Genomic Focus: Epigenetics Laura Bergeron Corey Henderson Cynthia Hansen Kyle Fowler

Introduction to 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 Types –H1 –H2A –H2b –H3 –H4 Species Conservation –Eukaryotes –Archea Nucleosome Formation –Chromatin Modifiable Tails –Methylation –Acetylation Histone Code Basic Packaging –VideoVideo 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 TE’s 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

The Histone Code Hypothesis Post-translational covalent modification of histone N-terminal tails –Acetylation –Methylation –Phosphorylation –Ubiquitination

“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 The Histone Code Hypothesis (Jenuwein and Allis, 2001)

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 The Histone Code Hypothesis

1.Chromatin cross-linked to DNA with formaldehyde 2.Fragmentation of chromatin 3.Immunoprecipitation with antibody specific for particular PTM 4.Chromatin-bound DNA extracted, purified, fluorescently labeled 5.Cohybridization of chromatin-bound DNA and non-immunoprecipitated DNA sample (labeled with different fluorescent marker) to microarray 6.Ratio of fluorescent signals  measure of enrichment due to ChIP Chromatin Immunoprecipitation (ChIP)

DNA 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) 2. Fragmentation of Chromatin Sonication to break apart nucleosomes Believed to fragment DNA at random  bp Exposure of antibody-binding epitopes

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

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

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

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

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 Methodology

Chromatin Immunoprecipitation (ChIP) ChIP on Chip ChIPs signal analysis Methods in Epigenetics Bound Input = enrichment for modification = absence of modification

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

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

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?