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Posts Tagged ‘Non-coding RNA’


Genome Jigsaws

Genome Jigsaws (Photo credit: dullhunk)

Sequencing became the household name.  In 2000s, it was thought to be the key of the Pandora’s box for cure.  Then, after completion of Human Genome Projects showed that there are less number of genes than expected.  This outcome induce to originate yet another set of sequencing programs and collaborations around the world, such as Human Protein Project, Human Microorganisms Projects, ENCODE, Transcriptome Sequencing and Consortiums etc.

It is in humankind to believe in magic and illusion.  The strength of biological diversity and complex mechanism of expression may chalanges the set up of a simple but informative specific essay.  Thus, there is a new developing field to mash rules of biology with mathematical formulas to develop the best bioinformatics or also called computational biology.  Predicting transcription start or termination sites, exon boundaries, possible binding sites of transcription regulators for chromatin modification activities, like histone acetylates and enhancer- and insulator-associated factors based on the human genome sequence.  Deep in mind, this assumption supports that the sequence contains signatures for chromatin modifications essential for gene regulation and development.

There are three primary colors, red, yellow and blue, however, an artist can create many shades. Recently, scientists combining and organizing more data to make sense of our blueprint of life to transfer info generation to generation with the hope to cure diseases of human kind.

Analyzing genome and transcriptome open the door.  These studies suggested that all eukaryotic cells has a rich portfolio of RNAs. Among these long non-coding RNAs has impact on protein coding gene expression, regulating multiple processes even including epigenetic gene expression.

Epigenetics, stemness and non-coding RNAs  play a great role to manipulate and correct the gene expression not only at a proper cell type but also location and time within genome without disturbing the host.

Main concern is differentiation of embryonic stem cells under these epigenetics and influencers.  The best known post-transcriptional modifications, which include methylation, acetylation, ubiquination, and SUMOylation of lysine residues, methylation of arginine residues, and phosphorylation of serines, occur on histone tails. “Epi” means “top” or
“above” so this mechanism give a new direction to the genetic pathways as long as the organism live sometime and may lead into evolutions.  It is critical to show the complexity of
mechanism and relativity of a gene role with a single example for each. 

For example,  DNA methylation occurs mostly on cytosine residues on the CpG islands usually located on promoter regions that are associated with tissue-specific gene expression.  However, there are many other forms of DNA methylations, such as  monoallelic methylation in gene imprinting and inactivation of the X chromosome,  in repetitive elements, like transposons.  There are two main mechanisms but this is not our main topic.  Yet, Myc and hypoxia-inducible factor-1α versus certain methyl-CpG-binding proteins, such as MBD1,MBD2, MBD4, MeCP2, and Kaiso works differently.

Stemness is an important factor for an intervention to correct a pathological condition. In terms of epigenetics, regulation and non-coding RNA Vascular endothelial growth factor A (VEGF-A) is an interesting example for differentiation of endothelial cells and morphogenesis of the vascular system during development with several reasons, epigenetics, gene interactions, time and space.  Everything has to be just right, because neither less nor too much can fulfill the destiny to become a complete adult cell or an organism.   For example, both having only one VEGF-A allele and having two-fold excess of VEGF-A results in death during early embryogenesis, since mice can’t develop proper vascular network.  However, explaining diverse mechanisms and functions of VEGF-A is require more information with specific details.  VEGF-A plays many roles in many pathological cases, such as cancer, inflammation, retinopathies, and arthritis because VEGF-A has also function in epigenetic reprogramming of the promoter regions of Rex1 and Oct4 genes, that are critical for a stem cell. Preferred mechanism is anti-angiogeneic state but tumor cells prefer hypermethylation to induce pro-angiogeneic state, thus VEGF-A stimulates PIGF in tumour cells among many other factors.

Now, let’s turn around to observe development of a cell with Polycomb repressive complexes (PRCs) because they are important chromatin regulators of embryonic stem (ES) cell function.  Originally, RYBP shown to function  as transcriptional repressor in reporter assays from both in tissue culture cells and in fruit fly (Drosophila melanogaster ) and as a direct interactor with Ring1A during embryogenesis through methylation. In addition, RYBP in epigenetic resetting during preimplantation development through repression of germ line genes and PcG targets before formation of pluripotent epiblast cells.  However, I do believe that the most important element is efficient repression of endogenous retroviruses (murine endogenous retrovirus called MuERV class),  preimplantation containing zygotic genome activation stage and germ line specific genes. The selective repressor activity of  RYBP  is in the ES cell state. When RYBP−/− ES cells were analyzed by measuring gene expression during differentiation as embryo bodies formed from mutant and wild-type cells, the result presented that  expression of pluripotency genes Oct4 and Nanog was usually downregulated. However, RYBP is able to bind genomic regions independently of H3K27me3 and there is no relation between altered RYBP binding in Dnmt1-mutant cells to DNA methylation status. In sum, RYBP has a large value in undifferentiated ES cells and may affect or even reset epigenetic landscape during early developmental stages. These are the gaps filled by long non coding RNAs.

We learn more compelling information by comparing and contrasting what is normal and what is abnormal. As a result, pathology is a key learning canvas for basic mechanisms in molecular genetics. Then peppered with functional genomics completes the story for an edible outcome.  We generally refer this as a Translational Research.  For example, recent foundlings suggest that H19 contributes to cancer, including hepatocellular carcinoma (HCC) after reviewing Oncomine resource.  According to these observations, in most HCC cases there is a lower expression of  H19 level is compared to the liver. Thus, in vitro and in vivo studies were undertaken with classical genetic analyzes based on loss- and gain-of-function on H19 to characterize two outcomes depend on H19, that are the effects on gene expression and on HCC metastasis. First, the expression of H19 showed gene expression variation since H19 expression was low in tumor cells than peripheral tumor cells.  Second, the metastasis of cancer based on alteration of miR-200 pathway contributing mesenchymal-to-epithelial transition by H19. Therefore, H19 and miR-200 are targets to be utilized during molecular diagnostics development and establishing targeted therapies in cancer.

Long story short, there is a circle of life where everything is connected even though they look different.  As a result, when we see a sunflower or a baby we remember to smile, because life is still an act to puzzle human.

References and Further Readings:

 

Non-coding RNAs as regulators of gene expression and epigenetics” Cardiovascular Res 1 June 2011: 430-440.

Epigenetic regulation of key vascular genes and growth factors” Cardiovasc Res 1 June 2011: 441-446.

Epigenetic Regulation by Long Noncoding RNAs” Science 14 December 2012: 1435-1439.

Epigenetic control of embryonic stem cell fate” JEM 25 October 2010: 2287-2295.

Transcribed dark matter: meaning or myth?” Hum Mol Genet 15 October 2010: R162-R168.

Epigenetic activation of the MiR-200 family contributes to H19-mediated metastasis suppression in hepatocellular carcinoma” Carcinogenesis 1 March 2013: 577-586.

Vernalization-Mediated Epigenetic Silencing by a Long Intronic Noncoding RNA” Science 7 January 2011: 76-79.

Predicting the probability of H3K4me3 occupation at a base pair from the genome sequence context” Bioinformatics 1 May 2013: 1199-1205.

