Genomic Focus: Epigenetics

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Genomic Focus Epigenetics
Corey Henderson
Laura Bergeron
Kyle Fowler
Cynthia Hansen
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Introduction to Epigenetics
http//en.wikipedia.org/wiki/Epigenetics
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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

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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/ImageNucleosomeKG.jp
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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

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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

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Discoveries for Epigenetics

• Filamentous Fungi
• RIP
• Repeat Induced Point Mutation
• MIP
• Methylation induced premeiotically
• Plants and Animals
• RIGS
• Repeat Induced Gene Silencing

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RNA

• Sequence-specific degradation
• RNAi
• AKA
• Cosuppression (plants)
• Quelling (Neurospora)
• Post Translational Gene Scilencing
• RNA/DNA interaction
• Methylation
• Feedback induced Epigentic regulation

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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

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Epigenetics
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Chromatin An Overview
http//www.sciencemuseum.org.uk/on-line/lifecycle/
133.asp
http//www.blackwellpublishing.com/11thhour/book5/
about/images/c5t2.gif
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The Histone Code Hypothesis

• Post-translational covalent modification of
  histone N-terminal tails
• Acetylation
• Methylation
• Phosphorylation
• Ubiquitination

http//www.umanitoba.ca/institutes/manitoba_instit
ute_cell_biology/MICB/davie_jim_2.htm
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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)

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The Histone Code Hypothesis

• Different PTMs bound by specific domains found in
  transcriptionally relevant genes
• Bromodomains ? acetylation
• Chromodomains ? methylation

(Jenuwein and Allis, 2001)
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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

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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

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• 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)

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
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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?datasheet21623
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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

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ChIP-on-Chip
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
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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

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Mass Spectrometry
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Methodology

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

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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.)

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Methodology
Schulze, S.
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Methodology

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

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Methodology
Public domain image
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Methods in Epigenetics

• Chromatin Immunoprecipitation (ChIP)
• ChIP on Chip
• ChIPs signal analysis

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Methods in Epigenetics
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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

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Data and Conclusions
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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

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Data and Conclusions
Example H3/H4 Acetylation ? H3 Lys4
Trimethylation
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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

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Data and Conclusions
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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

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Data and Conclusions

• H3-di-meK4 enrichment (blue) across chromosomal
  and EST arrays (grey boxgene)
• Signal for complete chromosomal arm (enrichment
  vs. signal)

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Future Work / Other Research

• Variant histones
• Euchromatin vs. heterochromatin
• Dosage compensation
• Implications (medicine, heredity, etc.)

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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.

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Chicken or the Egg?

• Yeast only has variants
• Diversity in variant function

www.cartoonstock.com
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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

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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
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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
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Epigenetics and medicine

• Cancer
• Heart failure
• Mood disorders

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Questions?