Chromatin Modification

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Chromatin Modification
Reading Seminar in Computational Biology Naomi
Habib 5.1.2006
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Chromatin Structure
Nucleosome (histone octamer wrapped with 146 bp
of DNA).
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Chromatin Roles

• Compactization
• Packing 2 meters of DNA in to a 10 micron
  diameter nucleus.
• Different compactization levels euchromatin and
  heterochromatin.
• Regulation of gene expression
• Influencing DNA accessibility through Nucleosome
  Occupancy.
• Histone variants.
• Nucleosome tail modifications.
• Co-regulation Effect common chromatin regions.
• Regulation of DNA replication, repair,
  recombination and more

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Nucleosome tail modifications

• Lysine acetylations.
• Histone Acetyl-Transferases (HAT) Histone
  Deacetylases (HDAC).
• Lysine and Argenine Metylations.
• Modified by histone-metyl-transferase.
• Phosphorilation.
• Ubiquitination.
• H2A ubiquitination affects 10-15 of this
  histone in most eukaryotic cells
• ADP-ribosylation.

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Possible regulation mechanisms

• Histone modifications modulate the accessibility
  of DNA through structural changes of the
  chromatin (Horn and Peterson, 2002 Tse et al.,
  1998).
• Modifications on different residues provide
  specific binding surfaces for transcription
  regulators. (Kurdistani and Grunstein, 2003
  Corona et al., 2002 Deuring et al., 2000).
• Histone code? number, variety, and
  interdependence of modifications.
• Part of the process of protein signaling, promote
  switch-like behavior and ensure robustness of the
  signal (Schreiber and Bernstei,2002).
• Epigenetic inheritance of information histone
  modifications pattern inherited partially by the
  daughter cell (Jenuwein T Allis C.D. 2001).

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Articles Outline

• Mapping Global Histone Acetylation Patterns to
  Gene ExpressionKurdistani,1 et al. Cell, June
  2004.
• Genome-wide Map of NucleosomeAcetylation and
  Methylation in YeastPokholok Et al. Cell, August
  2005.
• Genomic Maps and Comparative Analysis of Histone
  Modifications in Human and MouseBernstein et al.
  Cell January 2005.

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MethodsChip-Chip (Location analysis)
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Previous studies

• Yeast nucleosomes occupancy is less dense in
  intergenic regions than ORF. (Lee et al., 2004).
• Promotores and coding regions of transcribed
  genes had lower nucleosome occupancy (Lee et al.,
  2004).
• Many works focusing on one or two Lysine residues
  or on a few genomic loci
• Acetylation of Lysine residues on H3 and H4
  primarily associated with gene activation.
  (Grunstein,1997.Braunstein et at., 1993)
• histone methylation associated with
  transcriptional activity or repression, depending
  on the specific residue (Zhang and Reinberg,
  2001).

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Previous studies (continued)

• Genome wide analysis in D. melanogaster revealed
  parallel action of several active modifications
  all linked to one another and to transcription
  levels (Schubeler D. et al, 2004)
• Genome wide mapping of HATs and HDACs binding
  sites (review by Bas Van Steensel, 2005).
• HATs bind to all active promoters.
• HDACs have a preferences for distinct gene
  classes

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Mapping Global Histone Acetylation Patterns to
Gene ExpressionSiavash K. Kurdistani,1 Saeed
Tavazoie,2, Michael Grunstein1Cell, June 2004
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Description of the work

• Determining acetylation level of 11 Lysine
  residues in Saccharomyces cerevisiae.
• Location analysis Intergenic region ORF
  arrays.
• Normalizing the data for each array and
  variance-normalized across 11 sites arrays.
• Clustered the data into clusters of
  modifications state.
• Checked in each cluster
• Enrichment of co-expressed genes
• Enrichment of genes from specific functional
  categories.
• Search for unique cis DNA motifs.
• Enrichment for binding of specific transcription
  factors.