 

Further Readings specific to Embryonic Stem Cell Differentiation and Development :

“BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells” J. Biol. Chem. 2013 288:8053-8060

Abstract

“Regulation of DNA Methylation in Rheumatoid Arthritis Synoviocytes”  J. Immunol. 2013 190:1297-1303

Abstract

“DNA methylome signature in rheumatoid arthritis” Ann Rheum Dis 2013 72:110-117

Abstract

“The histone demethylase Kdm3a is essential to progression through differentiation” Nucleic Acids Res 2012 40:7219-7232

Abstract

“Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer” Nucleic Acids Res 2012 40:6725-6740

Abstract

“Yin Yang 1 extends the Myc-related transcription factors network in embryonic stem cells” Nucleic Acids Res 2012 40:3403-3418

Abstract

“RYBP Represses Endogenous Retroviruses and Preimplantation- and Germ Line-Specific Genes in Mouse Embryonic Stem Cells” Mol. Cell. Biol. 2012 32:1139-1149

Abstract

“Polycomb Repressor Complex-2 Is a Novel Target for Mesothelioma Therapy” Clin. Cancer Res. 2012 18:77-90

Abstract

“OCT4 establishes and maintains nucleosome-depleted regions that provide additional layers of epigenetic regulation of its target genes” Proc. Natl. Acad. Sci. USA 2011 108:14497-14502

Abstract

“Genome-wide promoter DNA methylation dynamics of human hematopoietic progenitor cells during differentiation and aging” Blood 2011 117:e182-e189

Abstract

“The CHD3 Chromatin Remodeler PICKLE and Polycomb Group Proteins Antagonistically Regulate Meristem Activity in the Arabidopsis” RootPlant Cell 2011 23:1047-1060

Abstract

“Chromatin structure of pluripotent stem cells and induced pluripotent stem cells” Briefings in Functional Genomics 2011 10:37-49

Abstract

Abbreviations used:

DNMT       DNA methyl transferase

ES             embryonic stem

JmjC         Jumonji C

lincRNA     long ncRNA

ncRNA       noncoding RNA

PcG          Polycomb group

PRC          Polycomb repressive complex

PRE          Polycomb repressive element

Previous Posts on Stem Cells:

…  Aviva Lev-Ari, PhD, RN New Life – The Healing Promise of Stem Cells View … p://www.technioniit.com/2012/09/new-life-healing-promise-of-stem-cells.html       Diseases and conditions where stem cell treatment is promising or emerging. Source: Wikipedia Since the …

…  Aviva Lev-Ari, PhD, RN Stem cells create new heart cells in baby mice, but not in adults, study …  picture on the left shows green c-kit+ precursor stem cells within an infarct (lower right) in a

14 January 2013  by Dr. Sudipta Saha on Pharmaceutical Intelligence
…  and Curator: Dr. Sudipta Saha, Ph.D. Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in …  from adult mouse ovaries. A fluorescence-activated cell sorting-based protocol has been standardized that can be used with adult …  compared to the ESC-derived or induced pluripotent stem cell-derived germline cells that are currently used as models for human …

…  PhD, RN The two leading therapy classes are: Cell-based Therapies for angiogenesis and myocardial …  Research Projects Stem Cell biology Embryonic stem cells in cardiovascular repairEarly differentiation of human endothelial …

…  Stem Cells with Unread Genome: microRNAs Author, Demet Sag, PhD Life is …  a coherent outcome. Thus, providing an engineered whole cell as a system of correction for “Stem Cell Therapy” may resolve unmet health problems.  Only 1% of the genome …

…  are not yet known. Some studies suggest a high rate of stem cell activity with differentiation of progenitors to cardiomyocytes. Other …

…  T-cells, said Dr. Margaret Goodell, director of the Stem Cells and Regenerative Medicine Center of Baylor College of Medicine. …  of pediatrics at BCM and a member of the Center for Cell and Gene Therapy at BCM, Texas Children¹s Hospital and The Methodist …  found that mice lacking the gene for this factor had a T-cell deficiency and in particular, too few of these early progenitor …

28 March 2013  by ritusaxena on Pharmaceutical Intelligence
…  and Curator: Ritu Saxena, Ph.D Although cancer stem cells constitute only a small percentage of the tumor burden, their …  after therapeutic target in cancer. The post on cancer stem cells published on the 22nd of March, 2013, describes the identity of CSCs, their functional characteristics, possible cell of origin and biomarkers. This post focuses on the therapeutic potential …

…  programs in the fields of personalized medicine, cell biology, cytogenetics, genotyping, and biobanking drive our …  by playing an important role in induced pluripotent stem (iPS) cell research. Induced pluripotent stem cells are powerful cells which can be made from skin or blood cells, and …

30 November 2012  by sjwilliamspa on Pharmaceutical Intelligence
…  seen in hematologic malignancies such as cutaneous T-cell lymphoma and peripheral T-cell lymphoma and little or no positive outcome …  resistance to chemotherapeutics, and similarity to cancer stem cells(6-10). Figure 1. HDACis led to the induction of EMT phemotype. (A …

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John Rinn - Genomic Garbage Man

John Rinn – Genomic Garbage Man (Photo credit: ChimpLearnGood)

DNA: One man’s trash is another man’s treasure, but there is no JUNK after all

Author: Demet Sag, PhD

One man’s trash is another man’s treasure, but there is no JUNK after all:

The JUNK has a meaning

 

Long non-coding RNAs recognized after transcriptome sequencing and studied more closely recently thanks to genomic tiling arrays, cDNA sequencing and RNA-Seq, which they have provided initial insights into the extent and depth of transcribed sequence across human and other genomes. How many are there in the genome? What are their mechanisms? How can we use them in molecular diagnostics and targeted therapies?  How do they effect the function in a disease? Is it possible to modulate gene expression at the level of stem cell to redirect the cell differentiation? These are the main questions that we are looking for.

In early 90s actually first lincRNA was described, Xist. The main function was dosage compensation. Then in 2000s FANTOM consortium project changed the perspective on these long transcripts. Then they are called natural antisense transcripts (NATs), because very large number of these transcripts is overlapping with, and is transcribed in the antisense direction, to protein-coding genes.  As a result of this study 11000 lincRNA discovered from full length cDNAs in mice. Later, yet another shift occur since these transcribed units are solely located in the introns or within “junk” DNA of protein-coding genes.  Another independent study quantified that about 40% of protein-coding genes express NATs. Proven that there is nothing junk about DNA. Then, it was found that there are 8000 lincRNAs and among these 4000 are determined since they provide cell identity with multi-exogenic, polyadenylated, capped, ether in the cytoplasm or in the nucleus. However, even more recent studies show that there are about 20,000 lincRNAs.  Furthermore, lincRNAs are classified under three distinct class: 1. Long-non-coding RNAs away from protein-coding genes, 2 NATs transcribed from the opposite strand of protein-coding genes, 3. Intronic lincRNAs expressed from within the introns of protein coding genes.

 

English: The human genome, categorized by func...

The human genome, categorized by function of each gene product, given both as number of genes and as percentage of all genes. (Photo credit: Wikipedia)

Their function is under study. However, keep in mind that they are redundant, so deleting or creating null mutations may or may not answer specific development questions. On the other hand, epigenetics, gene imprinting, and pathologies can be the best resource to identify their specific roles in biological functions and interactions.  Distinct gene regulation either as a cis or trans element, gene imprinting, modulating alternative splicing, nuclear organization, determining a chromatin structure are under study.  This will allow us to relate genome structure and function in health and disease better.  Identification of their function during biological responses require a long way to be completed due to complexity since lincRNAs also regulate microRNAs.  Regardless of many obstacles there is a progress.  Disregulation of these lincRNA mainly observed in several cancer types, prostate, breast, hepatocellular carcinoma, colorectal, glioma and melanoma, possibly more. Most of the studies are done in vitro. However, there are many great model organism work as well, such as mice, zebra fish, and worm.

