Transcript The Proteomics of Epigenetics

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?