinion, she proceeded with salpingo-oophorectomy, at age 39 years, a decade before her mother had developed cancer. But her delay was to allow her to recover and adjust emotionally to her ongoing situation, with a remaining risk for ovarian cancer.

in a  report in Carcinogenesis back in 2005[3] Lorena Losi, Benedicte Baisse, Hanifa Bouzourene and Jean Benhatter had shown some similar results in colorectal cancer as their abstract described:

“In primary colorectal cancers (CRCs), intratumoral genetic heterogeneity was more often observed in early than in advanced stages, at 90 and 67%, respectively. All but one of the advanced CRCs were composed of one predominant clone and other minor clones, whereas no predominant clone has been identified in half of the early cancers. A reduction of the intratumoral genetic heterogeneity for point mutations and a relative stability of the heterogeneity for allelic losses indicate that, during the progression of CRC, clonal selection and chromosome instability continue, while an increase cannot be proven.”

An article written by Drs. Andrei Krivtsov and Scott Armstrong entitled “Can One Cell Influence Cancer Heterogeneity”[4] commented on a study by Friedman-Morvinski[5] in Inder Verma’s laboratory discussed how genetic lesions can revert differentiated neurons and glial cells to an undifferentiated state [an important phenotype in development of glioblastoma multiforme].

In particular it is discussed that epigenetic state of the transformed cell may contribute to the heterogeneity of the resultant tumor.  Indeed many investigators (initially discovered and proposed by Dr. Beatrice Mintz of the Institute for Cancer Research, later to be named the Fox Chase Cancer Center) show the cellular microenvironment influences transformation and tumor development [6-8].

The mechanism by which tissue microecology influences invasion and metastasis is largely unknown. Recent studies have indicated differences in the molecular architecture of the metastatic lesion compared to the primary tumor, however, systemic analysis of the alterations within the activated protein signaling network has not been described. Using laser capture microdissection, protein microarray technology, and a unique specimen collection of 34 matched primary colorectal cancers (CRC) and synchronous hepatic metastasis, the quantitative measurement of the total and activated/phosphorylated levels of 86 key signaling proteins was performed. Activation of the EGFR-PDGFR-cKIT network, in addition to PI3K/AKT pathway, was found uniquely activated in the hepatic metastatic lesions compared to the matched primary tumors. If validated in larger study sets, these findings may have potential clinical relevance since many of these activated signaling proteins are current targets for molecularly targeted therapeutics. Thus, these findings could lead to liver metastasis specific molecular therapies for CRC.

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Schizophrenia genomics

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Histone Methylation at H3K9; Evidence for a Restrictive Epigenome in Schizophrenia

Schizophr Res. 2013 Sep; 149(0): 15–20.      doi:  10.1016/j.schres.2013.06.021

Epigenetic changes are stable and long-lasting chromatin modifications that regulate genomewide and local gene activity. The addition of two methyl groups to the 9th lysine of histone 3 (H3K9me2) by histone methyltransferases (HMT) leads to a restrictive chromatin state, and thus reduced levels of gene transcription. Given the numerous reports of transcriptional down-regulation of candidate genes in schizophrenia, we tested the hypothesis that this illness can be characterized by a restrictive epigenome.

METHODS   We obtained parietal cortical samples from the Stanley Foundation Neuropathology Consortium and lymphocyte samples from the University of Illinois at Chicago (UIC). In both tissues we measured mRNA expression of HMTs GLP, SETDB1 and G9a via real-time RT-PCR and H3K9me2 levels via western blot. Clinical rating scales were obtained from the UIC cohort.

RESULTS   A diagnosis of schizophrenia is a significant predictor for increased GLP, SETDB1 mRNA expression and H3K9me2 levels in both postmortem brain and lymphocyte samples. G9a mRNA is significantly increased in the UIC lymphocyte samples as well. Increased HMT mRNA expression is associated with worsening of specific symptoms, longer durations of illness and a family history of schizophrenia.

CONCLUSIONS   These data support the hypothesis of a restrictive epigenome in schizophrenia, and may associate with symptoms that are notoriously treatment resistant. The histone methyltransferases measured here are potential future therapeutic targets for small molecule pharmacology, and better patient prognosis.

Schizophrenia is conceptualized as a disorder of gene transcription and regulation. Consequently, chromatin is the ideal scaffold to examine this manifested pathophysiology of schizophrenia, as it constitutes the interface between the underlying genetic code and its surrounding biochemical environment. Through post-transcriptional modifications of histone proteins, gene expression can be either transcriptionally active in a ‘euchromatic’ environment, temporarily quieted in ‘facultative heterochromatin,’ or completely silenced in ‘constitutive heterochromatin’ (Zhang and Reinberg 2001). Post-translational modifications to lysine 9 of the H3 protein (H3K9) are uniquely able to reflect these three levels of transcriptional regulation. H3K9 modifications located in the promoter regions of actively transcribed genes are often acetylated (H3K9acetyl). Conversely, quieted transcription in gene-rich areas of the genome are often associated with mono- or dimethyl H3K9 (H3K9me2), while completely silenced areas of the genome are associated with trimethylated H3K9 (H3K9me3). In particular, the formation of H3K9me2 is catalyzed by histone methyltransferases (HMTs), including Eu-HMTase2 (G9a), Eu-HMTase1 (GLP), and SETDB1 (Krishnan et al. 2011) The different degrees of lysine methylation are possible due to the cooperation of these HMTs, which are able to form large heteromeric complexes (Fritsch et al. 2010).

H3K9 methylation has not been extensively studied in the brain, and until recently the regulation and role of the enzymes responsible for its formation were not known. Postnatal, neuronal-specific GLP/G9a knockdown produces a significant decrease in global H3K9me2 levels and inappropriate gene expression, leading to deficits in learning, reduction in exploratory behaviors and motivation in mice (Schaefer et al. 2009; Shinkai and Tachibana 2011;Tachibana et al. 2005;Tzeng et al. 2007). In humans, deletions or loss-of-function mutations of G9a results in Kleefstra Syndrome, characterized by a severe learning disability and developmental delay (Nillesen et al. 2011; Kleefstra et al. 2005). In humans, increased SETDB1 mRNA expression and resultant elevated H3K9me3 levels have been documented in Huntington Disease (HD) (Ryu et al. 2006; Fox et al. 2004).

A hallmark of schizophrenia is aberrant gene regulation, with the vast majority of studies reporting a down-regulation of gene transcription, suggesting that the epigenome of patients with schizophrenia is restrictive (Akbarian et al. 1995;Guidotti et al. 2000;Fatemi et al. 2005; Impagnatiello et al. 1998; Jindal et al. 2010). Postmortem brain studies indicate a reduction of an open histone modification, H3K4me3, and elevated expression of the histone deacetylase HDAC1 mRNA expression (Cheung et al. 2010; Sharma et al. 2008). The use of peripheral blood mononuclear cells as a reflection of overall chromatin state or at particular gene promoters has been successfully implemented in clinical studies of subjects afflicted depression, alcoholism, and schizophrenia. Peripheral blood cell studies have indicated that schizophrenia is associated with an abnormally condensed chromatin structure; (Issidorides et al. 1975; Kosower et al. 1995) specifically increased restrictive H3K9me2 and reduced H3K9 acetylation (Gavin et al. 2009b). Additionally, H3K9 acetylation in schizophrenia patients is less responsive to in vivo treatment with HDAC inhibitors when compared to both patients with bipolar disorder and nonpsychiatric controls (Sharma et al. 2006;Gavin et al. 2008). Finally, a correlation exists between age of onset of psychiatric symptoms of schizophrenia and baseline levels of H3K9me2 (Gavin et al. 2009b). It is the hypothesis of this paper that schizophrenia can be characterized by a restrictive epigenome, which is observable in both brain and peripheral blood, and has specific and observable effects on psychopathology. We have focused on levels of H3K9me2, indicative of facultative heterochromatin, and the enzymes that catalyze this modification, in patients with schizophrenia to examine their role in this illness.

3.1. mRNA Levels of HMT Gene Expression

We performed a multiple linear regression with each HMT gene of interest as the dependent variable. For postmortem brain tissue we examined sex, age, pH, RIN and diagnosis, whereas for lymphocytes we examined sex, age, and diagnosis as explanatory variables. In these two cohorts, we found that a diagnosis of schizophrenia is a significant predictor for GLP mRNA expression in both postmortem brain samples (β=0.44, F(1,24)=5.80, p<0.05), and in lymphocytes (β=−0.41, F(1,40)=7.91, p<0.01), indicating that patients with schizophrenia demonstrated increased levels compared to nonpsychiatric controls (Fig. 1a). Similarly, a diagnosis of schizophrenia is also a significant predictor for increased SETDB1 mRNA levels in both postmortem brain samples (β=0.39, F(1, 24)=4.33,p<0.05), and in lymphocytes (β=0.37, F(1,40)=6.19, p<0.05; Fig. 1b). A diagnosis of schizophrenia is not a significant predictor for elevated G9a mRNA levels in postmortem brain samples (β=0.22, F(1,24)=1.22, p=ns), but is for lymphocytes (β=−0.317, F(1,40)=4.46, p<0.05; Fig. 1c).

Interestingly, in both postmortem tissue (r=0.79, p<0.001) and lymphocytes (r=0.54, p<0.001), GLP and SETDB1 mRNA expression are positively correlated (data not shown).

Fig. 1

mRNA expression in both postmortem parietal cortical samples from the Stanley Foundation Neuropathology Consortium (on the left) and lymphocyte samples from University of Illinois at Chicago (on the right) and a. GLP mRNA levels, b. G9a mRNA levels and

To establish whether there exist differences in HMT mRNA among schizophrenic patients taking psychotropic medication, and those who were not, we performed a second multiple linear regression analysis on each individual cohort. The overall or type-specific use of antipsychotic, antidepressant or mood stabilizing medication are not significant predictors of HMT mRNA levels in either the postmortem or the lymphocyte cohorts.

3.2. H3K9me2 levels in the Postmortem Brain

In a previously published study we documented elevated global H3K9me2 levels in lymphocytes obtained from schizophrenia patients compared to nonpsychiatric controls (Gavin et al. 2009b). In the current study we attempted to discern whether this abnormality in a restrictive histone modification is present in brain tissue from the SFNC cohort as well. We performed a multiple linear regression with H3K9me2 levels as the dependent variable, with sex, age, and diagnosis as explanatory variables. We found that diagnosis of schizophrenia is a significant predictor of H3K9me2 levels extracted from postmortem brain tissue (β=0.40, F(1,24)=4.58, p<0.05; Fig. 2). GLP (r=0.65, p<0.001) and SETDB1 (r=0.44,p<0.05) are positively correlated with H3K9me2 levels, as discovered through a Pearson Correlation (data not shown).

Fig. 2

H3K9me2 levels are significantly increased parietal cortical samples from patients with schizophrenia when compared to nonpsychiatric controls. Below graph, a representative western blot image is shown. All data is shown as a ratio of optical density …     
3.3. Clinical Correlates with Lymphocyte HMT mRNA Levels

Lymphocyte levels of G9a mRNA demonstrated a positive correlation with the PANSS negative subscale total (r=0.61, p<0.05; Fig. 3a), GLP mRNA is positively correlated with the PANSS general subscale total, (r=0.64, p<0.01; Fig. 3b), and SETDB1 mRNA is more highly expressed in patients with longer durations of illness compared to both normal controls and patients in the ‘first episode psychosis’ group (ANOVA, F(2,30)=3.66, p<0.01; Fig. 3c). Patients with a family history of schizophrenia also had significantly increased levels of lymphocyte SETDB1 mRNA (t18=2.52, p<0.05; Fig. 3d).

Fig. 3

Clinical Correlates with Lymphocyte HMT mRNA Levels a. A rise in G9a mRNA is significantly correlated with increasing PANSS negative subscale totals; p<0.05. b. GLP mRNA is significantly increased upon worsening of PANSS general subscale scores;
4. Discussion

The current paper demonstrates an increase in GLP and SETDB1 mRNA in both postmortem parietal cortex and lymphocyte samples from patients with schizophrenia, as well as an increase in G9a mRNA in lymphocytes. G9a and GLP are responsible for the bulk of H3K9me2 modifications across the genome (Shinkai and Tachibana 2011; Tachibana et al. 2005), and SETDB1 is the only euchromatic HMT to specifically di- and tri-methylate H3K9 (Zee et al. 2010;Wang et al. 2003), but all three of these HMTs are able to form large heteromeric complexes, thus allowing for the sequential degrees of lysine methylation (Fritsch et al. 2010). Further, we demonstrate that the ultimate outcome of their catalytic activity, H3K9me2, is significantly increased in patients with schizophrenia as compared to nonpsychiatric controls. Moreover, GLP and SETDB1 mRNA are positively correlated with H3K9me2 levels. These findings add gravity to our previous demonstration of increased H3K9me2 levels in lymphocytes from schizophrenic patients (Gavin et al. 2009b).

Our investigations into the role of H3K9me2 in schizophrenia pathophysiology, as opposed to other H3K9 modifications, were motivated by the hypothesis that initial inactivation of gene promoter activity at various schizophrenia candidate genes can result in gradual entrenchment of the heterochromatin state as a result of disease chronicity and disuse (Sharma et al. 2012). Areas of H3K9me2 can then act as a platform for additional restrictive adaptors, thus resulting in the spreading of heterochromatin across previously unmodified gene rich areas. As such, the gene altering effects of medications are unable to overcome this restrictive burden, leading to repeated medication failures (Sharma et al. 2012). Support for this hypothesis has been previously demonstrated, (Sharma et al. 2008; Benes et al. 2007) including the finding that schizophrenia patients clinically treated for four weeks with the HDAC inhibitor, valproic acid, displayed no increase in peripheral blood cell acetylated histones 3 or 4 as compared to bipolar patients (Sharma et al. 2006). Here, we find an increase in both H3K9me2 levels and the enzymes which catalyze this modification, providing additional evidence supporting an increased heterochromatin state in schizophrenia.

The major role of the parietal cortex is to integrate and evaluate sensory information (Andersen & Buneo, 2003; Cohen & Andersen, 2002). It is one of the last areas of the human brain to fully mature, (Geschwind, 1965) thus early life environmental insults could have a profound effect. Disordered thought, a common symptom in schizophrenia, is most likely explainable through disruption of this system (Torrey, 2007). Patients with schizophrenia report either acute (McGhie & Chapman, 1961) or blunted (Freedman, 1974) sensitivity to sensory stimuli, and demonstrate overall impairment of sensory integration (Manschreck & Ames, 1984; Torrey, 1980). Similar patterns of transcriptional regulation are observed across the cortex, consequently, results from the parietal cortex likely reflect patterns of gene transcription in other brain regions (Hawrylycz et al., 2012).

Due to its heterogeneity, examining schizophrenia as a binary measurement of illness when examining biological relevancy can be limiting (Arango et al. 2000;Buchanan and Carpenter 1994). Through utilizing the PANSS, biological underpinnings that do not demarcate cleanly with diagnostic categories, can be correlated directly with specific symptomatology. Correlations between methyltransferase enzymes and clinical symptomatology indicate that these restrictive enzymes could contribute to specific facets of the illness, particularly negative and general symptoms, which are particularly resistant to improvement. Increased severity of negative symptoms are correlated with poorer disease prognosis, (Wieselgren et al. 1996) and are not alleviated through our current regimen of psychotropics.

Additionally, SETDB1 mRNA levels are also correlated with other markers of a worse disease prognosis, including a more chronic form of the illness, and a history of schizophrenia in the family. Pharmacological targeting of increased levels of SETDB1improves motor performance and extends survival in HD mice, indicating the promise of treatments that modulate gene silencing mechanisms in neuropsychiatric disorders (Ryu et al. 2006).

The main weakness of this current study was that clinical symptoms were correlated with mRNA extracted from peripheral tissue. Enzymes relating specifically to synaptic function were not examined, but rather overall mechanisms of epigenetic regulation that are not tissue specific. While postmortem investigations are able to serve as a useful snapshot at the time of death, the ability to measure and monitor histone marks over time as marker of disease progression, improvement, or as a predictor of pharmacological response are only possible using peripheral blood cells. A strong rationale for the use of blood chromatin ‘levels’ as a type of biosensor that registers the epigenetic milieu has been proposed elsewhere (Sharma 2012). Furthermore, previous studies have indicated the mRNA patterns of expression patterns in lymphocytes are capable of distinguishing between psychiatric diagnostic groups (Middleton et al. 2005).

The present study hypothesized that schizophrenia may be due to abnormal regulation of fundamental epigenetic mechanisms, thus, we chose to measure overall levels of H3K9me2 opposed to specific gene promoters, based on the assumption that while the individual genes silenced in the brain and blood may not be the same, similar global pathogenic processes may be occurring in both tissues.

The results of this paper indicate that chromatin is more restrictive in patients with schizophrenia, and may be significantly contributing to disease pathology. If, through pharmacological interventions, a reduction in this histone hyper-restrictive insult in schizophrenia can be relaxed, inducing a type of “genome softening,” then neuronal gene expression can be enhanced, thus allowing for increased plasticity and improved therapeutic response (Sharma 2005).

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Balancing Histone Methylation Activities in Psychiatric Disorders

Alterations in histone lysine methylation and other epigenetic regulators of gene expression contribute to changes in brain transcriptomes in mood and psychosis spectrum disorders, including depression and schizophrenia. Genetic association studies and animal models implicate multiple lysine methyltransferases (KMTs) and demethylases (KDMs) in the neurobiology of emotion and cognition. Here, we review the role of histone lysine methylation and transcriptional regulation in normal and diseased neurodevelopment and discuss various KMTs and KDMs as potential therapeutic targets in the treatment of neuropsychiatric disease.

Schizophrenia and depression are major psychiatric disorders that lack consensus neuropathology and, in a large majority of cases, a straightforward genetic risk architecture. Furthermore, many patients on the mood and psychosis spectrum show an incomplete response to conventional pharmacological treatments which are mainly aimed at monoamine signaling pathways in the brain (Box 1).

Box 1  Schizophrenia and Depression

Schizophrenia affects 1% of the general population and typically begins during young-adult years, although cognitive disturbances could be evident much earlier. The disease is, in terms of genetics and etiology, highly heterogeneous, and increasingly defined as different and partially independent symptom complexes: (i) psychosis with delusions, hallucinations and disorganized thought; (ii) cognitive dysfunction including deficits in attention, memory and executive function; and (iii) depressed mood and negative symptoms including inability to experience pleasure (anhedonia), social withdrawal and poor thought and speech output [42]. Currently prescribed antipsychotics, which are mainly aimed at dopaminergic and/or serotonergic receptor systems, exert therapeutic effects on psychosis in approximately 75% of patients. However, it is the cognitive impairment which is often the more disabling and persistent feature of schizophrenia [42]. Currently there are no established pharmacological treatments for this symptom complex. However, given that cognitive dysfunction is an important predictor for long-term outcome, this area is considered a high priority in schizophrenia research, as reflected by initiatives combining efforts from government agencies, academia and industry, including MATRICS (the Measurement and Treatment Research to Improve Cognition in Schizophrenia) [42].

Affective disorders as a group show, in terms of genetic risk architecture, some overlap with schizophrenia. For example, rare structural variants, including the balanced translocation at the Disrupted-in-Schizophrenia 1 (DISC-1) locus (1q42) or the 22q11 deletion are, in different individuals, associated with either mood disorder or schizophrenia [81, 82].

Depression, including its more severe manifestation, major depressive disorder which has a lifetime risk of 10–15% for the U.S. general population, is associated with excessive sadness, anhedonia, negative thoughts, and neurovegetative symptoms including changes in sleep pattern and appetite [1]. The disorder, which in more severe cases is accompanied by delusions, hallucinations and other symptoms of psychosis, often takes a chronic and recurrent course. Conventional antidepressant therapies primarily target monoamine metabolism and reuptake mechanisms at the terminals of serotonergic, noradrenergic and dopaminergic neurons. Unfortunately, up to 40% of cases show an insufficient response to these pharmacological treatments [1]. In addition, many antipsychotic and antidepressant drugs have significant side effect burden, including weight gain, diabetes and metabolic defects, extrapyramidal symptoms and sexual dysfunction [83, 84].

However, there is evidence that dysregulated gene transcription, indicative of compromised neural circuitry, contributes to disordered brain function in psychosis and mood spectrum disorder [1, 2]. While no single gene transcript is consistently affected, alterations in RNA levels contribute to defects in GABAergic inhibitory neurotransmission and more generally, synapse organization and function, metabolism and mitochondrial functions, and oligodendrocyte pathology [35]. While a number of transcriptional and post-transcriptional mechanisms may contribute to these changes, chromatin-associated proteins and epigenetic regulators invoked in sustained alterations of gene expression and function (Box 2) could play a critical role in the pathophysiology, or treatment of mental illness [6,7]. Indeed, there is evidence that changes in acetylation of histone lysine residues, which are broadly associated with active gene expression [8] and considered a potential therapeutic target for cancer and other medical conditions [9], also impact gene expression patterns in the brain and thereby influence emotional and cognitive functions. For example, mice or rats exposed to systemic treatment, or localized intracranial injections of class I/II histone deacetylase inhibitors (HDACi) exhibit behavioral changes reminiscent of those elicited by conventional antidepressant drugs [1013]. The short chain fatty acid derivative valproic acid, widely prescribed for its mood-stabilizing and anticonvulsant effects, induces brain histone hyperacetylation at a select set of gene promoters when administered to animals at comparatively high doses [14]. Conversely, overexpression of selected HDACs in neuronal structures implicated in the neurobiology of depression, including the hippocampus, elicit a pro-depressant behavioral phenotype [12]. Similarly, animals treated with class I/II HDACi often show improved performance in learning and memory paradigms and furthermore, drug-induced inhibition or activation of class III HDAC (also known as sirtuins) elicits changes in motivational and reward-related behaviors [15]. Therefore, the orderly balance between histone acetyl-transferase and deacetylase activities is critical for cognitive performance and synaptic and behavioral plasticity [16]. Likewise, However, HDACs interfere with acetylation of many non-histone proteins in the nucleus and cytoplasm [16], and moreover, some of these drugs carry a significant side effect burden [9]. Therefore, in light of the emerging role of epigenetic mechanisms in the neurobiology of these and other psychiatric conditions [6], the therapeutic potential of chromatin modifying drugs, other than the HDACi, warrants further investigations. This review will focus on histone lysine methylation, one of the most highly regulated chromatin markings in brain and other tissues. Multiple methyltransferases (KMTs) and demethylases (KDMs) were recently implicated in emotional and cognitive disorders (Fig. 1), and these types of chromatin modifying enzymes could emerge as novel targets in the treatment of mood and psychosis spectrum disorders.

Box 2  Epigenetic regulators and chromatin structure and function

Epigenetics, in the broader sense, applies both to dividing and postmitotic cells, and refers to a type of cellular memory that involves sustained changes in chromatin structure and function, including gene expression, in the absence of DNA sequence alterations (For in depth discussion, see [85]). Chromatin is essentially a repeating chain of nucleosomes comprised of genomic DNA wrapped around an octamer of core histones H2A/H2B/H3/H4. The histone proteins are intensely decorated with epigenetic information, with more than 70 (amino acid) residue-specific sites subject to various types of post-translational modifications (PTM). These include lysine (K) acetylation, methylation and poly ADP-ribosylation, arginine (R) methylation, and serine (S), threonine (T), tyrosine (Y) and histidine (H) phosphorylation [86]. In addition, a subset of the histone H2A, H2B and H4 lysines are covalently linked to the small protein modifiers ubiquitin and SUMO [87, 88]. Finally, epigenetic markings in genomic DNA include 5-methyl-cytosine and the related form, 5-hydroxy-methyl-cytosine [85]. These DNA and nucleosomal histone markings define the functional architecture of chromatin (see main text).

Proteins associated with methylation and other histone PTM are typically defined either as ‘writers’, ‘erasers’ or ‘readers’, essentially differentiating between the process of establishing or removing a mark as opposed to providing a docking site for chromatin remodeling complexes that regulate transcription, or induce and maintain chromatin condensation [18, 86, 89]. As it pertains to the brain, especially in the context of neuropsychiatric disease, a substantial body of knowledge has been generated for a select set of site-specific (K) methyltransferases and demethylases (Fig. 1A). In contrast, many PTMs are recognized by large numbers of reader proteins [90], but to date only very few of these readers have been explored in the brain. To mention just two examples, there are approximately 75 reader proteins specifically associated with histone H3-trimethyl-lysine 4 (H3K4me3), including several components of the SAGA complex ascribed with a key role for transcriptional initiation at RNA polymerase II target genes [90]. In contrast, H3K9me3, generally considered a repressive mark, provides a central hub for heterochromatin (associated) proteins including several members of the HP1 family and zinc finger domain containing molecules [90]. There is additional complexity because pluripotent stem cells and additional cell types decorate many of their promoters with ‘bivalent domains’ which include both open chromatin-associated (methylated H3K4 and H3/H4 acetylation) and repressive (methylated H3K27) marks [91, 92].

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Regulation of histone (K) methylation. (A) Listings of residue-specific KMTs and KDMs for H3K4/9/27/36/79 and H4K20. The majority of KMT and KDM are highly specific for a single histone residue, while a few enzymes target multiple residues, as indicated. Red marked KMT/KDM are implicated in neurodevelopment or psychiatric disease as discussed in main text. The non-catalytic JARID2 regulates activity and function of related KMTs. (B) Simplified scheme for selected mono- and trimethylated histone lysine markings implicated in transcriptional regulation, silencing and enhancer function.

The methylation of lysine and arginine residues, like other histone PTM, define chromatin states and function [8, 17]. To date, more than 20 methyl-marks on K and R residues have been described [18, 19]. As it pertains to the lysines, the majority of studies focused on the regulation and methylation-related functions of six specific sites: H3K4, H3K9, H3K27, H3K36, H3K79 and H4K20 [18]. For H3K4 and H3K9/K27, there is additional complexity because specific information is also conveyed (i) for H3K4, the unmethylated lysine effectively serving as a DNA methylation signal [20, 21], and (ii) for H3K9/K27 acetylation as an alternative PTM [22, 23] (Fig. 1A). For the aforementioned H3/H4 residues, specific biological functions and their interrelations with functional chromatin states, including transcriptional initiation and elongation, heterochromatic silencing and other mechanisms, have been described for the trimethyl-, and for some of the mono- and dimethyl-modifications (Representative examples are provided in Fig. 1B. See ref [17] for a detailed description of the histone methylation code and its relation to other types of histone PTM).

The following examples further illustrate the complex regulation of histone lysine methylation. Monomethylation of histone H3-lysine 4 (H3K4me1) plays an important role in neuronal activity-induced transcription at enhancer sequences [24], but the related forms, H3K4me3/2 are primarily found at the 5′ end of genes, with H3K4me3 mostly arranged as distinct and sharp peaks within 1–2Kb of transcription start sites. The H3K4me3 mark provides a docking site at the 5′ end of genes for chromatin remodeling complexes that either facilitate or repress transcription [25]. Furthermore, mono-methyl-H4-K20 shows strong positive correlation with gene expression at promoters enriched with CpGs, which contrasts to the trimethylated form of the same residue which generally is associated with repressed chromatin [23]. Taken together, these examples illustrate that even closely related histone lysine methylation markings are potentially associated with very different chromatin states.

To date, H3K4, H4K9, H3K27 and H4K20 methylation signals were measured at specific loci and genome-wide in human brain, essentially confirming that each of these epigenetic markings defines the same type of chromatin as in the peripheral tissues or animal brain [2630]. Interestingly, a subset of psychotherapeutic drugs including the mood-stabilizer valproate, the atypical antipsychotic clozapine and some monoamine oxidase inhibitors and stimulant drugs interfere with brain histone methylation (Table 1).

Molecular mechanisms of histone (lysine) methylation

A complex system of site-specific methyltransferases, which transfer the methyl-group of S-Adenosyl-Methionine (SAM) to lysine residues, has evolved in the vertebrate cell. There are an estimated 70 human genes harboring the Su(var)3–9,Enhancer of Zeste,Trithorax (SET) domain, which spans approximately 130 amino acids essential for KMT enzymatic activity [31]. The only known exception is the H3K79-specific methyltransferase, KMT4/DOT1L [31, 32], which lacks a SET. Each of the histone K residues discussed above is the preferential target of a distinct set of methyltransferase proteins (Fig. 1A)[19]. Of note, these histone-modifying enzymes are thought not to access histone substrates directly unless recruited by DNA-bound activators and repressors, a mechanism which could target each methyltransferase to a highly specific set of genomic loci [19].

An equally complex system exists for the site-specific lysine demethylases (Fig. 1A). There are at least two different mechanisms for active histone demethylation. The first enzyme type, represented by lysine-specific demethylase 1 (LSD1/KDM1A), contains an amine oxidase domain and requires flavin adenine dinucleotide (FAD) as a cofactor to demethylate di- and mono-methylated lysines. LSD1 and its homologue, LSD2, are primarily H3K4 demethylases, albeit depending on species and context, and activity against H3K9 also has been described [18]. Interestingly, monoamine oxidase inhibitors (MAOi) such as tranylcypromine or phenelzine — powerful antidepressants that exert their therapeutic effects mainly by elevating brain monoamine levels through inhibition of MAO-A/B — also block LSD1 type histone demethylases [18]. While LSD1 is thought to regulate histone methylation at promoters, LSD2 is bound to transcriptional elongation complexes and removes H3K4 methyl markings in gene bodies, thereby facilitating gene expression by reducing spurious transcriptional initiation outside of promoters [33]. The second type of demethylase, which in contrast to LSD1/LSD2 is capable of demethylating trimethyl markings, involves Fe2+-dependent dioxygenation by Jumonji-C (JmJC) domain-mediated catalysis [18]. Given that each of the KMTs and KDMs described has a different combinatorial set of functional domains and (protein) binding partners [18, 34], it is likely that the various site-specific methyltransferases and demethylases are largely non-redundant in function.

KMTs and KDMs with a role in cognition and neuropsychiatric disease

An increasing number of KMTs and KDMs are implicated in neurodevelopment and major psychiatric diseases (marked in red in Fig. 1A).

H3K4

The first histone lysine methyltransferase explored in the nervous system was KMT2A/MLL1, a member of the mixed-lineage leukemia (MLL) family of molecules. Mice heterozygous for an insertional (lacZ) loss-of-function Mll1mutation show distinct abnormalities in hippocampal plasticity and signaling [35], in conjunction with defects of learning and memory [36]. Of note, the hippocampus, and other portions of the forebrain including prefrontal cortex and ventral striatum, are frequently implicated in the neural circuitry of mood and psychosis spectrum disorders [1]. Furthermore, conditional deletion of Mll1resulted in defective neurogenesis during the early postnatal period [37]. While the full spectrum of MLL1 target genes in neurons and glia awaits further investigation, dysregulated expression of certain transcription factors such as DLX2, a key regulator for the differentiation of forebrain GABAergic neurons (which are essential for inhibitory neurotransmission and orderly synchronization of neural networks) [38], may contribute to the cognitive phenotype of the Mll1mutant mice. These observations may be relevant for the pathophysiology of schizophrenia, because some patients show in the prefrontal cortex a deficit in H3K4-trimethylation and gene expression at a subset of GABAergic promoters, including GAD1 encoding a GABA synthesis enzyme [28]. While the timing and age-of-onset for this ‘molecular lesion’ remains unknown, it is of interest that in the normal PFC, H3K4 methylation at the site of GABAergic genes progressively increases during the transition from fetal period to childhood to adulthood [28]. The epigenetic vulnerability of the Gad1 promoter during such prolonged developmental periods is further emphasized by recent animal studies demonstrating that Gad1-DNA methylation and histone acetylation are heavily influenced by the level of maternal care in the neonatal period/pre-weanling period [39].

There is additional evidence that epigenetic fine-tuning of the brain’s H3K4 methyl-markings is critical for orderly neurodevelopment. Of note, loss-of-function mutations in KDM5C/JARID1C/SMCX, an X-linked gene encoding a H3K4 demethylase, have been linked to mental retardation [40] and autism spectrum disorders [41]. The KDM5C gene product operates in a chromatin remodeling complex together with HDAC1/2 histone deacetylases and the transcriptional repressor REST, thereby poising neuron-restrictive silencer elements for H3K4 demethylation and decreased expression of target genes including synaptic proteins and sodium channels [40]. However, because this study was conducted with the HeLa cell line, it remains to be determined whether similar mechanisms operate in the nervous system.

In addition to its role in neurodevelopment, MLL-mediated H3K4 methylation could play a potential role for the treatment of psychosis. The atypical antipsychotic clozapine, which has a somewhat higher therapeutic efficacy when compared to conventional antipsychotics that function primarily as dopamine D2 receptor antagonists [42], upregulates H3K4 tri-methylation at the Gad1/GAD1GABA synthesis enzyme gene promoter. These effects were not mimicked in dopamine receptors D2/D3 (Drd2/3) compound null mutant mice, suggesting that blockade of dopamine D2-like receptors is not sufficient for clozapine-induced H3K4 methylation [28]. In the human PFC, GAD1-associated H3K4 methylation was increased in subjects exposed to clozapine, as compared to subjects treated with conventional antipsychotics. Conversely, mice heterozygous for the H3K4-specific KMT, mixed-lineage leukemia 1 (MLL1), exhibited decreased H3K4 methylation at brain Gad1 [28]. Therefore, it is possible that MLL1, which is highly expressed in GABAergic and other neurons of the adult cerebral cortex [28], will in the future emerge as a novel target for the treatment of psychosis. Questions that remain to be resolved include (i) the molecular pathways linking clozapine — a drug that impacts dopaminergic, serotonergic, muscarinic and other signaling pathways — to MLL1-mediated histone methylation, and (ii) whether or not the clozapine-induced changes in H3K4 methylation are restricted to GABAergic gene promoters or, alternatively, the reflection of more widespread epigenetic changes throughout the genome. Of note, clozapine’s effects on H3K4 methylation require intact brain circuitry and cannot be mimicked in cultured neurons differentiated from forebrain progenitor cells [43]. This finding is in good agreement with the recent observation that some of clozapine’s therapeutic effects require an intact serotonergic system, particularly its presynaptic components [44].

H3K9

The 9q34 subtelomeric deletion syndrome, which includes mental retardation and other developmental defects, is caused by deleterious mutations and haploinsufficiency of euchromatin histone methyltransferase 1 (EHMT1, also known as GLP and KMT1D) [45]. This gene encodes a H3K9-specific methyltransferase that operates in a multimeric complex that includes its closest homologue, G9a/KMT1C, and additional H3K9-specific HMTs [46]. Studies in mutant mice suggest that the GLP/G9a complex is important for suppression of non-neuronal and progenitor genes in mature neurons, and loss of this complex has deleterious effects on cognition and other higher brain functions [47]. Furthermore, G9a-mediated H3K9 methylation events within the reward circuitry, including the ventral striatum, are critical intermediates for the long-term effects of cocaine on reward behavior and neuronal morphology [48]. This would suggest that GLP/G9a, and proper regulation of H3K9 levels, is important for orderly brain function both in developing and mature brain.

Furthermore, changes in motivational and affective behaviors could be elicited by overexpression of the H3K9-HMT, SET domain bifurcated 1 (KMT1C/SETDB1/ESET), in adult forebrain neurons [49]. Interestingly, SETDB1 occupancy in neuronal chromatin is highly restricted, and may be confined to less than 0.75% of annotated genes [49]. However, among these are several NMDA and other ionotropic glutamate receptor subunit genes, including Grin2a/b (Nr2a/b)[49]. Mild to moderate inhibition of NMDA receptor-mediated (including Grin2b) neurotransmission elicits a robust improvement of depressive symptoms in some mood disorder patients [50], and, indeed, SETDB1-mediated H3K9 methylation and repressive chromatin remodeling at the Grin2b locus was associated with antidepressant-like behavioral phenotypes in the Setdb1 transgenic mice [49]. Of note, NMDA receptor antagonists, including GRIN2B-specific drugs, elicit significant therapeutic benefits even in subjects who failed multiple trials of selective serotonin reuptake inhibitors (SSRI) and other conventional antidepressants [50]. However, drugs directly acting at the NMDA receptor site have an unfavorable side effect profile, and therapeutic strategies aimed at SETDB1 expression and activity may therefore provide an alternative strategy.

Interestingly, mice with a genetic ablation of Kap1, encoding the SETDB1 binding partner KRAB-associated protein 1, also known as TRIM28/TIF1b/KRIP1)[51], show increased anxiety and deficits in cognition and memory [52], which are phenotypes that are broadly opposite from those observed in mice with increasedSetdb1 expression in brain [49]. These findings further speak to the therapeutic potential of the Kap1-Setdb1 repressor complex in the context of neuropsychiatric disease.

Finally, the H3K9-specific demethylase, KDM3A/Jmjd1A, showed increased H3K9-methylation at its own promoter in the ventral striatum of mice exposed to social defeat (a type of stressor associated with a depression-like syndrome in these animals), while mice that were treated with a conventional antidepressant or that were resilient to this type of stress did not show changes in KDM3A promoter methylation [53]. While it is unclear whether KDM3A or some other demethylase acitivity is altered in the depressed animals, the same study [53] reported widespread repressive histone methylation changes, including increased dimethyl-H3K9 and methylated H3K27 at hundreds of gene promoters in stress susceptible animals, which further emphasizes the importance of these PTMs for the epigenetics of mood disorder.

H3K27

The H3K27-selective methyltransferase, KMT6A, also known as Enhancer of zeste homolog2 (EZH2), is associated with the polycomb repressive chromatin remodeling complex 2 (PRC2) [54], and essential for cortical progenitor cell and neuron production. Consequently, loss of EZH2 function is associated with severe thinning of the cerebral cortex and a disproportionate loss of neurons residing in upper cortical layers I–IV [55]. Likewise, the H3K27-specific demethylase, JMJD3, is important for neurogenesis and neuronal lineage commitment [56]. Furthermore, H3K27 methylation is dynamically regulated in mature brain and involved in the neurobiology of major psychiatric disease. For example, changes in expression of brain-derived neurotrophic factor (Bdnf) in hippocampus of mice exposed to environmental enrichment or chronic stress are associated with opposite changes in the H3K27me3 mark at a subset of Bdnf gene promoters [12,57]. In addition, acute stress leads to an overall decrease in hippocampal H3K27me3 and H3K9me3 [58]. Furthermore, in the orbitofrontal cortex of suicide completers, alterations in H3K27 methylation were described at the TRKB gene, encoding the high affinity receptor for the nerve growth factor molecule, BDNF [27]. Changes in the balance between histone H3K4 and H3K27 methylation, or DNA cytosine and H3K27 methylation may also contribute to GABAergic gene expression deficits in schizophrenia [28, 43]. To date it remains unclear which of the various H3K27-specific KMTs and KDMs (Fig. 1) are involved in these disease-related alterations in postmortem brain tissue. Of note, the Jumonji and Arid containing protein 2 (JARID2), which by itself lacks catalytic activity but is crucial for subsequent H3K27 or H3K9 methylation by recruiting the polycomb PRC2 complex to its target genes [59, 60], is located within the schizophrenia susceptibility locus on chromosome 6p22 and confers genetic risk in multiple populations of different ethnic origin [61, 62]. While the biological functions of JARID2 have been studied primarily in the context of transcriptional regulation in stem cells [63, 64], this gene shows widespread expression in the mature nervous system [65], implying JARID2-mediated control over polycomb repressive chromatin remodeling in the adult brain.