It was also not surprising that their regulation possibly under control of hormones based on circadian clock of our body. So better to sleep eight hour a day is not a cliché.

 

Next topic will include understanding of lincRNA mechanisms and epigenetics followed by lincRNAs during disease and cellular genesis.

 

Mechanism, Genome and Genetics:

Long non-coding RNAs: insights into functions. Mercer TR, Dinger ME, Mattick JS Nat. Rev. Genet. 2009;10:155159. http://www.ncbi.nlm.nih.gov/pubmed/19188922

 

Long Noncoding RNAs: Past, Present, and Future” Genetics 1 March 2013: 651-669. http://www.genetics.org/content/193/3/651.abstract

 

“RNA-protein analysis using a conditional CRISPR nuclease” Proc. Natl. Acad. Sci. USA 2 April 2013: 5416-5421. http://www.pnas.org/content/110/14/5416.abstract

“Noncoding RNA and Polycomb recruitment” RNA 1 April 2013: 429-442. http://rnajournal.cshlp.org/content/19/4/429.abstract

 

“Emerging functional and mechanistic paradigms of mammalian long non-coding RNAs” Nucleic Acids Res 1 August 2012: 6391-6400. http://nar.oxfordjournals.org/content/40/14/6391.abstract

 

 

“Long noncoding RNAs regulate adipogenesis” Proc. Natl. Acad. Sci. USA 26 February 2013: 3387-3392. http://www.pnas.org/content/110/9/3387.abstract

 

“Circadian changes in long noncoding RNAs in the pineal gland” Proc. Natl. Acad. Sci. USA 14 August 2012: 13319-13324. http://www.pnas.org/content/109/33/13319.abstract

Animal and Development:

“Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis” Genome Res 1 March 2012: 577-591. http://genome.cshlp.org/content/22/3/577.abstract

 

“Genes for embryo development are packaged in blocks of multivalent chromatin in zebrafish sperm” Genome Res 1 April 2011: 578-589. http://genome.cshlp.org/content/21/4/578.abstract

Long noncoding RNAs in C. elegans” Genome Res 1 December 2012: 2529-2540. http://genome.cshlp.org/content/22/12/2529.abstract

A spatial and temporal map of C. elegans gene expression” Genome Res 1 February 2011: 325-341. http://genome.cshlp.org/content/21/2/325.abstract

 

“SFMBT1 functions with LSD1 to regulate expression of canonical histone genes and chromatin-related factors” Genes Dev. 1 April 2013: 749-766. http://genesdev.cshlp.org/content/27/7/749.abstract

Other related articles published on this Open Access Online Scientific Journal include the following:

…  therapies in a variety of animal models and contributed to regulatory CMC and IND-enabling safety and toxicology studies for inclusion in …  sequences sort a-cardiac and b-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J. Cell. Biol., …

…  and posttranscriptional mechanisms contributing to the regulatory network. We examined proinflammatory gene regulation in …  articles What about Circular RNAs? (pharmaceuticalintelligence.com) How Genes Function …

…  due to their broad scope and non-specificity in the human genome. “I am extremely pro-patent, but I simply believe that people …  believe that individuals have an innate right to their own genome, or to allow their doctor to look at that genome, just like the lungs or …

…  Intelligence https://pharmaceuticalintelligence.com/2013/03/02/genome-sequenc…of-the-healthy/ ‎ Key Issues in Genome Sequencing of Healthy Individuals Eric Topol, MD, Genomic Medicine I …  touching on important controversies in the use of whole genome …

6 February 2013  by Dr. Sudipta Saha on Pharmaceutical Intelligence
…  of recombination is highly uneven across the human genome, as in all studied organisms. Substantial recombination active regions …  this variation would require comparison of recombination genome-wide among many single genomes. Whole-genome amplification (WGA) of …

…  Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD Putting Genome Interpretation to the Test 01/30/2013 Ashley Yeager How well do methods for interpreting genome variation work? Ashley Yeager takes a look at a community experiment that is trying to assess just how useful genome interpretation tools in real-world situations. At the American …

…  genomes — through the end of this year, National Human Genome Research Institute estimates indicate. And in his book, The Creative …  the interpretation of an apparently healthy person’s genome and that of an individual who is already affected by a disease, whether …
Topics: Cardiovascular Pharmaceutical Genomics, Personalized Medicine & Genomic Research, Pharmaceutical R&D investment, CANCER BIOLOGY & Innovations in Cancer Therapy, Chemical Genetics, Cell Biology, Signaling & Cell Circuits, Computational Biology/Systems and Bioinformatics, Medical and Population Genetics, genome biology, Biological Networks, Gene Regulation and Evolution, Population Health Management, Genetics & Pharmaceut, human genome, National Institutes of Health, Scripps Research Institute, Proteomics, Bio Instrumentation in Experimental Life Sciences Resea, Massachusetts General Hospital, DNA, FDA Regulatory Affairs, Clinical Trials and IRB related issues, Biomarkers & Medical Diagnostics, metabolomics, Molecular Genetics & Pharmaceutical, Genomic Testing: Methodology for Diagnosis, Technology Transfer: Biotech and Pharmaceutical, Health Law & Patient Safety, Eric Topol, national human genome research institute, Encode, NIST

…  scary findings: the tale of John Lauerman’s whole genome sequencing FEBRUARY 15, 2012 Joe Thakuria draws John Lauerman’s blood for whole genome sequencing. By Madeleine Price Ball, licensed under …  scary findings: the tale of John Lauerman’s whole genome sequencing » Joe Thakuria draws John …

…  2: LEADERS in the Competitive Space of Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in …  Treatment https://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-drug-selection-in-cancer- …
Topics: Personalized Medicine & Genomic Research, Pharmaceutical R&D investment, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Medical and Population Genetics, genome biology, Disease Biology, Small Molecules in Development of Ther, Population Health Management, Genetics & Pharmaceut, Cancer, Foundation Medicine, Proteomics, DNA, DNA Sequencing, Biomarkers & Medical Diagnostics, metabolomics, AstraZeneca, Molecular Genetics & Pharmaceutical, Nature Medicine, Stem Cells for Regenerative Medicine, Genomic Testing: Methodology for Diagnosis, Technology Transfer: Biotech and Pharmaceutical, Full genome sequencing, Genomic Endocrinology, Preimplantation Genetic Diagnosi, Interviews with Scientific Leaders, Pharmacogenomics, Drug Delivery Platform Technology, Digene, Yuri Milner

3 February 2013  by sjwilliamspa on Pharmaceutical Intelligence
Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single …  of DNA replication and the ability to amplify a whole genome.  The amplicons are then sequenced either by whole-genome sequencing methods using Sanger-sequencing to verify any single …

…  Aviva Lev-Ari, RN Genome Biol. 2012 Dec 13;13(12):R115. [Epub ahead of print] Whole-genome reconstruction and mutational signatures in gastric cancer. Nagarajan …  read and DNA-PET sequencing to present the first whole-genome analysis of two gastric adenocarcinomas, one with chromosomal …

1 September 2012  by pkandala on Pharmaceutical Intelligence
…  by interpreting the mathematical patterns in the cancer genome. Researchers at the University of Oslo, Norway (UiO) have developed a …  Hospital and UiO. Finds the changed patterns in the genome There is much talk about finding the special cancer gene. In reality, …

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An illustration of the central dogma of molecu...

