Posts Tagged ‘Mutagenesis’

Reporter: Adina Hazan, PhD

Elizabeth Unger from the Tian group at UC Davis, Jacob Keller from the Looger lab from HHMI, Michael Altermatt from the Gradinaru group at California Institute of Technology, and colleagues did just this, by redesigned the binding pocket of periplasmic binding proteins (PBPs) using artificial intelligence, such that it became a fluorescent sensor specific for serotonin. Not only this, the group showed that it could express and use this molecule to detect serotonin on the cell, tissue, and whole animal level.

By starting with a microbial PBP and early version of an acetyl choline sensor (iAChSnFR), the scientists used machine learning and modeling to redesign the binding site to exhibit a higher affinity and specificity to serotonin. After three repeats of mutagenesis, modeling, and library readouts, they produced iSeroSnFR. This version harbors 19 mutations compared to iAChSnFR0.6 and a Kd of 310 µM. This results in an increase in fluorescence in HEK293T cells expressing the serotonin receptor of 800%. Of over 40 neurotransmitters, amino acids, and small molecules screened, only two endogenous molecules evoked some fluorescence, but at significantly higher concentrations.

To acutely test the ability of the sensor to detect rapid changes of serotonin in the environment, the researchers used caged serotonin, a technique in which the serotonin is rapidly released into the environment with light pulses, and showed that iSeroSnFR accurately and robustly produced a signal with each flash of light. With this tool, it was then possible to move to ex-vivo mouse brain slices and detect endogenous serotonin release patterns across the brain. Three weeks after targeted injection of iSeroSnFR to specifically deliver the receptor into the prefrontal cortex and dorsal striatum, strong fluorescent signal could be detected during perfusion of serotonin or electrical stimulation.

Most significantly, this molecule was also shown to be detected in freely moving mice, a tool which could offer critical insight into the acute role of serotonin regulation during important functions such as mood and alertness. Through optical fiber placements in the basolateral amygdala and prefrontal cortex, the team measured dynamic and real-time changes in serotonin release in fear-trained mice, social interactions, and sleep wake cycles. For example, while both areas of the brain have been established as relevant to the fear response, they reliably tracked that the PFC response was immediate, while the BSA displayed a delayed response. This additional temporal resolution of neuromodulation may have important implications in neurotransmitter pharmacology of the central nervous system.

This study provided the scientific community with several insights and tools. The serotonin sensor itself will be a critical tool in the study of the central nervous system and possibly beyond. Additionally, an AI approach to mutagenesis in order to redesign a binding pocket of a receptor opens new avenues to the development of pharmacological tools and may lead to many new designs in therapeutics and research.


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Rhodopsin role in ciliary trafficking

Jillian N Pearring
Department of Ophthalmology, Duke University School of Medicine, Durham, United States
No competing interests declared

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William J Spencer
Department of Ophthalmology, Duke University School of Medicine, Durham, United States
No competing interests declared

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>William J Spencer

Eric C Lieu
Department of Ophthalmology, Duke University School of Medicine, Durham, United States
No competing interests declared

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>Eric C Lieu, 

Vadim Y Arshavsky
Department of Ophthalmology, Duke University School of Medicine, Durham, United States
For correspondence: vadim.arshavsky@duke.edu
No competing interests declared

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>Vadim Y Arshavsky
eLife 2015;10.7554/eLife.12058   http://dx.doi.org/10.7554/eLife.12058

Sensory cilia are populated by a select group of signaling proteins that detect environmental stimuli. How these molecules are delivered to the sensory cilium and whether they rely on one another for specific transport remains poorly understood. Here, we investigated whether the visual pigment, rhodopsin, is critical for delivering other signaling proteins to the sensory cilium of photoreceptor cells, the outer segment. Rhodopsin is the most abundant outer segment protein and its proper transport is essential for formation of this organelle, suggesting that such a dependency might exist. Indeed, we demonstrated that guanylate cyclase-1, producing the cGMP second messenger in photoreceptors, requires rhodopsin for intracellular stability and outer segment delivery. We elucidated this dependency by showing that guanylate cyclase-1 is a novel rhodopsin-binding protein. These findings expand rhodopsin’s role in vision from being a visual pigment and major outer segment building block to directing trafficking of another key signaling protein.


Photoreceptor cells transform information entering the eye as photons into patterns of neuronal electrical activity. This transformation takes place in the sensory cilium organelle, the outer segment. Outer segments are built from a relatively small set of structural and signaling proteins, including components of the classical GPCR phototransduction cascade. Such a distinct functional and morphological specialization allow outer segments to serve as a nearly unmatched model system for studying general principles of GPCR signaling (Arshavsky et al., 2002) and, in more recent years, a model for ciliary trafficking (Garcia-Gonzalo and Reiter, 2012; Nemet et al., 2015; Pearring et al., 2013; Schou et al., 2015; Wang and Deretic, 2014). Despite our deep understanding of visual signal transduction, little is known how the outer segment is populated by proteins performing this function. Indeed, nearly all mechanistic studies of outer segment protein trafficking were devoted to rhodopsin (Nemet et al., 2015; Wang and Deretic, 2014), which is a GPCR visual pigment comprising the majority of the outer segment membrane protein mass (Palczewski, 2006). The mechanisms responsible for outer segment delivery of other transmembrane proteins remain essentially unknown. Some of them contain short outer segment targeting signals, which can be identified through site-specific mutagenesis (Deretic et al., 1998; Li et al., 1996; Pearring et al., 2014; Salinas et al., 2013; Sung et al., 1994; Tam et al., 2000; Tam et al., 2004). A documented exception is retinal guanylate cyclase 1 (GC-1), whose exhaustive mutagenesis did not yield a distinct outer segment targeting motif (Karan et al., 2011).

GC-1 is a critical component of the phototransduction machinery responsible for synthesizing the second messenger, cGMP (Wen et al., 2014). GC-1 is the only guanylate cyclase isoform expressed in the outer segments of cones and the predominant isoform in rods (Baehr et al., 2007; Yang et al., 1999). GC-1 knockout in mice is characterized by severe degeneration of cones and abnormal light-response recovery kinetics in rods (Yang et al., 1999). Furthermore, a very large number of GC-1 mutations found in human patients cause one of the most severe forms of early onset retinal dystrophy, called Leber’s congenital amaurosis (Boye, 2014; Kitiratschky et al., 2008). Many of these mutations are located outside the catalytic site of GC-1, which raises great interest to understanding the mechanisms of its intracellular processing and trafficking.

In this study, we demonstrate that, rather than relying on its own targeting motif, GC-1 is transported to the outer segment in a complex with rhodopsin. We conducted a comprehensive screen of outer segment protein localization in rod photoreceptors of rhodopsin knockout (Rho-/- ) mice and found that GC-1 was the only protein severely affected by this knockout. We next showed that this unique property of GC-1 is explained by its interaction with rhodopsin, which likely initiates in the biosynthetic membranes and supports both intracellular stability and outer segment delivery of this enzyme. These findings explain how GC-1 reaches its specific intracellular destination and also expand the role of rhodopsin in supporting normal vision by showing that it guides trafficking of another key phototransduction protein.


GC-1 is the outer segment-resident protein severely down-regulated in rhodopsin knockout rods

GC-1 stability and trafficking require the transmembrane core of rhodopsin but not its outer 119 segment targeting domain

GC-1 is a rhodopsin-interacting protein


The findings reported in this study expand our understanding of how the photoreceptor’s sensory cilium is populated by its specific membrane proteins. We have found that rhodopsin serves as an interacting partner and a vehicle for ciliary delivery of a key phototransduction protein, GC-1. This previously unknown function adds to the well-established roles of rhodopsin as a GPCR visual pigment and a major building block of photoreceptor membranes. We further showed that GC-1 is unique in its reliance on rhodopsin, as the other nine proteins tested in this study were expressed in significant amounts and faithfully localized to rod outer segments in the absence of rhodopsin.

Our data consolidate a number of previously published observations, including a major puzzle related to GC-1: the lack of a distinct ciliary targeting motif encoded in its sequence. The shortest recombinant fragment of GC-1 which localized specifically to the outer segment was found to be very large and contain both transmembrane and cytoplasmic domains (Karan et al., 2011). Our study shows that GC-1 delivery requires rhodopsin and, therefore, can rely on specific targeting information encoded in the rhodopsin molecule. Interestingly, we also found that this information can be replaced by an alternative ciliary targeting sequence from a GPCR not endogenous to photoreceptors. This suggests that the functions of binding/stabilization of GC-1 and ciliary targeting are performed by different parts of the rhodopsin molecule. Our findings also shed new light on the report that both rhodopsin and GC-1 utilize intraflagellar transport (IFT) for their ciliary trafficking and co-precipitate with IFT proteins (Bhowmick et al., 2009). The authors hypothesized that GC-1 plays a primary role in assembling cargo for the IFT particle bound for ciliary delivery. Our data suggest that it is rhodopsin that drives this complex, at least in photoreceptor cells where these proteins are specifically expressed. Unlike GC-1’s reliance on rhodopsin for its intracellular stability or outer segment trafficking, rhodopsin does not require GC-1 as its expression level and localization remain normal in rods of GC-1 knockout mice ((Baehr et al., 2007) and this study). The outer segment trafficking of cone opsins is not affected by the lack of GC-1 either (Baehr et al., 2007; Karan et al., 2008), although GC-1 knockout cones undergo rapid degeneration, likely because they do not express GC-2 – an enzyme with redundant function. The primary role of rhodopsin in guiding GC-1 to the outer segment is further consistent with rhodopsin directly interacting with IFT20, a mobile component of the IFT complex responsible for recruiting IFT cargo at the Golgi network (Crouse et al., 2014; Keady et al., 2011).