H3K36 and H4K20

Epigenetic dysregulation of nuclear receptor-binding SET domain containing protein 1/KMT3B could play a role in some neuro- and glioblastomas [66], but like for other H3K36 and H4K20 regulating enzymes (Fig. 1), to date little is known about their role in neurodevelopment, cognition and psychiatric disease. Strikingly, however, KMT3A/HYPB/SETD2, a member of the SET2 family of KMTs mediating H3K36 methylation [67], is also known as huntingtin-interacting protein 1 (HIP-1) or huntingtin(yeast)-interacting protein B (HYPB) [68]. Huntington’s is a triplet repeat disorder and chronic neurodegenerative condition with motor symptoms and cognitive defects, and significant changes in mood and affect [69]. Whether or not there is altered H3K36 methylation in the neuronal populations that are at risk for degeneration is unclear. Furthermore, the huntingtin/KMT3A interaction has been documented for yeast [68] but not brain. Of note, wildtype huntingtin is a facilitator of polycomb complex PCR2-mediated H3K27 methylation [70], and furthermore, H3K4 and H3K9 methylation changes have been reported in preclinical model systems and postmortem brains with Huntington’s disease [71, 72]. Therefore, it is possible that transcriptional dysregulation in this condition is associated with aberrant methylation patterns of multiple lysine residues.

KMTs and KDMs as Novel Drug Targets

Given the emerging role of histone methylation in the neurobiology of psychiatric disease, the next obvious question is whether this type of PTM could provide a target for a new generation of psychotropic therapeutics. In principle, KMTs and KDMs should provide fertile ground for the development of novel drugs, because these enzymes are considered more specific than, for example, HDACs, because each HDAC enzyme is likely to affect a much larger number of histone residues as compared to KMTs/KDMs [73]. However, like for other histone modifying enzymes, the specificity of KMTs and KDMs is not limited to histones but includes the (de)methylation of lysines of non-histone proteins, including the p53 tumor suppressor protein and the VEGF growth factor [74]. Druggable domains within the KMTs and KDMs could involve not only their catalytic sites, such as the SET domain for the KMTs or the amino oxidase and JmjC domains for the LSD1 and JMJD subtypes of KDMs, respectively, but also some of the many other functional domains that are specific to subsets of these proteins [75]. One potential candidate would be the bromodomain of the MLLs and other H3K4-specific methyltransferases [75]. Bromodomains, which are present in many different types of nuclear proteins, bind to acetylated histones and small molecules interfering with some of these interactions recently emerged as powerful modulators of systemic inflammation [76].

The catalytic activity of the SET domain containing KMTs requires the universal methyl donor, S-adenosyl-methionine (also known as AdoMET). Crystallographic and functional studies revealed that the SAM binding pocket of KMTs is different from the SAM pockets of other proteins, which may increase the chance to develop compounds which specifically target histone methyltransferases but not other enzymes and proteins [31]. Currently, however, no KMT or KDM related drug is in clinical trials. However, several of these compounds show therapeutic promise in preclinical studies. For example, the S-adenosylhomocysteine hydrolase inhibitor, 3-deazaneplanocin A (DZNep) induces apoptosis in breast cancer cells [77]. This drug alters H3K27 and H4K20 trimethylation via interference with polycomb PRC2 repressive chromatin remodeling [73]. Antioncogenic effects were also observed with BIX-01294, a drug that downregulates H3K9 methylation levels by binding to the SET domain of the G9a/GLP(EHMT1) methyltransferases [73]. The same drug was shown to alter addictive behaviors and H3K9 methylation when infused locally into the brain of cocaine-exposed mice [48]. As discussed above, while tranylcypromine and other monoamine oxidase inhibitors used for the treatment of depression are weak inhibitors of the LSD1 type of KDM, recently several compounds emerged with much stronger activity against LSD1/LSD2 [18]. It will be extremely interesting to explore these drugs in preclinical models for mood and psychosis spectrum disorders. Finally, microRNA-based therapeutic strategies, aimed at decreasing levels and expression of chromatin remodeling complexes, including some of the histone modifying enzymes discussed here, are gaining increasing prominence in the field of cancer therapy [73] and may in the future emerge as a novel therapeutic option in the context of neuropsychiatric disease.

Emerging Concept in DNA Methylation: Role of Transcription Factors in Shaping DNA Methylation Patterns
CLAIRE MARCHAL AND BENOIT MIOTTO*   Journal of Cellular Physiology Volume 230, Issue 4,  http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-4652

DNA methylation in mammals is a key epigenetic modification essential to normal genome regulation and development. DNA methylation patterns are established during early embryonic development, and subsequently maintained during cell divisions. Yet, discrete site-specific de novo DNA methylation or DNA demethylation events play a fundamental role in a number of physiological and pathological contexts, leading to critical changes in the transcriptional status of genes such as differentiation, tumor suppressor or imprinted genes. How the DNA methylation machinery targets specific regions of the genome during early embryogenesis and in adult tissues remains poorly understood. Here, we report advances being made in the field with a particular emphasis on the implication of transcription factors in establishing and in editing DNA methylation profiles. J. Cell. Physiol. 230: 743–751, 2015.

DNA methylation is a well-studied epigenetic modification in mammalian genomes, discovered in 1948. It is involved in a number of essential cellular processes such as transcription regulation, cellular differentiation, cellular identity maintenance, X inactivation, gene imprinting, and the cellular response to environmental changes (Klose and Bird, 2006; Guibert and Weber, 2013; Smith and Meissner, 2013; Subramaniam et al., 2014). DNA methylation has proved to be a dynamic process, requiring continuous regulation and potentially having an important regulatory role for tissuespecific differentiation or cellular signaling. Indeed, the analysis of the distribution of DNA methylation at the genome scale, and nowadays at the single-base resolution, in different physiological and pathological states, unraveled that local changes in DNA methylation contribute to cell-type specific variation in gene expression. Furthermore, aberrant DNA methylation patterns are documented in a number of human diseases from Immunodeficiency, Centromere instability, and Facial anomalies (ICF) syndrome to cancer, and contribute to the onset or development of these diseases (Smith and Meissner, 2013; Weng et al., 2013; Subramaniam et al., 2014). Needless to say, these discoveries also fuel the promising idea that therapeutic strategies targeting DNA methylation can be used in the prevention and the treatment of cancer and other human diseases, including neuro-developmental disorders (Weng et al., 2013; Subramaniam et al., 2014). As an example, antipsychotic drugs clozapine and sulpiride, combined with histone deacetylase inhibitor valproate, have a beneficial action in schizophrenia and bipolar patients, maybe because they revert the aberrant DNA methylation status at GABAergic gene promoters (Dong et al., 2008). In 2004, 5-azacytidine (VidazaTM, Celgene Corporation, Summit, NJ). A drug blocking DNA methylation, received approval by the Food and Drug Administration for the treatment of myelodysplastic syndromes (Kaminskas et al., 2005).

Figure 1. Overview of the DNA methylation and demethylation pathway. (A) DNMT1 is responsible for the maintenance of DNA methylation during DNA replication. It recognizes hemi-methylated CpG, thanks to its interaction with co-factor UHRF1, and it adds methylation on the un-methylated strand. Black bubbles: methylated CpG. Empty bubbles: un-methylated CpG. (B) DNMT3A/B are responsible for de novo DNA methylation. They establish new patterns of methylation directly from unmethylated CpG-containing sequences. In the embryo, their activity is modulated by a catalytically inactive family member DNMT3L. (C) Passive demethylation occurs through loss of DNMT1/3 activity in actively dividing cells. Loss can be attributed to post-translational modifications, gene mutations, gene silencing or any other mechanism that will eventually lead to DNMT activity inhibition. (D) Active DNA demethylation is catalyzed by the TET family of enzymes. TET1, 2 and 3 can oxydate 5mC into 5hmC (represented in grey bubbles), and eventually oxidate 5hmC into 5-formylcytosine and 5-carboxy-cytosine. None of these bases is recognized by DNMTs causing loss of DNA methylation during DNA replication. In addition, these oxidated bases are recognized by the base-excision repair (BER) pathway and catalytically removed.

Figure 2. Summary of the nuclear factors and epigenetic marks involved in the maintenance of DNA methylation status in different regions of the genome. The table recapitulates our current knowledge on transcription factors, chromatin remodellers and histone marks contributing to the establishment of DNA methylation and its erasure. The information is presented according to genomic features, sharing common regulators, such as promoters/enhancers, tumor suppressor genes, germline gene promoters, imprinted regions, DNA repeats, and retroviral elements and peri-centromeric regions.

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t KAP1/DNMTs control the maintenance of DNA methylation, independently of DNA replication, on a number of genomic targets. Yet, DNMTs have been shown to be recruited onto the chromatin by other chromatin remodelers, such as SETDB1 or G9a, or secondary to gene silencing (Gibbons et al., 2000; Dennis et al., 2001; Guibert and Weber, 2013; Pacaud et al., 2014). Thus, only the identification of the full-spectrum of transcription factors involved in the regulation of DNA methylation will tell whether this function is predominantly confer to KRAB-ZNF factors. This systematic analysis might help understand why only a limited number of factors per family are involved in the shaping of DNA methylation. In the case of ZNF factors several explanations have been postulated. The resolution of the structure of the ZNF fingers of Zfp57 bound onto methylated DNA indicated that a specific amino-acid sequence in the DNA binding ZNF fingers might be required for the recognition and binding of methylated CpG sequences (Liu et al., 2012; BuckKoehntop and Defossez, 2013). Using this knowledge, researchers have postulated that ZNF factors containing this motif might likely contribute to shape DNA methylation profile (Liu et al., 2013). An alternative hypothesis rely on the observation that KRAB-ZNF factors are present uniquely in vertebrate genomes and have expanded quite dramatically in mammalian genomes. As DNA repeats sequences also quickly evolved in mammalian genomes, it is suggested that humanspecific KRAB-ZNF factors might primarily contribute in DNA repeats silencing (Lukic et al., 2014).

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DNA methylation plays an important role in the control of gene expression and cell fate in mammals. Its regulation and function has been upon intense scrutiny since its discovery in mid-1900s. Yet, how DNA methylation patterns are established during embryogenesis, and edited in adult tissue, remains a matter of intense debate. Profiling of DNA methylation in many cell type, species and environmental set up indicates that the DNA methylation profile is thighly correlated with the cell type and its environment. As a consequence, de novo methylation and DNA demethylation events are not randomly distributed but are actually targeted to particular regulatory DNA elements in the genome, including promoters, enhancers or repeated DNAs. For this latter reason researchers have focused on the role of transcription factor in these DNA methylation events. Yet, it is also recognized that non-coding RNAs, short and long, contribute to the establishment and editing of DNA methylation profiles in mammals. Non-coding RNAs may directly interact and control methylation and demethylation activities and, as a consequence, the pattern of DNA methylation in the genome (Di Ruscio et al., 2013; Arab et al., 2014; Castro-Diaz et al., 2014; Molaro et al., 2014; Turelli et al., 2014). For instance, antisense long non-coding RNA TARID (TCF21 antisense RNA inducing demethylation), activates TCF21 expression by inducing promoter demethylation. TARID sequence is complementary to the sequence of the TCF21 promoter. Its transcription causes the anchoring of GADD45A (growth arrest and DNA-damageinducible, alpha), a regulator of DNA demethylation, at the TCF21 promoter and its subsequent chromatin remodelling (Arab et al., 2014). Understanding the interplay between noncoding RNAs and transcription factors in the establishment and the maintenance of DNA methylation is therefore an important challenge for the future.

The Expanding Role of MBD Genes in Autism: Identification of a MECP2 Duplication and Novel Alterations in MBD5, MBD6, and SETDB1

The methyl-CpG-binding domain (MBD) gene family was first linked to autism over a decade ago when Rett syndrome, which falls under the umbrella of autism spectrum disorders (ASDs), was revealed to be predominantly caused by MECP2mutations. Since that time, MECP2 alterations have been recognized in idiopathic ASD patients by us and others. Individuals with deletions across the MBD5 gene also present with ASDs, impaired speech, intellectual difficulties, repetitive behaviors, and epilepsy. These findings suggest that further investigations of the MBD gene family may reveal additional associations related to autism. We now describe the first study evaluating individuals with ASD for rare variants in four autosomal MBD family members, MBD5, MBD6, SETDB1, and SETDB2, and expand our initial screening in the MECP2 gene. Each gene was sequenced over all coding exons and evaluated for copy number variations in 287 patients with ASD and an equal number of ethnically matched control individuals. We identified 186 alterations through sequencing, approximately half of which were novel (96 variants, 51.6%). We identified seventeen ASD specific, nonsynonymous variants, four of which were concordant in multiplex families: MBD5 Tyr1269Cys, MBD6 Arg883Trp, MECP2 Thr240Ser, and SETDB1 Pro1067del. Furthermore, a complex duplication spanning the MECP2 gene was identified in two brothers who presented with developmental delay and intellectual disability. From our studies, we provide the first examples of autistic patients carrying potentially detrimental alterations in MBD6 and SETDB1, thereby demonstrating that the MBD gene family potentially plays a significant role in rare and private genetic causes of autism.

There is growing evidence of the involvement of the methyl-CpG-binding domain (MBD) genes in neurological disorders. To date, pathogenic mutations have been found in patients with clinical features along the autism continuum for two genes in this family, methyl-CpG-binding domain protein 5 (MBD5) and methyl-CpG-binding protein 2 (MECP2). Both genes carry an MBD domain, the unifying feature for the family that includes nine additional genes; BAZ1A, BAZ1B, MBD1,MBD2, MBD3, MBD4, MBD6, SETDB1 and SETDB2 (Roloff et al., 2003). The MBD genes are involved in a variety of functions, including chromatin remodeling (BAZ1A, BAZ1B, MBD1, MBD2, MBD3, and MECP2), DNA damage repair (BAZ1A and MBD4), histone methylation (SETBD1 and SETDB2), and X chromosome inactivation (MBD2, Roloff et al., 2003, Bogdanovic & Veenstra, 2009). There is also functional interplay among members of this family as they have been found to bind at the same promoter regions (MBD1, MBD2, MBD3, andMECP2), partner with each other in complexes (MBD1 and SETBD1), or act in the same complexes in a mutually exclusive manner (MBD2 and MBD3, Sarraf & I. Stancheva 2004; Ballestar et al., 2005; Le Guezennec et al., 2006; Matarazzo et al., 2007). Little is known thus far about the functions of MBD5 and MBD6; they each encode proteins that localize to chromatin but fail to bind methylated DNA (Laget et al., 2010).

One specific disorder in the autism spectrum, Rett syndrome, is caused almost exclusively by alterations in MECP2 (Amir et al., 1999). Due to the location ofMECP2 on the X chromosome, mutations in females can lead to Rett syndrome while males with the same genetic changes typically present with neonatal encephalopathy (Moretti & Zoghbi 2006). Further investigations have demonstrated that MECP2 misregulation can lead to a wide range of clinical features including autism, Angelman-like symptoms, mental retardation with or without infantile seizures, mild learning disabilities, and schizophrenia (Watson et al., 2001; Klauck et al., 2002; Carney et al., 2003; Shibayama et al., 2004;Coutinho et al., 2007; Harvey et al., 2007; Lugtenberg et al., 2009). Our group previously evaluated the MECP2 gene in a dataset of female ASD patients and identified two mutations reported in classic Rett syndrome patients; an Arg294X mutation and a 41 base pair deletion (Leu386fs) predicted to generate a truncated protein (Carney et al., 2003). Furthermore, while point mutations in MECP2 were first recognized to result in abnormal clinical phenotypes, increased expression of the wild type protein due to gene duplication also results in neurodevelopmental disorders (Meins et al., 2005; Van Esch et al., 2005; del Gaudio et al., 2006;Ramocki et al., 2009).

A second gene in the MBD family, MBD5, was tied to neurodevelopmental disorders following the identification of microdeletions on chromosome 2q22–2q23 (Vissers et al., 2003; Koolen et al., 2004; de Vries et al., 2005; Wagenstaller et al., 2007; Jaillard et al., 2008; van Bon et al., 2009; Williams et al., 2009; Chung et al., 2011; Talkowski et al., 2011; Noh & J. M. Graham Jr 2012). The minimal region for these nonrecurrent deletions covers only a single gene, MBD5 (van Bon et al., 2009; Williams et al., 2009; Talkowski et al., 2011). This suggests that the common features of ASDs, delayed or impaired speech, intellectual disability, epilepsy, and stereotypic hand movements found across microdeletion patients manifest due to a decreased expression of this critical gene (van Bon et al., 2009;Williams et al., 2009; Talkowski et al., 2011). Notably, two cases of individuals with duplications across the critical MBD5 region also present with autistic features and developmental delay (Chung et al., 2012). This demonstrates that precise regulation of both MBD5 and MECP2 must be maintained as either increased or decreased expression of each gene can result in a range of neurodevelopmental disorders.

Supplementing clinical evidence, mouse models have reiterated the potential significance of the MBD family in autism etiology. Mbd1 and Mecp2 null models have abnormal neurobehavioral phenotypes including increased anxiety, and impaired social interactions and synaptic plasticity (Guy et al., 2001; Shahbazian et al., 2002b; Zhao et al., 2003; Allan et al., 2008). Furthermore, a transgenicSetdb1 model established a link between this gene and behavior (Jiang et al., 2010a). Additionally, Setdb1 plays a role in the repression of Grin2b, a gene linked to autism, bipolar disorder, intellectual disability, and schizophrenia (Avramopoulos et al., 2007; Allen et al., 2008; Endele et al., 2010; Jiang et al., 2010a; Myers et al., 2011; O’Roak et al., 2011).

Studies have demonstrated that each of the MBD genes are expressed in the brain, while their specific functions having only been determined for a subset of genes (Shahbazian et al., 2002a, Bogdanovic & Veenstra, 2009, Jiang et al., 2010b, Laget et al., 2010, Safran et al., 2010). MeCP2 is a transcriptional regulator believed to act in neuronal maturation as levels increase over time (Shahbazian et al., 2002a,Chahrour et al., 2008). Stable levels of MeCP2 are required through adulthood, as elimination of this protein in adult mice mimics features seen in knockout Mecp2mice (McGraw et al., 2011). The H3K9 methyltransferase SETDB1 acts both in early development as well as later stages of life (Jiang et al., 2010a, Cho et al., 2012). Removal of Setdb1 in mice results in peri-implantation lethality (Dodge et al., 2004). Studies in the forebrain of transgenic Setdb1 mice demonstrate that it targets the NMDA receptors Grin2a and Grin2b as well as the glutamate receptorGrid2 (Yang et al., 2002, Jiang et al., 2010a).

While there is clinical evidence of MECP2 and MBD5 playing a role in autism, only two studies to date have evaluated patients with ASD for mutations in additional MBD family members (Li et al., 2005; Cukier et al., 2010). Previous work in our laboratory analyzed the coding regions of MBD1, MBD2, MBD3, andMBD4 in over 200 individuals with ASD of African and European ancestry and identified multiple variants that altered the amino acid sequence, were unique to patients with autism, and concordant with disease in multiplex families (Cukier et al., 2010). In contrast, a study by Li and colleagues was restricted to a dataset of 65 Japanese autistic patients and reported only a single variation that might be related to autism (Li et al., 2005). We now expand our initial study of MECP2 to a larger dataset that includes male patients and perform the first study evaluating patients with ASD for alterations in four additional MBD family members: MBD5MBD6, SETDB1 and SETDB2.

Sequencing across the five MBD genes in 287 patients with ASD and 288 ethnically matched control individuals identified a total of 186 unique variations (Table 1, Supplemental Tables 37). These variants included 177 single nucleotide polymorphisms (SNPs), five deletions and four insertions. Ninety (48.4%) of the variations have been previously reported in either the dbSNP 134 database (http://www.ncbi.nlm.nih.gov/projects/SNP/) or RettBASE (http://mecp2.chw.edu.au/), while the remaining 96 variants (51.6%) are novel. Fifty-six variations are predicted to alter the amino acid sequence. Fifty-three of the changes were found solely in patients with ASD and absent from controls. To determine variants most likely to contribute to ASD susceptibility, we prioritized changes that were either unique to affected individuals or that had an increased frequency in cases when compared to controls. The 17 most interesting variants were nonsynonymous and unique to our ASD population (Table 1). We utilized four distinct programs to characterize the variants; GERP (Cooper et al., 2005) and PhastCons (Siepel et al., 2005) to measure the level of amino acid conservation across species and PolyPhen (Adzhubei et al., 2010) and SIFT (Kumar et al., 2009) to predict which alterations might have the damaging consequences to protein function.

ASD Unique, Nonsynonymous Variations

The mutational burden between cases and controls of African or European ancestry for each gene was not statistically significant by the chi-squared test (Supplemental Table 8). This was determined for the overall load of all variants as well as nonsynonymous alterations (Supplemental Table 9).

MBD5

Thirty-two changes were identified in MBD5, 18 of which have been previously reported (Supplemental Table 3). A distinct set of 11 alterations were nonsynonymous, four of which were only identified in patients with ASD (Val443Met, Ile1247Thr, Tyr1269Cys, and Arg1299Gln, Figure 1A–D, Table 1). Three of these four alterations (75%) are predicted to be damaging by SIFT, as compared to only two of seven nonsynonymous variants (28.6%) identified solely in control individuals (Supplemental Table 3). One alteration of high interest, MBD5 Tyr1269Cys, was inherited paternally in all three ASD children in multiplex family 7763 (Figure 1C). Two of the affected individuals (0001 and 0100) were intellectually impaired with measured IQ in the moderate to severe range (Full Scale IQ: 40 and 50, respectively), while the remaining brother with autism (0101) had borderline intellectual functioning (Full Scale IQ=78). Furthermore, all three siblings had a delay in language and displayed self-injurious behaviors. Two individuals presented with macrocephaly (0100 and 0101), and individual 0100 has a history of epilepsy (recurrent non-febrile seizures).

Pedigrees of ASD families carrying alterations in MBD5 and MBD6

MBD6

A total of 44 alterations were detected in MBD6, two being single base pair insertions and the remainder of which were SNPs (Supplemental Table 4). Sixteen of the single nucleotide changes have been previously reported and 28 are novel. A subset of 17 alterations was identified only in individuals with ASD, seven of which are predicted to cause missense changes (Table 1, Figure 1E–K). While each of these changes was only identified in a single proband, three of the alterations have high PolyPhen and SIFT scores and are novel (Arg883Trp, Pro943Arg and Arg967Cys), suggesting a strong functional consequence. Furthermore, one of these alterations, Arg883Trp, was identified in multiplex family 7979 and passed maternally to both affected children (Figure 1I). Individual 0001 has a diagnosis of autism and is nonverbal with moderate intellectual disability. His sister (0100) has a diagnosis of Pervasive Developmental Disorder-Not Otherwise Specified and mild intellectual disability, displaying some phrase speech. Both siblings have a history self-injurious behavior. Their mother (1001), who also carries the alteration, was diagnosed with anxiety/panic disorders, depression, obsessive compulsive disorder, and has a history of epilepsy (adolescent onset seizures).

Along with novel variations of interest in MBD6, we found that two known SNPs occur at a higher frequency within our affected population compared to our control population. The first variation, rs61741508 (c.-2C>A), was recognized in sixteen patients with ASD and five controls and is located just upstream of the ATG start site in the Kozak consensus sequence. This variation also has high conservation scores (Supplemental Table 4). The second SNP, rs117084250 (c.2407-64C>T), falls within intron nine and was found in twelve individuals with ASD but only four controls. However, the conservation scores were relatively low, thereby making this a variant of lesser interest (Supplemental Table 4).

MECP2

Twenty-eight alterations were identified in MECP2 (Supplemental Table 5). Sixteen of these are currently in the dbSNP database and another one has been previously reported in RettBASE, leaving 11 novel variations. While none of the frequently recurring, classic Rett syndrome variations were identified in this study, there are two previously reported MeCP2 alterations of undetermined pathogenicity (Thr240Ser and Ala370Thr) that may cause clinical phenotypes. This first variation, MeCP2 Thr240Ser (rs61749738), was identified in two families of African ancestry (1072 and 17130) and absent in control individuals (Figure 2A,B). Further investigation into additional family members showed that the variation was inherited maternally in both cases and concordant with disease in multiplex family 1072. The second alteration, Ala370Thr (rs147017239), was also inherited maternally in a single proband of African ancestry (family18024, Figure 2C).

Pedigrees of ASD families carrying alterations in MeCP2

SETDB1

A total of 44 changes were found in SETDB1, comprised of 19 known and 25 novel alterations (Supplemental Table 6). Eight changes are predicted to be nonsynonymous, but only one of these, Pro1067del, was found solely in patients with ASD. This change is also the only ASD specific, nonsynonymous deletion identified in the entire study. The variant removes three nucleotides and predicts an in-frame deletion of a single amino acid. This deletion falls within the SET domain of the protein and was inherited maternally in both affected sons in family 17187 (Figure 3A).

Figure 3

Pedigrees of ASD families carrying alterations in SETDB1 and SETDB2

Another novel variation of interest in SETDB1 that we identified in a high proportion of cases versus controls, Pro529Leu, was identified in five ASD families of European ancestry and only a single control (Figure 3B–F). This variant was inherited paternally in one family and maternally in the remaining four families. In family 37265, the variation was passed from the father, who has dyslexia, to both the female proband with autism (0001) who was diagnosed with developmental and language delays as well as her brother (0100) who presented with ADHD, anxiety/panic disorder, language delay and macrocephaly (Figure 3E). In two of the families with maternal inheritance (17663 and 37673), the mothers presented with anxiety/panic disorder. In family 17663, the mother also presented with a history of seizures, sleep disorder and self-reported depression, while the mother in family 37673 reported history of adolescent onset Anorexia Nervosa. The increased incidence of this alteration in cases versus controls, along with neuropsychiatric and neurodevelopmental disorders in parents carrying the alteration, suggests that this variation may confer a variety of clinical consequences.

SETDB2

Thirty-eight single base pair alterations were identified in the SETDB2gene, 21 of which have been previously reported and the remaining 17 are novel (Supplemental Table 7). Eight SNPs are predicted to alter amino acids and three of these were unique to affected individuals: Ile425Thr, Thr475Met and Pro536Arg (Table 1, Figure 3C,G,H). However, these alterations are not predicted to have a highly detrimental effect on the protein and occur within singleton families, making it difficult to determine whether they may play a pathogenic role in ASD.

Along with isolating additional variations in MBD5 and MECP2 that may contribute to neuropsychiatric disease, this study is the first to report prospective pathogenic variations in MBD6 and SETDB1. These include two novel, nonsynonymous alterations in MBD6 (Arg883Trp and Pro943Arg) and one more in SETDB1 (Pro1067del). Furthermore, the MBD6 Arg883Trp and SetDB1 Pro1067del variations each segregated with ASD in the multiplex families. Potential for SETDB1 to play a role in neurobehavioral phenotypes is supported by results from transgenic Setdb1 mice demonstrating a role in mood behaviors (Jiang et al., 2010a).

To date, MBD5 mutations have been identified in individuals presenting a range of clinical phenotypes including ASD, developmental delay, intellectual disability, epilepsy, repetitive movements, and language impairments (Vissers et al., 2003;Koolen et al., 2004; de Vries et al., 2005; Wagenstaller et al., 2007; Jaillard et al., 2008; van Bon et al., 2009; Williams et al., 2009; Chung et al., 2011; Talkowski et al., 2011; Noh & Graham Jr 2012). These results suggest a significant role for theMBD5 isoform 1, which presents with increased expression in the brain (Laget et al., 2010). It has been estimated that between microdeletions and point mutations of MBD5, this gene may play a contributing genetic role in up to 1% of individuals with ASD (Talkowski et al., 2011). Of the nonsynonymous alterations identified in this study, ASD specific changes were more likely to be predicted to be damaging as compared to those variations found in control individuals (Supplemental Table 3). MBD5 Tyr1269Cys is a strong potentially pathogenic change due to its co-segregation with ASD in a multiplex family of three affected children, high conservation of this amino acid across species and altered function in the luciferase transcriptional activation assay. While this alteration does not fall in a known protein domain, it is specific to isoform 1, the isoform predominately expressed in brain (Laget et al., 2010). It seems likely that most alterations inMBD5 related to disease will be rare and unique, as the one alteration previously reported to have an increased frequency in patients with ASD, Gly79Glu, was only identified in a single control in the current study (Talkowski et al., 2011).

The role of MECP2 in developmental disorders is undisputed (Samaco & Neul 2011). Our study supports the possible pathogenicity of two specific MeCP2 alterations: Thr240Ser and Ala370Thr. The first variant, Thr240Ser was identified in two male probands from families of African ancestry, including the multiplex family 1072 where the variant segregated with ASD (Figure 2A,B). The maternal inheritance in family 17130 and presence of an unaffected carrier sister suggests that the variation may only present with a clinical phenotype in a hemizygous state. This variant falls within the transcriptional repression domain and has been previously reported in four studies; three cases of males with intellectual disability and one female with Rett syndrome (Yntema et al., 2002; Bourdon et al., 2003;Bienvenu & J. Chelly 2006; Campos et al., 2007; Bunyan & D. O. Robinson 2008). The second alteration, Ala370Thr, was identified in a singleton family of African ancestry and previously reported in three Chinese individuals: one female with Rett syndrome, her unaffected mother and a male presenting with epileptic encephalopathy (Figure 2C, Li et al., 2007; Wong & Li 2007). Both of these alterations must be further evaluated to isolate their potential functional consequences.

Finally, while we did identify variants of interest in four of the genes studied,SETDB2 alterations did not appear to be related to the occurrence of ASDs.

This is the first study to evaluate the coding regions of MBD5, MBD6, SETDB1, and SETDB2 for rare alterations in individuals with ASD. We identified novel point mutations predicted to be damaging and concordant with disease in multiplex families, as well as a complex duplication encompassing MECP2. Additional studies, ideally both in patients and animal models, are required to determine the precise consequences of these alterations. The results described here compound the evidence of MECP2 and MBD5’s involvement in ASDs and neurodevelopmental disorders and provide the first examples of autistic patients carrying potentially detrimental alterations in MBD6 and SETDB1. This study demonstrates the expanding role MBD genes play in autism etiology.

PI3K/Akt: getting it right matters

T F Franke1          Oncogene (2008) 27, 6473–6488;      http://dx.doi.org:/10.1038/onc.2008.313

The Akt serine/threonine kinase (also called protein kinase B) has emerged as a critical signaling molecule within eukaryotic cells. Significant progress has been made in clarifying its regulation by upstream kinases and identifying downstream mechanisms that mediate its effects in cells and contribute to signaling specificity. Here, we provide an overview of present advances in the field regarding the function of Akt in physiological and pathological cell function within a more generalized framework of Akt signal transduction. An emphasis is placed on the involvement of Akt in human diseases ranging from cancer to metabolic dysfunction and mental disease.

The molecular mechanisms of Akt regulation are summarized in Figure 1.

Figure 1.

Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorCanonical schematic depicting the present state of our understanding of Akt activation and regulation of downstream biological responses. Autophosphorylation of RTKs induces the recruitment of p85 regulatory subunits leading to PI3K activation. Once activated, p110 catalytic subunits phosphorylate plasma membrane-bound phosphoinositides (PI-4-P and PI-4,5-P2) on the D3-position of their inositol rings. The second messengers resulting from this PI3K-dependent reaction are PI-3,4-P2and PI-3,4,5-P3 (also called PIP3). PIP3, in turn, is the substrate for the phosphoinositide 3-phosphatase PTEN, an endogenous inhibitor of PI3K signaling in cells. The phosphoinositide products of PI3K form high-affinity binding sites for the PH domains of intracellular molecules. PDK1 and Akt are two of the many targets of PI3K products in cells. Following binding of the Akt PH domain to PI3K products, Akt is phosphorylated by PDK1 on a critical threonine residue in its kinase domain. mTORC2 is the main kinase activity that through phosphorylation of a C-terminal HM serine residue locks Akt enzyme into an active conformation. Other kinases such as DNA-PK and ILK1 are also capable of phosphorylating Akt at the HM site but may do so in a cell- or context-dependent manner. Akt activation is blunted by phosphatases including PP2A and PHLPP that inhibit Akt activity by dephosphorylation. Studies examining Akt-interacting proteins such as CTMP or second messengers such as Ins(1,3,4,5)P4 suggest that this common pathway of Akt regulation may be further specified within certain functional contexts or during development. Once activated, Akt activation is channeled into a plethora of downstream biological responses reaching from angiogenesis, cell survival, proliferation, translation to metabolism.

Full figure and legend (367K)

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Consequences of Akt activation include diverse biological responses, ranging from primarily metabolic functions such as glucose transport, glycolysis, glycogen synthesis and the suppression of gluconeogenesis to protein synthesis, increased cell size, cell-cycle progression and apoptosis suppression.

Insights into the molecular consequences of increased Akt activation were derived from seminal studies that ultimately identified the ‘orphan’ proto-oncogene as an obligate intermediate downstream of PI3K in the insulin-dependent metabolic control of glycogen synthesis. When searching for kinases that could regulate GSK3, the groups of Brian Hemmings and Phil Cohen realized that Akt inhibited GSK3 activity in an insulin-stimulated and PI3K-dependent manner by direct phosphorylation of an N-terminal regulatory serine residue (Cross et al., 1995). By systematically permutating the amino-acid sequence surrounding the Akt phosphorylation site in GSK3, Alessi et al. (1996b) derived an optimal peptide sequence for Akt phosphorylation (R-X-R-X-X-S/T; where R is an arginine residue, S is serine, T is threonine and X is any amino acid). This Akt consensus motif is a common feature of known substrates of Akt, and its presence predicts reasonably well whether a given protein may be phosphorylated by Akt enzyme in vitro (for review, see Manning and Cantley, 2007). Experiments using randomized permutations on the basis of the motif to optimize substrate peptides have defined the requirement for optimal phosphorylation by Akt further (Obata et al., 2000). The preferred phosphoacceptor for Akt-dependent phosphorylation is a serine residue, but a synthetic substrate peptide with a threonine residue as the phosphoacceptor instead (R-P-R-A-A-T-F; P=proline, A=alanine, F=phenylalanine) is also easily phosphorylated. For achieving optimal phosphorylation efficiency, the phosphoacceptor is best followed by a hydrophobic residue with a large side-chain in the p+1 position, and preceded by a serine or threonine at the p−2 position.

One of the first targets of Akt to be identified that has direct implications for regulating cell survival is the pro-apoptotic BCL2-antagonist of death (BAD) protein. BAD regulation by Akt has exemplified the molecular pathways linking survival factor signaling to apoptosis suppression (for review, see Franke and Cantley, 1997). When BAD is not phosphorylated, it will inhibit Bcl-xL and other anti-apoptotic Bcl-2 family members by direct binding of its Bcl-2 homology domain to their hydrophobic grooves (Gajewski and Thompson, 1996). Once phosphorylated, these phospho-serine residues of BAD form high-affinity binding sites for cytoplasmic 14-3-3 molecules. As a result, phosphorylated BAD is retained in the cytosol where its pro-apoptotic activity is effectively neutralized (Zha et al., 1996). The importance of BAD as an integration point of survival signaling is underscored by the fact that it is a substrate for multiple independent kinase pathways in cells, not all of which phosphorylate BAD at the same site(s) as Akt (Datta et al., 2000). The mechanisms of 14-3-3-dependent regulation of BAD function hereby resemble the Akt-dependent inhibition of FoxO transcription factors that regulate the transcription of pro-apoptotic genes (Brunet et al., 1999).

The function of Akt extends beyond maintaining mitochondrial integrity to keep cytochrome c and other apoptogenic factors in the mitochondria (Kennedy et al., 1999). Akt activity also mitigates the response of cells to the release of cytochrome c into the cytoplasm. Although caspase-9 is an Akt substrate in human cells, where it may explain cytochrome c resistance (Cardone et al., 1998), it may not be the only, or even the most important, target because Akt-dependent cytochrome c resistance can be observed in animal species where caspase-9 lacks a potential Akt phosphorylation site (Fujita et al., 1999; Zhou et al., 2000). Not surprisingly, other components of the post-mitochondrial machinery such as the X-linked inhibitor of apoptotic proteins (XIAP) have been suggested as potential Akt substrates (Dan et al., 2004).

Another important class of Akt targets are proteins involved in the stress-activated/mitogen activated protein kinase (SAPK/MAPK) cascades. Growing experimental evidence points to a close functional relationship between the Akt survival pathway and SAPK/MAPK cascades that are activated by various cellular stresses and are linked to apoptosis. Increased Akt activity has been shown to suppress the JNK and p38 pathways (Berra et al., 1998; Cerezoet al., 1998; Okubo et al., 1998). It has been shown that apoptosis signal-regulating kinase 1 (ASK1) is regulated by Akt and contains an Akt-specific phosphorylation site (Kim et al., 2001). These findings have been confirmed independently by other groups (Yuan et al., 2003; Mabuchi et al., 2004). Thus, ASK1 is likely to be one of the points of convergence between PI3K/Akt signaling and stress-activated kinase cascades, although probably not the only one. Akt also phosphorylates the small G protein Rac1 (Kwon et al., 2000), the MAP2K stress-activated protein kinase kinase-1 (SEK1; also known as JNKK1 or MKK4) (Park et al., 2002) and the MAP3K mixed lineage kinase 3 (MLK3) (Barthwal et al., 2003; Figueroa et al., 2003). Using yeast-2-hybrid screens to identify interacting partners for Akt kinases, Figueroa et al. (2003) found binding of Akt2 to the JNK adaptor POSH. These authors showed that the binding of Akt2 to POSH results in an inhibition of JNK activity, and that this inhibition is mediated by phosphorylation of the upstream kinase MLK3 and leads to the disassembly of the JNK signaling complex. In turn, POSH is also an Akt substrate (Lyons et al., 2007). Taken together, these findings point to an intriguing model for the regulation of the JNK pathway by Akt, in which the Akt-dependent phosphorylation of specific components can block signal transduction through the stress-regulated kinase cascade. In spite of this, it has been reported that Akt also blocks the pro-apoptotic activity of other MAP3Ks such as MLK1 and MLK2 that act in parallel to MLK3 but do not contain a typical Akt consensus phosphorylation motif (Xu et al., 2001). Thus, phosphorylation-based mechanisms may be limited in explaining the role of Akt in blocking JNK signaling.

