EPIGENETICS Textbook Fall 2013

Major Headings in Text Epigenetic gene regulation Chromatin 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 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

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 multi-complex 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