It was also reported that GC-1 trafficking requires participation of chaperone proteins, most importantly DnaJB6 (Bhowmick et al., 2009). Our data suggest that GC-1 interaction with DnaJB6 is transient, most likely in route to the outer segment, since we were not able to co-precipitate DnaJB6 with GC-1 from whole retina lysates (Figure 5). In contrast, the majority of GC-1 co-precipitates with rhodopsin from these same lysates, suggesting that these proteins remain in a complex after being delivered to the outer segment. Although our data do not exclude that the mature GC-1-rhodopsin complex may contain additional protein component(s), our attempts to identify such components by mass spectrometry have not yielded potential candidates.

Interestingly, GC-1 was previously shown to stably express in cell culture where it localizes to either ciliary or intracellular membranes (Bhowmick et al., 2009; Peshenko et al., 2015). This strikes at the difference between the composition of cellular components supporting membrane protein stabilization and transport in cell culture models versus functional photoreceptors. The goal of future experiments is to determine whether these protein localization patterns would be affected by co-expressing GC-1 with rhodopsin, thereby gaining further insight into the underlying intracellular trafficking mechanisms.

Finally, GC-1 trafficking was reported to depend on the small protein, RD3, thought to stabilize both guanylate cyclase isoforms, GC-1 and GC-2, in biosynthetic membranes (Azadi et al., 2010; Zulliger et al., 2015). In the case of GC-1, this stabilization would be complementary to that by rhodopsin and potentially could take place at different stages of GC-1 maturation and trafficking in photoreceptors. Another proposed function of RD3 is to inhibit the activity of guanylate cyclase isoforms outside the outer segment in order to prevent undesirable cGMP synthesis in other cellular compartments (Peshenko et al., 2011a).

In summary, this study explains how GC-1 reaches its intracellular destination without containing a dedicated targeting motif, expands our understanding of the role of rhodopsin in photoreceptor biology and extends the diversity of signaling proteins found in GPCR complexes to a member of the guanylate cyclase family. Provided that the cilium is a critical site of GPCR signaling in numerous cell types (Schou et al., 2015), it would be interesting to learn whether other ciliary GPCRs share rhodopsin’s ability to stabilize and deliver fellow members of their signaling pathways


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DNA mutagenesis and DNA repair

DNA mutagenesis and DNA repair

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence – Series E (2; 2.11)

Evelyn M. Witkin, born Evelyn Maisel is an American geneticist who was awarded the National Medal of Science for her work on DNA mutagenesis and DNA repair.

She earned her Ph.D. in 1947 with Theodosius Dobzhansky at Columbia University for her Drosophila research. Her interests evolved from Drosophila genetics to bacterial genetics, and she spent the summer of 1944 at Cold Spring Harbor, where she isolated a radiation-resistant mutant of E. coli. Witkin remained at the Carnegie Institution Department of Genetics at Cold Spring Harbor until 1955.

In 1971, she was appointed Professor of Biological Sciences at Douglass College, Rutgers University, and was named Barbara McClintock Professor of Genetics in 1979. Witkin moved to the Wakeman Institute at Rutgers University in 1983. Among her many honors are membership in the National Academy of Sciences (1977), Fellow of the American Association for the Advancement of Science (1980), American Women of Science Award for Outstanding Research, and Fellow of the American Academy of Microbiology. She was largely responsible for creating the field of DNA mutagenesis and DNA repair, which has played an important role in the biochemical sciences and in clinical radiation therapy for cancer. She is a member of the National Academy of Sciences, a Fellow of the American Association for the Advancement of Science, and the American Academy of Arts and Sciences. Dr. Witkin has also received the Thomas Hunt Morgan Medal of the Genetics Society of America in 2000 and the the Wiley Prize.

Oral History

The reason that I got into genetics in the first place, this is while I was an undergraduate [was that] a friend of mine, my first boyfriend, was a Harvard student who was quite a radical and he had gotten hold of [Trofim Denisovich] Lysenko’s papers. And we would read them and you know, Lysenko didn’t believe in the gene. He thought Mendel was a bourgeois nothing and didn’t believe in the gene and all you had to do was manipulate the environment and you could produce anything. And he was in power. You know, to Stalin this sounded great. You know the story. But at any rate, I didn’t know any genetics yet. So this sounded very nice. It would be wonderful to show that you could just manipulate the environment and change things. And I got intrigued and I thought this would be something to look into and my idea was in going into genetics was to test this! Actually it took about a month of genetics at Columbia to realize that he [Lysenko] was completely a fraud, but I never told Dobzhansky until his fiftieth [birthday] party. It was why I got into genetics and why I wanted a Russian advisor! I confessed, and that’s why—he thought it was absolutely hilarious.

When the Demerec lab was built we all had the opportunity to design our own laboratories. That was fun! And mine was right next to Hershey’s and that was a pleasure.

Well, he was an interesting man too. He was rather—you’ve probably heard from others—he was rather reserved and very focused on his work and not very sociable, but extremely generous and kind. And obviously very brilliant! He was very helpful to me.

I could tell you one story about the 1946 symposium, which I don’t know whether you’ve heard. It’s a story about [Salvador] Luria. He was one of the organizers of the program. That was the first symposium after the war and it was the first one on microorganisms and he and some others were getting the program together and he had seen a paper by a woman named Mary Bunting which was published in 1938. [She] worked with bacteria. He was very impressed by that paper. He said that was really the only paper he knows of that does real bacterial genetics before his work with Delbrück. And he said, “She has to be on the program. We have to find her and get her to come to the symposium.” Nobody knew where Mary Bunting was and he did some real hunting. He got other people to work on it, and I remember he was saying at one point, “We can’t have the symposium without her. We have to find her!” Well, they did find her. Her husband was on the faculty at Yale Medical School and they located her there. And she had three young children and was expecting a fourth. That was her Ph.D. Thesis—this paper [that Luria spoke about] She hadn’t been doing any scientific work for awhile and when Luria proposed that she come talk about this work at the symposium she was horrified and said she couldn’t possibly, and doesn’t even remember and “Go away!” And he was very insistent and she did turn up at the symposium and gave this wonderful talk. And it really changed her life forever. I don’t know if you [know], [but] she became the Dean of Radcliffe College.

But what happened at the symposium was, since she was bogged down with babies at Yale, some of the people here who were at Yale—[such as] Ed Tatum, I remember—they arranged for her to have some lab space at Yale and invited her to come and do some work whenever she had time. She came to seminars and caught up with things. And then she got a job when her children were a little bigger. I think her husband died around there too. She became the Dean of the Douglas College at Rutgers and she had done some very good work there in microbiology. And then she went on to become the Dean of Radcliffe and started the Mary Bunting Institute there, which is an institute designed to make it possible for women to resume their scientific careers after they’ve had a break for family reasons. She told me—I called her at one point to verify this because I mentioned this at some meeting, and I think I talked about it at the phage meeting in ’95—and she verified the fact and she said she would never have gone back to work if not for Luria’s having dug her out. Yes, it really shows things about Luria that I’m not sure are known. One is that he’s—he takes it for granted that men and women are equal. It didn’t occur to him that there’s a reason why she shouldn’t be there. And he also is very generous scientifically. He wanted her to have the credit for doing the first real genetics in bacteria.

Well, I guess I met Barbara [McClintock] my very first day here. I stayed at the dormitory and she was living there at the time. I guess I met here in the living room of the dormitory the very first day that I came and we started talking. I found her absolutely fascinating; she told me quite a lot about what she was doing. We became really good friends. And I spent a lot of time visiting her lab, and she developed a habit of calling me whenever she had something especially exciting. These were the years when she was beginning to discover transposition. I would just sort of drop everything and run over and she would show me something that was beginning to make sense to her and it was just such a privilege to be in that relation[ship] with her—to watch this story develop. It was unmistakably convincing as you explained it. You know, not having known very much about maize genetics it wasn’t easy for me to follow. But she was very patient about describing the experiments and she really was very confident about what she was doing.

Harvard geneticist wins Lasker Award for DNA work

Stephen J. Elledge Ph.D. is a Professor of Genetics at the Harvard Medical School. He earned his B.Sc. in chemistry from the University of Illinois at Urbana–Champaign and his Ph.D. in biology from MIT.

Education: Massachusetts Institute of Technology

Awards: Gairdner Foundation International Award, NAS Award in Molecular Biology, Lasker Award 2015

Harvard geneticist Stephen Elledge started his scientific career trying to figure out how to tinker with the DNA of human cells. Instead, he ended up eavesdropping on the process cells use to fix genetic mistakes.

This accidental work led to profound insights into DNA repair relevant to human birth defects, cancer, and aging.

Stephen J. Elledge, the Gregor Mendel Professor of Genetics and of Medicine at Harvard Medical School and Brigham and Women’s Hospital, is a co-recipient, with Evelyn Witkin of Rutgers University, of the 2015 Albert Lasker Basic Medical Research Award. The award, widely considered to be among the most respected in biomedicine, will be presented on Sept. 18 in New York City.

Elledge and Witkin are being honored for their seminal discoveries that have illuminated the DNA damage response, a cellular pathway that senses when DNA is altered and sets in a motion a series of responses to protect the cell. This pathway is critical to a better understanding of many diseases and conditions, such as cancer.

As cells divide and reproduce, they have to make precise copies of their DNA. Typos can doom a fetus, lead to birth defects, and cause cancer as well as symptoms of aging.

Elledge, who is also affiliated with Brigham and Women’s Hospital, uncovered a sequence of events, called a pathway, that protect a cell once its DNA has been damaged or incorrectly copied.

Harvard Medical School and Brigham and Women’s Hospital scientist recognized for discovering DNA repair

“Steve is an amazing scientist, mentor, and colleague,” said Jeffrey S. Flier, dean of Harvard Medical School (HMS). “His insights into the basic mechanisms of the DNA damage response have profoundly enriched our understanding not only of the fundamental genetics of all cellular life, but also of how we conceptualize many diseases and conditions, especially cancer. This distinction is richly deserved, and I am delighted that Steve is being honored for this extraordinary body of work.”

Stephen J. Elledge: Driven by Questions

Harvard geneticist Stephen J. Elledge has been recognized with the Lasker Award for a discovery that illuminates the DNA damage response. This pathway is critical to a better understanding of many diseases and conditions, such as cancer. Co-produced by Harvard Medical School and Brigham and Women’s Hospital.

