Genomic Focus: Epigenetics

Laura Bergeron Cynthia Hansen

Corey Henderson
Kyle Fowler

Introduction to Epigenetics

http://en.wikipedia.org/wiki/Epigenetics

Epigenetics: Defining the Science

The study of heritable changes in gene expression that occur without a change in DNA sequence.
Regulation differentiation From Structure not sequence
Heritable

Defensive tactics (the micro-arm-bar)

Repeat induced modification
Methylation

Histones: More than Just Packing

http://en.wikipedia.org/wiki/Histone

Types
H1 H2A H2b H3 H4

Species Conservation
Eukaryotes Archea

Nucleosome Formation
Chromatin

Modifiable Tails
Methylation Acetylation

Histone Code Basic Packaging

Video

http://en.wikipedia.org/wiki/Ima ge:NucleosomeKG.jpg

Chromatin
DNA + Protien Enables extraordinary condensation and packaging of eukaryotic genomes Fundamental unit: NUCLEOSOME Gene expression in Eukaryotes takes place with highly packaged chromatin Regulation of gene expression by chromatin structure is epigenetic regulation

Heterochromatin
Highly condensed DNA
Lots of repeats

Danger of TEs But: Neccesary

In absence of heterochromatin chromosomes fail to segregate properly

Effect of structure over sequence

Moving a euchromatic gene next to or within heterochromatin will show variable scilencing w/ no change in coding

Discoveries for Epigenetics

Filamentous Fungi
RIP
Repeat Induced Point Mutation

MIP
Methylation induced premeiotically

Plants and Animals

RIGS
Repeat Induced Gene Silencing

RNA
Sequence-specific degradation
RNAi
AKA
Cosuppression (plants) Quelling (Neurospora)

Post Translational Gene Scilencing RNA/DNA interaction

Methylation
Feedback induced Epigentic regulation

DNA Processes Developmental Control

X-inactivation Genomic Imprinting
Expression of alleles different depending on parental origin
Overlapping sense and antisense causing dsRNA causing allele specific repression

Histone modification of transcribed and untrasncribed regions of DNA

Epigenetics

Chromatin: An Overview

http://www.sciencemuseum.org.uk/on-line/lifecycle/133.asp

http://www.blackwellpublishing.com/11thhour/book5/about/images/c5t2.gif

The Histone Code Hypothesis

Post-translational covalent modification of histone N-terminal tails
Acetylation

Methylation
Phosphorylation Ubiquitination
http://www.umanitoba.ca/institutes/manitoba_institute_cell_biology/MICB/davie_jim_2.htm

The Histone Code Hypothesis

http://www.benbest.com/health/cancer.html

Code regulates accessibility of DNA and transcription of genes Language by which information about chromatin and underlying genes is conveyed to other protein complexes Combinatorial (Jenuwein and Allis, 2001)

The Histone Code Hypothesis

Different PTMs bound by specific domains found in transcriptionally relevant genes
Bromodomains acetylation Chromodomains methylation
(Jenuwein and Allis, 2001)

The Histone Code Hypothesis

In general, acetylation is associated with active genes

H4K12 acetylation heterochromatin in many organisms

Methylation associated with silenced genes

H3K4 methylation euchromatin in many organisms

In this study: H3-K4 dimethylation, H3-K4 trimethylation, H3-Ac, H4-Ac, and H3-K79 dimethylation

Chromatin Immunoprecipitation (ChIP)

1. 2. 3. 4. 5. Chromatin cross-linked to DNA with formaldehyde Fragmentation of chromatin Immunoprecipitation with antibody specific for particular PTM Chromatin-bound DNA extracted, purified, fluorescently labeled Cohybridization of chromatin-bound DNA and non-immunoprecipitated DNA sample (labeled with different fluorescent marker) to microarray Ratio of fluorescent signals measure of enrichment due to ChIP

6.

1.