Although many of its substrates are involved in clearly defined biological functions within a circumscribed context such as cell proliferation, a more thorough analysis of Akt signaling has suggested that the boundaries between metabolic processes and apoptosis suppression may be artificial. For example, the Akt target GSK3 has been implicated both in the regulation of glucose metabolism and cell survival (Pap and Cooper, 1998). These findings suggest that the distinctions between cell growth, survival, metabolism and apoptosis regulation do not properly reflect functional interactions between concurrent biological processes in cells. This shift in perception has been fueled by studies from the Korsmeyer laboratory that have demonstrated a canonical function for the pro-apoptotic Bcl-2 family member BAD in the regulation of glucokinase activity (Danial et al., 2003). It is conceivable that findings of PKA-dependent regulation of BAD in glucose metabolism can be extrapolated to BAD inhibition by Akt. Still, a formal confirmation for a role of Akt in this process has yet to be presented (for review, see Downward, 2003).

The critical importance of Akt signaling for neuronal function is implied from several lines of in vitro evidence using neuronal cell lines and dispersed primary neuronal cultures that have demonstrated a requirement for Akt in the protection against trophic factor deprivation, oxidative stress and ischemic injury (Dudek et al., 1997; Salinas et al., 2001; Noshita et al., 2002). Dysregulation of Akt activity is observed in neurodegenerative diseases including Alzheimer’s disease (Rickle et al., 2004; Ryderet al., 2004), Parkinson’s disease (Hashimoto et al., 2004) and Huntington’s disease (Humbert et al., 2002), and it is also associated with the pathobiological mechanisms underlying spinocerebellar ataxia (Chen et al., 2003). A mechanistic involvement of impaired Akt signaling in neurodegeneration is further supported by the Akt-dependent phosphorylation of the disease-related proteins huntingtin (Humbert et al., 2002) and ataxin (Chen et al., 2003).

Other studies suggest that the involvement of Akt in brain function extends beyond the protection of neuronal cells against apoptotic insults. Indeed, pathological changes in Akt signal transduction have been described that are associated with mental diseases. Significantly decreased Akt1 expression has been reported in patients suffering from familial schizophrenia (Emamian et al., 2004). Decreased Akt1 levels are correlated with increased GSK3 activity, presumably because of the lack of the Akt-dependent inhibitory input on GSK3. In support of AKT1 being a susceptibility gene for schizophrenia, Akt1(−/−) mice exhibit increased sensitivity to the sensorimotor disruptive effect of amphetamine, which is partly reversed by the treatment of mutant mice with the antipsychotic drug haloperidol (Emamian et al., 2004). Additional support for a contribution of impaired Akt signaling in the pathogenesis of schizophrenia derives from the finding of mutant PI3K signaling in schizophrenia (for review, see Arnold et al., 2005). A direct involvement of Akt in dopaminergic action is indicated by the observation that Akt1(−/−) mutant mice exhibit a behavioral phenotype resembling enhanced dopaminergic transmitter function (Emamian et al., 2004). By interacting with the GSK3 pathway, Akt modulates the suppression of dopamine (DA)-associated behaviors after treatment with the mood stabilizer lithium (Beaulieu et al., 2004). Furthermore, a β-arrestin 2-mediated kinase/phosphatase scaffold of Akt and protein phosphatase A (PP2A) is required for the regulation of Akt downstream of DA receptors (Beaulieu et al., 2005). Still, the role of Akt in dopaminergic responses by far exceeds actions downstream of DA receptors: the insulin-dependent regulation of DA transporter also depends on Akt activity (Garcia et al., 2005).

Since the field of Akt signaling in psychiatric disorders is still emerging, it may be too early to speculate about the molecular involvement of Akt in regulating higher brain function. Possible functional outlets of Akt include some of the substrates mentioned above, including mTORC1 and GSK3. In addition, substrates of Akt related to synaptic plasticity and transmission have been described. One such novel substrate of Akt related to neuronal excitability is the β2-subunit of the type A γ-aminobutyric acid receptor (GABAA-R) (Lin et al., 2001). In support of a direct involvement of Akt in synaptic function, studies directed at working memory performance performed in Akt1(−/−) mice (Lai et al., 2006) and in healthy individuals carrying the AKT1 coding variation observed in familial schizophrenia (Tan et al., 2008) find a strong correlation with cognitive performance. Additional roles for Akt in higher brain function are suggested by studies from the Nestler laboratory that have explored the IRS2-Akt pathway during the development of tolerance to opiate reward (Russo et al., 2007). By using viral-mediated gene transfer to express mutant Akt in midbrain neurons, these authors demonstrate that downregulation of the IRS2-Akt pathway mediates morphine-induced decreases in cell size of DA neurons in brain regions that are critically involved in the reward circuitry and affected in individuals addicted to drugs of abuse.

Finally, TSC patients show an increased incidence of autism spectrum disorders (ASD) ranging from 25 to 50% (for review, seeWiznitzer, 2004). Individuals with macrocephaly due to Lhermitte–Duclos disease are prone to ASD and show a pronounced incidence of mutations in the PTEN tumor suppressor gene (Butler et al., 2005). Additional experimental support for a possible involvement of PTEN/Akt in ASD is provided by data from the Parada laboratory examining the morphology and behavior of mutant mice with neuron-specific knockout of PTEN (Kwon et al., 2006). Future studies will be required to clarify the function of Akt in cognition and characterize the underlying molecular mechanisms. In spite of this, these initial studies suggest a complex function of Akt in conditions affecting brain function and mental health.

The emerging involvement of Akt in higher brain function is summarized in Figure 2.

Figure 2.

Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Akt kinase regulates diverse aspects of neuronal cell function. Akt activation in neuronal cells follows similar mechanisms to those outlined in Figure 1, including activation of PI3K by RTKs including IGF-1/insulin and nerve growth factor (BDNF/NT-3) receptors. Other mechanisms governing Akt activity in neuronal cells include G-coupled receptors for the monoamine neurotransmitters serotonin (5-HT) (for review, see Raymond et al., 2001) and dopamine (DA) (for review, see Beaulieu et al., 2007). Depending on receptor type (D2-DA and 5-HT1A receptors vs D1-DA receptors), binding of 5-HT or DA decreases or increases the activity of adenylyl cyclase (AC), respectively. Changes in cAMP second messenger levels, in turn, alter PKA and PP2A activity. PP2A is inhibited by increased PKA activity, thus maintaining Akt in an activated state after 5-HT1Areceptor simulation (Hsiung et al., 2008). After binding of DA to D2-DA receptor, following initial inhibition of AC, a secondary internalization complex is formed between β-arrestin 2, PP2A and Akt leading to the inhibition of Akt. In neuronal cells, activated Akt regulates diverse targets that have been implicated in the regulation of protein translation and cell size (mTORC1), axonal outgrowth (GSK3), apoptosis suppression (BAD) and synaptic plasticity (GABAA-R). Details regarding functional consequences of Akt regulation for higher brain function are discussed in the text.

Full figure and legend (306K)

 

When considering the present understanding of all the signals leading to and from Akt, we face a growing complexity that is in part compounded by the intersection of multiple signaling cascades. Many substrates of Akt are shared with other kinases that have similar specificities. Moreover, signals originating from activated Akt do not simply lead to changes in the biological activity of specific downstream substrates, but affect entire signaling networks. In spite of this, there is hope that there is order to the far-reaching physiological involvement of Akt. One possibility is that differential regulation of the binding partners of Akt may determine cell- and context-specific signaling by Akt. Studies are now needed to elucidate the physiological functions of the binding partners of Akt in mammalian physiology.

A second challenge that the field is facing arises from the involvement of Akt in multiple areas of physiology. These now exceed cancer and diabetes and, as briefly outlined above, include higher brain functions related to cognition.

SETDB1 in Early Embryos and Embryonic Stem Cells

Yong-Kook Kang

The histone methyltransferase SETDB1 contributes to the silencing of local chromatin and the target specificity appears to be determined through various proteins that SETDB1 interacts with. This fundamental function endows SETDB1 with specialized roles in embryonic cells. Keeping the genomic and transcriptomic integrity via proviral silencing and maintaining the pluripotency by repressing the differentiation-associated genes have been demonstrated as the roles of SETDB1 in embryonic stem cells. In early developing embryos, SETDB1 exhibits characteristic nuclear mobilizations that might account for its pleiotropic roles in these rapidly changing cells as well. Early lethality of SETDB1-null embryos, along with other immunolocalization findings, suggests that SETDB1 is necessary for reprogramming and preparing the genomes of zygotes and pluripotent cells for the post-implantation developmental program.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706629/

Exome sequencing of probands with autism have revealed broadly similar results:de novo mutations in a large set of genes occur in a significant fraction of patients, with relatively high OR’s for damaging mutations in genes expressed in the brain9,1921. Most interestingly, CHD8, which like CHD7 reads H3K4me marks, is frequently mutated in autism22, raising the question of whether the H3K4me pathway may play a role in many congenital diseases. Among 249 protein-alteringde novo mutations in CHD (Supplementary Table 4) and 570 such mutations in autism9,19,20,23, there were two genes, CUL3 and NCKAP1, with damaging mutations in both CHD and autism and none in controls (P = 0.001, Monte Carlo simulation), and several others with mutations in both (e.g., SUV40H1 and CHD7). Similarly, rare copy number variants at 22q11.2, 1q21, and 16p11 are found in patients with autism, CHD or both diseases2426. These observations suggest variable expressivity of mutations in key developmental genes. Identification of the complete set of these developmental genes and the full spectrum of the resulting phenotypes will likely be important for patient care and genetic counseling.

 

Context-specific microRNA function in developmental complexity

Adam P. Carroll1,2Paul A. Tooney1,2 and Murray J. Cairns1,2,*      http://jmcb.oxfordjournals.org/content/early/2013/03/01/jmcb.mjt004.full     J Mol Cell Biol (2013)    doi: 10.1093/jmcb/mjt004

Since their discovery, microRNAs (miRNA) have been implicated in a vast array of biological processes in animals, from fundamental developmental functions including cellular proliferation and differentiation, to more complex and specialized roles such as longterm potentiation and synapse-specific modifications in neurons. This review recounts the history behind this paradigm shift, which has seen small non-coding RNA molecules coming to the forefront of molecular biology, and introduces their role in establishing developmental complexity in animals. The fundamental mechanisms of miRNA biogenesis and function are then considered, leading into a discussion of recent discoveries transforming our understanding of how these molecules regulate gene network behaviour throughout developmental and pathophysiological processes. The emerging complexity of this mechanism is also examined with respect to the influence of cellular context on miRNA function. This discussion highlights the absolute imperative for experimental designs to appreciate the significance of context-specific factors when determining what genes are regulated by a particular miRNA. Moreover, by establishing the timing, location, and mechanism of these regulatory events, we may ultimately understand the true biological function of a specific miRNA in a given cellular environment.

It was once considered the central dogma of molecular biology that gene expression was regulated in a unidirectional manner whereby cellular instructions were encoded in DNA to be transcribed to produce RNA, which simply acted as a messenger molecule to produce the protein end-products that executed these cellular instructions. In fact, signs of a biological phenomenon whereby non-protein-coding RNA molecules could interfere with this very process were not even realized until the 1970s and early 1980s, when exogenous oligonucleotides complementary to ribosomal RNA were found to interfere with ribosome function (Taniguchi and Weissmann, 1978; Eckhardt and Luhrmann, 1979;Jayaraman et al., 1981). A number of experiments in both prokaryotes and eukaryotes further supported the notion of antisense RNA as an antagonist to RNA function (Chang and Stoltzfus, 1985; Ellison et al., 1985; Harland and Weintraub, 1985; Izant and Weintraub, 1985; Melton, 1985), and one such experiment elegantly demonstrated that the introduction of synthetic oligonucleotides complementary to 3′- and 5′-terminal repeats of Rous sarcoma virus 35S RNA not only attenuated viral replication and cell transformation, but also inhibited viral RNA translation in vitro (Stephenson and Zamecnik, 1978; Zamecnik and Stephenson, 1978).

In addition to this, the successful inhibition of thymidine kinase gene expression by antisense RNA in eukaryotic cells precipitated the concept of antisense RNA not only as an experimental tool, but also as a therapeutic design (Izant and Weintraub, 1984). Determining the functionality of a previously identified gene sequence without identifying, isolating, or characterizing the protein product; interfering with RNAs that are never translated; and silencing the expression of disease-associated transcripts in a sequence-specific manner: these were some very appealing prospects. By the late 1980s and early 1990s, a variety of techniques had evolved in the field of molecular and applied genetics whereby various antisense DNA and RNA construct designs were employed to efficiently downregulate target gene expression (Fire et al., 1991).

Meanwhile, the scientific community was also beginning to appreciate a role for endogenous antisense RNA. Short antisense transcripts were found to form an RNA–RNA duplex with the 5′ end of the replication primer of the ColE1 plasmid (Tomizawa et al., 1981; Tomizawa and Itoh, 1982). Endogenous antisense RNA control in prokaryotes was also linked with various biological processes such as plasmid replication, transposition, temporal bacteriophage development, and catabolite repression in bacteria (Light and Molin, 1983; Simons and Kleckner, 1983; Kumar and Novick, 1985). Evidence was also beginning to mount to implicate antisense control mechanisms in eukaryotic organisms (Adeniyi-Jones and Zasloff, 1985; Farnham et al., 1985; Heywood, 1986; Spencer et al., 1986; Williams and Fried, 1986; Stevens et al., 1987), including the demonstration that antisense transcripts in the bovine papillomavirus type 1 (BPV-1) genome prevented episomal replication (Bergman et al., 1986).

It was only a matter of time before phenomena of gene silencing began to unfold in animals. Previous work in the 1980s with Caenorhabditis elegans had established that mutations in the genes for lin-4, lin-14, lin-28, lin-29, and lin-41 altered the heterochronic lineage of developing larvae, resulting in a failure to control temporal aspects of post-embryonic development (Chalfie et al., 1981; Ambros and Horvitz, 1984; 1987; Ambros, 1989); thus, these genes were referred to as being ‘heterochronic’. However, in 1993 it was discovered that lin-4 was located within an intron and was thus unlikely to encode a protein. More significantly, two lin-4 transcripts ∼22 and 61 nucleotides in length were identified that exhibited complementarity to a repeat sequence element in the 3′ untranslated region (UTR) of lin-14 mRNA (Lee et al., 1993). With another report soon replicating this finding in C. elegans andCaenorhabditis briggsae (Wightman et al., 1993), the notion was set forth that the 22-nucloetide lin-4 transcript represented an active mature form of the 61-nucelotide transcript and functioned to control worm larval development by binding to the 3′-UTR of lin-14, thereby negatively regulating its function via an antisense RNA–RNA interaction. Furthermore, lin-4 exhibited complementarity to seven regions within the 3′-UTR of lin-14, demonstrating that gene expression was more potently inhibited as more of these non-coding transcripts bound to the mRNA (He and Hannon, 2004). Retrospectively, we can identify the lin-4 gene in C. elegans as the pioneer of a new class of small, non-coding RNAs called microRNA (miRNA) (Lee et al., 1993), which utilize the RNA interference (RNAi) pathway to regulate the expression of protein-encoding genes at post-transcriptional level (He and Hannon, 2004).

The following few years were somewhat quiet at the forefront of miRNA research, with lin-4 mechanism assumed to be a unique event. Meanwhile, RNAi was coming to prominence in 1998 with Fire and Mello (along with their colleagues) reporting double-stranded RNA (dsRNA) to be far more potent at mediating gene suppression in C. elegans than single-stranded antisense RNA (Fire et al., 1998). Interestingly, only small quantities of dsRNA were required to induce post-transcriptional gene silencing (PTGS), and it was hypothesized that an endogenous catalytic or amplification component was mediating mRNA degradation prior to translation (Montgomery et al., 1998). RNAi was soon thereafter reported as an ATP-dependent process in an in vitroDrosophila embryo lysate system where dsRNA was processed into 21–23-nucleotide species that appeared to guide sequence-specific mRNA cleavage (Zamore et al., 2000). When dsRNA was shown by the Tuschl laboratory to be processed into 21–22-nucleotide short interfering RNA (siRNA) by a ribonuclease III enzyme to mediate sequence-specific RNAi in human embryonic kidney HEK-293 cells, the prospect was set forth for exogenous 21–22-nucleotide siRNA to be developed as gene-specific therapeutic molecules (Elbashir et al., 2001a).

With incredible excitement surrounding the implications of RNAi, Ruvkun and colleagues discovered a second miRNA inC. elegans in 2000. Like lin-4, the newly discovered let-7 exhibited complementarity to the 3′-UTR of heterochronic genes, in this case lin-14, lin-28, lin-41, lin-42, and daf-12 (Reinhart et al., 2000). Moreover, they discovered that let-7 was highly conserved in its temporal regulation across phylogeny (Pasquinelli et al., 2000), refuting the widely believed concept that lin-4 and let-7 were a worm-specific oddity and propelling miRNA to significance as native endogenous clients of the RNAi machinery. This catalysed intense genome-wide searches for the discovery of more endogenous small regulatory RNAs in numerous species, to the point that miRBase Release 19 currently contains sequence data for 25141 mature miRNA products in 193 organism species (Kozomara and Griffiths-Jones, 2011).

The significance of non-coding RNA was further illuminated in 2001 when the completion of the human genome project revealed that <2% of the human genome encoded proteins (Lander et al., 2001). It has been realized that the ratio of non-coding to protein-coding DNA in the genome correlates with developmental complexity (Mattick, 2004), and a recent publication has reported on the exponential correlation of miRNA gene number and 3′-UTR length—but not 5′-UTR or coding sequence length—with morphological complexity in animals (Chen et al., 2012). This was measured according to the number of cell types within each organism, and also confirmed earlier observations that 3′-UTR length in housekeeping genes has remained short across organisms, thereby minimizing miRNA-binding site potential and reducing the complexity with which these constitutively expressed genes are regulated (Stark et al., 2005). Today we certainly have a stronger appreciation for RNA molecules to function not only as messengers of protein production, but also as complex regulatory molecules facilitating the intricate control of gene expression required for developmental complexity (Kosik, 2009).

Mechanisms of miRNA function

When considering non-coding RNA function, miRNAs constitute one of the largest classes of endogenous, non-coding regulatory RNA molecules in animals. In their mature form they are ∼19–22 nucleotides in length, and they interact via Watson–Crick binding with regions of complementarity primarily within the 3′-UTR of mRNA transcripts. In doing so, miRNAs act as sequence-specificity guides for the RNAi machinery to mediate repression of target gene expression at post-transcriptional level by negatively regulating mRNA stability and/or protein translation.

miRNA biogenesis

miRNAs are typically transcribed by RNA polymerase II (pol II) as long primary miRNA (pri-miRNA) transcripts, which undergo sequential cleavage into a precursor miRNA (pre-miRNA) transcript before being cleaved again into the mature miRNA duplex (Figure 1). These pri-miRNA transcripts range in length from several hundred nucleotides to several kilobases, can contain either a single miRNA or clusters of several miRNAs, and originate from intronic regions of protein-coding and non-coding genes, as well as from intergenic and exonic regions (Rodriguez et al., 2004; Saini et al., 2007). The microprocessor complex is responsible for mediating pri-miRNA cleavage, with the dsRNA-binding protein DGCR8 (DiGeorge syndrome critical region gene 8) binding the pri-miRNA and positioning the catalytic site of Drosha—a ribonuclease III (RNase III) dsRNA-specific endonuclease—11 nucleotides from the base of the duplex stem to mediate nuclear processing to the pre-miRNA transcript (Denli et al., 2004; Han et al., 2006). This produces a pre-miRNA hairpin typically 55–70 nucleotides in length with a two-nucleotide 3′ overhang, characteristic of RNase III-mediated cleavage (Lee et al., 2003). This two-nucleotide overhang facilitates the subsequent exportation of the pre-miRNA from the nucleus to the cytoplasm by a RanGTP/Exportin5-dependent mechanism and is suspected to also facilitate subsequent cleavage by the RNase III endonuclease Dicer (Yi et al., 2003; Bohnsack et al., 2004; Lund et al., 2004). This cleavage requires the interaction of Dicer with the dsRNA-binding protein TRBP [HIV-1 transactivating response (TAR) RNA-binding protein] (Forstemann et al., 2005), and as a result of Dicer processing the terminal base pairs and the loop of the pre-miRNA are excised. This produces a 19–22-nucleotide mature miRNA duplex, which possess two-nucleotide overhangs at each 3′ end (Lee et al., 2002).

 

Figure 1

http://jmcb.oxfordjournals.org/content/early/2013/03/01/jmcb.mjt004/F1.medium.gif

Figure 1

A model for canonical miRNA biogenesis and function in animals. After their transcription by RNA polymerase II, pri-miRNAs are cleaved in the nucleus by Drosha, which forms a microprocessor complex with DGCR8. This generates the pre-miRNA, which is actively exported into the cytoplasm via a RanGTP/Exportin 5-dependent mechanism. In the cytoplasm, Dicer binds the base of the pre-miRNA stem defined in the nucleus by Drosha. Dicer cleavage liberates a mature miRNA duplex that exhibits imperfect complementarity. This miRNA duplex is assembled into the miRISC loading complex, in which the passenger strand is discarded. The miRNA guides the mature miRISC to regions of complementarity within mRNA transcripts, thereby mediating post-transcriptional gene silencing through translational repression and/or mRNA degradation.

miRISC loading

After their maturation into small RNA duplexes, miRNAs are loaded into ribonucleoprotein (RNP) complexes, often referred to as miRNA-induced silencing complexes (miRISCs), RISCs, or miRNPs. The signature components of each miRISC are the miRNA and an Argonaute (AGO) protein. In humans, there are four AGO proteins (AGO1-4), each consisting of the highly conserved P-element-induced wimpy testes (PIWI), middle (MID), and PIWI-AGO-Zwille (PAZ) domains, along with a less-conserved terminal domain. The loading of the miRNA into this protein complex has been proposed to occur in tandem with Dicer-mediated miRNA maturation (Gregory et al., 2005;Maniataki and Mourelatos, 2005) and requires ATP hydrolysis with additional chaperone proteins to create an open conformation to facilitate loading of the miRNA duplex (Liu et al., 2004; Yoda et al., 2010).

A key feature of miRNA is that while both strands of a small RNA duplex are capable of activating the miRISC, typically only one strand will induce silencing (Khvorova et al., 2003). This asymmetry is primarily governed by the relative thermodynamic properties of the RNA duplex, such that the miRISC-associated helicase preferentially unwinds the miRNA duplex from the end with least resistance in terms of inter-strand hydrogen bonding. The strand with its 5′ end at this less thermodynamically stable end is selected as the guide strand, and proteins such as TRBP or protein kinase, interferon-inducible dsRNA-dependent activator (PACT) are proposed to interact with Dicer to sense this thermodynamic asymmetry (Schwarz et al., 2003; Noland et al., 2011). In doing so, the guide strand is retained in the miRISC, while the other strand (the passenger, or the miRNA* strand) is discarded (Hutvagner, 2005; Matranga et al., 2005). miRNA strand selection also appears to be independent of Dicer processing polarity (Preall et al., 2006), where both ends of a duplex have similar thermodynamic properties, both the miRNA and miRNA* act as the guide strand with similar frequencies (Schwarz et al., 2003). However, strand selection does not always occur according to the axiom of thermodynamic strand asymmetry, with tissue-specific factors appearing to play a role in enabling both the miRNA and miRNA* strands to co-accumulate and function as the guide strand (Ro et al., 2007). For this reason, miRNA nomenclature has advanced beyond the miRNA* system, with the adoption of miRNA-5p and -3p names to indicate whether the mature miRNA sequence is derived from the 5′ or 3′ end of the pre-miRNA transcript.

Once the mature miRNA strand has been isolated in the mature miRISC, the AGO protein functions as an interface for the miRNA to interact with its mRNA targets. Recent characterization of human AGO2 has revealed that the 3′ hydroxyl of the miRNA inserts into a hydrophobic pocket of AGO such that the terminal nucleotide stacks against the aromatic ring of a conserved phenylalanine residue in the AGO PAZ domain (Jinek and Doudna, 2009). Meanwhile, the MID domain forms a binding pocket that anchors the miRNA 5′ phosphate such that this terminal nucleotide is distorted and does not interact with the target mRNA (Ma et al., 2005; Parker et al., 2005).

…….

Since being discovered as regulators of developmental timing in C. elegans, it has become widely established that miRNA-mediated regulation of gene expression is a fundamental biological phenomenon required to facilitate key developmental processes such as cellular proliferation, programmed cell death, and cell lineage determination and differentiation (Bartel, 2009; Ambros, 2011). Their significance is such that 60% of the human genome is predicted to be regulated by miRNA function (Friedman et al., 2009), each miRNA estimated to regulate around 200 target genes (Krek et al., 2005).

Figure 2

http://jmcb.oxfordjournals.org/content/early/2013/03/01/jmcb.mjt004/F2.medium.gif

Figure 2

Characteristic miRNA associated with the proliferation and differentiation of specialized cell types. A number of distinct miRNAs are expressed at specific stages through development to play a vital role in mediating cell proliferation, specification, and differentiation. A number of miRNAs involved in the establishment of specialized cell types are illustrated for neurogenesis (Smirnova et al., 2005;Makeyev et al., 2007; Shen and Temple, 2009; Shi et al., 2010; Zhao et al., 2010), myogenesis (Chen et al., 2006; Kim et al., 2006), haematopoiesis (Chen et al., 2004; Georgantas et al., 2007;Vasilatou et al., 2010), oligodendrocyte differentiation (Lau et al., 2008; Dugas et al., 2010), as well as induced pluripotent stem (iPS) cell reprogramming (Miyoshi et al., 2011).

 

miRNAs play a central role in establishing the spatiotemporal gene expression patterns required to establish specialized cell types and promote developmental complexity. The inherent complexity of miRNA function, however, requires a scientific approach in which context-specific miRNA function must be acknowledged if advancements are to be made in understanding how these small regulatory RNA molecules function in various developmental and pathophysiological processes. While this requires an appreciation for mechanistic aspects such as non-redundant miRISC function and the dynamic regulatory outcomes this facilitates, arguably the greatest challenge facing miRNA biology is the identification of the many genes that each miRNA targets and an understanding of the context-specific factors that determine when and how these genes are regulated.

 

 

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RNA Modification

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New RNA Modification Added to Epitranscriptomic Library   

GEN News Highlights  Feb 17, 2016    http://www.genengnews.com/gen-news-highlights/new-rna-modification-added-to-epitranscriptomic-library/81252376/

 

http://www.genengnews.com/Media/images/GENHighlight/109124_web3461772372.jpg

 

In 1956, Francis Crick—co-discoverer of DNA’s helical structure—postulated what is now considered to be a central doctrine of the biological sciences stating that “The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid.” What Crick was suggesting was that DNA makes RNA and, in turn, RNA makes protein.

In the time since the initial proposal of the central dogma, scientists have come to understand that there are not only instances of reverse information flow from RNA to DNA, but chemical alterations to RNA structures that can have a profound effect on gene regulation. The discovery of these alterations has added a critical dimension to how scientists view the genetic code and recently spawned an entirely new field of study within molecular biology: the epitranscriptome.

Now, a recent study by scientists at the University of Chicago and Tel Aviv University has revealed evidence that provides a promising new lever in the control of gene expression. The researchers describe a small chemical modification to RNA that can significantly boost the conversion of genes to proteins.

The findings from this study were published recently in Nature through an article entitled “The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA.”

“Epigenetics, the regulation of gene expression beyond the primary information encoded by DNA, was thought until recently to be mediated by modifications of proteins and DNA,” explained co-senior study author Gidi Rechavi, Ph.D., chair in oncology at Tel Aviv University’s Sackler Faculty of Medicine and head of the Cancer Research Center at Sheba Medical Center. “The new findings bring RNA to a central position in epigenetics.”

“This discovery further opens the window on a whole new world of biology for us to explore,” added co-senior study author Chuan He, Ph.D., professor in the department of chemistry and investigator within the Howard Hughes Medical Institute at the University of Chicago. “These modifications have a major impact on almost every biological process.”

Previously, Dr. He’s laboratory discovered the first RNA demethylase that reverses the most prevalent mRNA methylation N6-methyladenosine (m6A), implying that the addition and removal of the methyl group could dramatically affect these messengers and the outcome of gene expression—as also seen for DNA and histones—which subsequent research found to be true.

In the current study, the investigators described a second functional mRNA methylation, N1-methyladenosine (m1A). Like m6A, the small modification is evolutionarily conserved and common, present in humans, rodents, and yeast. However, its location and effect on gene expression reflect a new form of epitranscriptome control.

“The discovery of m1A is extremely important, not only because of its own potential in affecting biological processes but also because it validates the hypothesis that there is not just one functional modification,” Dr. He stated. “There could be multiple modifications at different sites where each may carry a distinct message to control the fate and function of mRNA.”

From their findings, the research team estimates that that m1A may be present on transcripts of more than one out of three expressed human genes—suggesting that m1A, like m6A, may be a mechanism by which cells rapidly boost the expression of hundreds or thousands of specific genes.

“mRNA is the perfect place to regulate gene expression because they can code information from transcription and directly impact translation—you can add a consensus sequence to a group of genes and use a modification of the sequence to readily control several hundred transcripts simultaneously,” Dr. He said. “If you want to rapidly change the expression of several hundred or a thousand genes, this offers the best way.”

The scientists were excited by their findings and have plans for future studies that will examine the role of m1A methylation in human development, for diseases such as diabetes and cancer, and its potential as a target for therapeutic uses.

“This study represents a breakthrough discovery in the exciting, nascent field of the ‘epitranscriptome,’ which is how RNAs are regulated, akin to the genome and the epigenome,” commented Christopher Mason, Ph.D., associate professor at Weill Cornell Medicine, who was not affiliated with the study. “What is important about this work is that m6A was recently found to enrich at the ends of genes, and now we know that m1A is what is helping regulate the beginning of genes, and this opens up many questions about revealing the ‘epitranscriptome code’ just like the histone code or the genetic code.”

 

The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA

Dan DominissiniSigrid NachtergaeleSharon Moshitch-MoshkovitzNitzan Kol, et al.
Nature(2016 10 Feb )      http://dx.doi.org:/10.1038/nature16998      http://www.nature.com/nature/journal/vaop/ncurrent/full/nature16998.html

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N6-methyladenosine (m6A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N1-methyladenosine (m1A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m1A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m1A in promoting translation of methylated mRNA.

 

Figure 1: Development of m1A-seq to map a newly identified constituent of mammalian mRNA.

Development of m1A-seq to map a newly identified constituent of mammalian mRNA.

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a, Chemical structures of m1A and m6A. Methyl groups (-CH3) are in red and the positive charge (+) on m1A is in blue. b, LC-MS/MS quantitation of m1A, m6A and Ψ in human and mouse mRNA isolated from the indicated cell types. …

 

Figure 3: m1A occurs in GC-rich sequence contexts and in genes with structured 5′ UTRs.

m1A occurs in GC-rich sequence contexts and in genes with structured 5′ UTRs.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16998-f3.jpg

a, Sequence frequency logo for a set of 192 adenosines in peak areas that have a higher mismatch rate in immunoprecipitation relative to input (FC ≥ 6) in HepG2 demonstrates the GC-rich context of m1A. b, Length-adjusted minimum free energy…

 

Figure 5: m1A in mRNA is a dynamic modification that responds to changing physiological and stress conditions, and varies between tissues.

m1A in mRNA is a dynamic modification that responds to changing physiological and stress conditions, and varies between tissues.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature16998-f5.jpg

a, LC-MS/MS quantification of m1A (left, grey) and m6A (right, black) in mRNA of untreated and glucose-starved (upper panels) or heat shock-treated (lower panels) HepG2 cells, presented as percentage of unmodified A. Mean values ± s.e.m…

 

RNA modification discovery suggests new code for control of gene expression

A new cellular signal discovered by a team of scientists at the University of Chicago and Tel Aviv University provides a promising new lever in the control of gene expression.    Gene expression study

The study, published online Feb. 10 in the journal Nature, describes a small chemical modification that can significantly boost the conversion of genes to proteins. Together with other recent findings, the discovery enriches a critical new dimension to the “Central Dogma” of molecular biology: the epitranscriptome.

“This discovery further opens the window on a whole new world of biology for us to explore,” said Chuan He, the John T. Wilson Distinguished Service Professor in Chemistry, Howard Hughes Medical Institute investigator and senior author of the study. “These modifications have a major impact on almost every biological process.”

The central dogma of molecular biology describes the cellular pathway where genetic information from DNA is copied into temporary RNA “transcripts,” which provide the recipe for the production of proteins. Since Francis Crick first postulated the theory in 1956, scientists have discovered a multitude of modifications to DNA and proteins that regulate this process.

Only recently, however, have scientists focused on investigating dynamic modifications that specifically target the RNA step. In 2011, He’s group discovered the first RNA demethylase that reverses the most prevalent mRNA methylation N6-methyladenosine, or m6A, implying that the addition and removal of the methyl group could dramatically affect these messengers and impact the outcome of gene expression, as also seen for DNA and histones. Subsequently, scientists discovered that the dynamic and reversible methylation of m6A dramatically controlled the metabolism and function of most cellular messenger RNA, and thus, the production of proteins.

In the new Nature study, researchers from UChicago and Tel Aviv University describe a second functional mRNA methylation, N1-methyladenosine, or m1A. Like m6A, the small modification is evolutionarily conserved and common, and present in humans, rodents and yeast, the authors found. But its location and effect on gene expression reflect a new form of epitranscriptome control and suggest an even larger cellular “control panel.”

“The discovery of m1A is extremely important, not only because of its own potential in affecting biological processes, but also because it validates the hypothesis that there is not just one functional modification,” He said. “There could be multiple modifications at different sites where each may carry a distinct message to control the fate and function of mRNA.”

The researchers estimated that m1A was present on transcripts of more than one out of three expressed human genes. Methylated genes exhibited enhanced translation compared to unmethlyated genes, producing protein levels nearly twice as high in all cell types. This increase suggests that m1A, like m6A, may be a mechanism by which cells rapidly boost the expression of hundreds or thousands of specific genes, perhaps during important processes such as cell division, differentiation or under stress.

“mRNA is the perfect place to regulate gene expression, because they can code information from transcription and directly impact translation; you can add a consensus sequence to a group of genes and use a modification of the sequence to readily control several hundred transcripts simultaneously,” He said. “If you want to rapidly change the expression of several hundred or a thousand genes, this offers the best way.”

However, despite their complementary effects, m1A and m6A exert their influence on mRNA through different pathways. While studies have found that m6A localizes predominantly to the tail of messenger RNA molecules, increasing their translation and turnover rate, m1A was found largely near the start codon of mRNA transcripts, where protein translation begins. The different mechanisms could allow for finer tuning of post-transcriptional gene expression, or the selective activation of particular genes in different physiological situations.

“This study represents a breakthrough discovery in the exciting, nascent field of the ‘epitranscriptome,’ which is how RNAs are regulated, akin to the genome and the epigenome,” said Christopher Mason, associate professor at Weill Cornell Medicine, who was not affiliated with the study. “What is important about this work is that m6A was recently found to enrich at the ends of genes, and now we know that m1A is what is helping regulate the beginning of genes, and this opens up many questions about revealing the ‘epitranscriptome code’ just like the histone code or the genetic code.”

Future studies will examine the role of m1A methylation in human development, diseases such as diabetes and cancer, and its potential as a target for therapeutic uses.


Citation: “The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA,” Nature, Feb. 10, 2016, by Chuan He, Dan Dominissini, Sigrid Nachtergaele, Qing Dai, Dali Han, Wesley Clark, Guanqun Zheng, Tao Pan and Louis Dore from the University of Chicago, and Sharon Moshitch-Moshkovitz, Eyal Peer, Nitkan Kol, Moshe Shay Ben-Haim, Ayelet Di Segni, Mali Salmon-Divon, Oz Solomon, Eran Eyal, Vera Hershkovitz, Ninette Amariglio and Gideon Rechavi from Tel Aviv University. DOI: 10.1038/nature16998

Funding: National Institutes of Health, Howard Hughes Medical Institute, Flight Attendant Medical Research Institute, Israel Science Foundation, Israeli Centers of Excellence Program, Ernest and Bonnie Beutler Research Program, Chicago Biomedical Consortium, Damon Runyon Cancer Research Foundation and Kahn Family Foundation.

– See more at: http://news.uchicago.edu/article/2016/02/16/rna-modification-discovery-suggests-new-code-control-gene-expression#sthash.HX6wUgKW.dpuf

RNA modifications and epitranscriptomics conference   
University of Chicago, Chicago, Illinois, US   September 8-9, 2016
The meeting is aimed at bringing in students and postdocs as well as faculty involved in RNA modification and epitranscriptome research.  In addition to talks, there will be a poster session and reception.

Topics

  • M6A mRNA methylation
  • Biological functions of m6A RNA methylation
  • Dynamic RNA modifications

Registration will open on March 1, 2016

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Methylation and cancer epigenomics

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

2015 DNA methylation research grant winners

https://www.diagenode.com/landingPages/display/dna-methylation-grant-applications

 

Persistency of Transgene Expression Mediated by Lentiviral Gene Delivery in Pluripotent Cell Lines

Suleiman Alhaji
Ph.D. Student, University Putra, Malaysia

My general objectives are to (1) determine the duration of reporter gene expression from pluripotent cell lines transduced by lentivirus and (2) to assess the epigenetic effects on the provirus. More specifically, I plan to (1) produce LV carrying Green Fluorescent Protein (GFP) reporter gene, (2) obtain and maintain the required cell lines including establishing primary mouse fibroblast as a control, 3) measure the duration of GFP expression from pluripotent and control cell lines transduced with the lentivirus, (4) exclude the loss of the integrated provirus as the factor for GFP silencing in transduced non-pluripotent cell lines, and (5) study the effects of epigenetics on the GFP gene and the regulatory sequence of the provirus.

Transgene integration by lentiviral (LV) vector in the host cell’s genome would theoretically generate a prolonged or permanent transgene expression. However, several citations have reported a decline in transgene expression in early progenitor cells and stem cells transduced by LV. We hypothesized that prolonged transgene expression can be achieved if the transgene is introduced into the cells before epigenetic markers are established in the genome, i.e during the pluripotency period. Therefore, the proposed study seeks to determine if this phenomenon would occur in pluripotent cell lines, focusing on mouse induced pluripotent stem (iPS) cells as the target cell, in a gene therapy context.