“We are extremely proud of Steve, who is truly deserving of this recognition,” said Elizabeth G. Nabel, president of Brigham and Women’s Health Care. “Courageous and insatiably inquisitive, he represents the best of Brigham and Women’s and our mission of driving innovation in basic science to improve human health. As a devoted mentor, Steve is deeply committed to guiding the careers of young investigators, ensuring that the next generation of scientists is filled with curious, passionate, and talented researchers.”

Elledge often describes the process by which a cell duplicates itself as akin to the duplication of a small city. It is a vastly complex process that requires many levels of intricate coordination. Each cell contains a detailed blueprint for this entire process: DNA.

But not every duplication results in a perfect copy. That is because each time a cell makes a copy of itself, DNA is vulnerable to damage, not only from faulty cellular processes, but also from such entities as environmental chemicals. As DNA damage accumulates, it profoundly complicates a cell’s ability to make a faithful copy of itself. This can lead to serious illnesses, birth defects, cancer, and other health problems.

Witkin discovered how bacteria respond to DNA damage, detailing the response to UV radiation. Elledge uncovered a DNA-damage-response pathway that operates in more complex organisms, including humans.

Over the years, Elledge and his colleagues elucidated a signaling network that informs a cell when DNA sustains an injury. Called the DNA damage response, this network senses the problem and sends a signal to the rest of the cell so it can properly repair itself; otherwise, severe mutations could occur. As a result, this pathway helps keep the genome stable and suppresses adverse events such as tumor development. When individuals are born with mutations in this pathway, they often have severe developmental defects. If the pathway is interfered with later in life, cancer can result.

In addition to the award in basic medical research, the Lasker Foundation is also presenting awards to individuals in clinical research and in public service.

According to Claire Pomeroy, president of the Albert and Mary Lasker Foundation, this year’s recipients “remind us all that investing in biological sciences and medical research is crucial for our future.”

Joseph L. Goldstein of the University of Texas Southwestern Medical Center and chair of the Lasker Medical Research Awards Jury, added, “The 2015 Lasker winners had bold ideas and pursued novel questions that they tested through fearless experimentation.”

Over the past century, researchers have invested substantial efforts toward understanding the cell cycle. However, only recently have these studies gained a molecular foothold. Leading the research in this field is Stephen J. Elledge, professor of genetics at Harvard Medical School and Brigham and Women’s Hospital in Boston. Playing the dual roles of inventor and investigator, Elledge developed original techniques to define what drives the cell cycle and how cells respond to DNA damage. By using these tools, he and his colleagues have identified multiple genes involved in cell-cycle regulation.

Elledge’s work has earned him many awards, including a 2001 Paul Marks Prize for Cancer Research and a 2003 election to the National Academy of Sciences. In his Inaugural Article (1), published in this issue of PNAS, Elledge and his colleagues describe the function of Fbw7, a protein involved in controlling cell proliferation. These findings add to the growing cache of cell-cycle knowledge with implications for cancer research.

During his junior year abroad at the University of Southampton in England, Elledge gave biology a try by taking an introductory course and a semester of genetics. The classes sparked an interest, which he kept alive by taking a biochemistry class on his return to the United States. It was during his biochemistry lectures that Elledge first heard about recombinant DNA. “I just thought it was fabulous,” he said. “Once biology got down to being molecular, then it intersected with my interests.”

After receiving his bachelor’s degree in 1978, Elledge applied to graduate programs in biology and chemistry. Although he had not yet decided on which field to focus, he chose to continue his studies at the Massachusetts Institute of Technology (MIT) Biology Department. “I didn’t know what I wanted to do, but they had a lot of people, so I figured I’d be able to sort it out,” he said. Elledge ended up working with bacterial geneticist Graham Walker. For his thesis, Elledge studied the error-prone DNA repair mechanism in Escherichia coli called SOS mutagenesis. His work identified and described the regulation of a group of enzymes now know as errorprone polymerases, the first members of which were the umuCD genes in E. coli (2–4).

Elledge’s schedule at MIT allowed him time for side projects, and he used the opportunity to develop a new cloning tool. His creation was spurred by the frustration of unsuccessfully trying to use two existing tools, lambda phage and bacterial plasmid libraries, to clone the umuC gene, which produces proteins necessary for UV and chemical mutagenesis in E. coli. By combining the tools, Elledge invented a technique that allowed him to approach future cloning problems of this type with great rapidity (5). With the new technique, “you could make large libraries in lambda that behave like plasmids. We called them `phasmid’ vectors, like plasmid and phage together,” said Elledge. The phasmid cloning method was an early cornerstone for molecular biology research.

In 1984, Elledge began a postdoctoral fellowship at Stanford University (Stanford, CA) with mentor Ronald Davis. “Davis is an inventor,” said Elledge. “We had a lot in common because I’m interested in developing new technologies and so is he.”

Elledge soon began working on homologous recombination, an important niche in the field of eukaryotic genetics. Working with the yeast genome, Elledge searched for rec A, a gene that allows DNA to recombine homologously. Although he never located rec A, his work accidentally led him to a family of genes known as ribonucleotide reductases (RNRs), which are involved in DNA production (6). Rec A and RNRs share the same last 4 amino acids, which caused an antibody crossreaction in one of Elledge’s experiments. Initially disappointed with the false positives in his hunt for rec A, Elledge was later delighted with his luck. He found that RNRs are turned on by DNA damage (6), and that these genes are regulated by the cell cycle (7). “It was just serendipity,” he said.

Elledge’s work in this area led to a job offer from Baylor College of Medicine, Houston, in 1989. Prior to leaving Stanford, Elledge attended a talk at the University of California, San Francisco, by Paul Nurse, a leader in cell-cycle research who would later win the 2001 Nobel Prize in medicine. Nurse described his success in isolating the homolog of a key human cell-cycle kinase gene, Cdc2, by using a mutant strain of yeast (8). Although Nurse’s methods were primitive, Elledge was struck by the message he carried: that cell-cycle regulation was functionally conserved, and that many human genes could be isolated by looking for complimentary genes in yeast. Elledge then took advantage of his past successes in building phasmid vectors to build a versatile human cDNA library that could be expressed in yeast.

In his first experiments after setting up a laboratory at Baylor, he introduced this library into yeast, screening for complimentary cell-cycle genes. He quickly identified the same Cdc2 gene isolated by Nurse. However, Elledge also discovered a related gene known as Cdk2. Elledge subsequently found that Cdk2 controlled the G1 to S cell-cycle transition, a step that often goes awry in cancer. These results were published in theEMBO Journal in 1991 (9). “It was one of the biggest papers I’ve had,” said Elledge.

Elledge also continued to capitalize on his unexpected discovery of RNRs and used them to perform genetic screens to identify genes involved in sensing and responding to DNA damage. He subsequently worked out the signal transduction pathways in both yeast and humans that recognize damaged DNA and replication problems (1012). These “checkpoint” pathways are central to the prevention of genomic instability and a key to understanding tumorigenesis.

Elledge’s research caught the attention of Wade Harper, a new member of Baylor’s biochemistry faculty. Combining their efforts, Harper and Elledge studied the regulation of Cdk2. “I was a geneticist and Wade was a biochemist. Together we were able to accomplish much more than either alone,” said Elledge. Elledge revamped a method for detecting protein interactions, known as the “two-hybrid system,” into a cloning method by combining it with his lambda cloning techniques. By using the new method, Harper and Elledge succeeded in isolating a gene known as p21, which they later identified as part of a family of Cdk2inhibitors. The gene also was cloned by Bert Vogelstein’s laboratory at Johns Hopkins University (Baltimore, MD), who discovered p21 was regulated by the cancer gene p53. Elledge and Vogelstein realized the similarity of their findings after chatting on the phone and published articles back-to-back in Cell in 1993 (13,14).

Elledge and his laboratory continued to look for other human genes that complimented yeast cell-cycle regulators. In 1996, his team identified a conserved motif, the F-box, that is present in some proteins. This motif recognizes specific protein sequences and tags them with ubiquitin for destruction. The buildup of certain proteins can sabotage the cell cycle and bring it to a halt; thus, destroying these proteins keeps cells dividing. Further investigation showed that the F-box sequence is ubiquitous throughout evolution. “There were so many F-box proteins that we figured it was going to be very central,” he said. Since Elledge’s laboratory published its first article on the F-box in 1998 (15), almost a thousand articles have reported investigations of F-box proteins and related ubiquitin ligases. The F-box has been implicated in numerous pathways, including gene expression, the immune response, cell morphology, cancer, and circadian rhythms.

Elledge’s focus still centers on the F-box motif and the roles played by its multitude of variations. In his Inaugural Article, found on page 3338, Elledge and his colleagues (1) describe mouse knockouts missing the gene to create an F-box protein known as Fbw7. Previous research suggested that Fbw7 controls the degradation of cyclin E, a protein that drives cell proliferation. By studying the knockouts, Elledge’s team showed that Fbw7 controls not only the abundance of cyclin E but also Notch protein. Both of these proteins play key roles in regulating mammalian development.

Elledge’s findings add to the growing body of knowledge on how F-box proteins operate in cells. However, with the function of hundreds of different F-box proteins currently unknown, Elledge and his collaborators, including Wade Harper, will have their work cut out for decades more. He and his laboratory plan to continue studying the genetics and genomics of different F-box proteins, elucidating their roles in cell proliferation. Elledge expects that this vast mystery, combined with his regular discoveries, will keep his passion alive. “I’m a scientist. I want to discover new things, and I want to develop new ways of looking at things. That’s what makes me excited, and that’s what I’m interested in,” he said.

This is a Biography of a recently elected member of the National Academy of Sciences to accompany the member’s Inaugural Article on page 3338.


Tetzlaff, M. T., Yu, W., Li, M., Zhang, P., Finegold, M., Mahon, K., Harper, J. W., Schwartz, R. J. & Elledge, S. J. (2004) Proc. Natl. Acad. Sci. USA 100 , 3338-3345.