An illustration of the central dogma of molecular biology annotated with the processes ncRNAs are involved in. (Photo credit: Wikipedia)

X-ray structure of the tRNA Phe from yeast. Da...

X-ray structure of the tRNA Phe from yeast. Data was obtained by PDB 1ehz and rendered with PyMOL. violet: acceptor stem wine red: D-loop blue: anticodon loop orange: variable loop green: TPsiC-loop yellow: CCA-3′ of the acceptor stem grey: anticodon (Photo credit: Wikipedia)

 Our genome must be packed tightly to fit into the nucleus. Genome is the blue print of a living organism whether made up off a single or multiple cell.   Recently, the genome seen as a functional network of physical contacts within (cis) and between (trans) chromosomes.  It became necessary to map these physical DNA contacts at high-resolution with technologies such as the “chromosome conformation capture” (3C) and other 3C-related methods including 3C-Carbon Copy (5C) and Hi-C.  Yet, we all know that in vivo conformation, gene to gene interactions from a long distance, histones and 3D have an impact on gene regulation and expression.  The game is not just a sequence but functional genomics with a correct translation of sequence for development so that proper molecular diagnostics can be applied not only for prevention but also for monitoring the efficacy of the intervention. Thus, we can provide a targeted therapy for personalized medicine.

On the other hand, we still know very little about genome organization at the molecular level, although spatial genome organization can critically affect gene expression.  It is important to recognize who is there to be present and who is there to create the functional impact for regulation in a specific tissue and time.  In addition, mediation of these chromatin contacts based on a specific tissue is quite essential.  For example, during long-range control mechanism specific enhancers and distal promoters needed to be invited to a close physical proximity to each other by transcription factors that has been found at other loci.  Furthermore, chromatin-binding proteins such as the CCCTC-binding factor (CTCF) and cohesin seem to have critical roles in genome organization and gene expression.  Let’s not forget about epigenetics, since there are so many methods to regulate chromatin interactions like cytosine methylation, maternal gene, gradient level, post-translational modifications and non-coding RNAs.

The non-coding RNAs (ncRNAs) are silent but they have the 99% power because ncRNAs are a broad class of transcripts consisting of structural (rRNAs, tRNAs, snRNAs, snoRNAs, etc.), regulatory (miRNAs, piRNAs, etc.), and of sense/antisense transcripts.  Among these an interesting class is the latter group.   This class includes transcriptional “features” (eRNAs, tiRNAs), and a very large number of long non-coding RNAs (lncRNAs), length from 200 nt to 100 kb.  The magnificent future of lncRNAs comes from their production, as they can be transcribed nearby known protein-coding genes or from their introns. As a result, because of their intergenical production they are also called as “lincRNAs (long intergenical non-coding RNAs).  They are abundant and specific as microRNAs.  Hence, their inclusion into the biomarker list and assuming their roles during targeted therapy don’t require us to be a wizard but a functional genomicist knowing evolution, development and molecular genetics and plus signaling.

lincRNA can both activate and repress the gene either cis or trans acting to effect gene regulation will be discussed next.

As a result, one gene expression regulation needs from twenty to several hundred genes. As they say raising a child needs a village.

References:

“Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs”.

Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY.  Cell. 2007 Jun 29; 129(7):1311-23.

“Long noncoding RNA as modular scaffold of histone modification complexes”

Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, Shi Y, Segal E, Chang HYScience. 2010 Aug 6; 329(5992):689-93.

“Capturing Chromosome Conformation”.

Dekker J, Rippe K, Dekker M, Kleckner N.Science.2002;295:1306–1311.

“Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements”.

Dostie J, Richmond TA, Arnaout RA, Selzer RR, Lee WL, Honan TA, Rubio ED, Krumm A, Lamb J, Nusbaum C, Green RD, Dekker J.Genome Res. 2006;16:1299–1309.

“Chromosome conformation capture carbon copy technology”.

Dostie J, Zhan Y, Dekker J. Curr. Protoc. Mol. Biol. 2007 Chapter 21, Unit 21 14.

“Comprehensive mapping of long-range interactions reveals folding principles of the human genome”.

Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J.  Science. 2009;326:289–293.

“Chromatin conformation signatures: ideal human disease biomarkers?”

Crutchley JL, Wang XQ, Ferraiuolo MA, Dostie J.Biomark. Med. 2010;4:611–629.

“Relationship between CAD risk genotype in the chromosome 9p21 locus and gene expression. Identification of eight new ANRIL splice variants”.

Folkersen L, Kyriakou T, Goel A, Peden J, Mälarstig A, Paulsson-Berne G, Hamsten A, Hugh Watkins, Franco-Cereceda A, Gabrielsen A, Eriksson P, PROCARDIS consortia

PLoS One. 2009 Nov 2; 4(11):e7677.

” A myelopoiesis-associated regulatory intergenic noncoding RNA transcript within the human HOXA cluster”.

Zhang X, Lian Z, Padden C, Gerstein MB, Rozowsky J, Snyder M, Gingeras TR, Kapranov P, Weissman SM, Newburger PE.  Blood. 2009 Mar 12; 113(11):2526-34.

Monk M.   Genes Dev. 1988 Aug; 2(8):921-5.

Hox genes specify vertebral types in the presomitic mesoderm

Marta Carapuço,1 Ana Nóvoa,1 Nicoletta Bobola,2 and Moisés Mallo1,3 .  Genes Dev. 2005 September 15; 19(18): 2116–2121.

Krumlauf R.  Cell. 1994 Jul 29; 78(2):191-201.

“Noncoding RNA synthesis and loss of Polycomb group repression accompanies the colinear activation of the human HOXA cluster”.

Sessa L, Breiling A, Lavorgna G, Silvestri L, Casari G, Orlando V.  RNA. 2007 Feb; 13(2):223-39.

“Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs”.

Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY.  Cell. 2007 Jun 29; 129(7):1311-23.

“Long noncoding RNAs with enhancer-like function in human cells”.

Ørom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G, Lai F, Zytnicki M, Notredame C, Huang Q, Guigo R, Shiekhattar R

“Histone modifications at human enhancers reflect global cell-type-specific gene expression”.

Heintzman ND, Hon GC, Hawkins RD, Kheradpour P, Stark A, Harp LF, Ye Z, Lee LK, Stuart RK, Ching CW, Ching KA, Antosiewicz-Bourget JE, Liu H, Zhang X, Green RD, Lobanenkov VV, Stewart R, Thomson JA, Crawford GE, Kellis M, Ren B.   Nature. 2009 May 7; 459(7243):108-12.

“Tiny RNAs associated with transcription start sites in animals”.

Taft RJ, Glazov EA, Cloonan N, Simons C, Stephen S, Faulkner GJ, Lassmann T, Forrest AR, Grimmond SM, Schroder K, Irvine K, Arakawa T, Nakamura M, Kubosaki A, Hayashida K, Kawazu C, Murata M, Nishiyori H, Fukuda S, Kawai J, Daub CO, Hume DA, Suzuki H, Orlando V, Carninci P, Hayashizaki Y, Mattick JS.  Nat Genet. 2009 May; 41(5):572-8.

“Chromatin modifications and their function”.

Kouzarides T.   Cell. 2007 Feb 23; 128(4):693-705.