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Results

• Lysines Acetylations are positively and
  negatively correlated.
• H4K8 and K12 are strongly correlated in IGRs and
  ORFs.
• Some significant differences between IGRs and
  ORFs.
• hyper- and hypoacetylation lysines associated
  with transcription
• hyperacetylation of histone H3K9/18/27 but
  hypoacetylation of H4K16 and H2BK11/16 are
  correlated with transcription.

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Results (continued)

• Acetylation patterns define groups of
  biologically related genes.
• co-expressed, functional categories enrichment,
  Unique DNA motifs, Specific transcription factors
  binding.
• Clusters distinguish groups with similar
  expression in one condition and differentially
  expressed in others.
• TF Bdf1 binding is associated with acetylation
  (specifically H4K16). ? Model Acetylation
  patterns used as surfaces for specific protein
  binding.

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Genome-wide Map of NucleosomeAcetylation and
Methylation in Yeast

• Dmitry K. Pokholok, Christopher T. Harbison,
• Stuart Levine, Megan Cole, Nancy M. Hannett, P.
  Alex Rolfe, Elizabeth Herbolsheimer,Julia
  Zeitlinger,Fran Lewitter, David K. Gifford,and
  Richard A. Young.
• Cell, August 2005

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General Description

• Genome-wide location analysis of nucleosome
  acetylation and methylation in Yeast.
• Location analysis, array design
• Tile array of 44,00 probes (85 of the genome).
• 60-mer oligonucleotide, average probe density of
  266 bp.
• Improved resolution and accuracy.
• Compared Histone-modifications-antibody data to a
  control with core-histone-antibody.

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Checking Resolution -Gcn4 Location Analysis

• Gcn4 TF of amino acid-biosynthetic genes.
• FPR of 1 and FNR of 25 over a set of 84
  positive and 945 negatives genes.
• Gene sets chosen according to location analysis
  data, DNA binding site motif and 2-fold change in
  mRNA levels dependent on Gcn4.

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Nucleosome Occupancy

• 20 reduction on average in histone occupancy in
  intergenic sequences relative to genic sequences.
• No difference after normalization using control
  no-antibody data
• 40 of promoters have difference from their gene.

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Occupancy Expression

• Nucleosome occupancy inversely correlates with
  transcription.
• Occupancy is reduced maximally at promoters of
  active genes.
• In cells after oxidative stress, nucleosome
  occupancy dropped at genes know to be activated.
  ? Model gene activation leads to reduced
  nucleosome density.

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Histone Acetylation

• Histone acetylation enriched at promoter regions
  and transcriptional start sites of active genes.
• Acetylation at Gcn5 targets H3K9 and H3K14 and
  Esa1 targets H4 at Lys 5,8,12 and16.
• ? Model transcriptional activators recruit Gcn5
  and Esa1 to promoters of genes upon their
  Activation (Robert et al., 2004) .

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Histone Acetylation

• In general strong correlation between
  acetylation of histone H3 and H4 (targeted by
  Gcn5 and Esa1) and transcriptional activity.
• In contrast to Kurdistani et al.
• After oxidative stress, histone acetylation
  increased at genes know to be activated and
  targeted by Gcn5 or Esa1.

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Histone Metylation (H3K4me)

• Previously shown methyltransferase Set1
  recruited to the 5 end of actively transcribed
  genes targets H3K4.
• H3K4me3 peaks at beginning of genes, with high
  correlation to transcription rates.

• H3K4me2 is enriched in the middle of genes, and
  H3K4me at the end of genes.

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Histone Metylation (H3K36me3)

• Previously shown H3K36 trimethylation targeted
  by Set2 (associated with the later stages of
  elongation).
• H3K36me3 enriched throughout the coding region,
  peaking near the 3 ends, correlated with
  transcription.
• ? model Set2 is recruited by the elongation
  apparatus and that it methylates during active
  transcription.

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Histone Metylation (H3K79me3)

• Previously shown The Dot1 histone
  methyltransferase modifies H3K79, within the core
  domain in 90 of all H3.
• H3K79me3 enriched within the transcribed regions
  of genes with little correlation to
  transcription.