Elledge, S. J. & Walker, G. C. (1983) J. Mol. Biol. 164 , 175-192.

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  1. Elledge, S. J. & Walker, G. C. (1983) J. Bacteriol. 155 , 1306-1315.

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Perry, K., Elledge, S. J., Mitchell, B. B., Marsh, L. & Walker, G. C. (1985) Proc. Natl. Acad. Sci. USA 82 , 4331-4335.

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Elledge, S. J. & Walker, G. C. (1985) J. Bacteriol. 162 , 777-783.

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Elledge, S. J. & Davis, R. W. (1987) Mol. Cell. Biol. 7 , 2783-2793.

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Elledge, S. J. & Davis, R.W. (1990) Genes Dev. 4, 740-751.

Abstract/FREE Full Text

Lee, M. G. & Nurse, P. (1987) Nature 32 , 31-35.

Elledge, S. & Spottswood, M. (1991) EMBO J. 10 , 2653-2659.

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Zhou, Z. & Elledge, S. J. (1993) Cell 75, 1119-1127.

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Abstract/FREE Full Text

Zou, L. & Elledge, S. J. (2003) Science 300, 1542-1548.

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Harper, J. W., Adami, G., Wei, N., Keyomarsi, K. & Elledge, S. J. (1993) Cell 75 , 805-816.

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El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W. & Vogelstein, B. (1993) Cell 75, 817-825.

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Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J.W. & Elledge, S. J. (1996) Cell 86, 263-274.

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The DNA Damage Response—Self-awareness for DNAThe 2015 Albert Lasker Basic Medical Research Award

Stephen J. Elledge, PhD1

The 2015 Albert Lasker Basic Medical Research Award has been presented to Stephen J. Elledge, PhD, for discoveries concerning the DNA-damage response—a fundamental mechanism that protects the genomes of all living organisms. This Viewpoint provides a summary of the role of the DNA damage response in physiologic responses and the importance in human health.

One of the remarkable properties of nature’s most remarkable molecule, DNA, is self-awareness: it can detect information about its own integrity and transmit that information back to itself. The pathway responsible for this impressive ability is known as the DNA damage response (DDR). The first thoughts many scientists have about DNA damage involve the stereotypical DNA repair pathways such as nucleotide excision repair or base excision repair, which identify damaged bases, excise them, and perfectly patch the DNA. However, there is a much higher-level orchestrator of the cellular response to damaged DNA that deals with nonstereotypical and supremely deleterious alterations of DNA structure and distribution of information about their existence.

Particularly deleterious are the events that break both strands of the DNA or disrupt the most vulnerable aspect of the DNA molecule, its replication. These events require the cell to possess the ability to distinguish the myriad possible structures resulting from these events. Furthermore, cells require this knowledge to properly resolve these problems. If this fails, the integrity of the genome is lost and significantly deleterious events can ensue. Much like the brain, which takes sensory input and transduces that information through neural circuitry to effect the proper response, the DDR acts as a sensor that transduces information on the status of the integrity of the genome to elicit the proper response.

The DDR is a form of chemical intelligence. It ensures that the enzymes that have the ability to remodel the structure of DNA—enzymes that are actually dangerous to DNA if used inappropriately—are activated and deployed at the right time and right place to resolve a particular altered DNA structure to maintain genomic integrity.1 Morever, it is not only the repair enzymes that are the recipients of this information but also many aspects of cellular and organismal physiology.


The notion that cells respond to DNA damage has it roots in basic genetic research dating back to the 1940s, in work by Jean Weigle and Evelyn Witkin that contributed to knowledge of the SOS response in bacteria.2Years later my laboratory and others, through genetic research using the yeasts Saccharomyces cerevisiaeand Schizosaccharomyces pombe,discovered the major components of a very complex eukaryotic DDR.3Genetic analysis of the DNA damage–induced transcriptional induction of genes and radiation-sensitive mutants that control cell cycle transitions uncovered the core DDR genes. This provided a foothold for the transition to human cells, in which the significance of this pathway to human health emerged.


The core of the DDR is a pair of parallel protein kinase cascades that sense and distribute information about different classes of DNA structures. The ATM branch senses double-strand breaks and the ATR branch senses stalled replication structures and certain double-strand breaks that occur during S phase. ATM and ATR are both protein kinases of the PIKK family and when activated in response to damage, they phosphorylate downstream checkpoint kinases, Chk2 and Chk1, respectively, to transduce the damage signal.

The effect of activation of these pathways is substantial. More than 1000 proteins are altered by the DDR in response to structural alterations in DNA, profoundly altering cellular physiology.4 Beyond repair, communicating the information of DNA damage is critical for multicellular organisms in many other ways. The relevance of the DDR to human health is demonstrated by the more than 30 human disease syndromes that result from mutations in DDR genes spanning developmental disorders, neural degeneration, immune dysregulation, progeria, cancer, and other critical diseases.1 Below are several examples of some of these connections.


Immune Cell Function

Cells undergoing immunoglobulin and T-cell receptor rearrangement experience programmed double-strand breaks and require the DDR to properly execute these recombination events. ATM activation after initial cleavage of one allele results in the repositioning of the other allele to the nuclear periphery to ensure monoallelic recombination.1 ATM also arrests the cell cycle in response to programmed breaks to ensure recombination prior to S-phase entry. If S-phase entry occurs before the breaks are repaired, it can promote translocations. Immune deficiency also arises from mutations in several DDR genes including the RNF168gene responsible for RIDDLE syndrome, characterized by radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties.

Brain Development and Neural Degeneration

Mutations in multiple DDR components lead to developmental defects. Brain development in particular appears to be especially sensitive to defects in DNA repair and DDR function. Hypomorphic alleles of ATRcause Seckel syndrome, which is characterized by dwarfism, severe microcephaly, and facial malformation and mental retardation. Microcephaly is also associated with mutations in NBN and MRE11, both regulators of the ATM branch of the DDR, and mutations in MCPH1/BRIT1.1

Mutations in the ATM gene result in ataxia telangiectasia, a debilitating disease in which progressive loss of purkinjee cells in the cerebellum leads to ataxia. Patients with ataxia telangiectasia also develop weakened immune systems and high rates of infection and premature mortality.

Hematopoetic Disorders

Mutations in the Fanconi anemia pathway, which is controlled by ATR phosphorylation, results in numerous developmental defects including hematologic abnormalities and bone marrow failure. Patients with Fanconi anemia experience increased frequencies of myelodysplastic syndrome, and many develop acute myelogenous leukemia.

Responding to Viral Infections

A complex relationship exists between viral infection and the DDR. Many DNA viruses, including adenovirus, Kaposi sarcoma–associated herpesvirus, hepatitis B virus, and Epstein-Barr virus, activate the DDR because viral replication intermediates resemble DNA damage. Viruses may also indirectly activate the DDR by expressing oncoproteins that force cells into S phase and generate replicative stress. The DDR can signal directly to the immune system by inducing ligands for the NKG2D and DNAMA-1 receptors5expressed on natural killer cells and CD8+ T cells, both of which are capable of killing cells and contributing to antiviral immunity. In some cases, viruses such as SV40 have grown to depend on the DDR, and other viruses such as adenoviruses go to great lengths to inactivate the DDR, underscoring its role in controlling viral function.

The DDR and Cancer

The DDR is highly relevant to all aspects of cancer.6 Most critically, DDR function promotes genomic stability. Loss of a large number of DDR genes result in increased frequencies of cancer; these include ATM,NBS1p53BRCA1BRCA2PALB2BRIPBLMWRNMCPH153BP1ATRCHK1CHK2, and numerous Fanconi anemia genes whose loss enhance the frequencies of alterations in classical tumor suppressors and oncogenes. Second, the DDR is also relevant to the effectiveness of classic therapeutic treatments, such as radiotherapy and chemotherapy, because these therapies are based on induction of DNA damage, which triggers DDR-dependent cell death, particularly in proliferating cells. Because many tumors become defective in some aspect of the DDR, they become more dependent on other DDR or DNA repair components, and cancer therapies directed at inhibiting key components like CHK1, ATR, or PARP are being evaluated in clinical trials.6

Aging and Telomeres

Another critical sensory event occurs when somatic cells exceed their intended proliferative lifetimes, such as in the early stages of cancer. In these cells telomeric ends of chromosomes shorten and are sensed as DNA damage, activating the DDR. Telomere shortening also occurs in a normal physiological setting with aging. Under these conditions, DDR activation informs the cell such that it may choose to undergo apoptosis or a differentiation pathway called senescence, both potent tumor suppressive mechanisms. Senescence relies on 2 of the most potent tumor suppressors, p53 and p16.7 Importantly, the accumulation of senescent cells has been implicated in acceleration of aging and age-related diseases. Senescent cells secrete cytokines and chemokines and contribute to progressive chronic inflammation, which may contribute to aging and age-related diseases. Removing senescent cells reduces aging in mice.7 Thus, the DDR is a 2-edged sword. On the one hand it acts as a barrier to tumorigenesis and on the other hand it acts to promote aging and aging-related diseases.


The significance of the DDR to human health is clear. Further highlighting its importance is that the loss of the most central components make it impossible to make, not only an organism, but even a cell. Thus, the self-awareness afforded to the DNA molecule by the sensory and information distribution system known as the DDR is critical for the health and survival of the human species. This once again underscores the importance to human health of basic research in model organisms.


Corresponding Author: Stephen J. Elledge, PhD, Department of Genetics, Harvard Medical School, 77 Ave Louis Pasteur, Room 158D, NRB, Boston, MA 02115 (selledge@genetics.med.harvard.edu).

Published Online: September 8, 2015. doi:10.1001/jama.2015.10387.

Conflict of Interest Disclosures: The author has completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.