Two epigenetic analyses that will be performed on the promoter and transgene of the provirus are DNA methylation and chromatin modification. For DNA methylation profiling, the genomic DNA of the cell will be treated with bisulfite prior to PCR and sequencing of the proviral DNA. The cells may also be analyzed for 5-hmC using 5-hmC monoclonal antibody (C15200200-200) or hMeDIP Kit (C02010031) to assess the level of hydroxymethylation. We may also consider using Diagenode’s MethylCap kit (C02020010) to fractionate the methylated DNA by CpG density.

For the chromatin modification analysis, the cells will be treated with trichostatin A (TSA) before chromatin immunoprecipitation (ChIP) analysis by chromatin IP – bisulfite – sequencing (ChIP-Bis-Seq) and as well Combining chromatin IP and DNA methylation profiles in one assay using the Premium Bisulfite kit, Diagenode. We may also perform pull-down methylated DNA analysis by using specific antibodies such as (1) H3K4 monoclonal antibody (C1541065) (2) H3K4 polyclonal antibody (C15410037) (3) H3K9 polyclonal antibody (C15410004). Beads only will be used as a control.

 

Epigenome-wide methylation pattern discovery for irradiated skin in radiotherapy

Maxwell Johnson, Ph.D.
Research Fellow, University of Southern California, Department of Plastic and Reconstructive Surgery

Radiotherapy is utilized in neoadjuvant, definitive, and palliative treatment of a wide variety of cancers. From a reconstructive perspective, however, irradiated fields pose significant challenges as the tissue is often stiff, brittle, and heals poorly. Little is known about the mechanism by which irradiation produces these changes. The objective of our research is to reveal the epigenetic changes that occur in irradiated skin in order to identify potential targets for therapy. We aim to translate our findings into interventional studies in both cell culture and a mouse model to assess their efficacy in vitro and in vivo.

We have access to a bank of paired samples of irradiated and non-irradiated tissues from patients who have undergone reconstructive procedures after cancer treatment. We have used the Illumina Infinium Human Methylation450 BeadChip array to assess epigenome-wide methylation status of eight paired samples, and have identified a signature methylation pattern for irradiated skin. We would like to utilize the Diagenode Premium WGBS Kit for bisulfite sequencing of additional paired samples for two purposes. First, we would like to confirm the findings of our BeadChip array utilizing a more robust method of assessing epigenome-wide methylation status. Second, we would like to assess methylation status at loci that are not evaluated by the BeadChip array. Using this information, we plan to identify loci with the greatest change in methylation status between irradiated and non-irradiated samples. By comparing these loci to literature, we intend to identify genes that are known to have an effect on wound healing. We then plan to design interventional studies to assess the effects of modulating the expression of these genes and/or supplementing gene products in cell culture and a mouse model. We would like to utilize the Diagenode Bisulfite Kit to confirm methylation status at target genes in these additional studies.

 

Next-generation sequencing based methylome study of primary breast tumours

Rajbir Batra, Ph.D. Researcher
Cancer Research UK Cambridge Institute, University of Cambridge

Breast cancer is one of the leading causes of cancer death in women, and is unanimously considered a heterogeneous disease displaying distinct therapeutic responses and outcomes. While recent advances have led to the refinement of the molecular classification of the disease, the epigenetic landscape has received less attention.

We are delighted to win the DNA methylation research grant award and intend to use it to conduct a next-generation sequencing based methylome study of primary breast tumours. DNA methylation markers will also be investigated in Patient Derived Tumour Xenografts (PDTXs) and in circulating tumour DNA (ctDNA) to identify potential prognostic and predictive methylation biomarkers in breast cancer.

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Deciphering the Epigenome

Curator: Larry H. Bernstein, MD, FCAP

 

UPDATED on 1/29/2016

 

RNA Epigenetics

DNA isn’t the only decorated nucleic acid in the cell. Modifications to RNA molecules are much more common and are critical for regulating diverse biological processes.

By Dan Dominissini, Chuan He and Gidi Rechavi | January 1, 2016

 

RNA SOUP: Newly transcribed messenger RNA exiting the nucleus via nuclear pores
© BENJAMIN CAMPILLO/SCIENCE SOURCE
http://www.the-scientist.com/January2016/feature2.jpg

For years, researchers described DNA and RNA as linear chains of four building blocks—the nucleotides A, G, C, and T for DNA; and A, G, C, and U for RNA. But these information molecules are much more than their core sequences. A variety of chemical modifications decorate the nucleic acids, increasing the alphabet of DNA to about a dozen known nucleotide variants. The alphabet of RNA is even more impressive, consisting of at least 140 alternative nucleotide forms. The different building blocks can affect the complementarity of the RNA molecules, alter their structure, and enable the binding of specific proteins that mediate various biochemical and cellular outcomes.

The large size of RNA’s vocabulary relative to that of DNA’s is not surprising. DNA is involved mainly with genetic information storage, while RNA molecules—mRNA, rRNA, tRNA, miRNA, and others—are engaged in diverse structural, catalytic, and regulatory activities, in addition to translating genes into proteins. RNA’s multitasking prowess, at the heart of the RNA World hypothesis implicating RNA as the first molecule of life, likely spurred the evolution of numerous modified nucleotides. This enabled the diversified complementarity and secondary structures that allow RNA species to specifically interact with other components of the cellular machinery such as DNA and proteins.

Methylating RNA

The nucleotide building blocks of RNA contain pyrimidine or purine rings, and each position of these rings can be chemically altered by the addition of various chemical groups. Most frequently, a methyl (–CH3) group is tacked on to the outside of the ring. Other chemical additions such as acetyl, isopentenyl, and threonylcarbamoyl are also found added to RNA bases.

Among the 140 modified RNA nucleotide variants identified, methylation of adenosine at the N6 position (m6A) is the most prevalent epigenetic mark in eukaryotic mRNA. Identified in bacterial rRNAs and tRNAs as early as the 1950s, this type of methylation was subsequently found in other RNA molecules, including mRNA, in animal and plant cells as well. In 1984, researchers identified a site that was specifically methylated—the 3′ untranslated region (UTR) of bovine prolactin mRNA.1 As more sites of m6A modification were identified, a consistent pattern emerged: the methylated A is preceded by A or G and followed by C (A/G—methylated A—C).

The alphabet of RNA consists of at least 140 alternative nucleotide forms.

Although the identification of m6A in RNA is 40 years old, until recently researchers lacked efficient molecular mapping and quantification methods to fully understand the functional implications of the modification. In 2012, we (D.D. and G.R.) combined the power of next-generation sequencing (NGS) with traditional antibody-mediated capture techniques to perform high-resolution transcriptome-wide mapping of m6A, an approach we termed m6A-seq.2 Briefly, the transcriptome is randomly fragmented and an anti-m6A antibody is used to fish out the methylated RNA fragments; the m6A-containing fragments are then sequenced and aligned to the genome, thus allowing us to locate the positions of methylation marks.

Analyzing the human transcriptome in this way, we identified more than 12,000 methylated sites in mRNA molecules derived from approximately 7,000 protein-coding genes. The transcripts of most expressed genes, in a variety of cell types, were shown to be methylated, indicating that m6A modifications are widespread. In addition, about 250 noncoding RNA sequences—including well-characterized long noncoding RNAs (lncRNAs), such as the XIST transcripts that have a key role in X-chromosome inactivation—are decorated by m6A. In almost all cases, the epigenetic mark was found on adenosines embedded in the predicted A/G—methylated A—C sequence. We found that this pattern was consistently preceded by an additional purine (A or G) and followed by a uracil (U), extending the known consensus sequence to A/G—A/G—methylated A—C—U.2

At the macro level, we found that m6A methylation sites were enriched at two distinct landmarks. The highest relative representation of m6A was found in the stop codon–3′ UTR segment of the RNA, with nearly a third of such methylation found in this sequence just beyond a gene’s coding region. Within the coding regions of the RNA molecules, m6A enrichment mapped to unusually long internal exons; 87 percent of the exonic methylation peaks were found in exons longer than 400 nucleotides. (The average human exon is only 145 nucleotides in length). This pattern of decoration of transcribed RNA suggests that m6A is involved in the mediation of splicing of long-exon transcripts. RNAs transcribed from single­-isoform genes were found to be relatively undermethylated, while transcripts that are known to have multiple isoforms, determined by alternative splicing patterns, were hypermethylated.2 Moreover, specific alternative splicing types, such as intron retention, exon skipping, and alternative first or last exon usage, were highly correlated with m6A decoration. And silencing the m6A methylating protein METTL3 affected global gene expression and alternative splicing patterns in both human and mouse cells.2

These findings clearly indicate the importance of m6A decoration in regulating the expression of diverse transcripts. Moreover, our parallel study of the human and mouse methylome by m6A-seq has uncovered a remarkable degree of conservation in both consensus sequence and areas of enrichment, further supporting the importance of m6A function.2 But research into understanding how m6A marks themselves are regulated, and how this affects various cellular processes, is only just beginning.

Writers, erasers, and readers

The accumulating findings regarding the cellular consequences of m6A transcriptome decoration led to the search for the mediators that enable m6A to exert its influence. Epigenetic marks are introduced by enzymes and cofactors known as “writers,” and m6A is no exception. This mark is added to RNA by a large protein complex that includes three well-characterized components: METTL3, METTL14, and WTAP.3,4 (See illustration on opposite page.)

The transcripts of most expressed genes, in a variety of cell types, were shown to be methylated.

The reverse process of RNA demethylation is performed by “erasers.” In 2011, one of us (C.H.) and an international group of colleagues identified the first m6A eraser: the fat mass and obesity–associated protein (FTO).5 Four years earlier, three independent studies had discovered that a single-nucleotide polymorphism in the first intron of Fto was strongly associated with body mass index and obesity risk, and studies of mouse models where Fto was deleted or overexpressed further demonstrated its link with altered body weight. The research from the C.H. group showed that silencing the Fto gene or protein increased total m6A levels, while overexpression decreased levels of the epigenetic mark.5 C.H.’s group later discovered that another protein from the same protein family as FTO, ALKBH5, behaves as an active m6A demethylase.6 In contrast to the ubiquitous expression of Fto in all tissues, the highest expression level of Alkbh5 was demonstrated in mouse testes. Indeed, Alkbh5-null male mice exhibit aberrant spermatogenesis, probably a result of m6A-mediated altered expression of spermatogenesis-related genes.6

 

RNA METHYLATION DYNAMICS: At least 140 alternative RNA nucleotide forms exist. On mRNA, the most common is the methylation of adenosine on the N6 position (m6A). This epigenetic mark is laid down by a “writer” protein complex that includes three well-characterized components: METTL3, METTL14, and WTAP. The reverse process of RNA demethylation is performed by “erasers,” such as the enzymes FTO and ALKBH5.

http://www.the-scientist.com/January2016/methylation2.jpg

See full infographic: WEB | PDF THE SCIENTIST STAFF
These writers and erasers facilitate the dynamic nature of m6A methylation, which was shown when we (D.D. and G.R.) demonstrated changes in response to environmental stimuli, such as UV irradiation, heat shock, and exposure to interferon gamma or hepatocyte growth factor.2 Once RNA epigenetic modifications are laid down, they are recognized by specific “reader” proteins that bind to the modified nucleotide and mediate enhancement or inhibition of gene expression. In 2012, the G.R. group used methylated and nonmethylated versions of synthetic RNA baits that include the m6A consensus sequence to identify such readers of m6A.2 By preferential binding to the methylated bait, we isolated several specific m6A-binding proteins, including members of the RNA-binding YTH domain family, whose function was previously unknown.2

The finding of the first m6A-binding reader proteins has accelerated the deciphering of the various molecular and cellular processes mediated by m6A marking. In 2014, for example, we (C.H. and colleagues) showed that the human YTH domain family 2 (YTHDF2) reader protein selectively recognizes m6A and mediates mRNA degradation.7 We identified more than 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs. Binding of YTHDF2 to m6A in mRNA results in the translocation of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies in the cytoplasm where mRNA turnover is regulated.

Recently, C.H. and colleagues identified another m6A reader protein, YTHDF1, with a very different function—stimulating protein synthesis by ramping up the efficiency of translation machinery.8 The dueling functions of YTHDF2 and YTHDF1 provide a mechanism by which cells can adjust gene expression promptly and precisely to environmental stimuli. Finally, G.R. and his group have identified an additional reader protein, the RNA-binding protein heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1),2 which directly binds a set of m6A decorated transcripts and mediates alternative splicing.9

Clearly, m6A plays diverse roles in regulating cellular function, starting with basic processes such as gene expression, translation, and alternative splicing. As work on this epigenetic mark continues, we will undoubtedly link m6A to numerous phenotypes, and its dysregulation may undergird various diseases and syndromes.

RNA epigenetics in action

Understanding the molecular mechanisms by which m6A regulation controls RNA stability, translation efficiency, and alternative splicing is helping researchers decipher the importance of this new epigenetic mark in physiological and pathological processes. For example, researchers recently showed that translation increases in stressed mice thanks to m6A decoration. In 2015, two studies from Cornell University and Weill Cornell Medical College found increased m6A methylation of specific 5′ UTR adenosines in newly transcribed mRNAs as a result of stress-induced nuclear localization of the m6A YTHDF2 reader. The researchers suggested that the nuclear YTHDF2 preserves the unique 5′ UTR m6A methylation of stress-induced transcripts by limiting the demethylation activity of the FTO eraser. Increased 5′ UTR m6A methylation in turn promotes translation of specific transcripts, such as those for the heat shock protein Hsp70. While conventional mRNA translation starts by binding of the ribosome components to a region of the 5′ UTR marked by the unusual nucleotide 7meG (the “cap”), under stress conditions initiation of translation can start farther downstream.10

DECIDING CELL FATE: Among its many roles in the cell, m6A methylation helps regulate the expression of RNA transcripts that mediate the transition from pluripotency to differentiation. The presence of m6A appears to decrease the stability of transcripts important for maintaining pluripotency, priming the cells for future differentiation. The loss of METTL3, an m6A methlyase component, in mouse embryonic stem cells leads to the cells’ inability to exit the pluripotent state, a lethal outcome in the early embryos.
http://www.the-scientist.com/January2016/feature2_21.jpg

See full infographic: WEB THE SCIENTIST STAFF

In a second study, Weill Cornell Medical College’s Samie Jaffrey, who collaborated on the previous study, led a team that showed m6A-methylated mRNAs can be translated in a cap-independent manner. The researchers showed that a specific 5′ UTR m6A binds the eukaryotic initiation factor 3 (eIF3), which recruits the ribosomal 43S complex and initiates cap-independent translation. This study also demonstrated increased m6A levels in the Hsp70 mRNA that enhanced its cap-independent translation following heat-shock stress.11

Other work has hinted at m6A’s role in the regulation of circadian rhythms. Researchers identified m6A sites on many transcripts of genes involved in the regulation of daily cycles. Inhibition of m6A methylation by silencing of the METTL3 writer led to circadian period elongation, with altered distribution and processing of the transcripts of the clock genes Per2 and Arntl.12

It’s quickly becoming clear that m6A decoration has diverse cellular and physiological functions. But perhaps the best illustration of its critical ability to precisely control processes at the cellular level is its involvement in early embryogenesis. Cell-fate decisions are coordinated by alterations in global gene expression, which are orchestrated by epigenetic regulation. Well-established epigenetic marks, such as DNA methylation and histone modifications, are known to mediate embryonic stem cell (ESC) cell-fate decisions, and it turns out that m6A modification is no different.

Dynamic m6A RNA markings, the new kid on the epigenetic block, herald the era of tripartite epigenetics where modifications of DNA, RNA, and proteins join hands to fine-tune gene expression and to execute prompt and precise responses to environmental stimuli and stresses.

We (G.R. and collaborators) and other groups recently demonstrated that the m6A writer METTL3 is also an essential regulator for termination of mouse embryonic stem cell pluripotency. Knocking out Mettl3 in preimplantation murine epiblasts and in undifferentiated ESCs led to depletion of m6A in mRNAs. Cell viability was not affected, suggesting that m6A decoration is not essential for the maintenance of the ESC naive state, but m6A marks were critical for early differentiation. The loss of this modification led to aberrant and restricted lineage priming at the post-implantation stage, resulting in early embryonic lethality.13 The presence of m6A also decreased mRNA stability, including in those transcripts important for maintaining pluripotency. These findings demonstrated, for the first time, an essential function for an mRNA modification in vivo.14

Beyond mRNA

While m6A methylation is most prevalent on mRNAs, this mark also decorates other RNA species. It is well established, for example, that m6A is abundant on rRNAs, tRNAs, and small nuclear RNAs (snRNAs), which mediate splicing and other RNA processing and protein synthesis reactions.

More recently, researchers found that the reader protein HNRNPA2B1 binds to m6A marks in a subset of primary microRNA (miRNA) transcripts, recruiting the miRNA-microprocessor complex and promoting primary miRNA processing that is essential for mature miRNA biogenesis.9 Not only is the biogenesis of miRNA regulated by m6A marking and recruitment of HNRNPA2B1, miRNAs themselves appear to play a role in the placement of the m6A epigenetic marks. MiRNAs regulate m6A modification in specific transcript sites using a sequence-pairing mechanism where the “seed” sequence of a specific miRNA binds a complementary target sequence in the 3′ UTR of mRNA and directs methylation.15 The interaction is bidirectional: manipulation of miRNA sequence or expression affects m6A modification also by reducing binding of the METTL3 writer to the target mRNA sites.

Similarly, m6A appears to be involved in structural alterations of mRNAs and lncRNAs to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant RNA-binding protein responsible for mRNA processing. This novel mechanism, termed m6A-switch, was shown to affect alternative splicing and abundance of multiple target mRNAs.16 Taken together, these results demonstrate that m6A is an important mark on diverse RNA species.

Dynamic m6A RNA markings, the new kid on the epigenetic block, herald the era of tripartite epigenetics where modifications of DNA, RNA, and proteins join hands to fine-tune gene expression and to execute prompt and precise responses to environmental stimuli and stresses. Indeed, m6A is just one of 140 modified RNA nucleotides that likely affect the function of the nucleic acid messenger and key cellular actor in diverse ways. Molecular approaches are paving the way for the study of additional RNA modifications.

As the list of RNA epigenetic marks continues to expand, researchers will gain a clearer picture of how diverse cellular processes are regulated. The extremely large repertoire of such modifications is expected to reveal various RNA marks analogous to the known DNA and histone epigenetic marks, and the various modifications of DNA, RNA, and proteins can enrich the language that allows the development, adaptation, and diversity of complex organisms.

Dan Dominissini is a postdoctoral fellow in Chuan He’s group at the University of Chicago. Gidi Rechavi is a pediatric hematologist-oncologist and a researcher in genetics and genomics at the Chaim Sheba Medical Center in Tel Hashomer, Israel, and a Professor of Hematology at the Sackler School of Medicine at Tel Aviv University. Sharon Moshitch-Moshkovitz, a senior researcher in RNA biology at the Chaim Sheba Medical Center, also contributed to this article.

References

  1. S. Horowitz et al., “Mapping of N6-methyladenosine residues in bovine prolactin mRNA,” PNAS, 81:5667-71, 1984.
  2. D. Dominissini et al., “Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq,” Nature, 485:201-06, 2012.
  3. Y. Fu et al., “Gene expression regulation mediated through reversible m6A RNA methylation,” Nat Rev Genet, 15:293-306, 2014.
  4. K.D. Meyer, S.R. Jaffrey, “The dynamic epitranscriptome: N6-methyladenosine and gene expression control,” Nat Rev Mol Cell Biol, 15:313-26, 2014.
  5. G. Jia et al., “N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO,” Nat Chem Biol, 7:885-87, 2011.
  6. G. Zheng et al., “ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility,” Mol Cell, 49:18-29, 2013.
  7. X. Wang et al., “N6-methyladenosine-dependent regulation of messenger RNA stability,” Nature, 505:117-20, 2014.
  8. X. Wang et al., “N6-methyladenosine modulates messenger RNA translation efficiency,” Cell, 161:1388-99, 2015.
  9. C.R. Alarcón et al., “HNRNPA2B1 is a mediator of m6A-dependent nuclear RNA processing events,”Cell, 162:1299-308, 2015.
  10. J. Zhou et al., “Dynamic m6A mRNA methylation directs translational control of heat shock response,” Nature, 526:591-94, 2015.
  11. K.D. Meyer et al. “5′ UTR m6A promotes cap-independent translation,” Cell, 163:999-1010, 2015.
  12. J.-M. Fustin et al., “RNA-methylation-dependent RNA processing controls the speed of the circadian clock,” Cell, 155:793-806, 2013.
  13. S. Geula et al., “m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation,” Science, 347:1002-06, 2015.
  14. P.J. Batista et al., “m6A RNA modification controls cell fate transition in mammalian embryonic stem cells,” Cell Stem Cell, 15:707-19, 2014.
  15. T. Chen et al., “m6A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency,” Cell Stem Cell, 16:289-301, 2015.
  16. N. Liu et al., “N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions,” Nature, 518:560-64, 2015.

Tags

RNA methylationRNA epigeneticsrnamethylationepigenetics and epigenetic regulation

 

Telomerase Overdrive

Two mutations in a gene involved in telomere extension reverse the gene’s epigenetic silencing.

By Ashley P. Taylor | January 1, 2016

http://www.the-scientist.com//?articles.view/articleNo/44768/title/Telomerase-Overdrive/

EPIGENETIC ACTIVATION: A single base-pair mutation (lower allele) leads to epigenetic changes that promote expression of a telomerase gene.COURTESY OF JOSH STERN
http://www.the-scientist.com/January2016/shortlit2.jpg

EDITOR’S CHOICE IN GENETICS & GENOMICS

The paper
J.L. Stern et al., “Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERTexpression in multiple cancers,” Genes Dev, doi:10.1101/gad.269498, 2015.

The foundation
Chromosome ends are slightly shortened with each DNA replication. Terminal repetitive sequences called telomeres buffer coding DNA from this fate. In stem cells, telomerase extends the telomeres so that cell division can continue, perhaps indefinitely. In somatic cells, telomerase is inactive in part because the gene encoding telomerase’s catalytic sub­unit, telomerase reverse transcriptase (TERT), is epigenetically silenced. In most cancers, however, telomerase is again turned on and aids proliferation.

The mutations
In 2013, researchers found two mutations in the TERT promoter that occur frequently in cancer cell lines and are tied with TERT expression.

Regulation
To probe the mechanism of TERT activation, Josh Stern, a postdoctoral fellow in the lab of Thomas Cech at the University of Colorado Boulder, studied cancer cell lines that were heterozygous for one of these TERTmutations. Stern and his colleagues determined that the mutant TERT allele had histone methylation marks associated with gene activation and was transcribed, whereas the wild-type allele bore other histone methylation marks characteristic of gene silencing and was not transcribed.
“It’s very nice biochemical work to show that a single-base-pair mutation in the cancer genome activates the expression of the telomerase gene,” says Dana-Farber Cancer Institute’s Franklin Huang.

Application
“Telomerase is a fantastic therapeutic target for cancers because so many cancers are absolutely reliant on telomerase,” says Stern. “These TERT promoter mutations only occur in cancer, so if we can understand the mechanism, then we can potentially develop a highly specific cancer therapeutic.”

Tags

transcriptiontelomerestelomerasemutationliteraturegenetics & genomicsepigenetics and cancer

 

CRISPR Fixes Stem Cells Harboring Blindness-Causing Defect

http://www.genengnews.com/gen-news-highlights/crispr-fixes-stem-cells-harboring-blindness-causing-defect/81252293/

Marking yet another CRISPR-related first, scientists have replaced a defective gene associated with a sensory disease in stem cells that were derived from a patient’s tissue. The disease, retinitis pigmentosa (RP), is an inherited condition that degrades the retina and leads to blindness. A patient with the disease supplied a skin sample that was used to generate the stem cells, which were manipulated by means of the CRISPR/Cas9 gene-editing system.

CRISPR/Cas9, which zeroed in on a single disease-causing mutation in the RGPR gene, was able to make the necessary correction in 13% of the stem cells. This correction rate, according to the Columbia University and University of Iowa scientists who announced the results, is indicative of a practical approach—albeit one that still needs work. The Columbia/Iowa team added that they are working to show that their technique does not introduce any unintended genetic modifications in human cells, and that the corrected cells are safe for transplantation.

While the scientists freely acknowledge that their technique needs additional development before any cures are possible, they basked in the success of having accomplished a difficult genetic fix. The RGPR mutation that needed to be repaired sits in a highly repetitive sequence of the gene where it can be tricky to discriminate one region from another. In fact, it was not clear that CRISPR/Cas9 would be able to home in on and correct the point mutation.

The scientists described their work January 27 in the journal Scientific Reports in an article entitled “Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells.”

“Fibroblasts cultured from a skin-punch biopsy of an XLRP patient were transduced to produce [induced pluripotent stem cells (iPSCs)] carrying the patient’s c.3070G > T mutation,” the authors wrote. “The iPSCs were transduced with CRISPR guide RNAs, Cas9 endonuclease, and a donor homology template. Despite the gene’s repetitive and GC-rich sequences, 13% of RPGR gene copies showed mutation correction and conversion to the wild-type allele.”

The authors asserted that theirs was the first report of CRISPR/Cas9 being used to correct a pathogenic mutation in iPSCs derived from a patient with photoreceptor degeneration. This proof-of-concept finding, they added, supports the development of personalized iPSC-based transplantation therapies for retinal disease.

The authors also emphasized that because the corrections are made in cells derived from the patient’s own tissue, doctors can retransplant the cells with fewer fears of rejection by the immune system. Previous clinical trials have shown that generating retinal cells from embryonic stem cells and using them for transplantation is a safe and potentially effective procedure.

Recently, another group has used CRISPR to ablate a disease-causing mutation in rats with retinitis pigmentosa. Going forward, the first clinical use of CRISPR could be for treating an eye disease because compared to other body parts, the eye is easy to access for surgery, readily accepts new tissue, and can be noninvasively monitored.

 

 

Edited stem cells offer hope of precision therapy for blindness

http://www.rdmag.com/news/2016/01/edited-stem-cells-offer-hope-precision-therapy-blindness

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Skin cells from a patient with X-linked Retinitis Pigmentosa were transformed into induced pluripotent stem cells and the blindness-causing point mutation in the RPGR gene was corrected using CRISPR/Cas9. Credit: Vinit Mahajan, Univ.of Iowa Health Care

Using a new technology for repairing disease genes–the much-talked-about CRISPR/Cas9 gene editing–Univ. of Iowa researchers working together with Columbia Univ. Medical Center ophthalmologists have corrected a blindness-causing gene mutation in stem cells derived from a patient. The result offers hope that eye diseases might one day be treated by personalized, precision medicine in which patients’ own cells are used to grow replacement tissue.

With the aim of repairing the deteriorating retina in patients with an inherited blinding disease, X-linked Retinitis Pigmentosa (XLRP), Alexander Bassuk, MD, PhD, and Vinit Mahajan, MD, PhD, led a team of researchers who generated stem cells from patient skin cells and then repaired the damaged gene. The editing technique is so precise it corrected a single DNA change that had damaged the RPGR gene. More importantly, the corrected tissue had been derived from the patient’s own stem cells, and so could potentially be transplanted without the need for harmful drugs to prevent tissue rejection. The research was published Jan. 27 in the journal Scientific Reports.

“With CRISPR gene editing of human stem cells, we can theoretically transplant healthy new cells that come from the patient after having fixed their specific gene mutation, ” says Mahajan, clinical assistant professor of ophthalmology and visual sciences in the UI Carver College of Medicine. “And retinal diseases are a perfect model for stem cell therapy, because we have the advanced surgical techniques to implant cells exactly where they are needed.”

The study was a “proof-of-concept” experiment showing it is possible not only to repair a rare gene mutation, but that it can be done in patient stem cells. Use of stem cells is key because they can be re-programmed into retinal cells.

The CRISPR technology was able to correct the RPGR mutation in 13 percent of the stem cells, which is a practically workable correction rate.

Bassuk notes this result is particularly encouraging because the gene mutation sits in a highly repetitive sequence of the RPGR gene where it can be tricky to discriminate one region from another. In fact, initially determining the DNA sequence in this part of the gene was challenging. It was not clear that CRISPR/Cas9 would be able to home in on and correct the “point mutation.”

“We didn’t know before we started if we were going to be able to fix the mutation,” says Bassuk, associate professor in the Stead Family Department of Pediatrics at University of Iowa Children’s Hospital.

 

Epigenetics Research Reveals a Range of Clinical Possibilities

Advantageously Epigenetic Analyses Can Capture both Genetic Factors and Environmental Exposures

Richard A. Stein, M.D., Ph.D.

http://www.genengnews.com/gen-articles/epigenetics-research-reveals-a-range-of-clinical-possibilities/5650/

  • Over half a century ago, Conrad Hal Waddington introduced his model of the epigenetic landscape. He depicted a differentiating cell as a ball rolling down a landscape of bifurcating valleys and ridges, with each valley representing an alternative developmental path. Just as a ball may roll from valley to valley until it reaches the bottom of the landscape, a cell may progress from one developmental alternative to another until it reaches its fully differentiated state.

The model’s original purpose was to integrate concepts from genetics and developmental biology and to describe mechanisms that connect the genotype to the phenotype. Today, the model remains a compelling metaphor for epigenetics, which has developed into one of the most vibrant biomedical fields. Epigenetics has become indispensable for exploring development, differentiation, homeostasis, and diseases that span virtually every clinical discipline.

  • Analyzing Methylation Patterns

“Modern efforts toward explaining human disease purely based upon sequencing cannot possibly succeed in isolation,” says Andrew P. Feinberg, M.D., professor of medicine and director of the Center for Epigenetics at Johns Hopkins University School of Medicine. “At least half of human disease is caused by exposure to the environment.”

While the contribution of genetic factors to disease is more predictable and easier to study in the case of highly penetrant Mendelian disorders, most medical conditions involve multiple genes that may interact with one another and with environmental factors. Particularly for these conditions, capturing epigenetic changes becomes a crucial aspect of understanding pathogenesis and designing prophylactic and therapeutic interventions.

“In these cases,” notes Dr. Feinberg, “an approach not including epigenetics will be severely limited in what it can accomplish.”

In a recent study, Dr. Feinberg and colleagues reported that large blocks of the human genome are hypomethylated in the epidermis as a result of sun exposure, which together with aging represents a known risk factor for skin cancer. These hypomethylated regions overlap with regions that have methylation changes in patients with squamous cell carcinoma.

This overlap could explain the causal link between sun exposure and the increased risk of malignancy found in many epidemiological studies. Most of the methylation changes were observed in the epidermis, not in the dermis, pointing toward the combination between the genotype and exposure, acting on specific cell types, as a key factor in shaping disease.

“One of the advantages of epigenetic analyses is that they capture both genetic factors and environmental exposures,” explains Dr. Feinberg. In the study of complex diseases, the existence of many distinct genetic variants identified in different individuals makes it challenging to understand their roles in pathogenesis. “But if genetic variants converge on gene regulatory loci, then measuring methylation can still be informative about these variants,” continues Dr. Feinberg, “even if genetic changes are inconsistent across the patients.”

In combining data from genome-wide association analysis and epigenome-wide analysis, Dr. Feinberg and colleagues revealed that two single-nucleotide polymorphisms on human chromosome 11, located 100 kb apart and involved in different aspects of lipid metabolism, controlled DNA methylation at two CpG sites in a bidirectional promoter situated between two genes encoding the fatty acid desaturases FADS1 and FADS2. Genome-wide association studies alone would not capture the convergence of these two single-nucleotide polymorphisms as they regulate DNA methylation in the shared promoter region.

“Measuring DNA methylation,” concludes Dr. Feinberg, “can pick up the fact that these single nucleotide polymorphisms act through DNA methylation to regulate the genes.”

The image shows a cleavage-stage human embryo. This is around the same stage that DNA methylation is ‘set’ at metastable epialleles. [Instituto Bernabeu]

http://www.genengnews.com/Media/images/Article/Jan15_2016_RobertWaterland_CleavageStageHumanEmbryo3202193100.jpg

Identifying Metastable Epialleles

Over the years, genome-wide association studies provided opportunities to establish links between genetic variation and phenotypic changes. For these analyses, genetic material from any of an individual’s cells, such as a peripheral white blood cell, is informative about the individual’s genotype. However, for epigenetic changes, which vary across tissues and within the same tissue among different cells, it is much more challenging to examine associations with disease.

Robert A. Waterland, Ph.D., associate professor of pediatrics and molecular and human genetics at Baylor College of Medicine, thinks that identifying human metastable epialleles will help circumvent some of these challenges. “Getting investigators and the field interested in metastable epialleles is going to be an important first step in helping us understand how epigenetic dysregulation contributes to human disease,” says Dr. Waterland.

The term metastable epialleles refers to genomic loci with differential epigenetic regulation that are variably expressed in genetically identical individuals, and where the epigenetic state is established stochastically in the very early embryo, before gastrulation, and subsequently maintained. This leads to systemic (non-tissue-specific) interindividual epigenetic differences that are not genetically mediated.

The fact that DNA methylation at metastable epialleles is particularly sensitive to environmental influences makes these loci valuable in mechanistically exploring the developmental origins hypothesis, the concept that environmental exposures during critical periods of prenatal and early postnatal development can have long-term implications in the risk of disease. Previous studies have implicated epigenetic modifications as a mechanism by which environmental changes during pregnancy may lead to epigenetic changes that influence health later in life.

In the most recent genome-wide screen meant to identify metastable epialleles in humans, Dr. Waterland teamed up with Dr. Andrew Prentice and colleagues at the London School of Hygiene and Tropical Medicine and used two independent and complementary experimental approaches to identify DNA methylation changes that occur in the cleavage-stage embryo (shortly after the time of conception). The first approach involved a genome-wide screen for DNA methylation in multiple tissues from two healthy Caucasian adults. In parallel, genome-wide DNA methylation profiling was performed in a rural population from The Gambia to examine the link between the season of conception (a proxy for maternal nutritional status) and DNA methylation in the offspring and sought to capture the effect of maternal nutritional status on the epigenetic profile of the offspring.

“We identified the same genomic locus as the top hit in both screens, suggesting that this is likely to be a key indicator of early environmental influences on the epigenome,” explains Dr. Waterland. Both approaches identified VTRNA2-1 as the lead candidate for an environmentally-responsive epiallele.

VTRNA2-1, a genomically imprinted small noncoding RNA and a putative tumor suppressor gene, is preferentially methylated on the maternally inherited allele, and loss of imprinting at this locus promises to link the early embryonic environment to epigenetic changes that shape disease risk later in life. Besides VTRNA2-1, over 100 metastable epialleles were identified in the study.

“At metastable epialleles such as VTRNA2-1, DNA methylation in peripheral blood or in any easily accessible tissue can give an indication about the epigenetic regulation throughout the body,” concludes Dr. Waterland. “That is what is really different.”

http://www.genengnews.com/Media/images/Article/thumb_Jan15_2016_DavidBazettJones_ElectronSpectroscopic1381311078.jpg

Electron spectroscopic image of a region of the nucleus of a mouse embryonic fibroblast. Phosphorus and nitrogen maps allow chromatin (yellow) to be distinguished from protein-based structures (cyan). The arrow indicates the nuclear envelope. The large structure in the middle of the field, a chromocentre, is an accumulation of pericentric heterochromatin. It is surrounded by dispersed chromatin fibers. The heterochromatin mark, trimethlated H3K9, is immunolabelled and visualized with gold tags (white foci). [David Bazett-Jones]

Mapping Heterochromatin Domains

Electron spectroscopic image of a region of the nucleus of a mouse embryonic fibroblast. Phosphorus and nitrogen maps allow chromatin (yellow) to be distinguished from protein-based structures (cyan). The arrow indicates the nuclear envelope. The large structure in the middle of the field, a chromocentre, is an accumulation of pericentric heterochromatin. It is surrounded by dispersed chromatin fibers. The heterochromatin mark, trimethlated H3K9, is immunolabelled and visualized with gold tags (white foci). [David Bazett-Jones]

“For the first time, we found that a histone chaperone is implicated in organizing chromatin at a large scale,” says David Bazett-Jones, Ph.D., professor of biochemistry at the University of Toronto and senior scientist at the Hospital for Sick Children. The discovery and characterization of histone variants has been a vital facet of understanding chromatin organization and dynamics.

One of the most extensively studied histone variants is H3.3. Although H3.3 is 96% identical at the amino acid level to histone H3.1, histones H3.3 and H3.1 are functionally distinct. Histone H3.3 is expressed throughout the cell cycle, and it is enriched in transcriptionally active chromatin and in certain types of post-translational modifications. The death domain-associated protein DAXX, one of the proteins associated with histone H3.3 deposition, was recently identified as its chaperone.

Dr. Bazett-Jones and colleagues, including his graduate student Lindsy Rapkin, revealed that the loss of DAXX led to a global structural change in the chromatin landscape, characterized by genomic regions enriched in the trimethylated H3K9 epigenetic mark that were juxtaposed to large chromatin domains devoid of this modification.

“These major changes probably occur because the boundaries between heterochromatin domains and other regions were not being respected, leading to the inappropriate insertions of histone H3.3, and this exerted quite profound effects,” explains Dr. Bazett-Jones. The loss of DAXX led to the uncoupling of the epigenetic marks from the global chromatin architecture. “This shows that a major global reorganization of the chromatin was taking place,” Dr. Bazett-Jones continues.

To visualize chromatin changes that result from the loss of DAXX, Dr. Bazett-Jones and colleagues used electron spectroscopic imaging, an experimental approach that is based on the principle of electron energy loss spectroscopy. When a biological specimen is targeted with electrons and its atoms become ionized, the ionization energy is equal to the energy that is lost by the incident electrons that generated the event.

The electron microscope technique generates nitrogen and phosphorus maps, which are used to discriminate between nucleic-acid-rich and protein-rich cellular structures. These maps offer high-contrast images of chromatin and its three-dimensional organization in intact cells.

Another component of the DAXX deletion phenotype included the loss of nucleolar structural integrity, resulting in an increased number of cells containing mini-nucleoli, and the dispersal of ribosomal DNA genes outside the nucleolus. Collectively, these findings pointed toward a novel role that DAXX plays in the subnuclear organization of chromatin and in maintaining nucleolar structural integrity.

“Historically, we thought that the well-known epigenetic modifications dictate the compact character of heterochromatin,” notes Dr. Bazett-Jones. “But our findings, and those from other groups, reveal that a heterochromatin domain epigenetically marked with H3K9 trimethylation, for example, can be found in a structurally ‘open’ state, similar to euchromatin.”

