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EPIGENETICS Textbook

EPIGENETICS Textbook. Fall 2013. Major Headings in Text. Epigenetic gene regulation Basic mechanisms – histones and DNA methylation Additional mechanisms – other histone modifications Chromatin Gene activation and silencing Post-translational histone modification

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EPIGENETICS Textbook

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  1. EPIGENETICSTextbook Fall 2013

  2. Major Headings in Text • Epigenetic gene regulation • Basic mechanisms – histones and DNA methylation • Additional mechanisms – other histone modifications • Chromatin • Gene activation and silencing • Post-translational histone modification • Remodeling required for both activation and silencing (microarray data) • involves multiprotein complexes, uses ATP

  3. NOTE: TEXTBOOK SECTIONSNOT BEING COVERED • Recruiting Chromatin remodeling complexes • Mechanisms of Chromatin Remodeling

  4. Major Headings in Text • Is there a “histone code”? • Modifications at specific residues associated with different processes • Stages • Recruit modifying enzymes to target loci • Downstream effects of histone modifications • Direct or distant effects • Highly specific • Reversing the effects • remove activating histone modifications • deposit repressive marks

  5. Major Headings in Text • Maintaining histone transcription patterns – long term • Define cell identity and function – maintain differentiated state, • Complexes highly conserved in plants and animals; 1st described in Drosophila • Trithorax Group (trxG) maintains active transcription • Polycomb Group (PcG) maintains transcription repression

  6. DNA METHYLATION • Direct chemical modification of CpG or CpG islands, found on 70% of mammalian CpG • Methyl group sticks out into the major groove of DNA helix but does not interfere with G-C binding • Establish and maintain long term silencing

  7. DNA METHYLATION • 3 DNA methyl transferases maintain methyl groups, even through cell division • Dnmt1 maintains pattern – hemi-methylated template  fully methylated (Fig. 4.6) • Dnmt3a/Dnmt3b generates new CpG methylation pattern where there is none • Early embryogenesis - X chromosome inactivation (silencing by repressive histones) in XX • Pro-nuclei stage: • male pro- nuclei actively demethylated • Female pro-nuclei partially demethylated • Remethylation starts after implantation

  8. DNA Methylation & Gene Regulation • CpG islands • Found in 5’ promoter areas • not methylated on active and silent genes • EXCEPTIONS: • Silencing on X chromosome • When cells differentiate • Pathological processes, e.g., inactivation of tumor suppressor genes in some cancers

  9. DNA Methylation & Gene Regulation • MECHANISMS (See. Fig. 4.7) • DIRECT/SHORT REGIONS: Steric inhibition of transcription factor binding, i.e., transcriptional regulation • INDIRECT/LONGER REGIONS: mediated by “methyl binding domain” proteins acting in multi-complex units that also have histone modifying components, HMT, HDAC

  10. METHODOLOGY • Cells fixed with formaldehyde • Isolate chromatin and shear into 400-500 bp DNA • Perform chromatin immunoprecipitation (DNA is still attached) – Ab to histone protein or protein modification used to isolate associated DNA sequence • Heat to break DNA-protein cross-links • PCR DNA in immunopptd fragments (bound) and original sample (input)

  11. Genome-wide Chromatin Analysis • One way: • Uses microarray technology to measure genes and abundance, • expression microarray covers gene sequences • Genomic microarrays • Regions of CpG islands around promoters • “tiling arrays” - Selected regions along a chromosomal locus • Covers > 10,000 distinct genes

  12. DISEASES • Discussed cancer some already • Role in tumor suppression • Possibly tumor start & progression • Single gene mutations; multiple gene mutations over time • Epigenetic – inappropriate activation or silencing

  13. DISEASES !!! • Defective epigenetic regulators • Hybrid histone modifying enzymes (chromosomal rearrangements) • Acute Myelogenous Leukemia (AML) Chr11: 11q23 cuts gene for histone methyl transferase •  truncated enzyme •  new fusion proteins (N-term HAT fused to C-term of 2 other HATs •  no silencing of 2 genes, HOXA9 & MEIS1

  14. DISEASE • Absent or deregulated chromatin remodeling complexes • SW1/SNF binds to p53 to regulate the cell cycle • If mutated (absent) or deregulated  lack of control for cell growth • Defective methyl binding proteins – MeCP2  key neural gene no longer silenced and is over-expressed;  loss of neural development and function, Rett Syndrome

  15. MASSIVE EPIGENOMIC CONSORTIA • New tools and protocols being developed • Websites with information are freely accessible • Human Epigenetic Project (HEP) • Roadmap Epigenomics Project • Data already being published

  16. EXAMPLE EPIGENOMICS RESEARCHERS UNCOVER 67 NEW CHEMICAL MODIFICATIONS ON DNA ASSOCIATED PROTEINS http://www.roadmapepigenomics.org/

  17. WHAT BIOTECHNOLOGIES ARE BEING USED? • Microarray • PCR

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