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Elledge  SJ.  Cell cycle checkpoints: preventing an identity crisis. Science. 1996;274(5293):1664-1672.
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Matsuoka  S, Ballif  BA, Smogorzewska  A,  et al.  ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science. 2007;316(5828):1160-1166.
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Cerboni  C, Fionda  C, Soriani  A,  et al.  The DNA damage response: a common pathway in the regulation of NKG2D and DNAMA-1 ligand expression in normal, infected, and cancer cells. Front Immunol. 2014;4:508.
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Lord  CJ, Ashworth  A.  The DNA damage response and cancer therapy. Nature. 2012;481(7381):287-294.
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van Deursen  JM.  The role of senescent cells in ageing. Nature. 2014;509(7501):439-446.
PubMed   |  Link to Article

Novel Protein May Open Door to New Therapies for Infection and Cancer

GEN News Aug 28, 2015 http://www.genengnews.com/gen-news-highlights/novel-protein-may-open-door-to-new-therapies-for-infection-and-cancer/81251675/

Scientists at Florida State University say they have taken a critical step forward in the fight against cancer with a discovery that could open up the door for new research and treatment options.

Fanxiu Zhu, Ph.D., the FSU Margaret and Mary Pfeiffer Endowed Professor for Cancer Research, and his team uncovered a viral protein in the cell that inhibits the major DNA sensor and thus the body’s response to viral infection, suggesting that this cellular pathway could be manipulated to help a person fight infection, cancer, or autoimmune diseases. They named the protein KicGas.

“We can manipulate the protein and/or the sensor to boost or tune down the immune response in order to fight infectious and autoimmune diseases, as well as cancers,” said Dr. Zhu, whose study (“Inhibition of cGAS-cGAMP DNA-Sensing Signaling by a Herpesvirus Virion Protein”) was published in Cell Host and Microbe.

Dr. Zhu leads a research team investigating how DNA viruses can cause cancer. About 15% of human cancer cases are caused by viruses, so scientists have been seeking answers about how the body responds to viral infection and how some viruses maintain life-long infections.

In the past few years, researchers finally identified the major DNA sensor in cells, known as cGas. That spurred researchers to further examine this sensor in the context of human disease because ideally that sensor should have been alerting the body to fight disease brought by a DNA virus.

Although people are equipped with sophisticated immune systems to cope with viral infection, many viruses have co-evolved mechanisms to evade or suppress the body’s immune responses. So the discovery of this protein is critical to further exploration of how these DNA viruses work and how they can be thwarted.

To uncover this protein, Dr. Zhu’s team studies Kaposi’s sarcoma-associated herpesvirus (KSHV), a human herpesvirus that causes some forms of lymphoma and Kaposi’s sarcoma, a cancer commonly occurring in AIDS patients and other immunocompromised individuals.

In this study, researchers screened every protein in a KSHV cell (90 in total) and ultimately found that one of them directly inhibited the DNA sensor called cGAS. They infected human cell lines with the Kaposi’s sarcoma virus to mimic natural infection, and found when they eliminated the inhibitor protein (KicGas) the cells produced a much stronger immune response.

To do this work, Dr. Zhu collaborated with several scientists both in the U.S. and Germany, including Hong Li, Ph.D., FSU professor of chemistry and biochemistry.

Dr. Li, whose focuses are molecular biology and molecular biophysics, specifically examined how the protein inhibited the cGAS activity in test tubes. For the next phase of research, she is building a three-dimensional model of the interactions to help them better understand how the inhibitor functions.

“These are hard problems to solve, and there is still much to learn here,” Dr.  Li said. Learning how the inhibitor functions is a big next step, though. “Once we figure that out, we can hopefully design something to fight the disease,” according to Dr. Zhu.

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Loss of Gene Islands May Promote a Cancer Genome’s Evolution: A new Hypothesis on Oncogenesis

Loss of Gene Islands May Promote a Cancer Cell’s Survival, Proliferation and Evolution: A new Hypothesis (and second paper validating model) on Oncogenesis from the Elledge Laboratory

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

It is well established that a critical event in the transformation of a cell to the malignant state involves the mutation of hosts of oncogenes and tumor suppressor genes, which in turn, confer on a cell the inability to properly control its proliferation.    On a genomic scale, these mutations can result in gene amplifications, loss of heterozygosity (LOH), and epigenetic changes resulting in tumorigenesis.  The “two hit hypothesis”, proposed by Dr. Al Knudson of Fox Chase Cancer Center[1], proposes that two mutations in the same gene are required for tumorigenesis, initially proposed to explain the progression of retinoblastoma in children, indicating a recessive disease.

(Excerpts from a great article explaining the two-hit-hypothesis is given at the end of this post).

And, although many tumor genomes display haploinsufficeint tumor suppressor genes, and fit the two hit model quite nicely, recent data show that most tumors display hemizygous recurrent deletions within their genomes.  Tumors display numerous recurrent hemizygous focal deletions that seem to contain no known tumor suppressor genes. For instance a recent analysis of over three thousand tumors including breast, bladder, pancreatic, ovarian and gastric cancers averaged greater than 10 deletions/tumor and 82 regions of recurrent focal deletions,

It has been proposed these great number of hemizygous deletions may be a result of:

Note: some definitions of hemizygosity are given below.  In general at any locus, each parental chromosome can have 3 deletion states:

  1. wild type
  2. large deletion
  3. small deletion

Hemizygous deletions only involve one allele, not both alleles which is unlike the classic tumor suppressor like TP53

To see if it is possible that only one mutated allele of a tumor suppressor gene may be a casual event for tumorigenesis, Dr. Nicole Solimini and colleagues, from Dr. Stephen Elledge’s lab at Harvard, proposed a hypothesis they termed the cancer gene island model, after analyzing the regions of these hemizygous deletions for cancer related genes[2].  Dr. Soliin and colleagues analyzed whole-genome sequence data for 526 tumors in the COSMIC database comparing to a list generated from the Cancer Gene Census for homozygous loss-of-function mutations (mutations which result in a termination codon or frame-shift mutation: {this produces a premature stop in the protein or an altered sequence leading to a nonfunctional protein}.

Results of this analysis revealed:

  1. although tumors have a wide range of deletions per tumor (most epithelial high grade like ovarian, bladder, pancreatic, and esophageal adenocarcinomas had 10-14 deletions per tumor
  2. and although tumors exhibited a wide range (2- 16 ) loss of function mutations
  3. ONLY 14 of 82 recurrent deletions contained a known tumor suppressor gene and was a low frequency event
  4. Most recurrent cancer deletions do not contain putative tumor suppressor genes.

Therefore, as the authors suggest, an alternate method to the two-hit hypothesis may account for a selective growth advantage for these types of deletions, defining these low frequency hemizygous mutations in two general classes

  1. STOP genes: suppressors of tumor growth and proliferation
  2. GO genes: growth enhancers and oncogenes

Identifying potential STOP genes

To identify the STOP and GO genes the authors performed a primary screen of an shRNA library in telomerase (hTERT) immortalized human mammary epithelial cells using increased PROLIFERATION as a screening endpoint to determine STOP genes and decreased proliferation and lethality (essential genes) to determine possible GO genes. An initial screen identified 3582 possible STOP genes.  Using further screens and higher stringency criteria which focused on:

the authors were able to focus on and validate 878 genes to determine the molecular pathways involved in proliferation.

These genes were involved in cell cycle regulation, apoptosis, and autophagy (which will be discussed in further posts).

To further validate that these putative STOP genes are relevant in human cancer, the list of validated STOP genes found in the screen was compared to the list of loss-of-function mutations in the 526 tumors in the COSMIC databaseSurprisingly, the validated STOP gene list were significantly enriched for known and possibly NOVEL tumor suppressor genes and especially loss of function and deletion mutations but also clustered in gene deletions in cancer.  This not only validated the authors’ model system and method but suggests that hemizygous deletions in multiple STOP genes may contribute to tumorigenesis

as the function of the majority of STOP genes is to restrain tumorigenesis

A few key conclusions from this study offer strength to an alternative view of oncogenesis NAMELY:

A link to the supplemental data containing STOP and GO genes found in validation screens and KEGG analysis can be found at the following link:


A link to an interview with the authors, originally posted on Harvard’s site can be found here.

Cumulative Haploinsufficiency and Triplosensitivity Drive Aneuploidy Patterns and Shape the Cancer Genome; a new paper from the Elledge group in the journal Cell


A concern of the authors was the extent to which gene silencing could have on their model in tumors.  The validation of the model was performed in cancer cell lines and compared to tumor genome sequence in publicly available databases however a followup paper by the same group shows that haploinsufficiency contributes a greater impact on the cancer genome than these studies have suggested.

In a follow-up paper by the Elledge group in the journal Cell[3], Theresa Davoli and colleagues, after analyzing 8,200 tumor-normal pairs, show there are many more cancer driver genes than once had been predicted.  In addition, the distribution and potency of STOP genes, oncogenes, and essential genes (GO) contribute to the complex picture of aneuploidy seen in many sporadic tumors.  The authors proposed that, together with these and their previous findings, that haploinsufficiency plays a crucial role in shaping the cancer genome.

Hemizygosity and Haploinsufficiency

Below are a few definitions from Wikipedia:

Zygosity is the degree of similarity of the alleles for a trait in an organism.

Most eukaryotes have two matching sets of chromosomes; that is, they are diploid. Diploid organisms have the same loci on each of their two sets of homologous chromosomes, except that the sequences at these loci may differ between the two chromosomes in a matching pair and that a few chromosomes may be mismatched as part of a chromosomal sex-determination system. If both alleles of a diploid organism are the same, the organism is homozygous at that locus. If they are different, the organism is heterozygous at that locus. If one allele is missing, it is hemizygous, and, if both alleles are missing, it is nullizygous.

Haploinsufficiency occurs when a diploid organism has only a single functional copy of a gene (with the other copy inactivated by mutation) and the single functional copy does not produce enough of a gene product (typically a protein) to bring about a wild-type condition, leading to an abnormal or diseased state. It is responsible for some but not all autosomal dominant disorders.

