Transcript Diapositive 1 - Tsinghua University

Presented by 李连硕，王婷婷 and 郝志伟
2013.04.19
Motivation
1 genome
Chromatin
?
organization
Phenotypically
diverse cells
Red blood Fat (adipose) Striated
cells
cells
muscle cells
Stem cells
Paper’s content
• Map of chromatin modifications
in various human cells, including in vivo cells
• Analysis and observations
Histone modifications
Histones
Histone tail
H3K27me3:
Tri-methylation of lysine 27
on histone H3
DNA
Nucleosome
Modification types:
Methylation
Acetylation
Variants
…
Figures adapted from:
Marks et al., Nature Reviews Cancer, 2001
Cui and Miao, Eukaryotic Cell, 2000
Histone modification associations
Repressor
• H3K27me3
• H3K9me3
Enhancer
Promoter
• H3K4me1
• H3K27ac
• H3K4me3
• H3K9ac
Transcript
• H3K36me3
Experimental scope
Map of several histone modifcations:
– 9 types of modification:
– 29
H3K4me1, H3K4me3, H3K9me3, H3K27me3, H3K36me3,
H3K9ac, H3K27ac, H2AZ, WCE
300+ chromatin
state maps
tissues and cell types:
Fig 1 A
Experimental method
Chip-seq
Shear DNA strands
Attach antibodies to target modification
Precipitate
High throughput
Map to reference genome
Sequencing
& count
AGTTCTCAT
GCCTACACC
Experiment results
• Result: Density of each modification for each
sample along the whole genome (300 maps)
Tissue – Adipose
Brain – Mid-frontal lobe
H3K27me3
Blood – CD19+ B-cells
Culture – Keratinocytes
Tissue – Adipose
Brain – Mid-frontal lobe
H3K4me1
Blood – CD19+ B-cells
Culture – Keratinocytes
http://www.broadinstitute.org/pubs/epigenomicsresource/browser
etc.
Paper’s content
1.
2.
3.
4.
5.
6.
7.
Introduction to the chromatin state map
De novo annotation of regulatory elements
Observations during embryonic development
Brain cells specificities
Chromatin structure and nuclear architecture
Artifacts introduced by cell culture
Model for chromatin state transition
GENERAL VIEW ON THESE DATA
General view on these data
• Mapping reads to genome we can get reads count
distribution.
• Totally we have 203 (7modifications × 29 tissues)
such distributions.
• Are these distributions specific to different
modifications?
• Method
Quantification
Pairwise
correlation
Heatmap
2D clustering
Measure and visualize differences
between modifications
• The modifications organize
into separate clusters.
• Modifications associated
with
• Promoter: H3K4me3,
H3K9ac
• Transcript: H3K36me3
• Distal element:
H3K4me1, H3K27ac
• Repressive marks:
H3K27me3, H3K9me3
Measure and visualize differences
between cell types
• evident for modifications associated with regulatory activity
(H3K4me1, H3K27ac), which distinguish five groups of phenotypically
related tissue and cell types
• the marked separation of pluripotent stem cells from other cell and
tissue types evident in the H3K27me3 and H3K4me1 projections
portends a global reorganization that accompanies developmental
specification
MODIFICATIONS RELATED TO
ACTIVATORS
Enhancers and other distal regulatory
elements have cell type specific patterns
• Annotate candidate
regulatory elements by
calling H3K4me1 peaks in
30 cell types
• ~377,000 putative distal
regulatory elements, with a
median size of 1.2 kb
• Clustering on H3K4me1
patterns revealed 23 major
clusters of elements with
related cell-type
specificities
• each of the 23 clusters has
significantly enriched motifs
Different patterns between stem cells
and others
Further complexity
Global distributions of distal elements
• The proportion of the
genome that lies within
50 kb of an 3K4me1+
element in each cell
type
– Stem cells: 85%
– Others: ~50%
• Similar thing in
H3K36me3
– All are similar
MODIFICATIONS RELATED TO
REPRESSORS
Pluripotent Cell Chromatin Is Refractory to
Polycomb-Repressed State
The focal distribution of H3K27me3 in pluripotent cells could reflect reduced
Polycomb activity. However, EZH2 and other Polycomb factors are highly expressed
in ES cells.
So they consider an alternate model and mapped H2A.Z.
The conclusion is the title.