Tripathi V, Ellis JD, Shen Z, Song DY, Pan Q, Watt AT, Freier SM, Bennett CF, Sharma A, Bubulya PA, Blencowe BJ, Prasanth SG, Prasanth KV.   Mol Cell. 2010 Sep 24; 39(6):925-38.

Selected Further Reading

“Small and long non-coding RNAs in cardiac homeostasis and regeneration”

Ounzain, S.; Crippa, S.; Pedrazzini, T.  BBA – Molecular Cell Research vol. 1833 issue 4 April, 2013. p. 923-933

“Regulatory mechanisms of long noncoding RNAs in vertebrate central nervous system development and function.” 

Knauss, J.L.; Sun, T.  “Neuroscience vol. 235 April 3, 2013. p. 200-214

“Comparative genomics reveals ‘novel’ Fur regulated sRNAs and coding genes in diverse proteobacteria.”

Sridhar, J.; Sabarinathan, R.; Gunasekaran, P.; Sekar, K.   Gene vol. 516 issue 2 March 10, 2013. p. 335-344 DOI: 10.1016/j.gene.2012.12.057. ISSN: 0378-1119.

miRNAs Regulate Expression and Function of Extracellular Matrix Molecules”

Rutnam, Z.J.; Wight, T.N.; Yang,  B.B.Matrixixix Biology vol. 32 issue 2 March 11, 2013. p. 74-85 DOI: 10.1016/j.matbio.2012.11.003. ISSN: 0945-053X.

Transcript profiling of microRNAs during the early development of the maize brace root via Solexa sequencing

Liu, P.; Yan, K.; Lei, Y.x.; Xu, R.; Zhang, Y.m.; Yang, G.d.; Huang, J.g.; Wu, C.A.; Zheng, C.C.Genomics vol. 101 issue 2 February, 2013. p. 149-156 DOI: 10.1016/j.ygeno.2012.11.004. ISSN: 0888-7543.

Regulatory mechanisms of long noncoding RNAs in vertebrate central nervous system development and function

Knauss, J.L.; Sun, T.  Neuroscience vol. 235 April 3, 2013. p. 200-214 DOI: 10.1016/j.neuroscience.2013.01.022. ISSN: 0306-4522.

“The dynamic biliary epithelia: Molecules, pathways, and disease”

O’Hara, Steven P.; Tabibian, James H.; Splinter, Patrick L.; LaRusso, Nicholas F. Journal of Hepatology vol. 58 issue 3 March, 2013. p. 575-582 DOI: 10.1016/j.jhep.2012.10.011. ISSN: 0168-8278

ABBREVIATIONS

3C = Chromosome conformation capture

rRNAs = Ribosomal RNAs

tRNAs = Transfer RNAs

snRNAs = Small nuclear RNAs

snoRNAs = Small nucleolar RNAs

miRNAs = MicroRNAs

piRNAs = Piwi-interacting RNAs

eRNAs = Enhancer RNAs

tiRNAs = Transcription initiation RNAs

spliRNAs = Splice-site RNAs

lincRNAs = Long intergenic non-coding RNAs

lncRNPs = Long non-coding ribonucleoprotein complexes

Igf2r = Insulin-like growth factor II receptor

HMTs = Histone methyl transferases

TSSs = Transcriptional start sites

TFs = Transcription factors

RNAi = RNA interference

PTMs = Post-translational modifications

  • Patent. (postdocstreet.wordpress.com)

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The Development of siRNA-Based Therapies for Cancer

Author: Ziv Raviv, PhD

Background

The use of gene regulation technology in research and medicine had evolved rapidly since the discovery of post transcriptional gene silencing using RNA interference (RNAi). RNAi was first described in C. elegance in the 90s of the previous century. RNAi post transcriptional gene regulation is carried out by small non-coding RNA double strand RNA (dsRNA) molecules such as microRNA (miRNA; miR) and small interference RNA (siRNA), and has an important role in defending cells against parasitic nucleotide sequences (e.g. viruses) as well as in gene expression regulation.

In RNAi-mediated gene regulation, short dsRNA molecules are being transcribed in the nucleus (in the case of miRs) or introduced exogenously into the cell (in the case of synthetic siRNA or viruses), and are processed in the cytoplasm by an enzyme called Dicer that cleaves long dsRNA and pre-microRNA to produce short double-stranded RNA fragments of 21 base pairs long. The 21 nucleotides long double strand RNA is then being incorporated into the RNA-induced silencing complex (RISC) where it is unwound into two single strands RNA (ssRNA). The “guide” strand is then paired with its complementary targeted messenger RNA (mRNA) that is subsequently cleaved by Argonaute RISC-associated endonuclease. Consequently, the targeted gene protein expression is blocked, leading to its substantial reduced levels in the cell. This so called gene silencing or gene knockdown, hitting the message not the gene itself, will last as long as RNAi molecules are present. The mechanism of action of RNAi is illustrated in the following Video.

RNAi technology was then massively adapted for research allowing the evaluation of functional involvement of genes in various cellular processes because introducing synthetic siRNA into cells can selectively suppress any specific gene of interest.  Not only that RNAi serves as a valuable research tool both in cell culture and in vivo, RNAi has an extremely high potential for specific gene-targeting therapy, as many diseases consist gene deregulation. Synthetic siRNAs are perfectly and completely base pairing to a target (in contrast to endogenous miRs), leading to mRNA-induced cleavage in a single-specific manner that allows treatment without non-specific off-target side effects.

RNAi as therapeutic tool for cancer

All malignant conditions consist of gene deregulations in the form of mutations causing protein misfunction that lead to loss of cell growth regulation and consequently to cancer. Therefore, the fact that siRNA can selectively and specifically target any gene of interest creates a powerful tool to downregulate cancer-associated genes, that eventually will lead to a decrease and even abolishment of the malignant condition.

The advantages of using siRNA for therapy thus are:

  • RNAi technology represents a 3rd revolutionary step for pharmaceutics after small molecules and monoclonal antibodies (mAb), and has a strong commercial potential similar to mAb and even beyond.
  • The ability to target any gene of interest, by blocking specifically the message from DNA to protein consequently the protein is not allowed to be expressed and thus is not functioning.
  • Specificity – siRNA have strong potential to bind specifically to target mRNA, thus lowering unwanted side effects.
  • siRNAs are double stranded oligonucleotides, which are resistant to nucleases.
  • Fast pre-clinical development

General considerations for developing anti-cancer RNAi-based treatment

Given the great potential of siRNA as a therapeutic tool for cancer, one should bring into consideration some general aspects for the development of a siRNA anti-cancer drug:

  • Choosing the gene of interest to be silenced – A wide spectrum of genes could be considered as targets based upon gene of interest role in the cancer cell, type of cancer, and condition of the disease: (i) Oncogenes or central signaling molecules that are crucial for cancer cell growth (ii) Anti-apoptotic deregulated genes (iii) Cancer metabolism associated genes (iv) Angiogenic related genes (v) Metastatic condition related genes.
  • Considering the option of hitting combined target genes consist of different functions (e.g. an oncogene and an anti-apoptotic gene).
  • Basic research evaluation – To examine the effect of silencing the gene of interest in cancer cell based assays and in animal models.
  • Chemical modifications of the siRNA molecule – Modifications such as 2′OMe to increase protection from nuclease, decrease the immunogenicity, lower the incidence of off-target effects, and improve pharmacodynamics of the siRNA.
  • Drug delivery formulation – For an efficient transport of the siRNA. Such delivery system could be formulated using liposome-based nanoparticles (NP) or other nanocarriers to facilitate the siRNA effective systemic distribution.
  • PEGylation – PEGylation of the NPs carriers to reduce non-specific tissue interactions, increase serum stability and half life, and reduce immunogenicity of the siRNA molecule.
  • Site specific targeting – Target tissue-specific distribution of the siRNA drug could be performed by attaching on the outer surface of the nanocarrier a ligand that will direct the siRNA drug to the tumor site.
  • Preclinical – Efficiency and validity, as well as toxicity and pharmacokinetic studies for the siRNA-transporter formulation should be evaluated in animal models.
  • Personalized treatment – In first stages clinical trials, biomarkers should be developed and detected to direct the selection criteria for further treatment of patients with the selected siRNA.
  • Combined therapy – Conduct clinical trials using a combination of the siRNA drug together with a chemotherapy drug that is in-clinical use. Such combined therapy can result in synergism actions of the two combined drugs, and could lower the dosage and thus the side effects of the drugs. In addition, the use of established contemporary agents has practical industrial-related advantages as it is much easier to introduce a new mode of treatment on the background of an existing one.

