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Genomic Focus: Epigenetics Corey Henderson Laura Bergeron Cynthia Hansen 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 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 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. 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 1. Chromatin cross-linked to DNA with formaldehyde •Proteins not bound to DNA not crosslinked even at high concentrations DNA DNA http://publish.uwo.ca/~jkiernan/formglut.htm •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 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 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 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) Schulze,S Xist is expressed from both X chromosomes but only spreads and initiates silencing on ONE X 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?