Chromatin cross-linked to DNA with formaldehyde

Proteins not bound to DNA not crosslinked even at high concentrations Mild conditions can be used to reverse cross-links Effective in vivo or in vitro (Solomon and Varshavsky, 1985)

DNA

DNA
http://publish.uwo.ca/~jkiernan/formglut.htm

2. Fragmentation of Chromatin Sonication to break apart nucleosomes Believed to fragment DNA at random 500-700 bp Exposure of antibody-binding epitopes
http://openlearn.open.ac.uk/file.php/2645/formats/print.htm

3. Immunoprecipitation with antibody specific for particular PTM

Polyclonal usually preferred Polyclonal Acetylated Lysine Antibodies can be raised using peptides containing particular PTMs

ARTme2KQTARKSC IAQDFme2KTDLRF
http://www.creationsbydawn.net/pi/tutorials/rabbit.jpg

http://www.imgenex.com/images/antibody_image.jpg

http://www.abcam.com/index.html?datasheet=21623

4. Chromatin-bound DNA extracted, purified, fluorescently labeled

Reversal of formaldehyde cross-linking

Extract DNA from resulting suspension PCR to enrich bound DNA samples

Fluorescent labeling- Cy5 (red) and Cy3 (green)-labeled nucleotides incorporated in PCR

ChIP-on-Chip

5. Cohybridization of chromatin-bound DNA and nonimmunoprecipitated DNA sample (labeled with different fluorescent marker) to microarray 6. Ratio of fluorescent signals measure of enrichment due to ChIP

Mass Spectrometry

MS can be used to identify and quantify histone post-translational modifications (PTMs) on a proteomic scale If DNA sequence known, deviation from expected mass PTMs Identification of previously unknown modifications

Mass Spectrometry

Methodology
Histone Code
Post-translational covalent modifications to the histone tails

Methodology
Chromatin Immunoprecipitation (ChIP)
Isolation of DNA bound to chromatin Requires specific antibody or epitope tag to pull down protein (or modification) of interest Looked at relationship between genes and specific modifications (acetylation, methylation, phosphorylation, etc.)

Methodology

Schulze, S.

Methodology
Chromatin Immunoprecipitation (ChIP) ChIP on Chip (ChIPs!)
ChIP coupled with array Genomic approach to ChIP

Methodology

Public domain image

Methods in Epigenetics
Chromatin Immunoprecipitation (ChIP) ChIP on Chip ChIPs signal analysis

Bound

= enrichment for modification = absence of modification

Input

Methods in Epigenetics

Data and Conclusions

Are there relationships between genes and histone modifications? Relationships between various histone modifications? What did they hope to see?
Correlations! AKA, Thank god its linear

Data and Conclusions

Data and Conclusions

Found distinct correlations between certain histone modifications
Gene dimethylated at Lys 4 likely to also be dimethylated on Lys 79, and acetylated

Data and Conclusions

Example: H3/H4 Acetylation H3 Lys4 Trimethylation

Data and Conclusions

Found distinct correlations between certain histone modifications
Gene dimethylated at Lys 4 likely to also be dimethylated on Lys 79, and acetylated

Transcriptional state reflected in histone modifications

Genes actively transcribing vs. repressed Acetylation, H3-K4, H3-K79 associated with active genes

Data and Conclusions

More enrichment, More expression

Data and Conclusions

Transcriptional correlation beyond the gene level? Chromosome- & Genome-wide analysis
Entirety of H3-K4 methylation across chromosome 2L in Drosophila Related these data to cDNA array

Data and Conclusions

H3-di-meK4 enrichment (blue) across chromosomal and EST arrays (grey box=gene) Signal for complete chromosomal arm (enrichment vs. signal)
Sequences enriched for modification are above the line

Future Work / Other Research

Variant histones
Euchromatin vs. heterochromatin

Dosage compensation Implications (medicine, heredity, etc.)

Differences in Histones: Variant

All histones have variants except H4 The varients are subject to posttranslational modification as well Some are very similar with subtle differences (ex. H3 and H3.3) Others are very different (ex. H2A and macroH2A) Specific tasks: Transcription activating and silencing, damaged DNA detection, etc.

Chicken or the Egg?

Yeast only has variants

Diversity in variant function

www.cartoonstock.com

Histones and Viruses

Variant histones have been associated with viral stabilization Viruses make own chromatin through canonical histones Variant histones used as an entry point in DNA integration

Dosage Compensation

Xist: X inactive-specific transcript (non coding RNA) Xist is expressed from both X chromosomes but only spreads and initiates silencing on ONE X

Schulze,S

Similar Packaging to Heterochromatin

Incorporation of the histone variant macro H2A Evidence of DNA methylation, H3K9me, H3K27me3 Fewer active marks like acetylation of H3, H4

Schulze, S

Epigenetics and medicine

Cancer Heart failure Mood disorders

Questions?