This indicates that the boundaries between heterochromatin and euchromatin are much more fluid than previously envisioned, a concept that is crucial for understanding factors that dynamically shape the three-dimensional interaction between epigenetic changes. A key implication of these findings is that the epigenetic marks at a specific genomic locus depend on both the local environment and the three-dimensional context.

“We need to look at what loci come together in specific regions of the nucleus in three dimensions and how they affect each other,” concludes Dr. Bazett-Jones. “This is on top of capturing epigenetic marks, which are on top of the genomic sequences that we need to explore.”

Identifying Druggable Epigenetic Processes

“There is a big gap in understanding the biology of epigenetics,” says Chris J. Burns, Ph.D., laboratory head, Division of Chemical Biology, Walter and Eliza Hall Institute of Medical Research, Melbourne. “And this goes hand in hand with the need to learn how to generate small molecule probes or drugs.”

When interrogating epigenetic processes, researchers find it useful to integrate biological and chemical perspectives. For example, researchers have generated a large body of literature demonstrating that many epigenetic processes involve highly complicated protein complexes.

Historically, genetics studies have typically relied on knocking down or knocking out a gene and its protein product to examine the resulting phenotype. “In contrast, knocking down a protein that is part of a protein complex fundamentally alters that complex, and the phenotype could be quite different from the one that can be seen with a small molecule inhibition of a catalytic component of the protein complex,” notes Dr. Burns. This opens an acute need to identify small molecules that can selectively impact just one particular aspect of these protein complexes.

A major effort in Dr. Burns’ lab is focusing on identifying therapeutic agents that could target epigenetic processes. “Epigenetics in terms of drug discovery and development is still in an early stage,” explains Dr. Burns. While several drugs that target epigenetic processes have become available in recent years—drugs such as HDAC inhibitors and DNA methyl transferase inhibitors—many other drugs are still at early stages of development.

“Some epigenetic processes have not yet been drugged,” Dr. Burns points out. “For some of them, there may not be any therapeutic agents that are particularly good.”

Dr. Burns’ lab has collaborated with investigators led by Carl Walkley, Ph.D., joint head, Stem Cell Regulation Unit, St. Vincent’s Institute of Medical Research, Melbourne. Together, the research teams revealed that several bromodomain inhibitors exert powerful antitumor activity in human osteosarcoma cell lines and in osteosarcoma primary cells from mouse models of the disease.

The researchers’ findings were surprising. JQ1, one the bromodomain inhibitors tested, exerted its antiproliferative activity by inducing apoptosis, and not by mediating cell cycle arrest, as expected. Moreover, even though previous studies identified MYC as an oncogenic driver in osteosarcoma, the activity of JQ1 was exerted independently of MYC downregulation.

At the same time, this work revealed that downregulation of FOSL1, a gene previously implicated in osteoblast differentiation, is an important contributor to the effects of JQ1, marking the first time when this gene was implicated in osteosarcoma.

“Because we used primary cell from animals, these findings reflect the disease process better than cell lines, which may take on a number of other mutations,” concludes Dr. Burns. “This explains why our findings are contrary to previous reports in the literature.”

“We have shown that epigenetic drugs may work not only on protein-coding genes but also on the noncoding part of the genome,” says Claes Wahlestedt, M.D., Ph.D., professor and associate dean for therapeutic innovation at the University of Miami Miller School of Medicine.

A therapeutically promising class of epigenetic compounds consists of bromodomain inhibitors. These compounds have received increasing attention in recent years, and several leads have entered clinical trials for malignancies, atherosclerosis, and type 2 diabetes.

”One of our interests is to see if bromodomain inhibitors could be used for diseases of the nervous system,” notes Dr. Wahlestedt.

Using in vitro and in vivo approaches, investigators in Dr. Wahlestedt’s group, in collaboration with investigators led by Nagi Ayad, Ph.D., found that BET bromodomain inhibitors can inhibit glioblastoma cell proliferation by inducing a cyclin-dependent kinase inhibitor. These findings set the stage for subsequent experiments that used single molecule sequencing to profile long noncoding RNAs (lncRNAs) differentially expressed in glioblastoma multiforme. This helped identify a set of transcripts that are specific for this malignancy and could be regulated by bromodomain inhibitors.

In glioblastoma multiforme cells, the I-BET151 bromodomain inhibitor localized to the promoter of HOTAIR, a tumor-promoting lncRNA that acts as an epigenetic silencer and has been implicated in several cancers, decreased its expression, and restored the expression of several lncRNA species that are downregulated in this malignancy.

In another collaborative endeavor, Dr. Wahlestedt and colleagues conducted a semi-high-throughput gene-expression-based screen to identify small molecules that could increase the expression of C9ORF72. A GGGGCC hexanucleotide repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic cause for amyotrophic lateral sclerosis. Individuals without this condition harbor 2 to 25 of these repeats, but their number can reach up to several hundreds in ALS patients, reducing C9ORF72 expression, which has been implicated in the pathogenesis of this condition.

The gene-expression-based screen identified, in fibroblasts from affected and unaffected individuals, small interfering RNAs against the BRD3 bromodomain protein and several small molecule bromodomain inhibitors that were able to increase C9ORF72 expression. This effect occurred without changes in promoter CpG hypermethylation and trimethylated H3K9 marks, which are heterochromatin markers of the expanded C9ORF72 alleles.

“The mechanism of action of these compounds is probably broader than we thought before,” concludes Dr. Wahlestedt.

 

CRISPR Works Well but Needs Upgrades

More Effective and Reliable CRISPR Tools Will Have To Be Developed

MaryAnn Labant

http://www.genengnews.com/gen-articles/crispr-works-well-but-needs-upgrades/5652/

http://www.genengnews.com/Media/images/Article/thumb_UnivIllinois_Cas9DSB1921455173.jpg

In this image, which comes from the University of Illinois at Urbana-Champaign, Cas9 (green) is shown cutting DNA (white and brown) at the target sequence specified by the single guide RNA (red). The image was created from the Protein Data Bank file 4un3.pdb using Pymol, and it was enhanced using Photoshop.

The gene-editing technology known as CRISPR-Cas9 went through a disruptive phase when it first took the research world by storm.

Now, thousands of research articles later, it is starting to raise expectations in the therapeutic realm. In fact, CRISPR-Cas9 and other CRISPR systems are moving so close to therapeutic uses that the technology’s ethical implications are starting to attract notice. For example, people worry that CRISPR could be used to alter human germline cells, introducing genomic changes that could impact future generations.

Before any of that can happen, however, CRISPR will have to overcome a number of practical obstacles. If CRISPR is to be harnessed effectively and leveraged to its full potential, it will have to be better understood. Also, more effective and reliable CRISPR tools will have to be developed.

For example, little progress has been made in the area of targeted integration. “We effectively have the tools to cut, yet we lack efficient tools to paste. How the cells repair the double-strand break created by the RNA-guided nucleases, or RGNs, depends almost exclusively on the cells themselves in that there is no control over the repair mechanism. In addition to the RGNs, we deliver a vector that can function as a repair template, and hope the cells will use it,” explained Pablo Perez-Pinera, M.D., Ph.D., assistant professor, department of bioengineering, University of Illinois at Urbana-Champaign.

 

 

Fun with Lego (molecules)

http://www.rdmag.com/news/2016/01/fun-lego-molecules

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Depending on the relative amounts of different building-block molecules, it is possible to create different sandwich and wheel topologies (shown above in micrographs and below as models). Credit: American Chemical Society. Copyright 2016

A great childhood pleasure is playing with Legos and marveling at the variety of structures you can create from a small number of basic elements. Such control and variety of superstructures is a goal of polymer chemists, but it is hard to regulate their specific size and how the pieces fit together. This week in ACS Central Science, researchers report a simple system to make different nano-architectures with precision.

Using a variety of highly efficient chemical transformations and other techniques to ensure high yields and purity, Stephen Z. D. Cheng, Yiwen Li, Wen-Bin Zhang and coworkers designed systems to create giant molecules with ‘orthogonal’ ends, meaning that they only fit together with a specific partner just like Legos. Depending on the relative amounts of different building-block molecules, these molecules come together in different superstructures — ranging from cubes to wheels and sandwiches. Eventually, they could be employed in device-creation, where it is crucial to have precise control over the positions of the components.

 

Protein Expression Systems Proliferate

Bioprocessing Assembly Lines Are Being Retooled, Often At the Genomic Scale

Angelo DePalma, Ph.D.

http://www.genengnews.com/Media/images/Article/thumb_iStock_32784234_eColi1930160883.jpg

Despite some bells and whistles, most E. coli production systems have been the same. Now, new systems are being introduced that purport to express proteins more efficiently. [iStock/Scharvik]

Biomanufacturers enjoy a host of tools to optimize the production of therapeutic proteins, including expression systems, media, feeds, and gene-editing tools. Suffice it to say that protein expression is a growth industry.

Industry research firm Future Market Insights (FMI) breaks down the protein expression market into four product areas: competent cells, expression vectors, instruments, and reagents serving demand for research-grade and therapeutic proteins.

FMI has identified noteworthy growth drivers: the rising significance of biologics; innovations in proteomics; and patent expirations among small-molecule drugs. “These demands will boost the overall protein expression market in the coming future,” FMI literature states. “However, [attempts to contain rising costs] in various R&D activities in the fields of biotechnology and pharmaceutical industry as well as market consolidation of a high degree are some restraining factors for this market.”

The largest market for protein expression is expected to emerge in North America, given this region’s “well-established healthcare infrastructure.” North America is followed by Europe, and the Asia-Pacific region shows the highest growth. This information was derived from an FMI report (“Protein Expression Market: Global Industry Analysis and Opportunity Assessment 2015–2025”) that was issued last December.

 

 

Landmark Year

Through the efforts of scientists at Thermo Fisher Scientific, 2015 was a landmark year for transient protein production in CHO cells. The company’s ExpiCHO™ transient expression system achieved multiple g/L levels of protein expression previously thought possible only in stable cell lines, according to Jonathan Zmuda, Ph.D., associate director of cell biology at Thermo Fisher Scientific’s Gibco business unit.

“ExpiCHO allows drug developers to obtain meaningful quantities of protein from CHO cells at the very earliest stages of biologics development,” Dr. Zmuda asserts. “It allows CHO-derived protein to be used from discovery day one through the transition to stable cell lines, bioproduction, clinical trials, and product licensing.”

This has had the effect of streamlining drug development by eliminating the risk of starting a program with HEK 293-derived drug candidates, while also providing an alternative high-expressing system for proteins that are difficult to express in HEK 293.

New E. Coli Expression System

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New England BioLabs says that its SHuffle T7 E. coli expression system is able to express non-di-sulfide bonded proteins more efficiently than wild-type E. coli. The actual SHuffle strain expressing GFP is shown here.

Since E. coli was recruited for service around 1950, hundreds of thousands of publications have sung the praises of this bedrock expression system. But Mehmet Berkmen, Ph.D., staff scientist at New England BioLabs, notes that no more than a dozen distinct protein production strains exist. When production strains are examined closely, all are found to belong to just two basic strains, E. coli K-12 and E. coli B.

“Some strains have ‘bells and whistles,’ but the basic platform is the same,” Dr. Berkmen points out. “People are still looking for engineered lines that express protein more efficiently.”

Most expression systems are based on E. coli B, but that strain is not engineered specifically for protein production. The B strain is somewhat less domesticated than K-12, which has gone through numerous generations of selection for DNA manipulation. “E. coli B is more wild and tends to make protein better,” Dr. Berkmen notes. “But if you ask people why that is the case, they can’t provide an answer.”

New England BioLabs claims that its SHuffle® T7 E. coli expression system represents a breakthrough for microbial fermentation. The bacteria, which are chemically competent E. coli K-12 cells engineered to form proteins containing disulfide bonds in the cytoplasm, are suitable for T7-promoter-driven protein expression. The company has recently produced full-length antibodies, complete with disulfide bonds, in SHuffle organisms, which Dr. Berkmen calls “a significant step toward engineering and developing novel antibody formats and tools.”

New England BioLabs manufactures more than 500 proteins, 98% of them in E. coli. Perhaps even more interesting is the SHuffle system’s ability to express non-disulfide-bonded proteins more efficiently than wild-type E. coli. “SHuffle,” insists Dr. Berkmen, “represents a new chassis for protein production.”

The E. coli bacterium does not form disulfide bonds in its cytoplasm because two reducing pathways maintain the cytoplasmic proteome in its reduced state. Dr. Berkmen’s group knocked out those pathways and inserted a gene for a disulfide bond isomerase that increases fidelity of disulfide bond formation.

In addition to benefits already mentioned, SHuffle has a greatly diminished reducing capacity, permitting the formation of disulfide bonds for proteins that require it for folding and activity. Additionally, the cells, which are under oxidative stress, produce chaperones that also improve folding. For example, the activity of green fluorescent protein (GFP) expressed in SHuffle is much higher than protein produced in wild-type E. coli B.

It should be noted that a lack of glycosylation machinery persists in SHuffle cells. This problem, however, can be circumvented, as demonstrated in a seminal study carried out by Dr. Berkmen and colleagues. This study, which appeared last year in Nature Communications, described how IgG could be produced in SHuffle cells. Specifically, the investigators introduced mutations into the Fc portion of IgG. This resulted in efficient binding of aglycosylated IgG to its cognate receptor FcγRI.

Even in the absence of such ingenuity, E. coli remains a valuable expression system. It can be used to produce diagnostic and reagent proteins, or proteins for which glycosylation is noncritical.

“A Matter of Trying”

The principal advantages of using E. coli. are time and cost. “It takes basically one day, more or less, to obtain enough protein to suit many applications,” says David Chereau, Ph.D., CSO at Biozilla, a biotechnology contract research organization. As previously noted, the main disadvantages are lack of glycosylation apparatus and inability to support disulfide bond formation.

Workaround strategies can achieve stable disulfide bonds for some proteins. One strategy involves the following steps: Express the protein as an inclusion body, in insoluble form. Isolate the insoluble fraction. Solubilize this fraction with urea or some other suitable agent. Refold the protein.

“The process is relatively straightforward,” observes Dr. Chereau. “It’s much more difficult to find refolding conditions, which are normally determined empirically.” Refolding requires just the right buffer, salt concentrations, and additives. Also, refolding must be done in an oxidizing environment if disulfide bonds are to be achieved or maintained.

Dr. Chereau is philosophical about CHO cells’ inability to glycosylate: “Lack of glycosylation can be seen as an advantage or an inconvenience, depending.” E. coli is definitely out where glycosylation is a sine qua non. “But for the many applications where glycosylation isn’t needed, E. coli can be advantageous,” comments Dr. Chereau. Diagnostics and reagents are two such products. Additionally, obtaining a crystal structure during protein characterization is easier with glycans absent.

As part of its proof-of-concept services, Biozilla performs rapid screens to determine if E. coli is the right expression system for a particular product. Screening resembles design-of-experiment for mammalian cells, varying plasmids and vectors, as well as expression conditions.

Due to the success of CHO cells, bioprocessors tend to dismiss microbial fermentation, particularly for large proteins. “A lot of people think that expressing large proteins in E coli is difficult,” Dr. Chereau states, “but it’s often just a matter of trying. We have recently expressed a protein of 215 kDa in E. coli, which most people will tell you cannot be done. And we achieved it in very high yield.”

Rapid Prototyping

In June 2015, Invenra, a preclinical stage biotech company specializing in next-generation antibodies and antibody derivatives, entered an agreement with Oxford BioTherapeutics (OBT) to identify and characterize fully human therapeutic monoclonal antibodies (mAbs) against a novel cancer target that OBT has identified.

Invenra’s protein expression platform, through which it is capable of producing hundreds of thousands of full-length antibodies, uses cell-free expression to multiplex up to 10,000 protein variants simultaneously.

“We think of our technology as a rapid prototyping tool for proteins,” says Bryan Glaser, Ph.D., Invenra’s R&D director. “Once we have DNA, we can get protein in less than a day.” Invenra’s expression platform is suitable mainly for discovery and rapid protein prototyping. Yields are quite good: up to 500 μg/mL.

Other firms, such as Sutro Biopharma, are working on cell-free expression at much larger scales. Sutro claims that its Express CF™ technology can produce g/L yields in eight hours.

Cell-free expression involves E. coli extracts, typically S30 (used by most cell-free expression systems) and S12. The numbers reflect centrifugation speed. “Our system is based on S12, which is spun at lower speed than S30,” informs Dr. Glaser. “Our extract also does not undergo dialysis. We think of it as a ‘whole grain’ version.”

In addition to E. coli extracts, additives contain varying quantities of supplemental energy sources, nucleotides, and other small molecules that facilitate in vitro transcription and translation. Every vendor has its own unique blend.

Invenra’s standard mix, which is similar to off-the-shelf products from most commercial sources, is optimized for less complex molecules that don’t require disulfide bonds. Another mix has been optimized to include chaperones formulated to help expression and folding of IgGs and IgG-like molecules.

The upshot: fully functional, correctly folded IgGs and some bispecific antibodies, scFvs, and Fabs. More complex molecules are also possible, but each must be investigated independently. It is possible those could be made, but they would need to be optimized structure by structure. Dr. Glaser says expression capability depends to a large extent on amino acid sequence.

“We can fine-tune expression and folding conditions better than is possible in E. coli,” Dr. Glaser asserts. “We have better control over redox environment to facilitate disulfide bond formation, and we can add chaperones that are not present in E. coli organisms.” Still, the more disulfides the more complex the structure, and the lower the yield.

Dr. Glaser adds that antibody frameworks that express well in E. coli express well in cell-free systems, and ones that don’t express well in bacteria or mammalian cells tend not to express well cell-free. “It could be a framework sequence dependency,” he speculates. “It could be how well that framework folds. Perhaps the best-expressing molecules are those that do not require as much assistance from various chaperones and isomerases.”

Invenra’s expression system lends itself well to large-scale parallelism. The company has developed a credit-card-sized nanowell platform that expresses up to 10,000 unique antibodies per nanowell array. Cell-free expression of IgG using the Invenra nanowell platform system enables the incorporation of functional screening very early into the discovery process.

The ability to screen in excess of 100,000 IgG molecules can reduce the antibody display selection steps and preserve a larger diversity of epitope coverage. In addition, large combinations of binding partners can be empirically tested in various bispecific formats with relevant functional assays to identify the best pair and format for activity.

Getting the Bugs Out

Interest is growing for insect cell expression systems transiently transfected through the baculovirus expression vector system (BEVS). More and more clinical candidates are being generated in insect cells, including development-stage products for respiratory syncytial virus, Ebola virus, and norovirus.

 

A good deal of BEVS’ success is the ability of insect cells to produce multivalent, multisubunit vaccines through virus-like particles. These proteins can be made at large scale with BEVS for structural studies or to elucidate protein function.

Additionally, insect cells are ideal for making proteins that are toxic to mammalian or E. coli expression systems. BEVS shows its flexibility by providing rapid development cycles for treatments like seasonal influenza or pandemic infection vaccines. Because it is a transient system, BEVS allows for rapid turnaround times compared with mammalian cells, from identification of vaccine candidates to production.

 

Progress toward Therapeutic Epigenetics    

Epigenetic Targets Are Plentiful but Well Camouflaged

Angelo DePalma, Ph.D.

GEN  Jan 15, 2016 (Vol. 36, No. 2)   http://www.genengnews.com/gen-articles/progress-toward-therapeutic-epigenetics/5664/

  • Epigenetics is poised to become a cornerstone of drug development in oncology, diabetes, inflammation, developmental and metabolic disorders, cardiovascular and autoimmune diseases, pain, and neurological disorders.

    According to citations from PubMed Epigenetics, 40% year-on-year increases in epigenetics-related scientific publications occurred during the last decade, accompanied by a substantial increase in research funding. Data from ClinicalTrials.gov indicate that more than 40 different epigenetics-related drugs are undergoing clinical trials. Epigenetics will also likely affect developments in animal, plant, and environmental health.

    Jim Corbett, president of the human health business at PerkinElmer, notes that epigenetics research is currently limited by the number and availability of fully validated targets and preclinical disease models. “Another limitation stems from the relative dearth of fully selective antibodies for some of the writer and eraser targets to elucidate these complex signaling and modification events,” he points out. “Epigenetics research also suffers from a lack of a translational continuum for specific applications and for solutions from bench to bedside.”

    Nevertheless, the field is characterized by a high level of optimism. Research by Mordor Intelligence (“North America and Europe Epigenetics Market Growth, Trends and Forecasts, 2014–2020”) estimates that epigenetics will grow in market reach from approximately $2.9 billion in 2012 to about $12 billion in 2018.

    “I anticipate the development of second-generation epigenetic inhibitors with increased selectivity and targeting potential, standardization of epigenetic assays, and the validation of preclinical disease models leading to an improved understanding of epigenetic targets and mechanisms,” Corbett ventures. “The emergence of selective genome-editing technologies such as CRISPR will also apply in epigenetics and epigenome editing. I envision the future the emergence of personalized epigenetic profiles in patients.”

  • Computer Analogy

    Randy L. Jirtle, Ph.D., professor of epigenetics at North Carolina State University, describes epigenetics as a type of biological software. He explains an embryo’s combination of paternal and maternal genetic information, and eventual differentiation into 200–300 cell types, on the basis of cells running different programs.

    “The cell can be thought of as a programmable computer where the hardware is DNA and the software is the epigenome,” says Dr. Jirtle. “Very shortly after fertilization, this computer tells the cell how to work. And as with actual computers, things can go wrong because of viruses or—in the case of cells—mutations.”

    Dr. Jirtle demonstrated in 2003 that epigenetic modifications in utero may determine adult disease susceptibility, a notion that was not welcomed enthusiastically. “If you think of life as hardware, no known mechanism would explain this [connection],” asserts Dr. Jirtle. “But when you consider the ‘software,’ it becomes understandable.”

    Epigenetics can bring about positive effects as well. Through a process known as hormesis, low doses of a toxic agent or low doses of radiation can be administered strategically to improve an organism’s subsequent health. For example, mice exposed to low levels of ionizing radiation experienced a positive adaptive effect, which flies in the face of prevailing “no safe dosage” logic.

    In one strain of an experimental mouse bred to develop human-like diseases, 1 cGy of exposure—about the dose received from five X-rays—resulted in a decidedly positive hypermethylation of the epigenome. Exposed mice developed obesity, diabetes, and cancer at significantly lower rates than nonexposed mice. Negative effects occur at significantly higher doses as expected.

    Similarly positive epigenetic effects have been observed in plants exposed to very low doses of herbicides.

    Dr. Jirtle believes that the characterization of the repertoire of genes imprinted in humans, and their regulatory elements, the imprintome, will guide epigenome-based therapies. Imprinting is the process by which one parental copy of a gene is silenced. Thus, depending on the effectiveness of silencing, one could have two copies of a gene or none, either of which could potentially be deadly. In some cancers, for example, the inability to silence one parental gene for IGF2, which influences apoptosis, allows cancer cells to grow out of control.

    “There are probably around 150–500 disease-influencing genes that are regulated this way,” Dr. Jirtle points out.

  • Implementation Hurdles

    The connection between dysregulated DNA methylation and cancer is well established. Keith Booher, Ph.D., epigenetic service projects manager at Zymo Research, believes that modifying how methylation patterns change could allow a reset. Essentially, cells destined to become cancerous could be returned to a normal state.

    But significant hurdles block straightforward implementation. For example, getting drugs into cells, particularly solid tumors, is not easy. “It’s no coincidence that DNA methylation inhibitors have proved most successful for blood-based cancers, which are easier to target,” Dr. Booher tells GEN.

    Another hurdle is drug resistance, an issue with nearly all oncology agents. Moreover, drugs that alter the activity of the ubiquitous DNA methyl transferase will have broad activity on normal as well as abnormal cell processes.

    “Normal cells show low and high methylation levels,” Dr. Booher explains. “DNA methylation tends to limit gene expression, so you want to shut down those genes. And where DNA methylation is absent, genes tend to be expressed.

    “Methylation will change across the genome at different development stages, but in adult cells or developing blood cell you want methylation patterns to change in a regulated way. It’s difficult to limit the effect to diseased cells.”

    Finally, the way methylation inhibitors interact with DNA is in itself harmful. The original epigenetics-based drugs tested as broad chemotherapeutics, but their toxicology was high. It was only later, after the understanding the relationship between DNA methylation and carcinogenesis was better established, that the potential to use these agents at much lower doses became possible.

     

    Diagnostic Relevance

     

     

    This Circos plot from Swift Biosciences represents the methylation status of 1 Mb bins across chromo­somes 1–22 for Sample 8 (Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary).

    One of the most important advances in epigenetic research is the ability to obtain comprehensive, per-base methylation status of the methylome using next-generation sequencing (NGS). The significant drop in sequencing costs enables both whole-genome bisulfite sequencing and hybridization capture for targeted enrichment of the methylome.

    Initially, notes Laurie Kurihara, Ph.D., director of R&D at Swift Biosciences, these techniques were developed for microgram inputs of genomic DNA that undergo standard NGS library preparation followed by bisulfite conversion, a chemical process that converts nonmethylated cytosines to uracil. Subsequently, the polymerase chain reaction (PCR) process can be used to convert uracil to thymidine. “But the methylated cytosines are protected, thus demarcating methylation status when the DNA sequence is determined,” Dr. Kurihara observes. “The drawback is that bisulfite-induced DNA fragmentation destroys the bulk of the prepared NGS library. Hence the requirement for microgram DNA inputs.”

    To enable lower DNA inputs and improved methylome coverage and uniformity, Swift Biosciences has developed an NGS library preparation performed on bisulfite-converted DNA fragments. The underlying technology, Adaptase, is a proprietary NGS adapter attachment chemistry for single-stranded DNA.

    “By significantly improving sample recovery from bisulfite-converted DNA,” explains Dr. Kurihara, “more complete analysis of clinical samples is possible, particularly cell-free DNA from plasma that is limited to low-nanogram quantities of DNA.”

    Dr. Kurihara cites an example provided by Dennis Lo, M.D., Ph.D., professor of chemical pathology at the Chinese University of Hong Kong. Dr. Lo developed a noninvasive test for cancer by detection of genome-wide hypomethylation of cell-free DNA from patient plasma. Although this “liquid biopsy” does not uncover actionable cancer mutations, it may prove to be a sensitive blood test for early cancer detection as well as treatment monitoring.

    More recently, Dr. Lo’s group mapped the tissue of origin for cell-free plasma DNA using genome-wide bisulfite sequencing after mapping tissue-specific methylation patterns. Such noninvasive testing from blood may identify tissue- or organ-specific pathologies, including cancer, stroke, myocardial infarction, autoimmune disorders, and transplant rejection.

    “Given that advances in epigenetic technologies have enabled per-base methylation status from low DNA input clinical samples, proof of concept has been established that ‘liquid biopsy’ testing of patient blood may be a universal screen for a variety of diseases that may be pinpointed to individual organs or tissues,” Dr. Kurihara tells GEN. “Such universal testing could be particularly advantageous for early detection of cancer and other diseases where noninvasive screening has not previously been possible.”

  • NGS: An Enabling Technology

    The widespread adoption of next-generation genomic sequencing means that for the first time scientists can sequence large numbers of cancer patient genomes. Thus far, these studies have demonstrated that a large proportion of mutated cancer genes may be classified as epigenetic modifying factors.

    “Chromatin remodeling and modifying factors are involved in the regulation of gene expression,” says Ali Shilatifard, Ph.D., chairman of the department of biochemistry and molecular genetics at Northwestern University’s Feinberg School of Medicine. “The DNA methylation factors are highly mutated in most cancers characterized thus far.”

    Dr. Shilatifard provides the example of a family of mixed-lineage leukemia genes within the complexes known as COMPASS (complex proteins associated with Set1), which are highly mutated in a large number of cancers. “We’ve shown that MLL3/4, two members of the COMPASS family, modify regulatory elements known as enhancers,” notes Dr. Shilatifard. “The job of this COMPASS family member is to regulate these cis-regulatory elements during development.”

    It has been shown that MLL3/4 and another component of COMPASS, UTX, are some of the most mutated genes in cancer. “We propose that perhaps these mutations function through enhancer malfunction,” Dr. Shilatifard continues. “And enhancer malfunction through these family members could result in miscommunication of the regulatory elements and promoters and mis-regulation of the expression pattern, resulting in tissue-specific cancers. It’s now very clear that epigenetic regulation and enhancer malfunction are key events in cancer pathogenesis.”

    Dr. Shilatifard believes that over the next several years, academic labs and pharmaceutical companies will increasingly rely on agents that intervene epigenetically. For example, a recent study indicated that approximately 75% of patients with diffuse intrinsic pontine glioma (DIPG), a rare brain cancer in children, carried a single point mutation on histone H3, transforming lysine 27 into methionine. Many copies of histone H3 exist in these patients, but mutation in just one copy is sufficient to cause DIPG.

    After modeling this mutation in Drosophila, Dr. Shilatifard’s laboratory discovered that a single point mutation on one histone was associated with a global loss of histone methylation and an increase in histone acetylation.

    “Epigenetic regulators could be central for treating this disease,” comments Dr. Shilatifard. “Numerous examples in the literature suggest that inhibition of epigenetic regulators and interactors could be very important for treating cancer, and this may work for the treatment of DIPG through the inhibition of factors that bind to hyper-acetylated histones.”

 

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2.0 Genomics and Epigenetics: Genetic Errors and Methodologies – Cancer and Other Diseases

Writer and Curator: Larry H Bernstein, MD, FCAP

This is the second article in a series concerning genomic expression, The first of which was concerned with the expanded technologies in use for study of genomic expression.  This portion will also cover more of genetic errors as well as methodologies, but not all examples are in the realm of cancer.

I shall start with a New York Times editorial on July 24, 2015 by Angelina Jolie Pitt on her experience with BRCA1 gene and her family history.  It is very instructive on how she worked through her experience.

http://www.nytimes.com/2015/03/24/opinion/angelina-jolie-pitt-diary-of-a-surgery.html?

Two years ago she was found to have a positive test for BRCA1, carrying an 87 percent risk for breast cancer and a 50 percent risk for ovarian cancer.  At that time she had a preventive mastectomy.  The decision was not easy, but it also brought into consideration that her mother and grandmother both died of breast cancer.  She did not have an oophorectomy at that time because on considering the advice of medical experts, she would have been left with no estrogen support. She wanted to delay her early vegetative senescence.  She has reached the age of 39 years and on the advice of medical expert opinion, she proceeded with salpingo-oophorectomy, at age 39 years, a decade before  her  mother had developed cancer.  But her delay was to allow her to recover and adjust emotionally to her ongoing situation, with a remaining risk for ovarian cancer.

She tested negative for CA-1251-5 at this time prior to surgery. But the CA-125 test could well be negative with early onset ovarian cancer. It may be considered a better test for following treatment than for early diagnosis. Her choice was to sacrifice early menopause to the ability to live through her childrens’ childhood development.  This was a well thought out decision.  In addition, there were abnormal inflammatory markers that were not specific for cancer rsik, but were worth taking into account.  The procedure itself was simpler than the mastectomy.

23op-ed-thumbStandard

http://static01.nyt.com/images/2015/03/23/opinion/23op-ed/23op-ed-master315.jpg

2.1  CA-125 and Ovarian Cancer

2.1.1  lmmunoradiometric Assay of CA 125 in Effusions: Comparison with Carcinoembryonic Antigen

Marguerite M. Pinto, MD,‘ Larry H. Bernstein, MD,* Dennis A. Brogan, MPH, MT

and Elaine Criscuolo, CT(ASCP) CMIACS

The levels of CA 125 antigen were measured in 167 effusions from 150 patients using radioimmunoassay, and the results compared with the levels of carcinoembryonic antigen (CEA) in the fluids. The results indicate that an elevated fluid CA 125 level (>14,000 U/ml-68,000 U/ml) and a negative fluid CEA level (4 ng/ml) is suggestive of serous and endometrioid carcinoma of ovary, and adenocarcinoma of the endometrium and fallopian tube. Alternatively, an elevated fluid CEA level (14 ng/ml-600 ng/ml) and a negative CA 125 level (20-5000 U/ml) is seen in metastatic carcinomas of breast, lung, gastrointestinal tract, and mucinous ystadenocarcinoma. Lymphomas, melanomas, and benign effusions are negative for both antigens. The combined use of CEA and CA 125 antigen in fluids is useful in the differential diagnosis of adenocarcinoma of unknown primary. Cancer 59:218-222, 1987.

2.1.2 CA-125 in fine-needle aspirates of solid tumors: comparison with cytologic diagnosis and carcinoembryonic antigen (CEA) assay.

Marguerite M. Pinto, S Kotta

Diagnostic Cytopathology 03/1996; 14(2):121-5.
http://dx.doi.org:/10.1002/(SICI)1097-0339(199603)14:2<121::AID-DC4>3.0.CO;2-M

One hundred and twenty-two fine needle aspirates (FNA) from female patients were studied to determine whether CA-125 assay contributed to cytologic diagnosis and CEA assay. Cytologic examination was done on Papanicolaou-stained smears and cell blocks, CEA by EIA (Abbott Laboratory, > 5 ng/ml cutoff) and CA-125 by RIA (Abbott Laboratory, North Chicago, IL, > 66 mu/ml cutoff). Final diagnosis were correlated with histologic diagnosis when available, clinical, radiologic studies, and follow-up. Results: 29 benign, 93 malignant. Sensitivities and specificities: cytology, 91%, 100%; CEA: 59%, 86%; CA-125, 50%, 55%. CEA plus cytology sensitivity, 97%. CA-125 content was highest in endometrial/ovarian carcinoma (39,899 mu/ml) and < 5,000 mu/ml in other tumors and benign FNA in contrast to CEA which showed highest levels in carcinomas of colon, pancreas, and lung (> 280 ng/ml). While elevated CEA enhances the sensitivity of cytologic diagnosis of carcinomas of the colon, pancreas, and lung, low CEA and high CA-125 content supports an ovarian/endometrial primary.

2.1.3  Diagnostic efficiency of carcinoembryonic antigen and CA125 in the cytological evaluation of effusions.

Pinto MM, Bernstein LH, Rudolph RA, Brogan DA, Rosman M.
Arch Pathol Lab Med. 1992 Jun; 116(6):626-31.

In our previous study, the combination of the concentrations of carcinoembryonic antigen (CEA) and CA125 and the findings from cytological examination in 189 benign and malignant pleural and peritoneal effusions was useful in the diagnosis/classification of malignant effusions. Sensitivity of CEA (level, greater than 5 ng/mL) was 68%; specificity was 99% for the diagnosis of malignant effusions secondary to carcinoma of the lung, breast, gastrointestinal tract, and mucinous carcinoma of the ovary. Sensitivity of CA125 (level, greater than 5000 U/mL) was 85%; specificity was 96% for the diagnosis of malignant effusions in carcinoma of the ovary, fallopian tube, and endometrium. We now expanded the study to include 840 pleural and peritoneal effusions (benign, n = 520; malignant, n = 320) and analyzed the data by the statistical method of Rudolph and colleagues. Based on new cutoff values, ie, CEA level at 6.3 ng/mL and CA125 level at 3652 U/mL, the sensitivities for detection of malignant effusions secondary to carcinomas of the lung, breast, and gastrointestinal tract and mucinous carcinoma of the ovary varied between 75% and 100%; specificity was 98%. Sensitivity of CA125 for detection of malignant effusions from müllerian epithelial carcinoma was 71%; specificity was 99%. The elevated CEA fluid level alone helped to diagnose malignant effusions of the gastrointestinal tract in 54%, breast in 19%, and lung in 16%. The high CA125 fluid level was predictive of müllerian epithelial carcinoma. Adjunctive use of CEA and CA125 levels in fluid enhances the sensitivity of cytological diagnosis and may be predictive of the primary site in patients who present with carcinoma of an unknown primary source.

2.2 Carcinoembryonic antigen in diagnostics

2.2.1 Carcinoembryonic antigen content in fine needle aspirates of the lung. A diagnostic adjunct to cytology.

Pinto MM1, Ha DJ.
Acta Cytol. 1992 May-Jun; 36(3):277-82

Carcinoembryonic Antigen (CEA) was measured in 59 consecutive fine needle aspirates (FNAs) of the lung from 58 patients to determine if the CEA content would enhance the sensitivity of the cytologic diagnosis. Twenty-eight males and 30 females with tumors 1-40 cm in diameter were studied. Final diagnoses were correlated with the clinical history, radiologic studies, tissue (when available) and follow-up. Image-guided FNAs were performed by radiologists using a 22-gauge Chiba needle and 20-mL syringe with one to four passes per specimen. Cytologic examination included rapid assessment in the radiology suite and a final diagnosis in 24 hours. CEA was measured by enzyme immunoassay using monoclonal antibody. Nine benign aspirates and 50 malignant aspirates were diagnosed. The sensitivity of cytology was 86% and specificity, 100%. Using 5 ng/mL as the cutoff, the sensitivity of CEA for malignant aspirates was 50% and specificity, 90%. The combined sensitivity of CEA and cytology was 95%. The mean CEA in malignant aspirates was 131 ng/mL and in benign aspirates, 2.41. The highest mean CEA was seen in adenocarcinoma, 402.6 ng/mL. Lower CEA content was seen in epidermoid carcinoma (58.6 ng/mL), large cell carcinoma (8.09), oat cell carcinoma, metastatic carcinoma of the kidney and breast, thymoma and lymphoma (each less than 1 ng/mL). Elevated CEA alone was diagnostic in two aspirates of bronchioloalveolar carcinoma; carcinoma with an unknown primary source, three; and large cell carcinoma, one. The adjunctive use of CEA in FNAs of the lung enhances the sensitivity of the cytologic diagnosis.

2.2.2  Relationship between serum CA125 half life and survival in ovarian cancer

Table
Gupta and Lis Journal of Ovarian Research 2009 2:13
http://dx.doi.org:/10.1186/1757-2215-2-13

First Author, Year, Study Place Data Collection Study
Design
Sample
Size
RR/HR, (95% CI),
P-Value
Riedinger JM, 2006, France 1988 to
1996
R 553 2.04 (1.58-2.63), < 0.0001
Gadducci A, 2004, Italy 1996 to2002 R 71 3.11 (1.22-7.98), 0.0181
Munstedt K, 1997, Germany 1987 to1994 R 85 0.6184
Gadducci A, 1995, Italy 1986 to1992 R 225 2.13 (1.23-3.68), 0.0073
Rosman M, 1994, Connecticut 1985 to
1989
R 51 3.6 (1.8-7.4), < 0.001
Yedema C A, 1993, Netherlands 1984 to
1990
R 60 9.17 (1.49-56.3), 0.01
Hawkins RE, 1989, London NA P 29 3.7 (0.7-20.1), 0.001;27.8 (4.0-193), 0.001

1CA125 half-life was independent prognostic indicator for survival
2FIGO stage, tumor grade, residual disease, CA125
http://www.ovarianresearch.com/content/2/1/13/table/T6

3.3.0      DNA double strand breaks

2.3.1.  Collaboration and competition – DNA double-strand break repair pathways

Kass EM, Jasin M
FEBS Letters 2010; 584:3703-3708
http://dx.doi.org:/jfebslet.2010.07.057

DNA double-strand breaks occur in replication and exogenous sources pose risk to genome stability. There are two pathways to repair.  They are non-homologous end joining and homologous recombination. Both pathways cooperate and compete at double-strand break sites.