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Genomic Maps and Comparative Analysis ofHistone
Modifications in Human and Mouse

• Bradley E. Bernstein, Michael Kamal, Kerstin
  Lindblad-Toh, Stefan Bekiranov, Dione K. Bailey,
  Dana J. Huebert, Scott McMahon, Elinor K.
  Karlsson, Edward J. Kulbokas III, Thomas R.
  Gingeras, reStuart L. Schreiber, and Eric S.
  Lander.
• Cell, January 2005.

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General Description

• Maping the nonrepetitive portions of human
  chromosomes 21 and 22 for H3 Lys 4
  di-,tri-methylation and Lys 9/14 acetylation.
• Hepatoma cell line.
• Compared lysine 4 dimethylation for several
  orthologous human and mouse loci.
• cytokine cluster, IL-4 receptor region, and all
  four Hox clusters.
• human and mouse primary fibroblasts.
• Location analysis using tile arrays at 35 bp
  intervals.
• Assessing results and threshold reliability using
  real time PCR.

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Maps of Histone Modifications

• Genomic maps that detail more than 39Mbp.
• Modification sites cover 1 of the nonrepetitive
  portion of chromosome 21 and 22.
• ChiP sample compared to whole genome DNA.
• Relatively Uniform Histone Density across the
  Chromosomes.
• no depletion on active promoters detected.

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Di-,Tri-methylated H3K4

• Trimethyl Lys4 Correlate with 5 Ends of active
  Genes.
• 1 kb upstream to TSS of known genes / predicted
  genes / proximal to mRNA hybridization on the
  tile array.
• 5 end significantly enriched (6- to 8-fold) for
  polII Ser5-phosphorylated CTD. ? possible
  mechanism.
• Dimethyl Lys4 Sites in the Vicinity of Active
  Genes and dependent on cell type. ? novel markers
  for cell state?

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H3 Acetylation

• H3 Acetylation Enriched at 5 Ends of Genes and
  Strongly Correlates with Lys4 Methylation.
• Consistent with previous studies.
• ? Systematic colocalization of methyl and acetyl
  marks reflect recruitment of complexes containing
  methylases and acetylases, and/or crosstalk
  between modifications.

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Conservation
Sequence identity
cytokine and IL4R regions

• Lys4-Methylated Sites Show Stronger Conservation
  of Location than Underlying Sequence.
• Conserved 7-fold higher than expected at random.

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Unique Profile for HOX Clusters

• Broad Methylated Regions Overlay Substantial
  Portions of the Human and Mouse Hox Clusters
• For example 60kb region in HOXA with 85 K4me2.

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HOX clusters (continued)

• distinct expression patterns in other fibroblast
  lineage.
• ? lineage-dependent active chromatin domains
  created at differentiation to maintain
  expression.
• Detected transcription at 33 of methylated
  intergenic bases, 10 in nonmethylated.
• - hybridizing RNA to the tiling arrays.

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Summary

• Nucleosome Occupancy
• Human Uniform punctate Histone Density.
• Yeast Reduced histone occupancy in IGRs relative
  to ORFs.
• Histone acetylation
• Human Enriched at 5 Ends of Genes.
• Yeast Enriched at promoter regions and TSS of
  active genes.
• Histone methylation
• Human Yeast H3K4me3 peaks at 5 Ends of active
  Genes.
• Human H3K4me2 in the Vicinity of Active Genes. /
  Yeast H3K4me2 enriched in middle of genes
• Yeast Additional data regarding H3K4me 3K79me3
  and H3K36me3.
• Conservation
• Human Mouse H3K4me show stronger conservation
  of location than underlying sequence.
• HOX cluster
• Human Mouse Broad methylated
  lineage-dependent regions

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Open questions

• How are the acetylation patters established?
• In IGRs may involve recruitment of specific sets
  of HATs and HDACs.
• In ORFs determined by promoter regulatory motifs
  or association of HATs and HDACs with the RNAPII
  (elongating complex or Ser5-phosphorylated CTD).
• What is the regulatory mechanism? Does the
  histone code exist?
• ?

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(No Transcript)
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Kurdistani - additional data
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Kurdistani - additional data