1. EPIGENETICS Textbook
Fall 2013

2. 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

3. NOTE: TEXTBOOK SECTIONS NOT 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 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

17. WHAT BIOTECHNOLOGIES ARE BEING USED?
Microarray
PCR