2.3.2 DNA Double-Strand Break Repair Inhibitors as Cancer Therapeutics

Srivastava M, Rashavan SC
Chem & Biol 2015 Jan; pp17-29
http://dx.doi.org:/10.1016/jchembiol.2014.11.013

Homologous recombination and non-homologous end joining are the two major repair pathways expressed in eukaryotes.  For double-strand breaks, and the DSB repair gene is vulnerable to chemotherapy and radiation therapy, accounting for treatment resistance. Therefore, targeting DSB repair is attractive. Blocking the residual repair using inhibitors can potentiate treatment.

2.3.3  Animation published in DNA Repair: Helleday T, Lo J, van Gent DC, Engelward BP. DNA double-strand break repair: From mechanistic understanding to cancer treatment. DNA Repair. (14 Mar 2007)
2.3.3.1 http://web.mit.edu/engelward-lab/animations/DSBR.html

2.3.3.2 https://www.youtube.com/watch?v=eg8rpYFsqCA

2.3.4 Homology-dependent double strand break repair. Oxford Academic (Oxford University Press)

https://www.youtube.com/watch?v=86JCMM5kb2A

2.4.0 Managing DNA data sets

2.4.1 Bionimbus –  a cloud for managing, analyzing and sharing large genomics datasets

The Bionimbus Protected Data Cloud (PDC) is a collaboration between the Open Science Data Cloud (OSDC) and the IGSB (IGSB,) the Center for Research Informatics (CRI), the Institute for Translational Medicine (ITM), and the University of Chicago Comprehensive Cancer Center (UCCCC). The PDC allows users authorized by NIH to compute over human genomic data from dbGaP in a secure compliant fashion. Currently, selected datasets from the The Cancer Genome Atlas (TCGA) are available in the PDC.

https://bionimbus-pdc.opensciencedatacloud.org/

2.4.1.2 Accounting for uncertainty in DNA sequencing data

O’Rawe JA, Ferson S, Lyon GJ
Trends in Genetics 2015 Feb; 31(2):61-66
http://dx.doi.org:/10.101/jtig.2014.12.002

This article reviews uncertainty in quantification in DNA sequency applications and sources of error propagation, and it proposes methods to account for errors and uncertainties.

2.5.0 Linking Traits to Mechanisms and UPR response/proteostasis

2.5.1 Stress-Independent Activation of XBP1s and/or ATF6 Reveals –Three Linking traits based on their shared molecular mechanisms

Shoulders MD, Ryno LM, Genereux JC,…Wiseman BL
Cell Reports 2013 Apr; 3, pp 1279-1292
http://dx.doi.org:/10.1016/j.celrep.2013.03.024

The unfolded protein response (UPR) maintains ER proteostasis through the transcription factors XP1s and ATF6. This study measured orthogonal small molecule-mediated activation of transcription factors nXP1s and/or ATF6 using transcriptomics and quantitative proteomics. The finding is that three ER proteostasis environmants differentially influence

  1. Folding
  2. Traffiking, and
  3. Degradation of destabilized ER client proteins

Without affecting endogenous proteome. The proteostasis network is remodeled with the potential for selective restoration of the aberrant ER proteostasis.

2.5.2 Biological and chemical approaches to diseases of proteostasis deficiency.

Powers ET, Morimoto RI, Dillin A, Kelly JW, Balch WE
Annu Rev Biochem. 2009; 78:959-91.
http://dx.doi.org:/10.1146/annurev.biochem.052308.114844

Many diseases appear to be caused by the misregulation of protein maintenance. Such diseases of protein homeostasis, or “proteostasis,” include loss-of-function diseases (cystic fibrosis) and gain-of-toxic-function diseases (Alzheimer’s, Parkinson’s, and Huntington’s disease). Proteostasis is maintained by the proteostasis network, which comprises pathways that control protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation. The decreased ability of the proteostasis network to cope with inherited misfolding-prone proteins, aging, and/or metabolic/environmental stress appears to trigger or exacerbate proteostasis diseases. Herein, we review recent evidence supporting the principle that proteostasis is influenced both by an adjustable proteostasis network capacity and protein folding energetics, which together determine the balance between folding efficiency, misfolding, protein degradation, and aggregation. We review how small molecules can enhance proteostasis by binding to and stabilizing specific proteins (pharmacologic chaperones) or by increasing the proteostasis network capacity (proteostasis regulators). We propose that such therapeutic strategies, including combination therapies, represent a new approach for treating a range of diverse human maladies.

2.5.3 Extracellular Chaperones and Proteostasis

Amy R. Wyatt, Justin J. Yerbury, Heath Ecroyd, and Mark R. Wilson
Annual Review of Biochemistry 2013 Jun; 82: 295-322
http://dx.doi.org:/10.1146/annurev-biochem-072711-163904

There exists a family of currently untreatable, serious human diseases that arise from the inappropriate misfolding and aggregation of extracellular proteins. At present our understanding of mechanisms that operate to maintain proteostasis in extracellular body fluids is limited, but it has significantly advanced with the discovery of a small but growing family of constitutively secreted extracellular chaperones. The available evidence strongly suggests that these chaperones act as both sensors and disposal mediators of misfolded proteins in extracellular fluids, thereby normally protecting us from disease pathologies. It is critically important to further increase our understanding of the mechanisms that operate to effect extracellular proteostasis, as this is essential knowledge upon which to base the development of effective therapies for some of the world’s most debilitating, costly, and intractable diseases.

http://www.proteostasis.com/our-technology/proteostasis-network.html

proteostasis model

http://www.proteostasis.com/images/stories/technology/illustration1.gif

2.6.0 Transcription

2.6.1 Looping Back to Leap Forward. Transcription Enters a New Era

Levine M, Cattoglio C, Tijan R
Cell 2014 Mar; 157: 13-22.
http://dx.doi.org:/10.1016/j.cell.2014.02.009

Organism complexity is not in gene number, but lies in gene regulation. The human genbome contains hundreds of thousands of enhancers, and genes are embedded in a milieu of enhancers . Proliferation of cis-regulatory DNAs is accompanied by complexity and functional diversity of transcription machinery recognizing distal enhancers and promotors, and high-order spatial organization. This article reviews the dynamic communication of remote enhancers with target promoters.

2.6.2 Activating gene expression in mammalian cells with promoter-targeted duplex RNAs.

Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, Corey DR.
Nat Chem Biol. 2007 Mar; 3(3):166-73
http://dx.doi.org:/10.1038/nchembio860

The ability to selectively activate or inhibit gene expression is fundamental to understanding complex cellular systems and developing therapeutics. Recent studies have demonstrated that duplex RNAs complementary to promoters within chromosomal DNA are potent gene silencing agents in mammalian cells. Here we report that chromosome-targeted RNAs also activate gene expression. We have identified multiple duplex RNAs complementary to the progesterone receptor (PR) promoter that increase expression of PR protein and RNA after transfection into cultured T47D or MCF7 human breast cancer cells. Upregulation of PR protein reduced expression of the downstream gene encoding cyclooygenase 2 but did not change concentrations of estrogen receptor, which demonstrates that activating RNAs can predictably manipulate physiologically relevant cellular pathways. Activation decreased over time and was sequence specific. Chromatin immunoprecipitation assays indicated that activation is accompanied by reduced acetylation at histones H3K9 and H3K14 and by increased di- and trimethylation at histone H3K4. These data show that, like proteins, hormones and small molecules, small duplex RNAs interact at promoters and can activate or repress gene expression.
2.6.3 Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

M Gossen and H Bujard
Proc Natl Acad Sci U S A. 1992 Jun 15; 89(12): 5547–5551.

Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of “on/off” situations for such genes in a reversible way.

Diagrams of two regulatable gene expression systems.

Diagrams of two regulatable gene expression systems.

http://www.intechopen.com/source/html/16788/media/image5.jpeg

schematic-representation-of-transgenic-mouse-breeding-scheme-h2b-gfp-mice-should-not-express-gfp-in-the-absence-of-a-tetracycline-regulatable-transactivator

schematic-representation-of-transgenic-mouse-breeding-scheme-h2b-gfp-mice-should-not-express-gfp-in-the-absence-of-a-tetracycline-regulatable-transactivator

http://openi.nlm.nih.gov/imgs/512/321/2408727/2408727_pone.0002357.g001.png

2.7.0 Epigenetics and Cancer

2.7.1 Epigenetics and cancer metabolism

Johnson C, Warmoes MO, Shen X, Locasale JW
Cancer Letters 2015;  356:309-314.
http://dx.doi.org:/10.1016/j.canlet.2013.09.043

Cancer is characterized by adaptive metabolic changes for proliferation and survival of the neoplastic cell, which is accompanied by dysfunctional metabolic enzyme changes in a specific nutrient supplied environment. The oncogenic change uses epigenetic level enzymes that catalyze posttranslational modifications of the DNA/histone expression with metabolites including cofactors and substrates for reactions. This interaction of epigenetics and metabolism provides new insights for anti-cancer therapy.

2.7.2 Cancer Epigenetics. From Mechanism to Therapy

Dawson MA, Konzarides T
Cell 2012 Jul; 150:12-27
http://dx.doi.org:/10.1016/j.cell.2012.06.013

Carcinogenesis requires all of the following:

  • DNA methylation
  • Histone modification
  • Nucleosome remodeling
  • RNA mediated targeting

This article reviews basic principles of epigenetic pathways that are dysregulated in carcinogenesis.

2.7.4 A subway review of cancer pathways

Hahn WC, Weinberg RA
Nature Reviews: Cancer
http://www.nature.com/nrc/poster/subpathways/index.html

Cancer arises from the stepwise accumulation of genetic changes that confer upon an incipient neoplastic cell the properties of unlimited, self-sufficient growth and resistance to normal homeostatic regulatory mechanisms. Advances in human genetics and molecular and cellular biology have identified a collection of cell phenotypes � the main destinations in the subway map below � that are required for malignant transformation1. Specific molecular pathways (subway lines) are responsible for programming these behaviours. Although the connections between cancer-cell wiring and function remain incompletely explored and specified � hence the many lines under construction � the broad outlines of the molecular circuitry of the cancer cell can now be sketched. Further advances in understanding these pathways and their interconnections will accelerate the development of molecularly targeted therapies that promise to change the practice of oncology.

cancer subway map

cancer subway map

http://www.nature.com/nrc/poster/subpathways/images/map.gif

Subway map designed by Claudia Bentley.

Read Full Post »

Metabolomics Summary and Perspective


Metabolomics Summary and Perspective

Author and Curator: Larry H Bernstein, MD, FCAP 

 

This is the final article in a robust series on metabolism, metabolomics, and  the “-OMICS-“ biological synthesis that is creating a more holistic and interoperable view of natural sciences, including the biological disciplines, climate science, physics, chemistry, toxicology, pharmacology, and pathophysiology with as yet unforeseen consequences.

There have been impressive advances already in the research into developmental biology, plant sciences, microbiology, mycology, and human diseases, most notably, cancer, metabolic , and infectious, as well as neurodegenerative diseases.

Acknowledgements:

I write this article in honor of my first mentor, Harry Maisel, Professor and Emeritus Chairman of Anatomy, Wayne State University, Detroit, MI and to my stimulating mentors, students, fellows, and associates over many years:

Masahiro Chiga, MD, PhD, Averill A Liebow, MD, Nathan O Kaplan, PhD, Johannes Everse, PhD, Norio Shioura, PhD, Abraham Braude, MD, Percy J Russell, PhD, Debby Peters, Walter D Foster, PhD, Herschel Sidransky, MD, Sherman Bloom, MD, Matthew Grisham, PhD, Christos Tsokos, PhD,  IJ Good, PhD, Distinguished Professor, Raool Banagale, MD, Gustavo Reynoso, MD,Gustave Davis, MD, Marguerite M Pinto, MD, Walter Pleban, MD, Marion Feietelson-Winkler, RD, PhD,  John Adan,MD, Joseph Babb, MD, Stuart Zarich, MD,  Inder Mayall, MD, A Qamar, MD, Yves Ingenbleek, MD, PhD, Emeritus Professor, Bette Seamonds, PhD, Larry Kaplan, PhD, Pauline Y Lau, PhD, Gil David, PhD, Ronald Coifman, PhD, Emeritus Professor, Linda Brugler, RD, MBA, James Rucinski, MD, Gitta Pancer, Ester Engelman, Farhana Hoque, Mohammed Alam, Michael Zions, William Fleischman, MD, Salman Haq, MD, Jerard Kneifati-Hayek, Madeleine Schleffer, John F Heitner, MD, Arun Devakonda,MD, Liziamma George,MD, Suhail Raoof, MD, Charles Oribabor,MD, Anthony Tortolani, MD, Prof and Chairman, JRDS Rosalino, PhD, Aviva Lev Ari, PhD, RN, Rosser Rudolph, MD, PhD, Eugene Rypka, PhD, Jay Magidson, PhD, Izaak Mayzlin, PhD, Maurice Bernstein, PhD, Richard Bing, Eli Kaplan, PhD, Maurice Bernstein, PhD.

This article has EIGHT parts, as follows:

Part 1

Metabolomics Continues Auspicious Climb

Part 2

Biologists Find ‘Missing Link’ in the Production of Protein Factories in Cells

Part 3

Neuroscience

Part 4

Cancer Research

Part 5

Metabolic Syndrome

Part 6

Biomarkers

Part 7

Epigenetics and Drug Metabolism

Part 8

Pictorial

genome cartoon

genome cartoon

 iron metabolism

iron metabolism

personalized reference range within population range

personalized reference range within population range

Part 1.  MetabolomicsSurge

metagraph  _OMICS

metagraph _OMICS

Metabolomics Continues Auspicious Climb

Jeffery Herman, Ph.D.
GEN May 1, 2012 (Vol. 32, No. 9)

Aberrant biochemical and metabolite signaling plays an important role in

  • the development and progression of diseased tissue.

This concept has been studied by the science community for decades. However, with relatively

  1. recent advances in analytical technology and bioinformatics as well as
  2. the development of the Human Metabolome Database (HMDB),

metabolomics has become an invaluable field of research.

At the “International Conference and Exhibition on Metabolomics & Systems Biology” held recently in San Francisco, researchers and industry leaders discussed how

  • the underlying cellular biochemical/metabolite fingerprint in response to
  1. a specific disease state,
  2. toxin exposure, or
  3. pharmaceutical compound
  • is useful in clinical diagnosis and biomarker discovery and
  • in understanding disease development and progression.

Developed by BASF, MetaMap® Tox is

  • a database that helps identify in vivo systemic effects of a tested compound, including
  1. targeted organs,
  2. mechanism of action, and
  3. adverse events.

Based on 28-day systemic rat toxicity studies, MetaMap Tox is composed of

  • differential plasma metabolite profiles of rats
  • after exposure to a large variety of chemical toxins and pharmaceutical compounds.

“Using the reference data,

  • we have developed more than 110 patterns of metabolite changes, which are
  • specific and predictive for certain toxicological modes of action,”

said Hennicke Kamp, Ph.D., group leader, department of experimental toxicology and ecology at BASF.

With MetaMap Tox, a potential drug candidate

  • can be compared to a similar reference compound
  • using statistical correlation algorithms,
  • which allow for the creation of a toxicity and mechanism of action profile.

“MetaMap Tox, in the context of early pre-clinical safety enablement in pharmaceutical development,” continued Dr. Kamp,

  • has been independently validated “
  • by an industry consortium (Drug Safety Executive Council) of 12 leading biopharmaceutical companies.”

Dr. Kamp added that this technology may prove invaluable

  • allowing for quick and accurate decisions and
  • for high-throughput drug candidate screening, in evaluation
  1. on the safety and efficacy of compounds
  2. during early and preclinical toxicological studies,
  3. by comparing a lead compound to a variety of molecular derivatives, and
  • the rapid identification of the most optimal molecular structure
  • with the best efficacy and safety profiles might be streamlined.
Dynamic Construct of the –Omics

Dynamic Construct of the –Omics

Targeted Tandem Mass Spectrometry

Biocrates Life Sciences focuses on targeted metabolomics, an important approach for

  • the accurate quantification of known metabolites within a biological sample.

Originally used for the clinical screening of inherent metabolic disorders from dried blood-spots of newborn children, Biocrates has developed

  • a tandem mass spectrometry (MS/MS) platform, which allows for
  1. the identification,
  2. quantification, and
  3. mapping of more than 800 metabolites to specific cellular pathways.

It is based on flow injection analysis and high-performance liquid chromatography MS/MS.

Clarification of Pathway-Specific Inhibition by Fourier Transform Ion Cyclotron Resonance.Mass Spectrometry-Based Metabolic Phenotyping Studies F5.large

common drug targets

common drug targets

The MetaDisIDQ® Kit is a

  • “multiparamatic” diagnostic assay designed for the “comprehensive assessment of a person’s metabolic state” and
  • the early determination of pathophysiological events with regards to a specific disease.

MetaDisIDQ is designed to quantify

  • a diverse range of 181 metabolites involved in major metabolic pathways
  • from a small amount of human serum (10 µL) using isotopically labeled internal standards,

This kit has been demonstrated to detect changes in metabolites that are commonly associated with the development of

  • metabolic syndrome, type 2 diabetes, and diabetic nephropathy,

Dr. Dallman reports that data generated with the MetaDisIDQ kit correlates strongly with

  • routine chemical analyses of common metabolites including glucose and creatinine

Biocrates has also developed the MS/MS-based AbsoluteIDQ® kits, which are

  • an “easy-to-use” biomarker analysis tool for laboratory research.

The kit functions on MS machines from a variety of vendors, and allows for the quantification of 150-180 metabolites.

The SteroIDQ® kit is a high-throughput standardized MS/MS diagnostic assay,

  • validated in human serum, for the rapid and accurate clinical determination of 16 known steroids.

Initially focusing on the analysis of steroid ranges for use in hormone replacement therapy, the SteroIDQ Kit is expected to have a wide clinical application.

Hormone-Resistant Breast Cancer

Scientists at Georgetown University have shown that

  • breast cancer cells can functionally coordinate cell-survival and cell-proliferation mechanisms,
  • while maintaining a certain degree of cellular metabolism.

To grow, cells need energy, and energy is a product of cellular metabolism. For nearly a century, it was thought that

  1. the uncoupling of glycolysis from the mitochondria,
  2. leading to the inefficient but rapid metabolism of glucose and
  3. the formation of lactic acid (the Warburg effect), was

the major and only metabolism driving force for unchecked proliferation and tumorigenesis of cancer cells.

Other aspects of metabolism were often overlooked.

“.. we understand now that

  • cellular metabolism is a lot more than just metabolizing glucose,”

said Robert Clarke, Ph.D., professor of oncology and physiology and biophysics at Georgetown University. Dr. Clarke, in collaboration with the Waters Center for Innovation at Georgetown University (led by Albert J. Fornace, Jr., M.D.), obtained

  • the metabolomic profile of hormone-sensitive and -resistant breast cancer cells through the use of UPLC-MS.

They demonstrated that breast cancer cells, through a rather complex and not yet completely understood process,

  1. can functionally coordinate cell-survival and cell-proliferation mechanisms,
  2. while maintaining a certain degree of cellular metabolism.

This is at least partly accomplished through the upregulation of important pro-survival mechanisms; including

  • the unfolded protein response;
  • a regulator of endoplasmic reticulum stress and
  • initiator of autophagy.

Normally, during a stressful situation, a cell may

  • enter a state of quiescence and undergo autophagy,
  • a process by which a cell can recycle organelles
  • in order to maintain enough energy to survive during a stressful situation or,

if the stress is too great,

  • undergo apoptosis.

By integrating cell-survival mechanisms and cellular metabolism

  • advanced ER+ hormone-resistant breast cancer cells
  • can maintain a low level of autophagy
  • to adapt and resist hormone/chemotherapy treatment.

This adaptation allows cells

  • to reallocate important metabolites recovered from organelle degradation and
  • provide enough energy to also promote proliferation.

With further research, we can gain a better understanding of the underlying causes of hormone-resistant breast cancer, with

  • the overall goal of developing effective diagnostic, prognostic, and therapeutic tools.

NMR

Over the last two decades, NMR has established itself as a major tool for metabolomics analysis. It is especially adept at testing biological fluids. [Bruker BioSpin]

Historically, nuclear magnetic resonance spectroscopy (NMR) has been used for structural elucidation of pure molecular compounds. However, in the last two decades, NMR has established itself as a major tool for metabolomics analysis. Since

  • the integral of an NMR signal is directly proportional to
  • the molar concentration throughout the dynamic range of a sample,

“the simultaneous quantification of compounds is possible

  • without the need for specific reference standards or calibration curves,” according to Lea Heintz of Bruker BioSpin.

NMR is adept at testing biological fluids because of

  1.  high reproducibility,
  2. standardized protocols,
  3. low sample manipulation, and
  4. the production of a large subset of data,

Bruker BioSpin is presently involved in a project for the screening of inborn errors of metabolism in newborn children from Turkey, based on their urine NMR profiles. More than 20 clinics are participating to the project that is coordinated by INFAI, a specialist in the transfer of advanced analytical technology into medical diagnostics. The construction of statistical models are being developed

  • for the detection of deviations from normality, as well as
  • automatic quantification methods for indicative metabolites

Bruker BioSpin recently installed high-resolution magic angle spinning NMR (HRMAS-NMR) systems that can rapidly analyze tissue biopsies. The main objective for HRMAS-NMR is to establish a rapid and effective clinical method to assess tumor grade and other important aspects of cancer during surgery.

Combined NMR and Mass Spec

There is increasing interest in combining NMR and MS, two of the main analytical assays in metabolomic research, as a means

  • to improve data sensitivity and to
  • fully elucidate the complex metabolome within a given biological sample.
  •  to realize a potential for cancer biomarker discovery in the realms of diagnosis, prognosis, and treatment.

.

Using combined NMR and MS to measure the levels of nearly 250 separate metabolites in the patient’s blood, Dr. Weljie and other researchers at the University of Calgary were able to rapidly determine the malignancy of a  pancreatic lesion (in 10–15% of the cases, it is difficult to discern between benign and malignant), while avoiding unnecessary surgery in patients with benign lesions.

When performing NMR and MS on a single biological fluid, ultimately “we are,” noted Dr. Weljie,

  1. “splitting up information content, processing, and introducing a lot of background noise and error and
  2. then trying to reintegrate the data…
    It’s like taking a complex item, with multiple pieces, out of an IKEA box and trying to repackage it perfectly into another box.”

By improving the workflow between the initial splitting of the sample, they improved endpoint data integration, proving that

  • a streamlined approach to combined NMR/MS can be achieved,
  • leading to a very strong, robust and precise metabolomics toolset.

Metabolomics Research Picks Up Speed

Field Advances in Quest to Improve Disease Diagnosis and Predict Drug Response

John Morrow Jr., Ph.D.
GEN May 1, 2011 (Vol. 31, No. 9)

As an important discipline within systems biology, metabolomics is being explored by a number of laboratories for

  • its potential in pharmaceutical development.

Studying metabolites can offer insights into the relationships between genotype and phenotype, as well as between genotype and environment. In addition, there is plenty to work with—there are estimated to be some 2,900 detectable metabolites in the human body, of which

  1. 309 have been identified in cerebrospinal fluid,
  2. 1,122 in serum,
  3. 458 in urine, and
  4. roughly 300 in other compartments.

Guowang Xu, Ph.D., a researcher at the Dalian Institute of Chemical Physics.  is investigating the causes of death in China,

  • and how they have been changing over the years as the country has become a more industrialized nation.
  •  the increase in the incidence of metabolic disorders such as diabetes has grown to affect 9.7% of the Chinese population.

Dr. Xu,  collaborating with Rainer Lehman, Ph.D., of the University of Tübingen, Germany, compared urinary metabolites in samples from healthy individuals with samples taken from prediabetic, insulin-resistant subjects. Using mass spectrometry coupled with electrospray ionization in the positive mode, they observed striking dissimilarities in levels of various metabolites in the two groups.

“When we performed a comprehensive two-dimensional gas chromatography, time-of-flight mass spectrometry analysis of our samples, we observed several metabolites, including

  • 2-hydroxybutyric acid in plasma,
  •  as potential diabetes biomarkers,” Dr. Xu explains.

In other, unrelated studies, Dr. Xu and the German researchers used a metabolomics approach to investigate the changes in plasma metabolite profiles immediately after exercise and following a 3-hour and 24-hour period of recovery. They found that

  • medium-chain acylcarnitines were the most distinctive exercise biomarkers, and
  • they are released as intermediates of partial beta oxidation in human myotubes and mouse muscle tissue.

Dr. Xu says. “The traditional approach of assessment based on a singular biomarker is being superseded by the introduction of multiple marker profiles.”

Typical of the studies under way by Dr. Kaddurah-Daouk and her colleaguesat Duke University

  • is a recently published investigation highlighting the role of an SNP variant in
  • the glycine dehydrogenase gene on individual response to antidepressants.
  •  patients who do not respond to the selective serotonin uptake inhibitors citalopram and escitalopram
  • carried a particular single nucleotide polymorphism in the GD gene.

“These results allow us to pinpoint a possible

  • role for glycine in selective serotonin reuptake inhibitor response and
  • illustrate the use of pharmacometabolomics to inform pharmacogenomics.

These discoveries give us the tools for prognostics and diagnostics so that

  • we can predict what conditions will respond to treatment.

“This approach to defining health or disease in terms of metabolic states opens a whole new paradigm.

By screening hundreds of thousands of molecules, we can understand

  • the relationship between human genetic variability and the metabolome.”

Dr. Kaddurah-Daouk talks about statins as a current

  • model of metabolomics investigations.

It is now known that the statins  have widespread effects, altering a range of metabolites. To sort out these changes and develop recommendations for which individuals should be receiving statins will require substantial investments of energy and resources into defining the complex web of biochemical changes that these drugs initiate.
Furthermore, Dr. Kaddurah-Daouk asserts that,

  • “genetics only encodes part of the phenotypic response.

One needs to take into account the

  • net environment contribution in order to determine
  • how both factors guide the changes in our metabolic state that determine the phenotype.”

Interactive Metabolomics

Researchers at the University of Nottingham use diffusion-edited nuclear magnetic resonance spectroscopy to assess the effects of a biological matrix on metabolites. Diffusion-edited NMR experiments provide a way to

  • separate the different compounds in a mixture
  • based on the differing translational diffusion coefficients (which reflect the size and shape of the molecule).

The measurements are carried out by observing

  • the attenuation of the NMR signals during a pulsed field gradient experiment.

Clare Daykin, Ph.D., is a lecturer at the University of Nottingham, U.K. Her field of investigation encompasses “interactive metabolomics,”which she defines as

“the study of the interactions between low molecular weight biochemicals and macromolecules in biological samples ..

  • without preselection of the components of interest.

“Blood plasma is a heterogeneous mixture of molecules that

  1. undergo a variety of interactions including metal complexation,
  2. chemical exchange processes,
  3. micellar compartmentation,
  4. enzyme-mediated biotransformations, and
  5. small molecule–macromolecular binding.”

Many low molecular weight compounds can exist

  • freely in solution,
  • bound to proteins, or
  • within organized aggregates such as lipoprotein complexes.

Therefore, quantitative comparison of plasma composition from

  • diseased individuals compared to matched controls provides an incomplete insight to plasma metabolism.

“It is not simply the concentrations of metabolites that must be investigated,

  • but their interactions with the proteins and lipoproteins within this complex web.

Rather than targeting specific metabolites of interest, Dr. Daykin’s metabolite–protein binding studies aim to study

  • the interactions of all detectable metabolites within the macromolecular sample.

Such activities can be studied through the use of diffusion-edited nuclear magnetic resonance (NMR) spectroscopy, in which one can assess

  • the effects of the biological matrix on the metabolites.

“This can lead to a more relevant and exact interpretation

  • for systems where metabolite–macromolecule interactions occur.”

Diffusion-edited NMR experiments provide a way to separate the different compounds in a mixture based on

  • the differing translational diffusion coefficients (which reflect the size and shape of the molecule).

The measurements are carried out by observing

  • the attenuation of the NMR signals during a pulsed field gradient experiment.

Pushing the Limits

It is widely recognized that many drug candidates fail during development due to ancillary toxicity. Uwe Sauer, Ph.D., professor, and Nicola Zamboni, Ph.D., researcher, both at the Eidgenössische Technische Hochschule, Zürich (ETH Zürich), are applying

  • high-throughput intracellular metabolomics to understand
  • the basis of these unfortunate events and
  • head them off early in the course of drug discovery.

“Since metabolism is at the core of drug toxicity, we developed a platform for

  • measurement of 50–100 targeted metabolites by
  • a high-throughput system consisting of flow injection
  • coupled to tandem mass spectrometry.”

Using this approach, Dr. Sauer’s team focused on

  • the central metabolism of the yeast Saccharomyces cerevisiae, reasoning that
  • this core network would be most susceptible to potential drug toxicity.

Screening approximately 41 drugs that were administered at seven concentrations over three orders of magnitude, they observed changes in metabolome patterns at much lower drug concentrations without attendant physiological toxicity.

The group carried out statistical modeling of about

  • 60 metabolite profiles for each drug they evaluated.

This data allowed the construction of a “profile effect map” in which

  • the influence of each drug on metabolite levels can be followed, including off-target effects, which
  • provide an indirect measure of the possible side effects of the various drugs.

Dr. Sauer says.“We have found that this approach is

  • at least 100 times as fast as other omics screening platforms,”

“Some drugs, including many anticancer agents,

  • disrupt metabolism long before affecting growth.”
killing cancer cells

killing cancer cells

Furthermore, they used the principle of 13C-based flux analysis, in which

  • metabolites labeled with 13C are used to follow the utilization of metabolic pathways in the cell.

These 13C-determined intracellular responses of metabolic fluxes to drug treatment demonstrate

  • the functional performance of the network to be rather robust,
conformational changes leading to substrate efflux.

conformational changes leading to substrate efflux.

leading Dr. Sauer to the conclusion that

  • the phenotypic vigor he observes to drug challenges
  • is achieved by a flexible make up of the metabolome.

Dr. Sauer is confident that it will be possible to expand the scope of these investigations to hundreds of thousands of samples per study. This will allow answers to the questions of

  • how cells establish a stable functioning network in the face of inevitable concentration fluctuations.

Is Now the Hour?

There is great enthusiasm and agitation within the biotech community for

  • metabolomics approaches as a means of reversing the dismal record of drug discovery

that has accumulated in the last decade.

While the concept clearly makes sense and is being widely applied today, there are many reasons why drugs fail in development, and metabolomics will not be a panacea for resolving all of these questions. It is too early at this point to recognize a trend or a track record, and it will take some time to see how this approach can aid in drug discovery and shorten the timeline for the introduction of new pharmaceutical agents.

Degree of binding correlated with function

Degree of binding correlated with function

Diagram_of_a_two-photon_excitation_microscope_

Diagram_of_a_two-photon_excitation_microscope_

Part 2.  Biologists Find ‘Missing Link’ in the Production of Protein Factories in Cells

Biologists at UC San Diego have found

  • the “missing link” in the chemical system that
  • enables animal cells to produce ribosomes

—the thousands of protein “factories” contained within each cell that

  • manufacture all of the proteins needed to build tissue and sustain life.
‘Missing Link’

‘Missing Link’

Their discovery, detailed in the June 23 issue of the journal Genes & Development, will not only force

  • a revision of basic textbooks on molecular biology, but also
  • provide scientists with a better understanding of
  • how to limit uncontrolled cell growth, such as cancer,
  • that might be regulated by controlling the output of ribosomes.

Ribosomes are responsible for the production of the wide variety of proteins that include

  1. enzymes;
  2. structural molecules, such as hair,
  3. skin and bones;
  4. hormones like insulin; and
  5. components of our immune system such as antibodies.

Regarded as life’s most important molecular machine, ribosomes have been intensively studied by scientists (the 2009 Nobel Prize in Chemistry, for example, was awarded for studies of its structure and function). But until now researchers had not uncovered all of the details of how the proteins that are used to construct ribosomes are themselves produced.

In multicellular animals such as humans,

  • ribosomes are made up of about 80 different proteins
    (humans have 79 while some other animals have a slightly different number) as well as
  • four different kinds of RNA molecules.

In 1969, scientists discovered that

  • the synthesis of the ribosomal RNAs is carried out by specialized systems using two key enzymes:
  • RNA polymerase I and RNA polymerase III.

But until now, scientists were unsure if a complementary system was also responsible for

  • the production of the 80 proteins that make up the ribosome.

That’s essentially what the UC San Diego researchers headed by Jim Kadonaga, a professor of biology, set out to examine. What they found was the missing link—the specialized

  • system that allows ribosomal proteins themselves to be synthesized by the cell.

Kadonaga says that he and coworkers found that ribosomal proteins are synthesized via

  • a novel regulatory system with the enzyme RNA polymerase II and
  • a factor termed TRF2,”

“For the production of most proteins,

  1. RNA polymerase II functions with
  2. a factor termed TBP,
  3. but for the synthesis of ribosomal proteins, it uses TRF2.”
  •  this specialized TRF2-based system for ribosome biogenesis
  • provides a new avenue for the study of ribosomes and
  • its control of cell growth, and

“it should lead to a better understanding and potential treatment of diseases such as cancer.”

Coordination of the transcriptome and metabolome

Coordination of the transcriptome and metabolome

the potential advantages conferred by distal-site protein synthesis

the potential advantages conferred by distal-site protein synthesis

Other authors of the paper were UC San Diego biologists Yuan-Liang Wang, Sascha Duttke and George Kassavetis, and Kai Chen, Jeff Johnston, and Julia Zeitlinger of the Stowers Institute for Medical Research in Kansas City, Missouri. Their research was supported by two grants from the National Institutes of Health (1DP2OD004561-01 and R01 GM041249).

Turning Off a Powerful Cancer Protein

Scientists have discovered how to shut down a master regulatory transcription factor that is

  • key to the survival of a majority of aggressive lymphomas,
  • which arise from the B cells of the immune system.

The protein, Bcl6, has long been considered too complex to target with a drug since it is also crucial

  • to the healthy functioning of many immune cells in the body, not just B cells gone bad.

The researchers at Weill Cornell Medical College report that it is possible

  • to shut down Bcl6 in diffuse large B-cell lymphoma (DLBCL)
  • while not affecting its vital function in T cells and macrophages
  • that are needed to support a healthy immune system.

If Bcl6 is completely inhibited, patients might suffer from systemic inflammation and atherosclerosis. The team conducted this new study to help clarify possible risks, as well as to understand

  • how Bcl6 controls the various aspects of the immune system.

The findings in this study were inspired from

  • preclinical testing of two Bcl6-targeting agents that Dr. Melnick and his Weill Cornell colleagues have developed
  • to treat DLBCLs.

These experimental drugs are

  • RI-BPI, a peptide mimic, and
  • the small molecule agent 79-6.

“This means the drugs we have developed against Bcl6 are more likely to be

  • significantly less toxic and safer for patients with this cancer than we realized,”

says Ari Melnick, M.D., professor of hematology/oncology and a hematologist-oncologist at NewYork-Presbyterian Hospital/Weill Cornell Medical Center.

Dr. Melnick says the discovery that

  • a master regulatory transcription factor can be targeted
  • offers implications beyond just treating DLBCL.

Recent studies from Dr. Melnick and others have revealed that

  • Bcl6 plays a key role in the most aggressive forms of acute leukemia, as well as certain solid tumors.

Bcl6 can control the type of immune cell that develops in the bone marrow—playing many roles

  • in the development of B cells, T cells, macrophages, and other cells—including a primary and essential role in
  • enabling B-cells to generate specific antibodies against pathogens.

According to Dr. Melnick, “When cells lose control of Bcl6,

  • lymphomas develop in the immune system.

Lymphomas are ‘addicted’ to Bcl6, and therefore

  • Bcl6 inhibitors powerfully and quickly destroy lymphoma cells,” .

The big surprise in the current study is that rather than functioning as a single molecular machine,

  • Bcl6 functions like a Swiss Army knife,
  • using different tools to control different cell types.

This multifunction paradigm could represent a general model for the functioning of other master regulatory transcription factors.

“In this analogy, the Swiss Army knife, or transcription factor, keeps most of its tools folded,

  • opening only the one it needs in any given cell type,”

He makes the following analogy:

  • “For B cells, it might open and use the knife tool;
  • for T cells, the cork screw;
  • for macrophages, the scissors.”

“this means that you only need to prevent the master regulator from using certain tools to treat cancer. You don’t need to eliminate the whole knife,” . “In fact, we show that taking out the whole knife is harmful since

  • the transcription factor has many other vital functions that other cells in the body need.”

Prior to these study results, it was not known that a master regulator could separate its functions so precisely. Researchers hope this will be a major benefit to the treatment of DLBCL and perhaps other disorders that are influenced by Bcl6 and other master regulatory transcription factors.

The study is published in the journal Nature Immunology, in a paper titled “Lineage-specific functions of Bcl-6 in immunity and inflammation are mediated by distinct biochemical mechanisms”.

Part 3. Neuroscience

Vesicles influence function of nerve cells 
Oct, 06 2014        source: http://feeds.sciencedaily.com

Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.

Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.

Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.

Tiny vesicles containing protective substances

  • which they transmit to nerve cells apparently
  • play an important role in the functioning of neurons.

As cell biologists at Johannes Gutenberg University Mainz (JGU) have discovered,

  • nerve cells can enlist the aid of mini-vesicles of neighboring glial cells
  • to defend themselves against stress and other potentially detrimental factors.

These vesicles, called exosomes, appear to stimulate the neurons on various levels:

  • they influence electrical stimulus conduction,
  • biochemical signal transfer, and
  • gene regulation.

Exosomes are thus multifunctional signal emitters

  • that can have a significant effect in the brain.
Exosome

Exosome

The researchers in Mainz already observed in a previous study that

  • oligodendrocytes release exosomes on exposure to neuronal stimuli.
  • these are absorbed by the neurons and improve neuronal stress tolerance.

Oligodendrocytes, a type of glial cell, form an

  • insulating myelin sheath around the axons of neurons.

The exosomes transport protective proteins such as

  • heat shock proteins,
  • glycolytic enzymes, and
  • enzymes that reduce oxidative stress from one cell type to another,
  • but also transmit genetic information in the form of ribonucleic acids.

