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Transcript
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