Development of transport methods for siRNA

As mentioned above, an important aspect in applying siRNA-based therapy is the development of a suitable delivery method that should carry the siRNA molecule systemically to the site of the tumor. In addition, the siRNA-transporter formulation should provide protection from serum nucleases to the siRNA and should decrease its immunogenicity by blocking response of the innate immune system. Examples of such NPs are illustrated in Figure 1. Indeed, several clinical trials were conducted to evaluate the efficacy, validity, and safety use of such transporters for clinical use (Table I).

Figure 1: Various types of nanoparticles for siRNA delivery

Taken from: Cho K et al. Clin Cancer Res 2008;14:1310-1316

Table IClinical trials examining siRNA delivery methods

T1Click on table to enlarge

Table resources: nmOK drug database and clinicaltrials.gov

Download table with active links: Development of siRNA-Based Therapies for Cancer_Table I

Current development status of RNAi-based cancer therapies  

The potential use of RNAi technology to treat cancer is versatile as for any gene of interest it is easy to synthesize a siRNA molecule and the pre-clinical development of siRNA agent is fast. Several companies specialized in siRNA technology have begun recently developing RNAi-based therapies to various cancer associated genes (as well as to other diseases) and to conduct clinical trials. Table II summaries the current clinical trials status of such siRNA-based anti-cancer agents.

Table II: Current clinical trials of siRNA therapies for cancer

T2Click on table to enlarge

Table resources: nmOK drug database, clinicaltrials.gov, and World Health Organization (WHO)

Download table with active links: Development of siRNA-Based Therapies for Cancer_Table II

Conclusion remarks

The power of siRNA-based therapeutics resides in the ability to target and silence any desired gene. Pharmaceutical and biotech companies have started to conduct clinical trials of siRNA therapies for cancer. Most of these clinical trials are in the early preclinical and phase I stages. The results expected from these experiments should further direct the development of siRNA-based anti-cancer therapies and phase II and III trials should consequently emerge. Other target genes should be evaluated as well for siRNA-anti cancer therapy in addition to those that are currently in evaluation, and accelerated efforts should be made in the direction of combining existing chemotherapy with the technology of siRNA. The next future to come will tell us if the potential of siRNA therapy for cancer had been fulfilled.

Related references:

  1. RNAi-Based Therapies for Cancer in Development. Anna Azvolinsky, PhD. Cancernetwork, March 3, 2011.
  2. siRNA-based approaches in cancer therapy. GR Devi. Cancer Gene Therapy (2006) 13, 819–829
  3. Therapeutic Effect of RNAi Gene Silencing Effective in Cancer Treatment, Study Suggests. Sciencedaily, Feb. 11, 2013.
  4. Kinesin Spindle Protein SiRNA Slows Tumor Progression. Marra E, Palombo F, Ciliberto G, Aurisicchio L. J Cell Physiol. 2013 Jan;228(1):58-64.
  5. First-in-Humans Trial of an RNA Interference Therapeutic Targeting VEGF and KSP in Cancer Patients with Liver Involvement. Josep Tabernero et al. Cancer Discov. 2013 Apr;3(4):406-417.

Chemical modification:

  1. Chemical Modification of siRNAs for In Vivo Use. Behlke MA. Oligonucleotides. 2008 Dec; 18(4):305-19.

Delivery Technology:

  1. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Schiffelers RM et al. Nucleic Acids Res. 2004 Nov 1;32(19):e149.
  2. Therapeutic Nanoparticles for Drug Delivery in Cancer.  Kwangjae Cho, Xu Wang, Shuming Nie, et al. Clin Cancer Res 2008;14:1310-1316.
  3. Liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer. Malam Y, Loizidou M, Seifalian AM. Trends Pharmacol Sci. 2009 Nov; 30(11):592-9.
  4. Silence-therapeutics delivery platform

Related articles on this Open Access Online Scientific Journal:

  1. MIT Team: Microfluidic-based approach – A Vectorless delivery of Functional siRNAs into Cells. Reporter: Aviva Lev-Ari, Ph.D., RN
  2. Targeted Tumor-Penetrating siRNA Nanocomplexes for Credentialing the Ovarian Cancer Oncogene ID4. Reporter and Curator: Sudipta Saha, Ph.D.
  3. Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part II. Curator and Reporter: Aviral Vatsa Ph.D., MBBS
  4. Nanotechnology and HIV/AIDS treatment. Author: Tilda Barliya, PhD

To download tables of this post (with active links) :

  1. Development of siRNA-Based Therapies for Cancer_Table I
  2. Development of siRNA-Based Therapies for Cancer_Table II

Databases:

http://www.nmok.net

http://www.clinicaltrials.gov/

http://apps.who.int/trialsearch/

Related Videos:

RNA interference mechanism of action

RNA interference (RNAi): by Nature video

RNAi Therapeutics and Cancer Treatment

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Reporter: Aviva Lev-Ari, PhD, RN

 

Brain Development Is Guided by Junk DNA that Isn’t Really Junk

By Jeffrey Norris on April 15, 2013

Fluorescent dyes track the presence of the RNA molecules and the genes they  affect in the developing mouse brain.

UCSF researchers have uncovered a role in brain development and in neurological

disease for little appreciated molecules called long noncoding RNA. In this image,

fluorescent dyes track the presence of the RNA molecules and the genes they

affect in the developing mouse brain. Image courtesy of Alexander Ramos

Specific DNA once dismissed as junk plays an important role in brain development and might be involved in several devastating neurological diseases, UC San Francisco scientists have found.

Their discovery in mice is likely to further fuel a recent scramble by researchers to identify roles for long-neglected bits of DNA within the genomes of mice and humans alike.

While researchers have been busy exploring the roles of proteins encoded by the genes identified in various genome projects, most DNA is not in genes. This so-called junk DNA has largely been pushed aside and neglected in the wake of genomic gene discoveries, the UCSF scientists said.

In their own research, the UCSF team studies molecules called long noncoding RNA (lncRNA, often pronounced as “link” RNA), which are made from DNA templates in the same way as RNA from genes.

“The function of these mysterious RNA molecules in the brain is only beginning to be discovered,” said Daniel Lim, MD, PhD, assistant professor of neurological surgery, a member of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF, and the senior author of the study, published online April 11 in the journal Cell Stem Cell.

