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Genomic Focus: Epigenetics

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Title: Genomic Focus: Epigenetics


1
Genomic Focus Epigenetics
Corey Henderson
Laura Bergeron
Kyle Fowler
Cynthia Hansen
2
Introduction to Epigenetics
http//en.wikipedia.org/wiki/Epigenetics
3
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

4
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
g
5
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

6
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

7
Discoveries for Epigenetics
  • Filamentous Fungi
  • RIP
  • Repeat Induced Point Mutation
  • MIP
  • Methylation induced premeiotically
  • Plants and Animals
  • RIGS
  • Repeat Induced Gene Silencing

8
RNA
  • Sequence-specific degradation
  • RNAi
  • AKA
  • Cosuppression (plants)
  • Quelling (Neurospora)
  • Post Translational Gene Scilencing
  • RNA/DNA interaction
  • Methylation
  • Feedback induced Epigentic regulation

9
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

10
Epigenetics
11
Chromatin An Overview
http//www.sciencemuseum.org.uk/on-line/lifecycle/
133.asp
http//www.blackwellpublishing.com/11thhour/book5/
about/images/c5t2.gif
12
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
13
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)

14
The Histone Code Hypothesis
  • Different PTMs bound by specific domains found in
    transcriptionally relevant genes
  • Bromodomains ? acetylation
  • Chromodomains ? methylation

(Jenuwein and Allis, 2001)
15
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

16
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

17
  • 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
18
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
19
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

20
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
21
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

22
Mass Spectrometry
23
Methodology
  • Histone Code
  • Post-translational covalent modifications to the
    histone tails

24
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.)

25
Methodology
Schulze, S.
26
Methodology
  • Chromatin Immunoprecipitation (ChIP)
  • ChIP on Chip (ChIPs!)
  • ChIP coupled with array
  • Genomic approach to ChIP

27
Methodology
Public domain image
28
Methods in Epigenetics
  • Chromatin Immunoprecipitation (ChIP)
  • ChIP on Chip
  • ChIPs signal analysis

29
Methods in Epigenetics
30
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

31
Data and Conclusions
32
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

33
Data and Conclusions
Example H3/H4 Acetylation ? H3 Lys4
Trimethylation
34
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

35
Data and Conclusions
36
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

37
Data and Conclusions
  • H3-di-meK4 enrichment (blue) across chromosomal
    and EST arrays (grey boxgene)
  • Signal for complete chromosomal arm (enrichment
    vs. signal)

38
Future Work / Other Research
  • Variant histones
  • Euchromatin vs. heterochromatin
  • Dosage compensation
  • Implications (medicine, heredity, etc.)

39
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.

40
Chicken or the Egg?
  • Yeast only has variants
  • Diversity in variant function

www.cartoonstock.com
41
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

42
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
43
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
44
Epigenetics and medicine
  • Cancer
  • Heart failure
  • Mood disorders

45
Questions?
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