Al Knudsen and The “Two-Hit Hypothesis” of Cancer

Excerpt from a Scientist article by Eugene Russo about Dr. Knudson’s Two hit Hypothesis;

for full article please follow the link http://www.the-scientist.com/?articles.view/articleNo/19649/title/-Two-Hit–Hypothesis/

The “two-hit” hypothesis was, according to many, among the more significant milestones in that rapid evolution of biomedical science. The theory explains the relationship between the hereditary and nonhereditary, or sporadic, forms of retinoblastoma, a rare cancer affecting one in 20,000 children. Years prior to the age of gene cloning, Knudson’s 1971 paper proposed that individuals will develop cancer of the retina if they either inherit one mutated retinoblastoma (Rb) gene and incur a second mutation (possibly environmentally induced) after conception, or if they incur two mutations or hits after conception.3 If only one Rb gene functions normally, the cancer is suppressed. Knudson dubbed these preventive genes anti-oncogenes; other scientists renamed them tumor suppressors.

When first introduced, the “two-hit” hypothesis garnered more interest from geneticists than from cancer researchers. Cancer researchers thought “even if it’s right, it may not have much significance for the world of cancer,” Knudson recalls. “But I had been taught from the early days that very often we learn fundamental things from unusual cases.” Knudson’s initial motivation for the model: a desire to understand the relationship between nonhereditary forms of cancer and the much rarer hereditary forms. He also hoped to elucidate the mechanism by which common cancers, such as those of the breast, stomach, and colon, become more prevalent with age.

According to the then-accepted somatic mutation theory, the more mutations, the greater the risk of cancer. But this didn’t jibe with Knudson’s own studies on childhood cancers, which suggested that, in the case of cancers such as retinoblastoma, disease onset peaks in early childhood. Knudson set out to determine the smallest number of cancer-inducing events necessary to cause cancer and the role of these events in hereditary vs. nonhereditary cancers. Based on existing data on cancer cases and some mathematical deduction, Knudson came up with the “two-hit” hypothesis.

Not until 1986, when researchers at the Whitehead Institute for Biomedical Research in Cambridge, Mass., cloned the Rb gene, would there be solid evidence to back up Knudson’s pathogenesis paradigm.4 “Even with the cloning of the gene, it wasn’t clear how general it would be,” says Knudson. There are, it turns out, several two-hit lesions, including polyposis, neurofibromitosis, and basal cell carcinoma syndrome. Other cancers show only some correspondence with the two-hit model. In the case of Wilm’s tumor, for example, the model accounts for about 15 percent of the cancer incidence; the remaining cases seem to be more complicated.


His seminal paper on the two-hit hypothesis[1]

A.G. Knudson, “Mutation and cancer: statistical study of retinoblastoma,” Proceedings of the National Academy of Sciences, 68:820-3, 1971.

The two hit hypothesis proposed by A.G. Knudson.  A description with video of Dr. Knudson talk at AACR can be found at the following link (photo creditied to A.G. Knudson and Fox Chase Cancer Center at the following link:http://www.fccc.edu/research/research-awards/knudson/index.html


1.            Knudson AG, Jr.: Mutation and cancer: statistical study of retinoblastoma. Proceedings of the National Academy of Sciences of the United States of America 1971, 68(4):820-823.

2.            Solimini NL, Xu Q, Mermel CH, Liang AC, Schlabach MR, Luo J, Burrows AE, Anselmo AN, Bredemeyer AL, Li MZ et al: Recurrent hemizygous deletions in cancers may optimize proliferative potential. Science 2012, 337(6090):104-109.

3.            Davoli T, Xu Andrew W, Mengwasser Kristen E, Sack Laura M, Yoon John C, Park Peter J, Elledge Stephen J: Cumulative Haploinsufficiency and Triplosensitivity Drive Aneuploidy Patterns and Shape the Cancer Genome. Cell 2013, 155(4):948-962.

Other papers on this site on CANCER and MUTATION include:

Cancer Mutations Across the Landscape

Salivary Gland Cancer – Adenoid Cystic Carcinoma: Mutation Patterns: Exome- and Genome-Sequencing @ Memorial Sloan-Kettering Cancer Center

Whole exome somatic mutations analysis of malignant melanoma contributes to the development of personalized cancer therapy for this disease

Breast Cancer and Mitochondrial Mutations

Winning Over Cancer Progression: New Oncology Drugs to Suppress Passengers Mutations vs. Driver Mutations

Hold on. Mutations in Cancer do good.

Rewriting the Mathematics of Tumor Growth; Teams Use Math Models to Sort Drivers from Passengers

How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis.

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Sensors and Signaling in Oxidative Stress

Sensors and Signaling in Oxidative Stress

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

Article XI Sensors and Signaling in Oxidative Stress

Image created by Adina Hazan 06/30/2021

This is article ELEVEN in the following series on Calcium Role in Cardiovascular Diseases

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease
Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
and Aviva Lev-Ari, PhD, RN

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
Aviva Lev-Ari, PhD, RN


Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary
Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure –
Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells:
The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction
(Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of
vesicles with cell membranes during Neurotransmission
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part XI: Sensors and Signaling in Oxidative Stress
Larry H. Bernstein, MD, FCAP

Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


This important article on oxidative stress was published in Free Radical Biol. and Med.

Nrf2:INrf2(Keap1) Signaling in Oxidative Stress

James W. Kaspar, Suresh K. Niture, and Anil K. Jaiswal*
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD

Free Radic Biol Med. 2009 Nov; 47(9): 1304–1309.           http://dx.doi.org/10.1016/j.freeradbiomed.2009.07.035

Nrf2:INrf2(Keap1) are cellular sensors of chemical and radiation induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor INrf2. INrf2 functions as an adapter for Cul3/Rbx1 mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2cysteine151 and/or PKC phosphorylation of Nrf2serine40 results in

Nrf2 is stabilized and

The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis and promotes cell survival.


Oxidative stress is induced by a vast range of factors including xenobiotics, drugs, heavy metals and ionizing radiation. Oxidative stress leads to the generation of Reactive Oxygen Species (ROS) and electrophiles. ROS and electrophiles generated can have a profound impact on survival, growth development and evolution of all living organisms [1,2] ROS include

ROS and electrophiles can cause diseases such as cancer, cardiovascular complications, acute and chronic inflammation, and neurodegenerative diseases [1]. Therefore, it is obvious that

Much of what we know about the mechanisms of protection against oxidative stress has come from the study of prokaryotic cells [4,5]. Prokaryotic cells utilize transcription factors OxyR and SoxRS to sense the redox state of the cell, and

Eukaryotic cells have similar mechanisms to protect against oxidative stress [Fig. 1; ref. 3,6–9]. Initial effect of oxidative/electrophilic stress leads to activation of a battery of defensive gene expression that leads to detoxification of chemicals and ROS and prevention of free radical generation and cell survival [Fig. 1].

Fig 1.  Chemical and radiation exposure and coordinated induction of defensive genes.

Fig. 1. Chemical and radiation exposure and coordinated induction of defensive genes.

Of these genes, some are enzymes such as NAD(P)H:quinine oxidoreductase 1 (NQO1), NRH:quinone oxidoreductase 2 (NQO2), glutathione S-transferase Ya subunit (GST Ya Subunit), heme oxygenase 1 (HO-1), and γ-glutamylcysteine synthetase (γ-GCS), also known as glutamate cysteine ligase (GCL). Other genes have end products that regulate a wide variety of cellular activities including

There is a wide variety of factors associated with the cellular response to oxidative stress. For example,

whereas activation of c-jun, N-terminal kinases (JNK), p38 kinase and TP53 may lead to cell cycle arrest and apoptosis [10]. The Nrf2 pathway is regarded as the most important in the cell to protect against oxidative stress. [3,6–9]. It is noteworthy that accumulation of ROS and/or electrophiles leads to oxidative/electrophile stress,

These changes lead to degeneration of tissues and premature aging, apoptotic cell death, cellular transformation and cancer.

Antioxidant Response Element and Nrf2

Promoter analysis identified a cis-acting enhancer sequence designated as the antioxidant response element (ARE) that

The ARE sequence is responsive to a broad range of structurally diverse chemicals apart from β-nafthoflavone and phenolic antioxidants [12]. Mutational analysis revealed GTGACA***GC to be the core sequence of the ARE [11,13–14]. This core sequence is present in all Nrf2 downstream genes that respond to antioxidants and xenobiotics [3,6–9]. Nrf2 binds to the ARE and regulates ARE-mediated antioxidant enzyme genes expression and induction in response to a variety of stimuli including antioxidants, xenobiotics, metals, and UV irradiation [6,15–21].

Nrf2 is ubiquitously expressed in a wide range of tissue and cell types [22–24] and belongs to a subset of basic leucine zipper genes (bZIP) sharing a conserved structural domain designated as a cap’n’collar domain which is highly conserved in Drosphila transcription factor CNC (Fig. 2; ref. 25].

Fig. 2. Schematic Presentation of Various Domains of Nrf (Nrf1, Nrf2, Nrf3) and INrf2

Fig. 2. Schematic Presentation of Various Domains of Nrf (Nrf1, Nrf2, Nrf3) and INrf2

Nrf, NF-E2 Related Factor; INrf2, Inhibitor of Nrf2; NTR, N-Terminal Region; BTB, Broad complex, Tramtrack, Bric-a-brac; IVR, Intervening/linker Region; DGR, Kelch domain/ diglycine repeats; CTR, C-Terminal Region.

The basic region, just upstream of the leucine zipper region,

ARE-mediated transcriptional activation requires heterodimerization of Nrf2 with other bZIP proteins including Jun (c-Jun, Jun-D, and Jun-B) and small Maf (MafG, MafK, MafF) proteins [18– 20,26–27].

Initial evidence demonstrating the role of Nrf2 in antioxidant-induction of detoxifying enzymes came from studies on

Nrf2 was subsequently shown to be involved in

Overexpression of Nrf2 cDNA was shown to upregulate the expression and induction of the NQO1 gene in response to antioxidants and xenobiotics [17]. In addition, Nrf2-null mice exhibited a marked

The importance of this transcription factor in upregulating ARE-mediated gene expression has been demonstrated by several in vivo and in vitro studies [reviewed in ref. 3]. The results indicate that Nrf2 is an important activator of phase II antioxidant genes [3,8].