Daniel Lim, MD, PhD

Alexander Ramos, a student enrolled in the MD/PhD program at UCSF and first author of the study, conducted extensive computational analysis to establish guilt by association, linking lncRNAs within cells to the activation of genes.

Ramos looked specifically at patterns associated with particular developmental pathways or with the progression of certain diseases. He found an association between a set of 88 long noncoding RNAs and Huntington’s disease, a deadly neurodegenerative disorder. He also found weaker associations between specific groups of long noncoding RNAs and Alzheimer’s disease, convulsive seizures, major depressive disorder and various cancers.

“Alex was the team member who developed this new research direction, did most of the experiments, and connected results to the lab’s ongoing work,” Lim said. The study was mostly funded through Lim’s grant – a National Institutes of Health (NIH) Director’s New Innovator Award, a competitive award for innovative projects that have the potential for unusually high impact.

LncRNA versus Messenger RNA

Unlike messenger RNA, which is transcribed from the DNA in genes and guides the production of proteins, lncRNA molecules do not carry the blueprints for proteins. Because of this fact, they were long thought to not influence a cell’s fate or actions.

Alexander Ramos

Nonetheless, lncRNAs also are transcribed from DNA in the same way as messenger RNA, and they, too, consist of unique sequences of nucleic acid building blocks.

Evidence indicates that lncRNAs can tether structural proteins to the DNA-containing chromosomes, and in so doing indirectly affect gene activation and cellular physiology without altering the genetic code. In other words, within the cell, lncRNA molecules act “epigenetically” — beyond genes — not through changes in DNA.

The brain cells that the scientists focused on the most give rise to various cell types of the central nervous system. They are found in a region of the brain called the subventricular zone, which directly overlies the striatum. This is the part of the brain where neurons are destroyed in Huntington’s disease, a condition triggered by a single genetic defect.

Ramos combined several advanced techniques for sequencing and analyzing DNA and RNA to identify where certain chemical changes happen to the chromosomes, and to identify lncRNAs on specific cell types found within the central nervous system. The research revealed roughly 2,000 such molecules that had not previously been described, out of about 9,000 thought to exist in mammals ranging from mice to humans.

In fact, the researchers generated far too much data to explore on their own. The UCSF scientists created a website through which their data can be used by others who want to study the role of lncRNAs in development and disease.

“There’s enough here for several labs to work on,” said Ramos, who has training grants from the California Institute for Regenerative Medicine (CIRM) and the NIH.

“It should be of interest to scientists who study long noncoding RNA, the generation of new nerve cells in the adult brain, neural stem cells and brain development, and embryonic stem cells,” he said.

Other co-authors who worked on the study include UCSF postdoctoral fellows Aaron Diaz, PhD, Abhinav Nellore, PhD, Michael Oldham, PhD, Jun Song, PhD, Ki-Youb Park, PhD, andGabriel Gonzales-Roybal, PhD; and MD/PhD student Ryan Delgado. Additional funders of the study included the Sontag Foundation and the Sandler Foundation.

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Epigenetic mechanisms

Epigenetic mechanisms

Image Source:http://nihroadmap.nih.gov/EPIGENOMICS/images/epigeneticmechanisms.jpg |Author=National Institute of Health |Date=2005

The Underappreciated EpiGenome

Author:  Demet Sag, PhD

Early 1990’s Kavai group developed a method called Restriction Landmark Genomic Scanning using Methylation-sensitive endonucleases (RLGS-M) to identify differential methylation during development based on CpG islands. In their study they showed that the appearance and disappearance of the spots were specific to tissue and affecting gene regulation.

English: Revised definition of gene and flow o...

Revised definition of gene and flow of genetic information (adapted from Mattick JS (2003). Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms. BioEssays 25:930. doi:10.1002/bies.10332).

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

There are three main routes of epigenetic modifications one

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

Comparative developmental biology studies and genetics in organisms give away clues that can be applied or open the door for a new discovery in human disease models.  Plants do utilize methylation and transposomes, which are viral particles that can affect the genome structure and present in all organisms including human, for their gene regulation and development extensively (The EMBO Journal, (22 March 2013) | doi:10.1038/emboj.2013.49). Thus, Arapsidosis is a good model.

The environment and gene expression define inheritable materials at transcriptional levels. He group (Cui-Jun Zhang et al, 2013, doi:10.1038/emboj.2013.49) suggested splicing machinery affecting RdDM and transcriptional silencing to control gene regulation during development since an RNA-directed DNA methylation (RdDM) pathway directs de novo methylation. Furthermore, these differences are highly regulated at RNA level with silencing, splicing, transposon activation/inactivation and modulation at epigenetic level.

In fruit fly with more than five hundreds years of accumulated data, the sex determination pathways, soma or germline, share common genes but they act differently in each pathway. Sxl (sex-lethal), which is key gene of somatic sex determination and is an RNA binding protein, regulates the genes through splicing in one its mechanisms (DOI: 10.1002/dvdy.23924). Yet, in germline ovo, which is a DNA binding protein, regulates the expression and development with different sets of rules yet these two paths always communicate to make the final outcome. The effects of RNA on epigenetics and gene regulation during development will be another topic to discuss. However, the three major epigenetic factors do not run in order necessarily, but always have three targeted outcome: initiate, differentiate and maintain. Thus, from environment to phenotype there are places/parts scientists can modulate or reprogram but there parts must be kept intact.

Yang et al, 2012 presented a study on PHD Finger Protein 7 (PHF7), which is an important factor for male germline sexual identity in Drosophila, and called this gene as a “epigenetic reader’ since the expression of this gene in XX soma started a female germline development. This type of epigenetic readers may also alter the outcome to balance cell metabolism towards desired phenotype in stem cells.

Adenine methylation

Adenine methylation (Photo credit: Allen Gathman)

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

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

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

Early in development things are short term and stop after the development seized but be able to maintain the short term phonotype during wound healing, coagulation, trauma, disease and immune responses. Some cells will loose their ability to differentiate to very low levels. Yet, in life everything is possible even with less than 1% chance because nothing is accidental.

X-chromosome has fascinating characteristics simply because they present unusual mechanisms among female and male differentiation in fruit flies and mammals but their distinct characteristics in evolution marry with surprising parallel mechanisms in regulation. These features are the importance of noncoding RNAs, and epigenetic spreading of chromatin-modifying activities, and at the end of the actions most part of the Y chromosome is lost and one of the X-chromosome is downregulated in the big picture.

Figure 2: DNA methylation analysis methods not...

DNA methylation analysis methods not based on methylation-specific PCR. Following bisulfite conversion, the genomic DNA is amplified with PCR that does not discriminate between methylated and non-methylated sequences. The numerous methods available are then used to make the discrimination based on the changes within the amplicon as a result of bisulfite conversion. (Photo credit: Wikipedia)

Revisiting RNA directed DNA methylation study once again shows that unread sequence has the word on gene expression; it can still create the diversity that may help rebirth of stem cells with a correct program and develop tools for unmet human diseases. This will be the next topic to discuss.

Personal Impression

While I was listening Dr. Ecker, I remembered these studies. The question becomes what we know then what we know now. “The Underappreciated Epigenome: Methylation of Brain” by Joseph Ecker, Ph.D. of Salk Institute gave a talk on differential expression in adult vs. fetal brain development at Future of Genomics VI Medicine on March 7, 2013.