“As we have now discovered in cell cultures, exosomes seem to have a whole range of functions,” explained Dr. Eva-Maria Krmer-Albers. By means of their transmission activity, the small bubbles that are the vesicles

  • not only promote electrical activity in the nerve cells, but also
  • influence them on the biochemical and gene regulatory level.

“The extent of activities of the exosomes is impressive,” added Krmer-Albers. The researchers hope that the understanding of these processes will contribute to the development of new strategies for the treatment of neuronal diseases. Their next aim is to uncover how vesicles actually function in the brains of living organisms.

http://labroots.com/user/news/article/id/217438/title/vesicles-influence-function-of-nerve-cells

The above story is based on materials provided by Universitt Mainz.

Universitt Mainz. “Vesicles influence function of nerve cells.” ScienceDaily. ScienceDaily, 6 October 2014. www.sciencedaily.com/releases/2014/10/141006174214.htm

Neuroscientists use snail research to help explain “chemo brain”

10/08/2014
It is estimated that as many as half of patients taking cancer drugs experience a decrease in mental sharpness. While there have been many theories, what causes “chemo brain” has eluded scientists.

In an effort to solve this mystery, neuroscientists at The University of Texas Health Science Center at Houston (UTHealth) conducted an experiment in an animal memory model and their results point to a possible explanation. Findings appeared in The Journal of Neuroscience.

In the study involving a sea snail that shares many of the same memory mechanisms as humans and a drug used to treat a variety of cancers, the scientists identified

  • memory mechanisms blocked by the drug.

Then, they were able to counteract or

  • unblock the mechanisms by administering another agent.

“Our research has implications in the care of people given to cognitive deficits following drug treatment for cancer,” said John H. “Jack” Byrne, Ph.D., senior author, holder of the June and Virgil Waggoner Chair and Chairman of the Department of Neurobiology and Anatomy at the UTHealth Medical School. “There is no satisfactory treatment at this time.”

Byrne’s laboratory is known for its use of a large snail called Aplysia californica to further the understanding of the biochemical signaling among nerve cells (neurons).  The snails have large neurons that relay information much like those in humans.

When Byrne’s team compared cell cultures taken from normal snails to

  • those administered a dose of a cancer drug called doxorubicin,

the investigators pinpointed a neuronal pathway

  • that was no longer passing along information properly.

With the aid of an experimental drug,

  • the scientists were able to reopen the pathway.

Unfortunately, this drug would not be appropriate for humans, Byrne said. “We want to identify other drugs that can rescue these memory mechanisms,” he added.

According the American Cancer Society, some of the distressing mental changes cancer patients experience may last a short time or go on for years.

Byrne’s UT Health research team includes co-lead authors Rong-Yu Liu, Ph.D., and Yili Zhang, Ph.D., as well as Brittany Coughlin and Leonard J. Cleary, Ph.D. All are affiliated with the W.M. Keck Center for the Neurobiology of Learning and Memory.

Byrne and Cleary also are on the faculty of The University of Texas Graduate School of Biomedical Sciences at Houston. Coughlin is a student at the school, which is jointly operated by UT Health and The University of Texas MD Anderson Cancer Center.

The study titled “Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase” received support from National Institutes of Health grant (NS019895) and the Zilkha Family Discovery Fellowship.

Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase

Source: Univ. of Texas Health Science Center at Houston

http://www.rdmag.com/news/2014/10/neuroscientists-use-snail-research-help-explain-E2_9_Cchemo-brain

Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase

Rong-Yu Liu*,  Yili Zhang*,  Brittany L. Coughlin,  Leonard J. Cleary, and  John H. Byrne   +Show Affiliations
The Journal of Neuroscience, 1 Oct 2014, 34(40): 13289-13300;
http://dx.doi.org:/10.1523/JNEUROSCI.0538-14.2014

Doxorubicin (DOX) is an anthracycline used widely for cancer chemotherapy. Its primary mode of action appears to be

  • topoisomerase II inhibition, DNA cleavage, and free radical generation.

However, in non-neuronal cells, DOX also inhibits the expression of

  • dual-specificity phosphatases (also referred to as MAPK phosphatases) and thereby
  1. inhibits the dephosphorylation of extracellular signal-regulated kinase (ERK) and
  2. p38 mitogen-activated protein kinase (p38 MAPK),
  3. two MAPK isoforms important for long-term memory (LTM) formation.

Activation of these kinases by DOX in neurons, if present,

  • could have secondary effects on cognitive functions, such as learning and memory.

The present study used cultures of rat cortical neurons and sensory neurons (SNs) of Aplysia

  • to examine the effects of DOX on levels of phosphorylated ERK (pERK) and
  • phosphorylated p38 (p-p38) MAPK.

In addition, Aplysia neurons were used to examine the effects of DOX on

  • long-term enhanced excitability, long-term synaptic facilitation (LTF), and
  • long-term synaptic depression (LTD).

DOX treatment led to elevated levels of

  • pERK and p-p38 MAPK in SNs and cortical neurons.

In addition, it increased phosphorylation of

  • the downstream transcriptional repressor cAMP response element-binding protein 2 in SNs.

DOX treatment blocked serotonin-induced LTF and enhanced LTD induced by the neuropeptide Phe-Met-Arg-Phe-NH2. The block of LTF appeared to be attributable to

  • overriding inhibitory effects of p-p38 MAPK, because
  • LTF was rescued in the presence of an inhibitor of p38 MAPK
    (SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]) .

These results suggest that acute application of DOX might impair the formation of LTM via the p38 MAPK pathway.
Terms: Aplysia chemotherapy ERK  p38 MAPK serotonin synaptic plasticity

Technology that controls brain cells with radio waves earns early BRAIN grant

10/08/2014

bright spots = cells with increased calcium after treatment with radio waves,  allows neurons to fire

bright spots = cells with increased calcium after treatment with radio waves, allows neurons to fire

BRAIN control: The new technology uses radio waves to activate or silence cells remotely. The bright spots above represent cells with increased calcium after treatment with radio waves, a change that would allow neurons to fire.

A proposal to develop a new way to

  • remotely control brain cells

from Sarah Stanley, a research associate in Rockefeller University’s Laboratory of Molecular Genetics, headed by Jeffrey M. Friedman, is

  • among the first to receive funding from U.S. President Barack Obama’s BRAIN initiative.

The project will make use of a technique called

  • radiogenetics that combines the use of radio waves or magnetic fields with
  • nanoparticles to turn neurons on or off.

The National Institutes of Health is one of four federal agencies involved in the BRAIN (Brain Research through Advancing Innovative Neurotechnologies) initiative. Following in the ambitious footsteps of the Human Genome Project, the BRAIN initiative seeks

  • to create a dynamic map of the brain in action,

a goal that requires the development of new technologies. The BRAIN initiative working group, which outlined the broad scope of the ambitious project, was co-chaired by Rockefeller’s Cori Bargmann, head of the Laboratory of Neural Circuits and Behavior.

Stanley’s grant, for $1.26 million over three years, is one of 58 projects to get BRAIN grants, the NIH announced. The NIH’s plan for its part of this national project, which has been pitched as “America’s next moonshot,” calls for $4.5 billion in federal funds over 12 years.

The technology Stanley is developing would

  • enable researchers to manipulate the activity of neurons, as well as other cell types,
  • in freely moving animals in order to better understand what these cells do.

Other techniques for controlling selected groups of neurons exist, but her new nanoparticle-based technique has a

  • unique combination of features that may enable new types of experimentation.
  • it would allow researchers to rapidly activate or silence neurons within a small area of the brain or
  • dispersed across a larger region, including those in difficult-to-access locations.

Stanley also plans to explore the potential this method has for use treating patients.

“Francis Collins, director of the NIH, has discussed

  • the need for studying the circuitry of the brain,
  • which is formed by interconnected neurons.

Our remote-control technology may provide a tool with which researchers can ask new questions about the roles of complex circuits in regulating behavior,” Stanley says.
Rockefeller University’s Laboratory of Molecular Genetics
Source: Rockefeller Univ.

Part 4.  Cancer

Two Proteins Found to Block Cancer Metastasis

Why do some cancers spread while others don’t? Scientists have now demonstrated that

  • metastatic incompetent cancers actually “poison the soil”
  • by generating a micro-environment that blocks cancer cells
  • from settling and growing in distant organs.

The “seed and the soil” hypothesis proposed by Stephen Paget in 1889 is now widely accepted to explain how

  • cancer cells (seeds) are able to generate fertile soil (the micro-environment)
  • in distant organs that promotes cancer’s spread.

However, this concept had not explained why some tumors do not spread or metastasize.

The researchers, from Weill Cornell Medical College, found that

  • two key proteins involved in this process work by
  • dramatically suppressing cancer’s spread.

The study offers hope that a drug based on these

  • potentially therapeutic proteins, prosaposin and Thrombospondin 1 (Tsp-1),

might help keep human cancer at bay and from metastasizing.

Scientists don’t understand why some tumors wouldn’t “want” to spread. It goes against their “job description,” says the study’s senior investigator, Vivek Mittal, Ph.D., an associate professor of cell and developmental biology in cardiothoracic surgery and director of the Neuberger Berman Foundation Lung Cancer Laboratory at Weill Cornell Medical College. He theorizes that metastasis occurs when

  • the barriers that the body throws up to protect itself against cancer fail.

But there are some tumors in which some of the barriers may still be intact. “So that suggests

  • those primary tumors will continue to grow, but that
  • an innate protective barrier still exists that prevents them from spreading and invading other organs,”

The researchers found that, like typical tumors,

  • metastasis-incompetent tumors also send out signaling molecules
  • that establish what is known as the “premetastatic niche” in distant organs.

These niches composed of bone marrow cells and various growth factors have been described previously by others including Dr. Mittal as the fertile “soil” that the disseminated cancer cell “seeds” grow in.

Weill Cornell’s Raúl Catena, Ph.D., a postdoctoral fellow in Dr. Mittal’s laboratory, found an important difference between the tumor types. Metastatic-incompetent tumors

  • systemically increased expression of Tsp-1, a molecule known to fight cancer growth.
  • increased Tsp-1 production was found specifically in the bone marrow myeloid cells
  • that comprise the metastatic niche.

These results were striking, because for the first time Dr. Mittal says

  • the bone marrow-derived myeloid cells were implicated as
  • the main producers of Tsp-1,.

In addition, Weill Cornell and Harvard researchers found that

  • prosaposin secreted predominantly by the metastatic-incompetent tumors
  • increased expression of Tsp-1 in the premetastatic lungs.

Thus, Dr. Mittal posits that prosaposin works in combination with Tsp-1

  • to convert pro-metastatic bone marrow myeloid cells in the niche
  • into cells that are not hospitable to cancer cells that spread from a primary tumor.
  • “The very same myeloid cells in the niche that we know can promote metastasis
  • can also be induced under the command of the metastatic incompetent primary tumor to inhibit metastasis,”

The research team found that

  • the Tsp-1–inducing activity of prosaposin
  • was contained in only a 5-amino acid peptide region of the protein, and
  • this peptide alone induced Tsp-1 in the bone marrow cells and
  • effectively suppressed metastatic spread in the lungs
  • in mouse models of breast and prostate cancer.

This 5-amino acid peptide with Tsp-1–inducing activity

  • has the potential to be used as a therapeutic agent against metastatic cancer,

The scientists have begun to test prosaposin in other tumor types or metastatic sites.

Dr. Mittal says that “The clinical implications of the study are:

  • “Not only is it theoretically possible to design a prosaposin-based drug or drugs
  • that induce Tsp-1 to block cancer spread, but
  • you could potentially create noninvasive prognostic tests
  • to predict whether a cancer will metastasize.”

The study was reported in the April 30 issue of Cancer Discovery, in a paper titled “Bone Marrow-Derived Gr1+ Cells Can Generate a Metastasis-Resistant Microenvironment Via Induced Secretion of Thrombospondin-1”.

Disabling Enzyme Cripples Tumors, Cancer Cells

First Step of Metastasis

First Step of Metastasis

Published: Sep 05, 2013  http://www.technologynetworks.com/Metabolomics/news.aspx?id=157138

Knocking out a single enzyme dramatically cripples the ability of aggressive cancer cells to spread and grow tumors.

The paper, published in the journal Proceedings of the National Academy of Sciences, sheds new light on the importance of lipids, a group of molecules that includes fatty acids and cholesterol, in the development of cancer.

Researchers have long known that cancer cells metabolize lipids differently than normal cells. Levels of ether lipids – a class of lipids that are harder to break down – are particularly elevated in highly malignant tumors.

“Cancer cells make and use a lot of fat and lipids, and that makes sense because cancer cells divide and proliferate at an accelerated rate, and to do that,

  • they need lipids, which make up the membranes of the cell,”

said study principal investigator Daniel Nomura, assistant professor in UC Berkeley’s Department of Nutritional Sciences and Toxicology. “Lipids have a variety of uses for cellular structure, but what we’re showing with our study is that

  • lipids can send signals that fuel cancer growth.”

In the study, Nomura and his team tested the effects of reducing ether lipids on human skin cancer cells and primary breast tumors. They targeted an enzyme,

  • alkylglycerone phosphate synthase, or AGPS,
  • known to be critical to the formation of ether lipids.

The researchers confirmed that

  1. AGPS expression increased when normal cells turned cancerous.
  2. inactivating AGPS substantially reduced the aggressiveness of the cancer cells.

“The cancer cells were less able to move and invade,” said Nomura.

The researchers also compared the impact of

  • disabling the AGPS enzyme in mice that had been injected with cancer cells.

Nomura. observes -“Among the mice that had the AGPS enzyme inactivated,

  • the tumors were nonexistent,”

“The mice that did not have this enzyme

  • disabled rapidly developed tumors.”

The researchers determined that

  • inhibiting AGPS expression depleted the cancer cells of ether lipids.
  • AGPS altered levels of other types of lipids important to the ability of the cancer cells to survive and spread, including
    • prostaglandins and acyl phospholipids.

“What makes AGPS stand out as a treatment target is that the enzyme seems to simultaneously

  • regulate multiple aspects of lipid metabolism
  • important for tumor growth and malignancy.”

Future steps include the

  • development of AGPS inhibitors for use in cancer therapy,

“This study sheds considerable light on the important role that AGPS plays in ether lipid metabolism in cancer cells, and it suggests that

  • inhibitors of this enzyme could impair tumor formation,”

said Benjamin Cravatt, Professor and Chair of Chemical Physiology at The Scripps Research Institute, who is not part of the UC.

Agilent Technologies Thought Leader Award Supports Translational Research Program
Published: Mon, March 04, 2013

The award will support Dr DePinho’s research into

  • metabolic reprogramming in the earliest stages of cancer.

Agilent Technologies Inc. announces that Dr. Ronald A. DePinho, a world-renowned oncologist and researcher, has received an Agilent Thought Leader Award.

DePinho is president of the University of Texas MD Anderson Cancer Center. DePinho and his team hope to discover and characterize

  • alterations in metabolic flux during tumor initiation and maintenance, and to identify biomarkers for early detection of pancreatic cancer together with
  • novel therapeutic targets.

Researchers on his team will work with scientists from the university’s newly formed Institute of Applied Cancer Sciences.

The Agilent Thought Leader Award provides funds to support personnel as well as a state-of-the-art Agilent 6550 iFunnel Q-TOF LC/MS system.

“I am extremely pleased to receive this award for metabolomics research, as the survival rates for pancreatic cancer have not significantly improved over the past 20 years,” DePinho said. “This technology will allow us to

  • rapidly identify new targets that drive the formation, progression and maintenance of pancreatic cancer.

Discoveries from this research will also lead to

  • the development of effective early detection biomarkers and novel therapeutic interventions.”

“We are proud to support Dr. DePinho’s exciting translational research program, which will make use of

  • metabolomics and integrated biology workflows and solutions in biomarker discovery,”

said Patrick Kaltenbach, Agilent vice president, general manager of the Liquid Phase Division, and the executive sponsor of this award.

The Agilent Thought Leader Program promotes fundamental scientific advances by support of influential thought leaders in the life sciences and chemical analysis fields.

The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis

Ping Xie, Minghua Zhang, Shan He, Kefeng Lu, Yuhan Chen, Guichun Xing, et al.
Nature Communications
  2014; 5(3733).  http://dx.doi.org:/10.1038/ncomms4733

Neddylation, the covalent attachment of ubiquitin-like protein Nedd8, of the Cullin-RING E3 ligase family

  • regulates their ubiquitylation activity.

However, regulation of HECT ligases by neddylation has not been reported to date. Here we show that

  • the C2-WW-HECT ligase Smurf1 is activated by neddylation.

Smurf1 physically interacts with

  1. Nedd8 and Ubc12,
  2. forms a Nedd8-thioester intermediate, and then
  3. catalyses its own neddylation on multiple lysine residues.

Intriguingly, this autoneddylation needs

  • an active site at C426 in the HECT N-lobe.

Neddylation of Smurf1 potently enhances

  • ubiquitin E2 recruitment and
  • augments the ubiquitin ligase activity of Smurf1.

The regulatory role of neddylation

  • is conserved in human Smurf1 and yeast Rsp5.

Furthermore, in human colorectal cancers,

  • the elevated expression of Smurf1, Nedd8, NAE1 and Ubc12
  • correlates with cancer progression and poor prognosis.

These findings provide evidence that

  • neddylation is important in HECT ubiquitin ligase activation and
  • shed new light on the tumour-promoting role of Smurf1.
 Swinging domains in HECT E3

Swinging domains in HECT E3

Subject terms: Biological sciences Cancer Cell biology

Figure 1: Smurf1 expression is elevated in colorectal cancer tissues.

Smurf1 expression is elevated in colorectal cancer tissues.

Smurf1 expression is elevated in colorectal cancer tissues.

(a) Smurf1 expression scores are shown as box plots, with the horizontal lines representing the median; the bottom and top of the boxes representing the 25th and 75th percentiles, respectively; and the vertical bars representing the ra

Figure 2: Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer.

Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer

Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer

(a) Representative images from immunohistochemical staining of Smurf1, Ubc12, NAE1 and Nedd8 in the same colorectal cancer tumour. Scale bars, 100 μm. (bd) The expression scores of Nedd8 (b, n=283 ), NAE1 (c, n=281) and Ubc12 (d, n=19…

Figure 3: Smurf1 interacts with Ubc12.

Smurf1 interacts with Ubc12

Smurf1 interacts with Ubc12

(a) GST pull-down assay of Smurf1 with Ubc12. Both input and pull-down samples were subjected to immunoblotting with anti-His and anti-GST antibodies. Smurf1 interacted with Ubc12 and UbcH5c, but not with Ubc9. (b) Mapping the regions…

Figure 4: Nedd8 is attached to Smurf1through C426-catalysed autoneddylation.

Nedd8 is attached to Smurf1through C426-catalysed autoneddylation

Nedd8 is attached to Smurf1through C426-catalysed autoneddylation

(a) Covalent neddylation of Smurf1 in vitro.Purified His-Smurf1-WT or C699A proteins were incubated with Nedd8 and Nedd8-E1/E2. Reactions were performed as described in the Methods section. Samples were analysed by western blotting wi…

Figure 5: Neddylation of Smurf1 activates its ubiquitin ligase activity.

Neddylation of Smurf1 activates its ubiquitin ligase activity.

Neddylation of Smurf1 activates its ubiquitin ligase activity.

(a) In vivo Smurf1 ubiquitylation assay. Nedd8 was co-expressed with Smurf1 WT or C699A in HCT116 cells (left panels). Twenty-four hours post transfection, cells were treated with MG132 (20 μM, 8 h). HCT116 cells were transfected with…

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The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response

M Simicek, S Lievens, M Laga, D Guzenko, VN. Aushev, et al.
Nature Cell Biology 2013; 15, 1220–1230    http://dx.doi.org:/10.1038/ncb2847

The RAS-like GTPase RALB mediates cellular responses to nutrient availability or viral infection by respectively

  • engaging two components of the exocyst complex, EXO84 and SEC5.
  1. RALB employs SEC5 to trigger innate immunity signalling, whereas
  2. RALB–EXO84 interaction induces autophagocytosis.

How this differential interaction is achieved molecularly by the RAL GTPase remains unknown.

We found that whereas GTP binding

  • turns on RALB activity,

ubiquitylation of RALB at Lys 47

  • tunes its activity towards a particular effector.

Specifically, ubiquitylation at Lys 47

  • sterically inhibits RALB binding to EXO84, while
  • facilitating its interaction with SEC5.

Double-stranded RNA promotes

  • RALB ubiquitylation and
  • SEC5–TBK1 complex formation.

In contrast, nutrient starvation

  • induces RALB deubiquitylation
  • by accumulation and relocalization of the deubiquitylase USP33
  • to RALB-positive vesicles.

Deubiquitylated RALB

  • promotes the assembly of the RALB–EXO84–beclin-1 complexes
  • driving autophagosome formation. Thus,
  • ubiquitylation within the effector-binding domain
  • provides the switch for the dual functions of RALB in
    • autophagy and innate immune responses.

Part 5. Metabolic Syndrome

Single Enzyme is Necessary for Development of Diabetes

Published: Aug 20, 2014 http://www.technologynetworks.com/Metabolomics/news.aspx?ID=169416

12-LO enzyme promotes the obesity-induced oxidative stress in the pancreatic cells.

An enzyme called 12-LO promotes the obesity-induced oxidative stress in the pancreatic cells that leads

  • to pre-diabetes, and diabetes.

12-LO’s enzymatic action is the last step in

  • the production of certain small molecules that harm the cell,

according to a team from Indiana University School of Medicine, Indianapolis.

The findings will enable the development of drugs that can interfere with this enzyme, preventing or even reversing diabetes. The research is published ahead of print in the journal Molecular and Cellular Biology.

In earlier studies, these researchers and their collaborators at Eastern Virginia Medical School showed that

  • 12-LO (which stands for 12-lipoxygenase) is present in these cells
  • only in people who become overweight.

The harmful small molecules resulting from 12-LO’s enzymatic action are known as HETEs, short for hydroxyeicosatetraenoic acid.

  1. HETEs harm the mitochondria, which then
  2. fail to produce sufficient energy to enable
  3. the pancreatic cells to manufacture the necessary quantities of insulin.

For the study, the investigators genetically engineered mice that

  • lacked the gene for 12-LO exclusively in their pancreas cells.

Mice were either fed a low-fat or high-fat diet.

Both the control mice and the knockout mice on the high fat diet

  • developed obesity and insulin resistance.

The investigators also examined the pancreatic beta cells of both knockout and control mice, using both microscopic studies and molecular analysis. Those from the knockout mice were intact and healthy, while

  • those from the control mice showed oxidative damage,
  • demonstrating that 12-LO and the resulting HETEs
  • caused the beta cell failure.

Mirmira notes that fatty diet used in the study was the Western Diet, which comprises mostly saturated-“bad”-fats. Based partly on a recent study of related metabolic pathways, he says that

  • the unsaturated and mono-unsaturated fats-which comprise most fats in the healthy,
  • relatively high fat Mediterranean diet-are unlikely to have the same effects.

“Our research is the first to show that 12-LO in the beta cell

  • is the culprit in the development of pre-diabetes, following high fat diets,” says Mirmira.

“Our work also lends important credence to the notion that

  • the beta cell is the primary defective cell in virtually all forms of diabetes and pre-diabetes.”

A New Player in Lipid Metabolism Discovered

Published: Aug18, 2014  http://www.technologynetworks.com/Metabolomics/news.aspx?ID=169356

Specially engineered mice gained no weight, and normal counterparts became obese

  • on the same high-fat, obesity-inducing Western diet.

Specially engineered mice that lacked a particular gene did not gain weight

  • when fed a typical high-fat, obesity-inducing Western diet.

Yet, these mice ate the same amount as their normal counterparts that became obese.

The mice were engineered with fat cells that lacked a gene called SEL1L,

  • known to be involved in the clearance of mis-folded proteins
  • in the cell’s protein making machinery called the endoplasmic reticulum (ER).

When mis-folded proteins are not cleared but accumulate,

  • they destroy the cell and contribute to such diseases as
  1. mad cow disease,
  2. Type 1 diabetes and
  3. cystic fibrosis.

“The million-dollar question is why don’t these mice gain weight? Is this related to its inability to clear mis-folded proteins in the ER?” said Ling Qi, associate professor of molecular and biochemical nutrition and senior author of the study published online July 24 in Cell Metabolism. Haibo Sha, a research associate in Qi’s lab, is the paper’s lead author.

Interestingly, the experimental mice developed a host of other problems, including

  • postprandial hypertriglyceridemia,
  • and fatty livers.

“Although we are yet to find out whether these conditions contribute to the lean phenotype, we found that

  • there was a lipid partitioning defect in the mice lacking SEL1L in fat cells,
  • where fat cells cannot store fat [lipids], and consequently
  • fat goes to the liver.

During the investigation of possible underlying mechanisms, we discovered

  • a novel function for SEL1L as a regulator of lipid metabolism,” said Qi.

Sha said “We were very excited to find that

  • SEL1L is required for the intracellular trafficking of
  • lipoprotein lipase (LPL), acting as a chaperone,” .

and added that “Using several tissue-specific knockout mouse models,

  • we showed that this is a general phenomenon,”

Without LPL, lipids remain in the circulation;

  • fat and muscle cells cannot absorb fat molecules for storage and energy combustion,

People with LPL mutations develop

  • postprandial hypertriglyceridemia similar to
  • conditions found in fat cell-specific SEL1L-deficient mice, said Qi.

Future work will investigate the

  • role of SEL1L in human patients carrying LPL mutations and
  • determine why fat cell-specific SEL1L-deficient mice remain lean under Western diets, said Sha.

Co-authors include researchers from Cedars-Sinai Medical Center in Los Angeles; Wageningen University in the Netherlands; Georgia State University; University of California, Los Angeles; and the Medical College of Soochow University in China.

The study was funded by the U.S. National Institutes of Health, the Netherlands Organization for Health Research and Development National Institutes of Health, the Cedars-Sinai Medical Center, Chinese National Science Foundation, the American Diabetes Association, Cornell’s Center for Vertebrate Genomics and the Howard Hughes Medical Institute.

Part 6. Biomarkers

Biomarkers Take Center Stage

Josh P. Roberts
GEN May 1, 2013 (Vol. 33, No. 9)  http://www.genengnews.com/

While work with biomarkers continues to grow, scientists are also grappling with research-related bottlenecks, such as

  1. affinity reagent development,
  2. platform reproducibility, and
  3. sensitivity.

Biomarkers by definition indicate some state or process that generally occurs

  • at a spatial or temporal distance from the marker itself, and

it would not be an exaggeration to say that biomedicine has become infatuated with them:

  1. where to find them,
  2. when they may appear,
  3. what form they may take, and
  4. how they can be used to diagnose a condition or
  5. predict whether a therapy may be successful.

Biomarkers are on the agenda of many if not most industry gatherings, and in cases such as Oxford Global’s recent “Biomarker Congress” and the GTC “Biomarker Summit”, they hold the naming rights. There, some basic principles were built upon, amended, and sometimes challenged.

In oncology, for example, biomarker discovery is often predicated on the premise that

  • proteins shed from a tumor will traverse to and persist in, and be detectable in, the circulation.

By quantifying these proteins—singularly or as part of a larger “signature”—the hope is

  1. to garner information about the molecular characteristics of the cancer
  2. that will help with cancer detection and
  3. personalization of the treatment strategy.

Yet this approach has not yet turned into the panacea that was hoped for. Bottlenecks exist in

  • affinity reagent development,
  • platform reproducibility, and
  • sensitivity.

There is also a dearth of understanding of some of the

  • fundamental principles of biomarker biology that we need to know the answers to,

said Parag Mallick, Ph.D., whose lab at Stanford University is “working on trying to understand where biomarkers come from.”

There are dogmas saying that

  • circulating biomarkers come solely from secreted proteins.

But Dr. Mallick’s studies indicate that fully

  • 50% of circulating proteins may come from intracellular sources or
  • proteins that are annotated as such.

“We don’t understand the processes governing

  • which tumor-derived proteins end up in the blood.”

Other questions include “how does the size of a tumor affect how much of a given protein will be in the blood?”—perhaps

  • the tumor is necrotic at the center, or
  • it’s hypervascular or hypovascular.

He points out “The problem is that these are highly nonlinear processes at work, and

  • there is a large number of factors that might affect the answer to that question,” .

Their research focuses on using

  1. mass spectrometry and
  2. computational analysis
  • to characterize the biophysical properties of the circulating proteome, and
  • relate these to measurements made of the tumor itself.

Furthermore, he said – “We’ve observed that the proteins that are likely to

  • first show up and persist in the circulation, ..
  • are more stable than proteins that don’t,”
  • “we can quantify how significant the effect is.”

The goal is ultimately to be able to

  1. build rigorous, formal mathematical models that will allow something measured in the blood
  2. to be tied back to the molecular biology taking place in the tumor.

And conversely, to use those models

  • to predict from a tumor what will be found in the circulation.

“Ultimately, the models will allow you to connect the dots between

  • what you measure in the blood and the biology of the tumor.”

Bound for Affinity Arrays

Affinity reagents are the main tools for large-scale protein biomarker discovery. And while this has tended to mean antibodies (or their derivatives), other affinity reagents are demanding a place in the toolbox.

Affimers, a type of affinity reagent being developed by Avacta, consist of

  1. a biologically inert, biophysically stable protein scaffold
  2. containing three variable regions into which
  3. distinct peptides are inserted.

The resulting three-dimensional surface formed by these peptides

  • interacts and binds to proteins and other molecules in solution,
  • much like the antigen-binding site of antibodies.

Unlike antibodies, Affimers are relatively small (13 KDa),

  • non-post-translationally modified proteins
  • that can readily be expressed in bacterial culture.

They may be made to bind surfaces through unique residues

  • engineered onto the opposite face of the Affimer,
  • allowing the binding site to be exposed to the target in solution.

“We don’t seem to see in what we’ve done so far

  • any real loss of activity or functionality of Affimers when bound to surfaces—

they’re very robust,” said CEO Alastair Smith, Ph.D.

Avacta is taking advantage of this stability and its large libraries of Affimers to develop

  • very large affinity microarrays for
  • drug and biomarker discovery.

To date they have printed arrays with around 20–25,000 features, and Dr. Smith is “sure that we can get toward about 50,000 on a slide,” he said. “There’s no real impediment to us doing that other than us expressing the proteins and getting on with it.”

Customers will be provided with these large, complex “naïve” discovery arrays, readable with standard equipment. The plan is for the company to then “support our customers by providing smaller arrays with

  • the Affimers that are binding targets of interest to them,” Dr. Smith foretold.

And since the intellectual property rights are unencumbered,

  • Affimers in those arrays can be licensed to the end users
  • to develop diagnostics that can be validated as time goes on.

Around 20,000-Affimer discovery arrays were recently tested by collaborator Professor Ann Morgan of the University of Leeds with pools of unfractionated serum from patients with symptoms of inflammatory disease. The arrays

  • “rediscovered” elevated C-reactive protein (CRP, the clinical gold standard marker)
  • as well as uncovered an additional 22 candidate biomarkers.
  • other candidates combined with CRP, appear able to distinguish between different diseases such as
  1. rheumatoid arthritis,
  2. psoriatic arthritis,
  3. SLE, or
  4. giant cell arteritis.

Epigenetic Biomarkers

Methylation of adenine

Sometimes biomarkers are used not to find disease but

  • to distinguish healthy human cell types, with
  •  examples being found in flow cytometry and immunohistochemistry.

These widespread applications, however, are difficult to standardize, being

  • subject to arbitrary or subjective gating protocols and other imprecise criteria.

Epiontis instead uses an epigenetic approach. “What we need is a unique marker that is

  • demethylated only in one cell type and
  • methylated in all the other cell types,”

Each cell of the right cell type will have

  • two demethylated copies of a certain gene locus,
  • allowing them to be enumerated by quantitative PCR.

The biggest challenge is finding that unique epigenetic marker. To do so they look through the literature for proteins and genes described as playing a role in the cell type’s biology, and then

  • look at the methylation patterns to see if one can be used as a marker,

They also “use customized Affymetrix chips to look at the

  • differential epigenetic status of different cell types on a genomewide scale.”

explained CBO and founder Ulrich Hoffmueller, Ph.D.

The company currently has a panel of 12 assays for 12 immune cell types. Among these is an assay for

  • regulatory T (Treg) cells that queries the Foxp3 gene—which is uniquely demethylated in Treg
  • even though it is transiently expressed in activated T cells of other subtypes.

Also assayed are Th17 cells, difficult to detect by flow cytometry because

  • “the cells have to be stimulated in vitro,” he pointed out.

Developing New Assays for Cancer Biomarkers

Researchers at Myriad RBM and the Cancer Prevention Research Institute of Texas are collaborating to develop

  • new assays for cancer biomarkers on the Myriad RBM Multi-Analyte Profile (MAP) platform.

The release of OncologyMAP 2.0 expanded Myriad RBM’s biomarker menu to over 250 analytes, which can be measured from a small single sample, according to the company. Using this menu, L. Stephen et al., published a poster, “Analysis of Protein Biomarkers in Prostate and Colorectal Tumor Lysates,” which showed the results of

  • a survey of proteins relevant to colorectal (CRC) and prostate (PC) tumors
  • to identify potential proteins of interest for cancer research.

The study looked at CRC and PC tumor lysates and found that 102 of the 115 proteins showed levels above the lower limit of quantification.

  • Four markers were significantly higher in PC and 10 were greater in CRC.

For most of the analytes, duplicate sections of the tumor were similar, although some analytes did show differences. In four of the CRC analytes, tumor number four showed differences for CEA and tumor number 2 for uPA.

Thirty analytes were shown to be

  • different in CRC tumor compared to its adjacent tissue.
  • Ten of the analytes were higher in adjacent tissue compared to CRC.
  • Eighteen of the markers examined demonstrated  —-

significant correlations of CRC tumor concentration to serum levels.

“This suggests.. that the Oncology MAP 2.0 platform “provides a good method for studying changes in tumor levels because many proteins can be assessed with a very small sample.”

Clinical Test Development with MALDI-ToF

While there have been many attempts to translate results from early discovery work on the serum proteome into clinical practice, few of these efforts have progressed past the discovery phase.

Matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry on unfractionated serum/plasma samples offers many practical advantages over alternative techniques, and does not require

  • a shift from discovery to development and commercialization platforms.

Biodesix claims it has been able to develop the technology into

  • a reproducible, high-throughput tool to
  • routinely measure protein abundance from serum/plasma samples.

“.. we improved data-analysis algorithms to

  • reproducibly obtain quantitative measurements of relative protein abundance from MALDI-ToF mass spectra.

Heinrich Röder, CTO points out that the MALDI-ToF measurements

  • are combined with clinical outcome data using
  • modern learning theory techniques
  • to define specific disease states
  • based on a patient’s serum protein content,”

The clinical utility of the identification of these disease states can be investigated through a retrospective analysis of differing sample sets. For example, Biodesix clinically validated its first commercialized serum proteomic test, VeriStrat®, in 85 different retrospective sample sets.

Röder adds that “It is becoming increasingly clear that

  • the patients whose serum is characterized as VeriStrat Poor show
  • consistently poor outcomes irrespective of
  1. tumor type,
  2. histology, or
  3. molecular tumor characteristics,”

MALDI-ToF mass spectrometry, in its standard implementation,

  • allows for the observation of around 100 mostly high-abundant serum proteins.

Further, “while this does not limit the usefulness of tests developed from differential expression of these proteins,

  • the discovery potential would be greatly enhanced
  • if we could probe deeper into the proteome
  • while not giving up the advantages of the MALDI-ToF approach,”

Biodesix reports that its new MALDI approach, Deep MALDI™, can perform

  • simultaneous quantitative measurement of more than 1,000 serum protein features (or peaks) from 10 µL of serum in a high-throughput manner.
  • it increases the observable signal noise ratio from a few hundred to over 50,000,
  • resulting in the observation of many lower-abundance serum proteins.

Breast cancer, a disease now considered to be a collection of many complexes of symptoms and signatures—the dominant ones are labeled Luminal A, Luminal B, Her2, and Basal— which suggests different prognose, and

  • these labels are considered too simplistic for understanding and managing a woman’s cancer.

Studies published in the past year have looked at

  1. somatic mutations,
  2. gene copy number aberrations,
  3. gene expression abnormalities,
  4. protein and miRNA expression, and
  5. DNA methylation,

coming up with a list of significantly mutated genes—hot spots—in different categories of breast cancers. Targeting these will inevitably be the focus of much coming research.

“We’ve been taking these large trials and profiling these on a variety of array or sequence platforms. We think we’ll get

  1. prognostic drivers
  2. predictive markers for taxanes and
  3. monoclonal antibodies and
  4. tamoxifen and aromatase inhibitors,”
    explained Brian Leyland-Jones, Ph.D., director of Edith Sanford Breast Cancer Research. “We will end up with 20–40 different diseases, maybe more.”

Edith Sanford Breast Cancer Research is undertaking a pilot study in collaboration with The Scripps Research Institute, using a variety of tests on 25 patients to see how the information they provide complements each other, the overall flow, and the time required to get and compile results.

Laser-captured tumor samples will be subjected to low passage whole-genome, exome, and RNA sequencing (with targeted resequencing done in parallel), and reverse-phase protein and phosphorylation arrays, with circulating nucleic acids and circulating tumor cells being queried as well. “After that we hope to do a 100- or 150-patient trial when we have some idea of the best techniques,” he said.

Dr. Leyland-Jones predicted that ultimately most tumors will be found

  • to have multiple drivers,
  • with most patients receiving a combination of two, three, or perhaps four different targeted therapies.

Reduce to Practice

According to Randox, the evidence Investigator is a sophisticated semi-automated biochip sys­tem designed for research, clinical, forensic, and veterinary applications.

Once biomarkers that may have an impact on therapy are discovered, it is not always routine to get them into clinical practice. Leaving regulatory and financial, intellectual property and cultural issues aside, developing a diagnostic based on a biomarker often requires expertise or patience that its discoverer may not possess.

Andrew Gribben is a clinical assay and development scientist at Randox Laboratories, based in Northern Ireland, U.K. The company utilizes academic and industrial collaborators together with in-house discovery platforms to identify biomarkers that are

  • augmented or diminished in a particular pathology
  • relative to appropriate control populations.

Biomarkers can be developed to be run individually or

  • combined into panels of immunoassays on its multiplex biochip array technology.

Specificity can also be gained—or lost—by the affinity of reagents in an assay. The diagnostic potential of Heart-type fatty acid binding protein (H-FABP) abundantly expressed in human myocardial cells was recognized by Jan Glatz of Maastricht University, The Netherlands, back in 1988. Levels rise quickly within 30 minutes after a myocardial infarction, peaking at 6–8 hours and return to normal within 24–30 hours. Yet at the time it was not known that H-FABP was a member of a multiprotein family, with which the polyclonal antibodies being used in development of an assay were cross-reacting, Gribben related.