Negative Regulation of Nrf2 mediated by INrf2

A cytosolic inhibitor (INrf2), also known as Keap1 (Kelch-like ECH-associating protein 1), of Nrf2 was identified and reported [Fig. 2; ref. 34–35]. INrf2, existing as a dimer [36], retains Nrf2 in the cytoplasm. Analysis of the INrf2 amino acid sequence and domain structure-function analyses have revealed that

Keap1 has three additional domains/regions:

  1. the N-terminal region (NTR),
  2. the invervening region (IVR), and
  3. the C-terminal region (CTR) [8].

The BTB/POZ domain has been shown to be

In the Drosophila Kelch protein, and in IPP,

The main function of INrf2 is to serve as

Cul3 serves as a scaffold protein that forms the E3 ligase complex with Rbx1 and recruits a cognate E2 enzyme [8].


  1. via its N-terminal BTB/POZ domain binds to Cul3 [44] and
  2. via its C-terminal Kelch domain binds to the substrate Nrf2

Under normal cellular conditions, the cytosolic INrf2/Cul3-Rbx1 complex is constantly degrading Nrf2. When a cell is exposed to oxidative stress Nrf2 dissociates from the INrf2 complex, stabilizes and translocates into the nucleus leading to activation of ARE-mediated gene expression [3,6–9]. An alternative theory is that Nrf2 in response to oxidative stress escapes INrf2 degradation, stabilizes and translocates in the nucleus [49–50]. We suggested the theory of escape of Nrf2 from INrf2 [49] and similar suggestion was also made in another report [50]. However, the follow up studies in our laboratory could not support the escape theory. Escape theory is a possibility but has to be proven by experiments before it can be adapted. Therefore, we will use the release of Nrf2 from INrf2 in the rest of this review.

Numerous reports have suggested that

A model Nrf2:INrf2 signaling from antioxidant and xenobiotic to activation of ARE-mediated defensive gene expression is shown in Fig. 3.

Fig. 3. Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Fig. 3. Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Since the metabolism of antioxidants and xenobiotics results in the generation of ROS and electrophiles [51], it is thought that these molecules might act as second messengers, activating ARE-mediated gene expression. Several protein kinases including PKC, ERK, MAPK, p38, and PERK [49,52– 56] are known to modify Nrf2 and activate its release from INrf2. Among these mechanisms,

  1. oxidative/electrophilic stress mediated phosphorylation of Nrf2 at serine40 by PKC is necessary for Nrf2 release from INrf2, but
  2. is not required for Nrf2 accumulation in the nucleus [49,52–53].

In addition to post-translational modification in Nrf2, several crucial residues in INrf2 have also been proposed to be important for activation of Nrf2. Studies based on

INrf2 itself undergoes ubiquitination by the Cul3 complex, via a proteasomal independent pathway,

It has been suggested that normally INrf2 targets Nrf2 for ubiquitin mediated degradation but

The redox modulation of cysteines in INrf2

In addition to cysteine151 modification,

Serine104 of INrf2 is required for dimerization of INrf2, and

Recently, Eggler at al. demonstrated that modifying specific cysteines of the electrophile-sensing human INrf2 protein is insufficient to disrupt binding to the Nrf2 domain Neh2 (58). Upon introduction of electrophiles, modification of INrf2C151 leads to a change in the conformation of the BTB domain by means of perturbing the homodimerization site, disrupting Neh2 ubiquitination, and causing ubiquitination of INrf2. Modification of INrf2 cysteines by electrophiles does not lead to disruption of the INrf2–Nrf2 complex. Rather, the switch of ubiquitination from Nrf2 to INrf2 leads to Nrf2 nuclear accumulation.

More recently, our laboratory demonstrated that phosphorylation and de-phosphorylation of tyrosine141 in INrf2 regulates its stability and degradation, respectively [59]. The de-phosphorylation of tyrosine141 caused destabilization and degradation of INrf2 leading to the release of Nrf2. Furthermore, we showed that prothymosin-α mediates nuclear import of the INrf2/Cul3-Rbx1 complex [60]. The INrf2/Cul3-Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2 resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α mediated nuclear import of INrf2/Cul3-Rbx1 complex leads to ubiquitination and degradation of nuclear Nrf2 presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression. An auto-regulatory loop also exists within the Nrf2 pathway [61]. An ARE was identified in the INrf2 promoter that facilitates Nrf2 binding causing induction of the INrf2 gene. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by serving as an adaptor for degradation.

Other Regulatory Mediators of Nrf2

Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1) is a transcription repressor [62] that is ubiquitously expressed in tissues [63–64] and distantly related to Nrf2 [8]. In the absence of cellular stress, Bach1 heterodimers with small Maf proteins [65] that bind to the (ARE) [66] repressing gene expression. In the presence of oxidative stress, Bach1 releases from the ARE and is replaced by Nrf2. Bach1 competes with Nrf2 for binding to the ARE leading to suppression of Nrf2 downstream genes [66].

Nuclear import of Nrf2, from time of exposure to stabilization, takes roughly two hours [67]. This is followed by activation of a delayed mechanism involving Glycogen synthase kinase 3 beta (GSK3f3) that controls switching off of Nrf2 activation of gene expression (Fig. 3). GSK3f3 is a multifunctional serine/threonine kinase, which plays a major role in various signaling pathways [68]. GSK3f3 phosphorylates Fyn, a tyrosine kinase, at unknown threonine residue(s) leading to nuclear localization of Fyn [69]. Fyn phosphorylates Nrf2 tyrosine 568 resulting in nuclear export of Nrf2, binding with INrf2 and degradation of Nrf2 [70].

The negative regulation of Nrf2 by Bach1 and GSK3f3/Fyn are important in repressing Nrf2 downstream genes that were induced in response to oxidative/electrophilic stress. The tight control of Nrf2 is vital for the cells against free radical damage, prevention of apoptosis and cell survival [3,6–9,70].

Nrf2 in Cytoprotection, Cancer and Drug Resistance

Nrf2 is a major protective mechanism against xenobiotics capable of damaging DNA and initiating carcinogenesis [71]. Inducers of Nrf2 function as blocking agents that prevents carcinogens from reaching target sites, inhibits parent molecules undergoing metabolic activation, or subsequently preventing carcinogenic species from interacting with crucial cellular macromolecules, such as DNA, RNA, and proteins [72]. A plausible mechanism by which blocking agents impart their chemopreventive activity is the induction of detoxification and antioxidant enzymes [73]. Oltipraz, 3H-1,2,-dithiole-3-thione (D3T), Sulforaphane, and Curcumin can be considered potential chemopreventive agents because

Studies have shown a role of Nrf2 in protection against cadmium and manganese toxicity [82]. Nrf2 also plays an important role in reduction of methyl mercury toxicity [83]. Methylmercury activates Nrf2 and the activation of Nrf2 is essential for reduction of methylmercury by facilitating its excretion into extracellular space. In vitro and in vivo studies have shown a role of Nrf2 in neuroprotection and protection against Parkinson’s disease [84– 86]. Disruption of Nrf2 impairs the resolution of hyperoxia-induced acute lung injury and inflammation in mice [87]. Nrf2-knockout mice were more prone to

INrf2/Nrf2 signaling is also shown to regulate oxidative stress tolerance and lifespan in Drosophila [91].

A role of Nrf2 in drug resistance is suggested based on its property to induce detoxifying and antioxidant enzymes (92–97). The loss of INrf2 (Keap1) function is shown to

Studies have reported mutations resulting in dysfunctional INrf2 in lung, breast and bladder cancers (96–100). A recent study reported that somatic mutations also occur in the coding region of Nrf2, especially in cancer patients with a history of smoking or suffering from squamous cell carcinoma (101). These mutations abrogate its interaction with INrf2 and nuclear accumulation of Nrf2. This gives advantage to

However, the understanding of the mechanism of Nrf2 induced drug resistance remains in its infancy. In addition, the studies on Nrf2 regulated downstream pathways that contribute to drug resistance remain limited.

Future Perspectives

Nrf2 creates a new paradigm in cytoprotection, cancer prevention and drug resistance. Considerable progress has been made to better understand all mechanisms involved within the intracellular pathways regulating Nrf2 and its downstream genes. Preliminary studies demonstrate that

Further studies are needed to better understand the negative regulation of Nrf2. Also better understanding of the negative regulation of Nrf2 could help design a new class of effective chemopreventive compounds not only targeting Nrf2 activation, but also


Nrf2    NF-E2 related factor 2;  INrf2   Inhibitor of Nrf2 also known as Keap1;   ROS    Reactive oxygen species.

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How Mobile Elements in “Junk” DNA Promote Cancer – Part 1: Transposon-mediated Tumorigenesis

How Mobile Elements in “Junk” DNA Promote Cancer – Part 1: Transposon-mediated Tumorigenesis

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

How Mobile Elements in “Junk” DNA Promote Cancer – Part 1 Transposon-mediated Tumorigenesis

Word Cloud by Daniel Menzin


Landscape of Somatic Retrotransposition in Human Cancers. Science (2012); Vol. 337:967-971. (1)

Sequencing of the human genome via massive programs such as the Cancer Genome Atlas Program (CGAP) and the Encyclopedia of DNA Elements (ENCODE) consortium in conjunction with considerable bioinformatics efforts led by the National Center for Biotechnology Information (NCBI) have unlocked a myriad of yet unclassified genes (for good review see (2).  The project encompasses 32 institutions worldwide which, so far, have generated 1640 data sets, initially depending on microarray platforms but now moving to the more cost effective new sequencing technology.  Initially the ENCODE project focused on three types of cells: an immature white blood cell line GM12878, leukemic line K562, and an approved human embryonic cell line H1-hESC.  The analysis was rapidly expanded to another 140 cell types.  DNA sequencing had revealed 20,687 known coding regions with hints of 50 more coding regions.  Another 11,224 DNA stretches were classified as pseudogenes.  The ENCODE project reveals that many genes encode for an RNA, not protein product, so called regulatory RNAs.

