Transcript Chromatin Modification

Chromatin Modification
Reading Seminar in
Computational Biology
Naomi Habib 5.1.2006
Chromatin Structure
Nucleosome (histone octamer
wrapped with 146 bp of DNA).
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…
Nucleosome tail
modifications
• Lysine acetylations.
• Histone Acetyl-Transferases (HAT) & Histone Deacetylases (HDAC).
• Lysine and Argenine Metylations.
– Modified by histonemetyl-transferase.
• Phosphorilation.
• Ubiquitination.
– H2A ubiquitination affects
10-15% of this histone in
most eukaryotic cells
• ADP-ribosylation.
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).
Articles Outline
• Mapping Global Histone Acetylation Patterns
to Gene Expression
Kurdistani,1 et al. Cell, June 2004.
• Genome-wide Map of Nucleosome
Acetylation and Methylation in Yeast
Pokholok Et al. Cell, August 2005.
• Genomic Maps and Comparative Analysis of
Histone Modifications in Human and Mouse
Bernstein et al. Cell January 2005.
Methods:
Chip-Chip
(Location
analysis)
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).
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
Mapping Global Histone
Acetylation Patterns to Gene
Expression
Siavash K. Kurdistani,1 Saeed Tavazoie,2, Michael
Grunstein1
Cell, June 2004
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 variancenormalized 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.
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.
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.
Genome-wide Map of
Nucleosome
Acetylation 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
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.
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.
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.
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.
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) .
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.
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.
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.
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.
Genomic Maps and
Comparative Analysis of
Histone 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.
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.
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.
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 Ser5phosphorylated CTD.  possible mechanism.
• Dimethyl Lys4 Sites in the Vicinity of Active Genes and
dependent on cell type.  novel markers for cell state?
(Me2>>3)
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.
Conservation
cytokine and IL4R regions
Sequence identity
• Lys4-Methylated Sites Show Stronger Conservation of
Location than Underlying Sequence.
– Conserved 7-fold higher than expected at random.
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.
HOX clusters (continued)
• distinct expression patterns in other fibroblast lineage.
HOXA cluster.
 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.
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
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?
?
Kurdistani - additional data
Kurdistani - additional data