Randox developed monoclonal antibodies specific to H-FABP, funded trials investigating its use alone, and multiplexed with cardiac biomarker assays, and, more than 30 years after the biomarker was identified, in 2011, released a validated assay for H-FABP as a biomarker for early detection of acute myocardial infarction.

Ultrasensitive Immunoassays for Biomarker Development

Research has shown that detection and monitoring of biomarker concentrations can provide

  • insights into disease risk and progression.

Cytokines have become attractive biomarkers and candidates

  • for targeted therapies for a number of autoimmune diseases, including rheumatoid arthritis (RA), Crohn’s disease, and psoriasis, among others.

However, due to the low-abundance of circulating cytokines, such as IL-17A, obtaining robust measurements in clinical samples has been difficult.

Singulex reports that its digital single-molecule counting technology provides

  • increased precision and detection sensitivity over traditional ELISA techniques,
  • helping to shed light on biomarker verification and validation programs.

The company’s Erenna® immunoassay system, which includes optimized immunoassays, offers LLoQ to femtogram levels per mL resolution—even in healthy populations, at an improvement of 1-3 fold over standard ELISAs or any conventional technology and with a dynamic range of up to 4-logs, according to a Singulex official, who adds that

  • this sensitivity improvement helps minimize undetectable samples that
  • could otherwise delay or derail clinical studies.

The official also explains that the Singulex solution includes an array of products and services that are being applied to a number of programs and have enabled the development of clinically relevant biomarkers, allowing translation from discovery to the clinic.

In a poster entitled “Advanced Single Molecule Detection: Accelerating Biomarker Development Utilizing Cytokines through Ultrasensitive Immunoassays,” a case study was presented of work performed by Jeff Greenberg of NYU to show how the use of the Erenna system can provide insights toward

  • improving the clinical utility of biomarkers and
  • accelerating the development of novel therapies for treating inflammatory diseases.

A panel of inflammatory biomarkers was examined in DMARD (disease modifying antirheumatic drugs)-naïve RA (rheumatoid arthritis) vs. knee OA (osteoarthritis) patient cohorts. Markers that exhibited significant differences in plasma concentrations between the two cohorts included

  • CRP, IL-6R alpha, IL-6, IL-1 RA, VEGF, TNF-RII, and IL-17A, IL-17F, and IL-17A/F.

Among the three tested isoforms of IL-17,

  • the magnitude of elevation for IL-17F in RA patients was the highest.

“Singulex provides high-resolution monitoring of baseline IL-17A concentrations that are present at low levels,” concluded the researchers. “The technology also enabled quantification of other IL-17 isoforms in RA patients, which have not been well characterized before.”

The Singulex Erenna System has also been applied to cardiovascular disease research, for which its

  • cardiac troponin I (cTnI) digital assay can be used to measure circulating
  • levels of cTnI undetectable by other commercial assays.

Recently presented data from Brigham and Women’s Hospital and the TIMI-22 study showed that

  • using the Singulex test to serially monitor cTnI helps
  • stratify risk in post-acute coronary syndrome patients and
  • can identify patients with elevated cTnI
  • who have the most to gain from intensive vs. moderate-dose statin therapy,

according to the scientists involved in the research.

The study poster, “Prognostic Performance of Serial High Sensitivity Cardiac Troponin Determination in Stable Ischemic Heart Disease: Analysis From PROVE IT-TIMI 22,” was presented at the 2013 American College of Cardiology (ACC) Annual Scientific Session & Expo by R. O’Malley et al.

Biomarkers Changing Clinical Medicine

Better Diagnosis, Prognosis, and Drug Targeting Are among Potential Benefits

  1. John Morrow Jr., Ph.D.

Researchers at EMD Chemicals are developing biomarker immunoassays

  • to monitor drug-induced toxicity including kidney damage.

The pace of biomarker development is accelerating as investigators report new studies on cancer, diabetes, Alzheimer disease, and other conditions in which the evaluation and isolation of workable markers is prominently featured.

Wei Zheng, Ph.D., leader of the R&D immunoassay group at EMD Chemicals, is overseeing a program to develop biomarker immunoassays to

  • monitor drug-induced toxicity, including kidney damage.

“One of the principle reasons for drugs failing during development is because of organ toxicity,” says Dr. Zheng.
“proteins liberated into the serum and urine can serve as biomarkers of adverse response to drugs, as well as disease states.”

Through collaborative programs with Rules-Based Medicine (RBM), the EMD group has released panels for the profiling of human renal impairment and renal toxicity. These urinary biomarker based products fit the FDA and EMEA guidelines for assessment of drug-induced kidney damage in rats.

The group recently performed a screen for potential protein biomarkers in relation to

  • kidney toxicity/damage on a set of urine and plasma samples
  • from patients with documented renal damage.

Additionally, Dr. Zheng is directing efforts to move forward with the multiplexed analysis of

  • organ and cellular toxicity.

Diseases thought to involve compromised oxidative phosphorylation include

  • diabetes, Parkinson and Alzheimer diseases, cancer, and the aging process itself.

Good biomarkers allow Dr. Zheng to follow the mantra, “fail early, fail fast.” With robust, multiplexible biomarkers, EMD can detect bad drugs early and kill them before they move into costly large animal studies and clinical trials. “Recognizing the severe liability that toxicity presents, we can modify the structure of the candidate molecule and then rapidly reassess its performance.”

Scientists at Oncogene Science a division of Siemens Healthcare Diagnostics, are also focused on biomarkers. “We are working on a number of antibody-based tests for various cancers, including a test for the Ca-9 CAIX protein, also referred to as carbonic anhydrase,” Walter Carney, Ph.D., head of the division, states.

CAIX is a transmembrane protein that is

  • overexpressed in a number of cancers, and, like Herceptin and the Her-2 gene,
  • can serve as an effective and specific marker for both diagnostic and therapeutic purposes.
  • It is liberated into the circulation in proportion to the tumor burden.

Dr. Carney and his colleagues are evaluating patients after tumor removal for the presence of the Ca-9 CAIX protein. If

  • the levels of the protein in serum increase over time,
  • this suggests that not all the tumor cells were removed and the tumor has metastasized.

Dr. Carney and his team have developed both an immuno-histochemistry and an ELISA test that could be used as companion diagnostics in clinical trials of CAIX-targeted drugs.

The ELISA for the Ca-9 CAIX protein will be used in conjunction with Wilex’ Rencarex®, which is currently in a

  • Phase III trial as an adjuvant therapy for non-metastatic clear cell renal cancer.

Additionally, Oncogene Science has in its portfolio an FDA-approved test for the Her-2 marker. Originally approved for Her-2/Neu-positive breast cancer, its indications have been expanded over time, and was approved

  • for the treatment of gastric cancer last year.

It is normally present on breast cancer epithelia but

  • overexpressed in some breast cancer tumors.

“Our products are designed to be used in conjunction with targeted therapies,” says Dr. Carney. “We are working with companies that are developing technology around proteins that are

  • overexpressed in cancerous tissues and can be both diagnostic and therapeutic targets.”

The long-term goal of these studies is to develop individualized therapies, tailored for the patient. Since the therapies are expensive, accurate diagnostics are critical to avoid wasting resources on patients who clearly will not respond (or could be harmed) by the particular drug.

“At this time the rate of response to antibody-based therapies may be very poor, as

  • they are often employed late in the course of the disease, and patients are in such a debilitated state
  • that they lack the capacity to react positively to the treatment,” Dr. Carney explains.

Nanoscale Real-Time Proteomics

Stanford University School of Medicine researchers, working with Cell BioSciences, have developed a

  • nanofluidic proteomic immunoassay that measures protein charge,
  • similar to immunoblots, mass spectrometry, or flow cytometry.
  • unlike these platforms, this approach can measure the amount of individual isoforms,
  • specifically, phosphorylated molecules.

“We have developed a nanoscale device for protein measurement, which I believe could be useful for clinical analysis,” says Dean W. Felsher, M.D., Ph.D., associate professor at Stanford University School of Medicine.

Critical oncogenic transformations involving

  • the activation of the signal-related kinases ERK-1 and ERK-2 can now be followed with ease.

“The fact that we measure nanoquantities with accuracy means that

  • we can interrogate proteomic profiles in clinical patients,

by drawing tiny needle aspirates from tumors over the course of time,” he explains.

“This allows us to observe the evolution of tumor cells and

  • their response to therapy
  • from a baseline of the normal tissue as a standard of comparison.”

According to Dr. Felsher, 20 cells is a large enough sample to obtain a detailed description. The technology is easy to automate, which allows

  • the inclusion of hundreds of assays.

Contrasting this technology platform with proteomic analysis using microarrays, Dr. Felsher notes that the latter is not yet workable for revealing reliable markers.

Dr. Felsher and his group published a description of this technology in Nature Medicine. “We demonstrated that we could take a set of human lymphomas and distinguish them from both normal tissue and other tumor types. We can

  • quantify changes in total protein, protein activation, and relative abundance of specific phospho-isoforms
  • from leukemia and lymphoma patients receiving targeted therapy.

Even with very small numbers of cells, we are able to show that the results are consistent, and

  • our sample is a random profile of the tumor.”

Splice Variant Peptides

“Aberrations in alternative splicing may generate

  • much of the variation we see in cancer cells,”

says Gilbert Omenn, Ph.D., director of the center for computational medicine and bioinformatics at the University of Michigan School of Medicine. Dr. Omenn and his colleague, Rajasree Menon, are

  • using this variability as a key to new biomarker identification.

It is becoming evident that splice variants play a significant role in the properties of cancer cells, including

  • initiation, progression, cell motility, invasiveness, and metastasis.

Alternative splicing occurs through multiple mechanisms

  • when the exons or coding regions of the DNA transcribe mRNA,
  • generating initiation sites and connecting exons in protein products.

Their translation into protein can result in numerous protein isoforms, and

  • these isoforms may reflect a diseased or cancerous state.

Regulatory elements within the DNA are responsible for selecting different alternatives; thus

  • the splice variants are tempting targets for exploitation as biomarkers.
Analyses of the splice-site mutation

Analyses of the splice-site mutation

Despite the many questions raised by these observations, splice variation in tumor material has not been widely studied. Cancer cells are known for their tremendous variability, which allows them to

  • grow rapidly, metastasize, and develop resistance to anticancer drugs.

Dr. Omenn and his collaborators used

  • mass spec data to interrogate a custom-built database of all potential mRNA sequences
  • to find alternative splice variants.

When they compared normal and malignant mammary gland tissue from a mouse model of Her2/Neu human breast cancers, they identified a vast number (608) of splice variant proteins, of which

  • peptides from 216 were found only in the tumor sample.

“These novel and known alternative splice isoforms

  • are detectable both in tumor specimens and in plasma and
  • represent potential biomarker candidates,” Dr. Omenn adds.

Dr. Omenn’s observations and those of his colleague Lewis Cantley, Ph.D., have also

  • shed light on the origins of the classic Warburg effect,
  • the shift to anaerobic glycolysis in tumor cells.

The novel splice variant M2, of muscle pyruvate kinase,

  • is observed in embryonic and tumor tissue.

It is associated with this shift, the result of

  • the expression of a peptide splice variant sequence.

It is remarkable how many different areas of the life sciences are tied into the phenomenon of splice variation. The changes in the genetic material can be much greater than point mutations, which have been traditionally considered to be the prime source of genetic variability.

“We now have powerful methods available to uncover a whole new category of variation,” Dr. Omenn says. “High-throughput RNA sequencing and proteomics will be complementary in discovery studies of splice variants.”

Splice variation may play an important role in rapid evolutionary changes, of the sort discussed by Susumu Ohno and Stephen J. Gould decades ago. They, and other evolutionary biologists, argued that

  • gene duplication, combined with rapid variability, could fuel major evolutionary jumps.

At the time, the molecular mechanisms of variation were poorly understood, but today

  • the tools are available to rigorously evaluate the role of
  • splice variation and other contributors to evolutionary change.

“Biomarkers derived from studies of splice variants, could, in the future, be exploited

  • both for diagnosis and prognosis and
  • for drug targeting of biological networks,
  • in situations such as the Her-2/Neu breast cancers,” Dr. Omenn says.

Aminopeptidase Activities

“By correlating the proteolytic patterns with disease groups and controls, we have shown that

  • exopeptidase activities contribute to the generation of not only cancer-specific
  • but also cancer type specific serum peptides.

according to Paul Tempst, Ph.D., professor and director of the Protein Center at the Memorial Sloan-Kettering Cancer Center.

So there is a direct link between peptide marker profiles of disease and differential protease activity.” For this reason Dr. Tempst argues that “the patterns we describe may have value as surrogate markers for detection and classification of cancer.”

To investigate this avenue, Dr. Tempst and his colleagues have followed

  • the relationship between exopeptidase activities and metastatic disease.

“We monitored controlled, de novo peptide breakdown in large numbers of biological samples using mass spectrometry, with relative quantitation of the metabolites,” Dr. Tempst explains. This entailed the use of magnetic, reverse-phase beads for analyte capture and a MALDI-TOF MS read-out.

“In biomarker discovery programs, functional proteomics is usually not pursued,” says Dr. Tempst. “For putative biomarkers, one may observe no difference in quantitative levels of proteins, while at the same time, there may be substantial differences in enzymatic activity.”

In a preliminary prostate cancer study, the team found a significant difference

  • in activity levels of exopeptidases in serum from patients with metastatic prostate cancer
  • as compared to primary tumor-bearing individuals and normal healthy controls.

However, there were no differences in amounts of the target protein, and this potential biomarker would have been missed if quantitative levels of protein had been the only criterion of selection.

It is frequently stated that “practical fusion energy is 30 years in the future and always will be.” The same might be said of functional, practical biomarkers that can pass muster with the FDA. But splice variation represents a new handle on this vexing problem. It appears that we are seeing the emergence of a new approach that may finally yield definitive diagnostic tests, detectable in serum and urine samples.

Part 7. Epigenetics and Drug Metabolism

DNA Methylation Rules: Studying Epigenetics with New Tools

The tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.

Patricia Fitzpatrick Dimond, Ph.D.

http://www.genengnews.com/media/images/AnalysisAndInsight/Feb7_2013_24454248_GreenPurpleDNA_EpigeneticsToolsII3576166141.jpg

New tools may help move the field of epigenetic analysis forward and potentially unveil novel biomarkers for cellular development, differentiation, and disease.

DNA sequencing has had the power of technology behind it as novel platforms to produce more sequencing faster and at lower cost have been introduced. But the tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.

Among these mechanisms, DNA methylation, or the enzymatically mediated addition of a methyl group to cytosine or adenine dinucleotides,

  • serves as an inherited epigenetic modification that
  • stably modifies gene expression in dividing cells.

The unique methylomes are largely maintained in differentiated cell types, making them critical to understanding the differentiation potential of the cell.

In the DNA methylation process, cytosine residues in the genome are enzymatically modified to 5-methylcytosine,

  • which participates in transcriptional repression of genes during development and disease progression.

5-methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. DNA methylation affects gene transcription by physically

  • interfering with the binding of proteins involved in gene transcription.

Methylated DNA may be bound by methyl-CpG-binding domain proteins (MBDs) that can

  • then recruit additional proteins. Some of these include histone deacetylases and other chromatin remodeling proteins that modify histones, thereby
  • forming compact, inactive chromatin, or heterochromatin.

While DNA methylation doesn’t change the genetic code,

  • it influences chromosomal stability and gene expression.

Epigenetics and Cancer Biomarkers

multistage chemical carcinogenesis

multistage chemical carcinogenesis

And because of the increasing recognition that DNA methylation changes are involved in human cancers, scientists have suggested that these epigenetic markers may provide biological markers for cancer cells, and eventually point toward new diagnostic and therapeutic targets. Cancer cell genomes display genome-wide abnormalities in DNA methylation patterns,

  • some of which are oncogenic and contribute to genome instability.

In particular, de novo methylation of tumor suppressor gene promoters

  • occurs frequently in cancers, thereby silencing them and promoting transformation.

Cytosine hydroxymethylation (5-hydroxymethylcytosine, or 5hmC), the aforementioned DNA modification resulting from the enzymatic conversion of 5mC into 5-hydroxymethylcytosine by the TET family of oxygenases, has been identified

  • as another key epigenetic modification marking genes important for
  • pluripotency in embryonic stem cells (ES), as well as in cancer cells.

The base 5-hydroxymethylcytosine was recently identified as an oxidation product of 5-methylcytosine in mammalian DNA. In 2011, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2′-deoxycytidine (5hmdC) and 5-methyl-2′-deoxycytidine (5mdC) in genomic DNA, scientists at the Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California investigated

  • whether levels of 5hmC can distinguish normal tissue from tumor tissue.

They showed that in squamous cell lung cancers, levels of 5hmdC showed

  • up to five-fold reduction compared with normal lung tissue.

In brain tumors,5hmdC showed an even more drastic reduction

  • with levels up to more than 30-fold lower than in normal brain,
  • but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1, the enzyme that converts 5hmC to 5hmdC.

Immunohistochemical analysis indicated that 5hmC is “remarkably depleted” in many types of human cancer.

  • there was an inverse relationship between 5hmC levels and cell proliferation with lack of 5hmC in proliferating cells.

Their data suggest that 5hmdC is strongly depleted in human malignant tumors,

  • a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue.

In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis.

Enzymatic Mapping

But according to New England Biolabs’ Sriharsa Pradhan, Ph.D., methods for distinguishing 5mC from 5hmC and analyzing and quantitating the cell’s entire “methylome” and “hydroxymethylome” remain less than optimal.

The protocol for bisulphite conversion to detect methylation remains the “gold standard” for DNA methylation analysis. This method is generally followed by PCR analysis for single nucleotide resolution to determine methylation across the DNA molecule. According to Dr. Pradhan, “.. bisulphite conversion does not distinguish 5mC and 5hmC,”

Recently we found an enzyme, a unique DNA modification-dependent restriction endonuclease, AbaSI, which can

  • decode the hydryoxmethylome of the mammalian genome.

You easily can find out where the hydroxymethyl regions are.”

AbaSI, recognizes 5-glucosylatedmethylcytosine (5gmC) with high specificity when compared to 5mC and 5hmC, and

  • cleaves at narrow range of distances away from the recognized modified cytosine.

By mapping the cleaved ends, the exact 5hmC location can, the investigators reported, be determined.

Dr. Pradhan and his colleagues at NEB; the Department of Biochemistry, Emory University School of Medicine, Atlanta; and the New England Biolabs Shanghai R&D Center described use of this technique in a paper published in Cell Reports this month, in which they described high-resolution enzymatic mapping of genomic hydroxymethylcytosine in mouse ES cells.

In the current report, the authors used the enzyme technology for the genome-wide high-resolution hydroxymethylome, describing simple library construction even with a low amount of input DNA (50 ng) and the ability to readily detect 5hmC sites with low occupancy.

As a result of their studies, they propose that

factors affecting the local 5mC accessibility to TET enzymes play important roles in the 5hmC deposition

  • including include chromatin compaction, nucleosome positioning, or TF binding.
  •  the regularly oscillating 5hmC profile around the CTCF-binding sites, suggests 5hmC ‘‘writers’’ may be sensitive to the nucleosomal environment.
  • some transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.

“We were able to do complete mapping in mouse embryonic cells and are pleased about what this enzyme can do and how it works,” Dr. Pradhan said.

And the availability of novel tools that make analysis of the methylome and hypomethylome more accessible will move the field of epigenetic analysis forward and potentially novel biomarkers for cellular development, differentiation, and disease.

Patricia Fitzpatrick Dimond, Ph.D. (pdimond@genengnews.com), is technical editor at Genetic Engineering & Biotechnology News.

Epigenetic Regulation of ADME-Related Genes: Focus on Drug Metabolism and Transport

Published: Sep 23, 2013

Epigenetic regulation of gene expression refers to heritable factors that are functionally relevant genomic modifications but that do not involve changes in DNA sequence.

Examples of such modifications include

  • DNA methylation, histone modifications, noncoding RNAs, and chromatin architecture.

Epigenetic modifications are crucial for

packaging and interpreting the genome, and they have fundamental functions in regulating gene expression and activity under the influence of physiologic and environmental factors.

In this issue of Drug Metabolism and Disposition, a series of articles is presented to demonstrate the role of epigenetic factors in regulating

  • the expression of genes involved in drug absorption, distribution, metabolism, and excretion in organ development, tissue-specific gene expression, sexual dimorphism, and in the adaptive response to xenobiotic exposure, both therapeutic and toxic.

The articles also demonstrate that, in addition to genetic polymorphisms, epigenetics may also contribute to wide inter-individual variations in drug metabolism and transport. Identification of functionally relevant epigenetic biomarkers in human specimens has the potential to improve prediction of drug responses based on patient’s epigenetic profiles.

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=157804

This study is published online in Drug Metabolism and Disposition

Part 8.  Pictorial Maps

 Prediction of intracellular metabolic states from extracellular metabolomic data

MK Aurich, G Paglia, Ottar Rolfsson, S Hrafnsdottir, M Magnusdottir, MM Stefaniak, BØ Palsson, RMT Fleming &

Ines Thiele

Metabolomics Aug 14, 2014;

http://dx.doi.org:/10.1007/s11306-014-0721-3

http://link.springer.com/article/10.1007/s11306-014-0721-3/fulltext.html#Sec1

http://link.springer.com/static-content/images/404/art%253A10.1007%252Fs11306-014-0721-3/MediaObjects/11306_2014_721_Fig1_HTML.gif

Metabolic models can provide a mechanistic framework

  • to analyze information-rich omics data sets, and are
  • increasingly being used to investigate metabolic alternations in human diseases.

An expression of the altered metabolic pathway utilization is the selection of metabolites consumed and released by cells. However, methods for the

  • inference of intracellular metabolic states from extracellular measurements in the context of metabolic models remain underdeveloped compared to methods for other omics data.

Herein, we describe a workflow for such an integrative analysis

  • emphasizing on extracellular metabolomics data.

We demonstrate,

  • using the lymphoblastic leukemia cell lines Molt-4 and CCRF-CEM,

how our methods can reveal differences in cell metabolism. Our models explain metabolite uptake and secretion by predicting

  • a more glycolytic phenotype for the CCRF-CEM model and
  • a more oxidative phenotype for the Molt-4 model,
  • which was supported by our experimental data.

Gene expression analysis revealed altered expression of gene products at

  • key regulatory steps in those central metabolic pathways, and

literature query emphasized the role of these genes in cancer metabolism.

Moreover, in silico gene knock-outs identified unique

  •  control points for each cell line model, e.g., phosphoglycerate dehydrogenase for the Molt-4 model.

Thus, our workflow is well suited to the characterization of cellular metabolic traits based on

  • -extracellular metabolomic data, and it allows the integration of multiple omics data sets
  • into a cohesive picture based on a defined model context.

Keywords Constraint-based modeling _ Metabolomics _ Multi-omics _ Metabolic network _ Transcriptomics

1 Introduction

Modern high-throughput techniques have increased the pace of biological data generation. Also referred to as the ‘‘omics avalanche’’, this wealth of data provides great opportunities for metabolic discovery. Omics data sets

  • contain a snapshot of almost the entire repertoire of mRNA, protein, or metabolites at a given time point or

under a particular set of experimental conditions. Because of the high complexity of the data sets,

  • computational modeling is essential for their integrative analysis.

Currently, such data analysis is a bottleneck in the research process and methods are needed to facilitate the use of these data sets, e.g., through meta-analysis of data available in public databases [e.g., the human protein atlas (Uhlen et al. 2010) or the gene expression omnibus (Barrett et al.  2011)], and to increase the accessibility of valuable information for the biomedical research community.

Constraint-based modeling and analysis (COBRA) is

  • a computational approach that has been successfully used to
  • investigate and engineer microbial metabolism through the prediction of steady-states (Durot et al.2009).

The basis of COBRA is network reconstruction: networks are assembled in a bottom-up fashion based on

  • genomic data and extensive
  • organism-specific information from the literature.

Metabolic reconstructions capture information on the

  • known biochemical transformations taking place in a target organism
  • to generate a biochemical, genetic and genomic knowledge base (Reed et al. 2006).

Once assembled, a

  • metabolic reconstruction can be converted into a mathematical model (Thiele and Palsson 2010), and
  • model properties can be interrogated using a great variety of methods (Schellenberger et al. 2011).

The ability of COBRA models

  • to represent genotype–phenotype and environment–phenotype relationships arises
  • through the imposition of constraints, which
  • limit the system to a subset of possible network states (Lewis et al. 2012).

Currently, COBRA models exist for more than 100 organisms, including humans (Duarte et al. 2007; Thiele et al. 2013).

Since the first human metabolic reconstruction was described [Recon 1 (Duarte et al. 2007)],

  • biomedical applications of COBRA have increased (Bordbar and Palsson 2012).

One way to contextualize networks is to

  • define their system boundaries according to the metabolic states of the system, e.g., disease or dietary regimes.

The consequences of the applied constraints can

  • then be assessed for the entire network (Sahoo and Thiele 2013).

Additionally, omics data sets have frequently been used

  • to generate cell-type or condition-specific metabolic models.

Models exist for specific cell types, such as

  1. enterocytes (Sahoo and Thiele2013),
  2. macrophages (Bordbar et al. 2010),
  3. adipocytes (Mardinoglu et al. 2013),
  4. even multi-cell assemblies that represent the interactions of brain cells (Lewis et al. 2010).

All of these cell type specific models, except the enterocyte reconstruction

  • were generated based on omics data sets.

Cell-type-specific models have been used to study

  • diverse human disease conditions.

For example, an adipocyte model was generated using

  • transcriptomic, proteomic, and metabolomics data.

This model was subsequently used to investigate metabolic alternations in adipocytes

  • that would allow for the stratification of obese patients (Mardinoglu et al. 2013).

The biomedical applications of COBRA have been

  1. cancer metabolism (Jerby and Ruppin, 2012).
  2. predicting drug targets (Folger et al. 2011; Jerby et al. 2012).

A cancer model was generated using

  • multiple gene expression data sets and subsequently used
  • to predict synthetic lethal gene pairs as potential drug targets
  • selective for the cancer model, but non-toxic to the global model (Recon 1),

a consequence of the reduced redundancy in the cancer specific model (Folger et al. 2011).

In a follow up study, lethal synergy between FH and enzymes of the heme metabolic pathway

  • were experimentally validated and resolved the mechanism by which FH deficient cells,
    e.g., in renal-cell cancer cells survive a non-functional TCA cycle (Frezza et al. 2011).

Contextualized models, which contain only the subset of reactions active in a particular tissue (or cell-) type,

  • can be generated in different ways (Becker and Palsson, 2008; Jerby et al. 2010).

However, the existing algorithms mainly consider

  • gene expression and proteomic data
  • to define the reaction sets that comprise the contextualized metabolic models.

These subset of reactions are usually defined

  • based on the expression or absence of expression of the genes or proteins (present and absent calls),
  • or inferred from expression values or differential gene expression.

Comprehensive reviews of the methods are available (Blazier and Papin, 2012; Hyduke et al. 2013). Only the compilation of a large set of omics data sets

  • can result in a tissue (or cell-type) specific metabolic model, whereas

the representation of one particular experimental condition is achieved

  • through the integration of omics data set generated from one experiment only (condition-specific cell line model).

Recently, metabolomic data sets have become more comprehensive and

  • using these data sets allow direct determination of the metabolic network components (the metabolites).

Additionally, metabolomics has proven to be stable, relatively inexpensive, and highly reproducible (Antonucci et al. 2012). These factors make metabolomic data sets particularly valuable for

  • interrogation of metabolic phenotypes.

Thus, the integration of these data sets is now an active field of research (Li et al. 2013; Mo et al. 2009; Paglia et al. 2012b; Schmidt et al. 2013).

Generally, metabolomic data can be incorporated into metabolic networks as

  • qualitative, quantitative, and thermodynamic constraints (Fleming et al. 2009; Mo et al. 2009).

Mo et al. used metabolites detected in the

  • spent medium of yeast cells to determine intracellular flux states through a sampling analysis (Mo et al. 2009),
  • which allowed unbiased interrogation of the possible network states (Schellenberger and Palsson 2009) and
  • prediction of internal pathway use.
Modes of transcriptional regulation during the YMC

Modes of transcriptional regulation during the YMC

Such analyses have also been used to reveal the effects of

  1. enzymopathies on red blood cells (Price et al. 2004),
  2. to study effects of diet on diabetes (Thiele et al. 2005) and
  3. to define macrophage metabolic states (Bordbar et al. 2010).

This type of analysis is available as a function in the COBRA toolbox (Schellenberger et al. 2011).

In this study, we established a workflow

  • for the generation and analysis of condition-specific metabolic cell line models
  • that can facilitate the interpretation of metabolomic data.

Our modeling yields meaningful predictions regarding

  • metabolic differences between two lymphoblastic leukemia cell lines (Fig. 1A).

Fig. 1

metabol leukem cell lines11306_2014_721_Fig1_HTML

metabol leukem cell lines11306_2014_721_Fig1_HTML

A Combined experimental and computational pipeline to study human metabolism.

  1. Experimental work and omics data analysis steps precede computational modeling.
  2. Model predictions are validated based on targeted experimental data.
  3. Metabolomic and transcriptomic data are used for model refinement and submodel extraction.
  4. Functional analysis methods are used to characterize the metabolism of the cell-line models and compare it to additional experimental data.
  5. The validated models are subsequently used for the prediction of drug targets.

B Uptake and secretion pattern of model metabolites. All metabolite uptakes and secretions that were mapped during model generation are shown.

  • Metabolite uptakes are depicted on the left, and
  • secreted metabolites are shown on the right.
  1. A number of metabolite exchanges mapped to the model were unique to one cell line.
  2. Differences between cell lines were used to set quantitative constraints for the sampling analysis.

C Statistics about the cell line-specific network generation.

D Quantitative constraints.

For the sampling analysis, an additional set of constraints was imposed on the cell line specific models,

  • emphasizing the differences in metabolite uptake and secretion between cell lines.

Higher uptake of a metabolite was allowed

  • in the model of the cell line that consumed more of the metabolite in vitro, whereas
  • the supply was restricted for the model with lower in vitro uptake.

This was done by establishing the same ratio between the models bounds as detected in vitro.

X denotes the factor (slope ratio) that distinguishes the bounds, and

  • which was individual for each metabolite.

(a) The uptake of a metabolite could be x times higher in CCRF-CEM cells,

(b) the metabolite uptake could be x times higher in Molt-4,

(c) metabolite secretion could be x times higher in CCRF-CEM, or

(d) metabolite secretion could be x times higher in Molt-4 cells.LOD limit of detection.

The consequence of the adjustment was, in case of uptake, that one model was constrained to a lower metabolite uptake (A, B), and the difference depended on the ratio detected in vitro. In case of secretion, one model

  • had to secrete more of the metabolite, and again
  • the difference depended on the experimental difference detected between the cell lines

2 Results

We set up a pipeline that could be used to infer intracellular metabolic states

  • from semi-quantitative data regarding metabolites exchanged between cells and their environment.

Our pipeline combined the following four steps:

  1. data acquisition,
  2. data analysis,
  3. metabolic modeling and
  4. experimental validation of the model predictions (Fig. 1A).

We demonstrated the pipeline and the predictive potential to predict metabolic alternations in diseases such as cancer based on

^two lymphoblastic leukemia cell lines.

The resulting Molt-4 and CCRF-CEM condition-specific cell line models could explain

^  metabolite uptake and secretion
^  by predicting the distinct utilization of central metabolic pathways by the two cell lines.
^  the CCRF-CEM model resembled more a glycolytic, commonly referred to as ‘Warburg’ phenotype,
^  our model predicted a more respiratory phenotype for the Molt-4 model.

We found these predictions to be in agreement with measured gene expression differences

  • at key regulatory steps in the central metabolic pathways, and they were also
  • consistent with additional experimental data regarding the energy and redox states of the cells.

After a brief discussion of the data generation and analysis steps, the results derived from model generation and analysis will be described in detail.

2.1 Pipeline for generation of condition-specific metabolic cell line models

integration of exometabolomic (EM) data

integration of exometabolomic (EM) data

2.1.1 Generation of experimental data

We monitored the growth and viability of lymphoblastic leukemia cell lines in serum-free medium (File S2, Fig. S1). Multiple omics data sets were derived from these cells.Extracellular metabolomics (exo-metabolomic) data,

integration of exometabolomic (EM) data

integration of exometabolomic (EM) data

^  comprising measurements of the metabolites in the spent medium of the cell cultures (Paglia et al. 2012a),
^ were collected along with transcriptomic data, and these data sets were used to construct the models.

2.1.4 Condition-specific models for CCRF-CEM and Molt-4 cells

To determine whether we had obtained two distinct models, we evaluated the reactions, metabolites, and genes of the two models. Both the Molt-4 and CCRF-CEM models contained approximately half of the reactions and metabolites present in the global model (Fig. 1C). They were very similar to each other in terms of their reactions, metabolites, and genes (File S1, Table S5A–C).

(1) The Molt-4 model contained seven reactions that were not present in the CCRF-CEM model (Co-A biosynthesis pathway and exchange reactions).
(2) The CCRF-CEM contained 31 unique reactions (arginine and proline metabolism, vitamin B6 metabolism, fatty acid activation, transport, and exchange reactions).
(3) There were 2 and 15 unique metabolites in the Molt-4 and CCRF-CEM models, respectively (File S1, Table S5B).
(4) Approximately three quarters of the global model genes remained in the condition-specific cell line models (Fig. 1C).
(5) The Molt-4 model contained 15 unique genes, and the CCRF-CEM model had 4 unique genes (File S1, Table S5C).
(6) Both models lacked NADH dehydrogenase (complex I of the electron transport chain—ETC), which was determined by the absence of expression of a mandatory subunit (NDUFB3, Entrez gene ID 4709).

Rather, the ETC was fueled by FADH2 originating from succinate dehydrogenase and from fatty acid oxidation, which through flavoprotein electron transfer

FADH2

FADH2

  • could contribute to the same ubiquinone pool as complex I and complex II (succinate dehydrogenase).

Despite their different in vitro growth rates (which differed by 11 %, see File S2, Fig. S1) and
^^^ differences in exo-metabolomic data (Fig. 1B) and transcriptomic data,
^^^ the internal networks were largely conserved in the two condition-specific cell line models.

2.1.5 Condition-specific cell line models predict distinct metabolic strategies

Despite the overall similarity of the metabolic models, differences in their cellular uptake and secretion patterns suggested distinct metabolic states in the two cell lines (Fig. 1B and see “Materials and methods” section for more detail). To interrogate the metabolic differences, we sampled the solution space of each model using an Artificial Centering Hit-and-Run (ACHR) sampler (Thiele et al. 2005). For this analysis, additional constraints were applied, emphasizing the quantitative differences in commonly uptaken and secreted metabolites. The maximum possible uptake and maximum possible secretion flux rates were reduced
^^^ according to the measured relative differences between the cell lines (Fig. 1D, see “Materials and methods” section).

We plotted the number of sample points containing a particular flux rate for each reaction. The resulting binned histograms can be understood as representing the probability that a particular reaction can have a certain flux value.

A comparison of the sample points obtained for the Molt-4 and CCRF-CEM models revealed

  • a considerable shift in the distributions, suggesting a higher utilization of glycolysis by the CCRF-CEM model
    (File S2, Fig. S2).

This result was further supported by differences in medians calculated from sampling points (File S1, Table S6).
The shift persisted throughout all reactions of the pathway and was induced by the higher glucose uptake (34 %) from the extracellular medium in CCRF-CEM cells.

The sampling median for glucose uptake was 34 % higher in the CCRF-CEM model than in Molt-4 model (File S2, Fig. S2).

The usage of the TCA cycle was also distinct in the two condition-specific cell-line models (Fig. 2). Interestingly,
the models used succinate dehydrogenase differently (Figs. 2, 3).

TCA_reactions

TCA_reactions

The Molt-4 model utilized an associated reaction to generate FADH2, whereas

  • in the CCRF-CEM model, the histogram was shifted in the opposite direction,
  • toward the generation of succinate.

Additionally, there was a higher efflux of citrate toward amino acid and lipid metabolism in the CCRF-CEM model (Fig. 2). There was higher flux through anaplerotic and cataplerotic reactions in the CCRF-CEM model than in the Molt-4 model (Fig. 2); these reactions include

(1) the efflux of citrate through ATP-citrate lyase,
(2) uptake of glutamine,
(3) generation of glutamate from glutamine,
(4) transamination of pyruvate and glutamate to alanine and to 2-oxoglutarate,
(5) secretion of nitrogen, and
(6) secretion of alanine.

energetics-of-cellular-respiration

energetics-of-cellular-respiration

The Molt-4 model showed higher utilization of oxidative phosphorylation (Fig. 3), again supported by
elevated median flux through ATP synthase (36 %) and other enzymes, which contributed to higher oxidative metabolism. The sampling analysis therefore revealed different usage of central metabolic pathways by the condition-specific models.

Fig. 2

Differences in the use of  the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).

Differences in the use of the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).

Differences in the use of the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).

The table provides the median values of the sampling results. Negative values in histograms and in the table describe reversible reactions with flux in the reverse direction. There are multiple reversible reactions for the transformation of isocitrate and α-ketoglutarate, malate and fumarate, and succinyl-CoA and succinate. These reactions are unbounded, and therefore histograms are not shown. The details of participating cofactors have been removed.

Figure 3.

Molt-4 has higher median flux through ETC reactions II–IV 11306_2014_721_Fig3_HTML

Molt-4 has higher median flux through ETC reactions II–IV 11306_2014_721_Fig3_HTML

Atp ATP, cit citrate, adp ADP, pi phosphate, oaa oxaloacetate, accoa acetyl-CoA, coa coenzyme-A, icit isocitrate, αkg α-ketoglutarate, succ-coa succinyl-CoA, succ succinate, fumfumarate, mal malate, oxa oxaloacetate,
pyr pyruvate, lac lactate, ala alanine, gln glutamine, ETC electron transport chain

Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes

Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes

metabolic pathways 1476-4598-10-70-1

metabolic pathways 1476-4598-10-70-1

Metabolic Systems Research Team fig2

Metabolic Systems Research Team fig2

Metabolic control analysis of respiration in human cancer tissue. fphys-04-00151-g001

Metabolic control analysis of respiration in human cancer tissue. fphys-04-00151-g001

Metabolome Informatics Research fig1

Metabolome Informatics Research fig1

Modelling of Central Metabolism network3

Modelling of Central Metabolism network3

N. gaditana metabolic pathway map ncomms1688-f4

N. gaditana metabolic pathway map ncomms1688-f4

protein changes in biological mechanisms

protein changes in biological mechanisms

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