However some of the most recent and interesting results focus on the noncoding regions of the human genome, previously discarded as uninteresting or “junk” DNA .  Only 2% of the human genome contains coding regions while 98% of this noncoding part of the genome is actually found to be highly active “with about 4 million constantly communicating switches” (3).  Some of these “switches” in the noncoding portion contain small, repetitive elements which are mobile throughout the genome, and can control gene expression and/or predispose to disease such as cancer.  These mobile elements, found in almost all organisms, are classified as transposable elements (TE), inserting themselves into far-reaching regions of the genome.  Retro-transposons are capable of generating new insertions through RNA intermediates.  These transposable elements are normally kept immobile by epigenetic mechanisms(4-6) however some TEs can escape epigenetic repression and insert in areas of the genome, a process described as insertional mutagenesis as the process can lead to gene alterations seen in disease(7).  In addition, this insertional mutagenesis can lead to the transformation of cells and, as described in Post 2, act as a model system to determine drivers of oncogenesis. This insertional mutagenesis is a different mechanism of genetic alteration and rearrangement seen in cancer like recombination and fusion of gene fragments as seen with the Philadelphia chromosome and BCR/ABL fusion protein (8).  The mechanism of transposition and putative effects leading to mutagenesis are described in the following figure:


Figure.  Insertional mutagenesis based on transposon-mediated mechanism.  A) Basic structure of  transposon contains gene/sequence flanked by two inverted repeats (IR) and/or direct repeats (DR).  An enzyme, the transposase (red hexagon) binds and cuts at the IR/DR and transposon is pasted at another site in DNA, containing an insertion site.  B)   Multiple transpositions may results in oncogenic events by inserting in promoters leading to altered expression of genes driving oncogenesis or inserting within coding regions and inactivating tumor suppressors or activating oncogenes.  Deep sequencing of the resultant tumor genomes ( based on nested PCR from IR/DRs) may reveal common insertion sites (CIS) and oncogenic mutations could be identified.

In a bioinformatics study Eunjung Lee et al.(1), in collaboration with the Cancer Genome Atlas Research Network, the authors had analyzed 43 high-coverage whole-genome sequencing datasets from five cancer types to determine transposable element insertion sites.  Using a novel computational method, the authors had identified 194 high-confidence somatic TE insertion sites present in cancers of epithelial origin such as colorectal, prostate and ovarian, but not in brain or blood cancers.  Sixty four of the 194 detected somatic TE insertions were located within 62 annotated genes. Genes with TE insertion in colon cancers have commonly high mutation rates and enriched genes were associated with cell adhesion functions (CDH12, ROBO2,NRXN3, FPR2, COL1A1, NEGR1, NTM and CTNNA2) or tumor suppressor functions (NELL1m ROBO2, DBC1, and PARK2).  None of the somatic events were located within coding regions, with the TE sequences being detected in untranslated regions (UTR) or intronic regions.  Previous studies had shown insertion in these regions (UTR or intronic) can disrupts gene expression (9). Interestingly, most of the genes with insertion sites were down-regulated, suggested by a recent paper showing that local changes in methylation status of transposable elements can drive retro-transposition (10,11).  Indeed, the authors found that somatic insertions are biased toward the hypomethylated regions in cancer cell DNA.  The authors also confirmed that the insertion sites were unique to cancer and were somatic insertions, not germline (germline: arising during embryonic development) in origin by analyzing 44 normal genomes (41 normal blood samples from cancer patients and three healthy individuals).

The authors conclude:

“that some TE insertions provide a selective advantage during tumorigenesis,

rather than being merely passenger events that precede clonal expansion(1).”

The authors also suggest that more bioinformatics studies, which utilize the expansive genomic and epigenetic databases, could determine functional consequences of such transposable elements in cancerThe following Post will describe how use of transposon-mediated insertional mutagenesis is leading to discoveries of the drivers (main genetic events) leading to oncogenesis.

1.            Lee, E., Iskow, R., Yang, L., Gokcumen, O., Haseley, P., Luquette, L. J., 3rd, Lohr, J. G., Harris, C. C., Ding, L., Wilson, R. K., Wheeler, D. A., Gibbs, R. A., Kucherlapati, R., Lee, C., Kharchenko, P. V., and Park, P. J. (2012) Science 337, 967-971

2.            Pennisi, E. (2012) Science 337, 1159, 1161

3.            Park, A. (2012) Don’t Trash These Genes. “Junk DNA may lead to valuable cures. in Time, Time, Inc., New York, N.Y.

4.            Maksakova, I. A., Mager, D. L., and Reiss, D. (2008) Cellular and molecular life sciences : CMLS 65, 3329-3347

5.            Slotkin, R. K., and Martienssen, R. (2007) Nature reviews. Genetics 8, 272-285

6.            Yang, N., and Kazazian, H. H., Jr. (2006) Nature structural & molecular biology 13, 763-771

7.            Hancks, D. C., and Kazazian, H. H., Jr. (2012) Current opinion in genetics & development 22, 191-203

8.            Sattler, M., and Griffin, J. D. (2001) International journal of hematology 73, 278-291

9.            Han, J. S., Szak, S. T., and Boeke, J. D. (2004) Nature 429, 268-274

10.          Reichmann, J., Crichton, J. H., Madej, M. J., Taggart, M., Gautier, P., Garcia-Perez, J. L., Meehan, R. R., and Adams, I. R. (2012) PLoS computational biology 8, e1002486

11.          Byun, H. M., Heo, K., Mitchell, K. J., and Yang, A. S. (2012) Journal of biomedical science 19, 13

Other research paper on ENCODE and Cancer were published on this Scientific Web site as follows:

Expanding the Genetic Alphabet and linking the genome to the metabolome

Junk DNA codes for valuable miRNAs: non-coding DNA controls Diabetes

ENCODE Findings as Consortium

Reveals from ENCODE project will invite high synergistic collaborations to discover specific targets

ENCODE: the key to unlocking the secrets of complex genetic diseases

Impact of evolutionary selection on functional regions: The imprint of evolutionary selection on ENCODE regulatory elements is manifested between species and within human populations

Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes

Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets

Commentary on Dr. Baker’s post “Junk DNA codes for valuable miRNAs: non-coding DNA controls Diabetes”

Cancer Genomics – Leading the Way by Cancer Genomics Program at UC Santa Cruz

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Usp9x: Promising therapeutic target for pancreatic cancer

Curator: Ritu Saxena, Ph.D.

Screen Shot 2021-07-19 at 7.43.43 PM

Word Cloud By Danielle Smolyar

Introduction and Research Relevance:

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer death in the United States with a median survival of <6 mo and a dismal 5-yr survival rate of 3%–5%. The cancer’s lethal nature stems from its propensity to rapidly disseminate to the lymphatic system and distant organs. This aggressive biology and resistance to conventional and targeted therapeutic agents leads to a typical clinical presentation of incurable disease at the time of diagnosis.


Also, it has been well documented that despite much progress in its molecular characterization, Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy.

Recent article published in the journal Nature talks about discovering the link between a gene and the prognosis of Pancreatic Ductal Adenocarcenoma (PDA). The discovery might have therapeutic relevance in PDA.

Although previous work had attributed a pro-survival role to USP9X in human neoplasia, the researchers found instead that loss of Usp9x protects pancreatic cancer cells from death. Thus, the study proposed USP9X to be a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.


News brief: (http://www.sanger.ac.uk/about/press/2012/120429.html)

29 April 2012

Gene against pancreatic cancer discovered

Study points to potential new treatment for deadly pancreatic cancer

In a study published in Nature (Sunday 29 April), researchers have identified a potential new therapeutic target for pancreatic cancer.

The team found that when a gene involved in protein degradation is switched-off through chemical tags on the DNA’s surface, pancreatic cancer cells are protected from the bodies’ natural cell death processes, become more aggressive, and can rapidly spread.

Pancreatic cancer kills around 8,000 people every year in the UK and, although survival rates are gradually improving, fewer than 1 in 5 patients survive for a year or more following their diagnosis.

Co-lead author Professor David Tuveson, from Cancer Research UK’s Cambridge Research Institute, said: “The genetics of pancreatic cancer has already been studied in some detail, so we were surprised to find that this gene hadn’t been picked up before. We suspected that the fault wasn’t in the genetic code at all, but in the chemical tags on the surface of the DNA that switch genes on and off, and by running more lab tests we were able to confirm this.”

The team expects this gene, USP9X, could be faulty in up to 15 per cent of pancreatic cancers, raising the prospect that existing drugs, which strip away these chemical tags, could be an effective way of treating some pancreatic cancers.

” This study strengthens our emerging understanding that we must also look into the biology of cells to identify all the genes that play a role in cancer. ” Dr David Adams

“Drugs which strip away these tags are already showing promise in lung cancer and this study suggests they could also be effective in treating up to 15 per cent of pancreatic cancers,” continues Professor Tuveson.

The researchers used a mouse model of pancreatic cancer to screen for genes that speed up pancreatic cancer growth using a technique called ‘Sleeping Beauty transposon mutagenesis’. This system uses mobile genetic elements that hop around the cell’s DNA from one location to the next. Cells that acquire mutations in genes that contribute to cancer development will grow out and ‘driver’ cancer genes may be identified.

By introducing the Sleeping Beauty transposon into mice pre-disposed to develop pancreatic cancer, the researchers were able to screen for a class of genes called a tumour suppressor that, under normal circumstances, would protect against cancer. These genes are a bit like the cell’s ‘brakes’, so when they become faulty there is little to stop the cell from multiplying out of control.

This approach uncovered many genes already linked to pancreatic cancer. But unexpectedly, USP9X, was identified.

Co-lead author Dr David Adams, from the Wellcome Trust Sanger Institute, said: “The human genome sequence has delivered many promising new leads and transformed our understanding of cancer. Without it, we would have only a small, shattered glimpse into the causes of this disease. This study strengthens our emerging understanding that we must also look into the biology of cells to identify all the genes that play a role in cancer.”


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