Developmental Specification Is Associated with
Progressive Chromatin Restriction
DEVELOPMENTAL SPECIFICATION IS
ASSOCIATED WITH PROGRESSIVE
CHROMATIN RESTRICTION
Developmental Specification Is Associated with
Progressive Chromatin Restriction
• Object: the locations and characteristics of loci with
H3K27me3 in the differentiated populations
promoters
distal elements
gene bodies
Figure 4. Epigenetic States Relate to Context-Specific
Genome Regulatory Programs(A) Chromatin states are
depicted for a set of 100 kb loci with variable activity
patterns.
the number of loci
Promoter, gene, and distal element activities are largely concordant within a locus, but are
correlate negatively with H3K27me3
Developmental Specification Is Associated with
Progressive Chromatin Restriction
Object: whether chromatin restriction proceed under certain contexts
Figure 4. Epigenetic States Relate to Context-Specific Genome Regulatory Programs
(B) Boxplots show H3K27me3 coverage of intergenic regions relative to gene bodies (left),
and H3K4me1 peak density in intergenic regions relative to gene bodies (right).(C)
Heatmap shows composite H3K4me1 profiles over genes and flanking regions (TSS ±25 kb
and TES ±25 kb; all genes >15 kb) for each cell type (rows). (D) Heatmap (left) shows the
distribution of highly conserved noncoding sequence elements over gene bodies and
flanking regions (TSS ±25 kb and TES ±25 kb) for all genes >15 kb (rows).
Brain sections are notable for a high prevalence of repressive chromatin throughout intergenic regions and a relative
confinement of H3K4me1 sites within genes
Brain sections display higher H3K4me1
signals in gene bodies
The top genes are strongly enriched for functional annotations related to
brain physiology, including axon guidance and synapse
Developmental Specification Is Associated with
Progressive Chromatin Restriction
• Developmental specification is accompanied chromatin
state transition regulated by polycomb repression.
• Chromatin restriction may also proceed significantly
further in certain specialized cells
RELATING MACROSCALE CHROMATIN
FEATURES TO NUCLEAR ARCHITECTURE
Relating Macroscale Chromatin Features to Nuclear Architecture
Object: to investigate macroscale patterns of histone modification
and the relationship between modification and nuclear architecture
four groups: (1) “active” loci
with high H3K36me3 and
H3K4me1 coverage, (2)
“Polycomb-repressed” loci with
high H3K27me3, (3)
Heterochromatic loci with high
H3K9me3, and (4) “Null” loci
devoid of histone modification
Figure 5. Macroscale Chromatin
Features and Nuclear Architecture
intervals that occupy inactive nuclear compartments show enrichment for nuclear
lamina contacts.
CULTURE ENVIRONMENTS TRIGGER
MACROSCALE CHROMATIN STATE
CHANGES
Culture Environments Trigger Macroscale Chromatin State Changes
Observation: H3K9me3 state varies markedly in its prevalence
Figure 6. Macroscale Chromatin Aberrations in Cultured Cells(A) H3K9me3 signal tracks for representative cell types are shown for a 3.5
Mb region of chromosome 16 that contains a culture-specific H3K9me3 domain.(B) Heatmap shows normalized H3K9me3 signals for 296
H3K9me3 domains (rows) in the indicated cell types (columns).
Preferential association of variable H3K9me3 domains with the culture environment
Culture Environments Trigger Macroscale Chromatin State Changes
Object: Why H3K9me3 state is rare in vivo models but ∼50-fold more prevalent in
cultured primary cells
serum
growth stimuli
(E) Boxplot (left) shows expression levels of genes within culture-specific H3K9me3 domains (cluster II) in
fibroblasts cultured in high or low serum. p value of Wilcoxon rank-sum test is shown. Median expression levels
for these genes are also shown for lung adenocarcinoma cells undergoing TGF-β-mediated EMT
EMT and serum-exposure both lead to subtle increases in the median expression of
genes underlying H3K9me3 domains
Culture Environments Trigger Macroscale Chromatin State Changes
Object: whether Suv39h1 mediates the culture-specific domains
Suv39h1: H3K9 methyltransferase
Suv39h1 is required for the
maintenance of H3K9me3 in the
culture-specific domains
(C) For each cluster in (B), heatmap shows H3K9me3 signals
in lung fibroblasts after 4 or 10 days of Suv39h1
knockdown.(D) H3K9me3 signal tracks for two culturespecific domains are shown for lung fibroblasts after 4 or
10 days of Suv39h1 knockdown
DISCUSSION
Discussion
Specification is accompanied by a stark transition in the epigenetic landscape from a
uniquely accessible state to increasingly restrictive configurations
Contributions
• Provide a new dataset for chromatin landscape in
in vivo cells
• Identified ~400,000 putative distal regulatory
elements
• Updated model for chromatin reorganization
during development
• Epigenomic landscape could shape the evolution
of genome sequence
(eg. conserved sequence in introns of genes with neuronal functions)
• Identified artifacts introduced by cell culture