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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 • Remodeling required for both activation and silencing (microarray data) – involves multiprotein complexes, uses ATP NOTE: TEXTBOOK SECTIONS NOT BEING COVERED • Recruiting Chromatin remodeling complexes • Mechanisms of Chromatin Remodeling Major Headings in Text • Is there a “histone code”? – Modifications at specific residues associated with different processes – Stages 1. Recruit modifying enzymes to target loci 2. Downstream effects of histone modifications a) Direct or distant effects b) Highly specific 3. Reversing the effects a) remove activating histone modifications b) deposit repressive marks 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 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 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 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 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 multicomplex units that also have histone modifying components, HMT, HDAC 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) 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 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 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 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 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 EXAMPLE EPIGENOMICS RESEARCHERS UNCOVER 67 NEW CHEMICAL MODIFICATIONS ON DNA ASSOCIATED PROTEINS http://www.roadmapepigenomics.org/ WHAT BIOTECHNOLOGIES ARE BEING USED? • Microarray • PCR