I like to give snapshot of his talk, and relating to the third wheel of the epigenetics: non coding RNAs for epigenetics, stem cell biology and development. He also reconnecting the dots and demonstrated that there is a linear relation between gene regulation region and methylation type.  As a result the plasticity of development takes place with the extensive mutation reconfigurations during early post natal stages up to two years at synaptogenesis. 

Ecker’s Study

The study focused onto inheritability of methylation in different organisms and comparative expression pattern. The data from chip sequencing for

  • histone modifications,
  • whole genome bisulfide sequencing for DNA modifications and
  • methylome profiling

projected a differential expression pattern between

  • adult and
  • fetal brain

for 5 hydroxymethyl cytosine hmC and 5 methyl cytosine mC.

Completion of base resolution of human methylation and aberrant epigenomic reprogramming in induced stem cells showed that the density of genic mCH is positively correlated with gene expression.

  • There was an increased mCH and an elevated gene expression pattern, unlike mCG that the gene is silenced at stem cell differentiation.
  • mCH expression was not only tissue specific but also cell specific based on comparative expression study.
  • There was no mCH expression in fetal frontal cortex unlike adult frontal cortex with accumulation of mCH during synaptogenesis. Also
  • deserts of methylation can be counted as heterochromatic regions and protective transfactors between mCG and mCH methylation.
  • In DNMT pattern showed neurons enriched with mCH but glia was depleted.  Furthermore, these
  • sites are not randomly but occurring with correlation.

Ecker group also published (Lister et al, 2009) the first genome-wide study in a mammalian genome, from both

  • human embryonic stem cells and
  • fetal fibroblasts, along with
  • comparative analysis of messenger RNA and
  • small RNA components of the transcriptome, several
  • histone modifications, and
  • sites of DNA–protein interaction for several key regulatory factors.

Like the related review paper by Spivakov and Fisher pointed out the search for molecular signatures of ‘stemness’ and pluripotency is becoming important for cell therapy. Thus, there is a huge effort on transcription machinery of key genes during early development and understanding of stem cells, but working on the epigenetic profiles and their interaction with transcription machinery is equally important. This poised but activable factors under the stem cell genome may open new doors for diagnostics and therapies.  As a result, “restricted” human diversity will open doors for a personalized medicine and delivery mechanisms.

Until human genome was sequenced the expected number of genes was high but only 1% of genome is read producing about 25000 genes. That brings up three modules to be concerned:

1. Use the RNA wisely as the ancestor of transferable genetic material that viruses used, even human has embedded over 90% natural viral in their genome;

2. Apply epigenetics with all three types from a scratch;

3. Inheritance.

RNA is regulating the methylation on genome through transposons and silencing the genes at transcriptional level to create intergenerational or transgenerational reprograming. This makes sense since after the decision is made there are two intentions passing onto next generation and maintaining the decision consistently.  If

  • in soma by mitosis to daughter cells, or
  • in germline by meiosis

the characters are transferred to the next generation. However, we also need to mind after the fact because no one is choosing what they get let alone not being able to choose their parents. Choosing the healthy tolerance levels in genome for future medicine is the key.

REFERENCES

Borge, F. and Martiense, R.A. “Establishing epigenetic variation during genome reprogramming” RNA Biology, Volume 10, Issue 4, April 2013,  doi: org/10.4161/rna.24085

Dalakouras,A. and Wassenegger, M. “Revisiting RNA-directed DNA methylation” RNA Biology, Volume 10, Issue 3 March 2013 Pages 453 – 455 http://dx.doi.org/10.4161/rna.23542

Piferrer, F. “Epigenetics of sex determination and gonadogenesis” Developmental Dynamics 8 FEB 2013 DOI: 10.1002/dvdy.23924

Yang SY, Baxter EM, Van Doren M. “Phf7 controls male sex determination in the Drosophila germline” Dev Cell. 2012 May 15;22(5):1041-51. doi: 10.1016/j.devcel.2012.04.013.

Yongkyu Park,  Mitzi I. KurodaEpigenetic Aspects of X-Chromosome Dosage Compensation” Science 10 August 2001: Vol. 293 no. 5532 pp. 1083-1085  DOI: 10.1126/science.1063073

Okano M, Xie S, Li E (July 1998). “Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases” Nat Genet 19 (3): 219–20. doi:10.1038/890    

Di Croce L, Raker VA, Corsaro M, et al. (2002). “Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor” Science 295 (5557): 1079–82. doi:10.1126/science.1065173

Jun Kawai1,+, Shinji Hirotsune2,3,Kenji Hirose1,3,+,Shinji Fushiki4, Sachihiko Watanabe1,+ and Yoshihide Hayashizaki2,3, “Methylation profiles of genomic DNA of mouse developmental brain detected by restriction landmark genomic scanning (RLGS) method” Nucl. Acids Res. (1993) 21 (24): 5604-5608. doi: 10.1093/nar/21.24.5604

Cui-Jun Zhang, Jin-Xing Zhou, Jun Liu, Ze-Yang Ma, Su-Wei Zhang, Kun Dou, Huan-Wei Huang, Tao Cai, Renyi Liu, Jian-Kang Zhu and Xin-Jian He. “The splicing machinery promotes RNA-directed DNA methylation and transcriptional silencing in Arabidopsis” The EMBO Journal , (22 March 2013) | doi:10.1038/emboj.2013.49

Ryan Lister1,9, Mattia Pelizzola1,9, Robert H. Dowen1, R. David Hawkins2, Gary Hon2, Julian Tonti-Filippini4, Joseph R. Nery1, Leonard Lee2, Zhen Ye2, Que-Minh Ngo2, Lee Edsall2, Jessica Antosiewicz-Bourget5,6, Ron Stewart5,6, Victor Ruotti5,6, A. Harvey Millar4, James A. Thomson5,6,7,8, Bing Ren2,3 & Joseph R. Ecker1 “Differential methylation between stem cells and adult stem cellsNature 462, 315-322 (19 November 2009) | doi:10.1038/nature08514

Mikhail Spivakov & Amanda G. Fisher “Epigenetic signatures of stem-cell identity” Nature Reviews Genetics 8, 263-271 (April 2007) | doi:10.1038/nrg2046

Louise C Laurent1,2,3,15, Caroline M Nievergelt4,15, Candace Lynch2,3, Eyitayo Fakunle2,3, Julie V Harness5, Uli Schmidt6, Vasiliy Galat7,8, Andrew L Laslett9,10,11, Timo Otonkoski12,13, Hans S Keirstead5, Andrew Schork4, Hyun-Sook Park14 & Jeanne F Loring2  “Restricted human ethnic diversity in human stem cell lines.” Nature Methods 7, 6 – 7 (2010) doi:10.1038/nmeth0110-06

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New Insight into How Genes Function

Reporter: Larry H Bernstein, MD, FCAP

 

New Insight into How Genes Function

GENNewsHighlights    Feb 17, 2013

Long segments of noncoding RNA are key to

  • physically manipulating DNA in order to activate certain genes.
  • These noncoding RNA-activators (ncRNA-a) have a crucial role in
An illustration of the central dogma of molecu...

Diagram of a eukaryotic gene

Diagram of a eukaryotic gene (Photo credit: Wikipedia)

This image shows the coding region in a segmen...

This image shows the coding region in a segment of eukaryotic DNA. (Photo credit: Wikipedia)

English: Sporulation involved ncRNA

English: Sporulation involved ncRNA (Photo credit: Wikipedia)

English: Genes required for ectodermal specifi...

English: Genes required for ectodermal specification during early embryogenesis (Photo credit: Wikipedia)

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