Genome-wide Chromatin State Transitions
Associated with Developmental
and Environmental Cues
Jiang Zhu,1,2,3,4,12Mazhar Adli,1,2,3,4,12,* James Y. Zou,1,2,3,5Griet Verstappen,1,6Michael Coyne,1Xiaolan Zhang,1
Timothy Durham,1Mohammad Miri,3Vikram Deshpande,3Philip L. De Jager,1,7David A. Bennett,8Joseph A. Houmard,9
Deborah M. Muoio,10Tamer T. Onder,11Ray Camahort,1,6Chad A. Cowan,1,6Alexander Meissner,1,6Charles B. Epstein,1
Noam Shoresh,1and Bradley E. Bernstein1,2,3,4,*
1Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
2Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
3Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
4Center for Systems Biology and Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02114, USA
5School of Engineering and Applied Sciences
6Department of Stem Cell and Regenerative Biology
Harvard University, Cambridge, MA 02138, USA
7Program in Translational NeuroPsychiatric Genomics, Institute for the Neurosciences, Departments of Neurology and Psychiatry,
Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02215, USA
8Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, IL 60612, USA
9Department of Exercise and Sport Science, East Carolina University, Greenville NC 27858, USA
10Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC 27710, USA
11Koc University School of Medicine, Istanbul, Turkey
12These authors contributed equally to this work
*Correspondence: email@example.com (M.A.), firstname.lastname@example.org (B.E.B.)
Differences in chromatin organization are key to the
multiplicity of cell states that arise from a single
genetic background, yet the landscapes of in vivo
tissues remain largely uncharted. Here, we mapped
chromatin genome-wide in a large and diverse
collection of human tissues and stem cells. The
maps yield unprecedented annotations of functional
genomic elements and their regulation across devel-
opmental stages, lineages, and cellular environ-
ments. They also reveal global features of the
epigenome, related to nuclear architecture, that
also vary across cellular phenotypes. Specifically,
developmental specification is accompanied by pro-
gressive chromatin restriction as the default state
transitions from dynamic remodeling to generalized
compaction. Exposure to serum in vitro triggers
a distinct transition that involves de novo estab-
lishment of domains with features of constitutive
heterochromatin. We describe how these global
chromatin state transitions relate to chromosome
and nuclear architecture, and discuss their implica-
tions for lineage fidelity, cellular senescence, and
Since the initial sequencing of the human genome a decade
ago, our understanding of the primary DNA sequence has
advanced profoundly (Lander, 2011). Sequence signals and
multispecies conservation have enabled precise annotation
of protein coding genes and the identification of increasing
numbers of noncoding RNAs, regulatory elements, and motifs.
Systematic genotyping studies have identified common variants
associated with complex diseases and recurrent mutations that
confer growth advantage in cancer.
However, entirely sequence-directed investigations cannot
address the fundamental question of how one genome can
give rise to a large and phenotypically diverse collection of
cells and tissues during embryonic development. Nor can they
explain how environmental conditions further shape these
phenotypes and affect disease risks (Feinberg, 2007). An under-
standing of the regulatory networks and epigenetic mechanisms
that underlie context-specific gene expression programs and
cellular phenotypes remains a critical scientific goal with broad
implications for human health.
Genomic DNA is organized into chromatin, which adopts
characteristic configurations when DNA interacts with transcrip-
tion factors (TFs), RNA polymerase, or other regulators (Mar-
gueron and Reinberg, 2010). Charting these configurations
with genome-wide maps of histone modifications (‘‘chromatin
state maps’’) thus represents an effective means for identifying
642 Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc.
functional DNA elements and assessing their activities in a given
cell population (Zhou et al., 2011). Signature patterns of ‘‘active’’
chromatin marks demarcate poised or active promoters, tran-
scribed regions, and candidate enhancers. Other modifications
reveal distinct modes of chromatin repression, such as those
mediated by Polycomb regulators or heterochromatin proteins.
Recent studies have applied chromatin profiling to charac-
terize enhancer dynamics and epigenetic regulatory mecha-
nisms in differentiation, cellular reprogramming, and disease
processes (Ernst et al., 2011; Hawkins et al., 2010b; ENCODE
Project Consortium, 2012). However, the overwhelming focus
of such studies on in vitro cells has constrained our ability to
detect and characterize regulatory elements in the human
genome and to understand how global features of the epige-
nome impact cellular phenotypes across different lineages,
developmental stages, and environmental conditions.
Here, we present a resource of over 300chromatin state maps
for a phenotypically diverse collection of human tissues, blood
lineages, and stem cells, produced by the NIH Roadmap Epige-
nomics Mapping Consortium (Bernstein et al., 2010). The maps
depict the distributions of major histone modifications and
provide a systematic view of the dynamic chromatin landscapes
of in vivo tissues. We use the maps to identify and characterize
?400,000 cell-type-specific distal regulatory elements, many
of which can be tied to upstream TFs or signaling pathways
and whose activity patterns provide a precise fingerprint of cell
phenotype. We also describe global chromatin state transitions
that distinguish groups of cells representative of different devel-
opmental stages or environmental conditions and investigate
their implications for lineage fidelity, nuclear architecture, cellu-
lar senescence, and reprogramming. This extensive catalog of
in vivo chromatin states thus presents a unique resource of
genomic annotations for biomedical research along with novel
epigenetic features that vary markedly across cellular states.
Charting Chromatin Landscapes of Human Tissues,
Blood Lineages, and Stem Cells
We acquired chromatin state maps for 29 tissues and cell
types spanning a wide range of developmental stages, lineages,
and derivations (Figure 1A). We used chromatin immunoprecip-
itation and high-throughput sequencing (ChIP-seq) to map
histone modifications associated with diverse regulatory and
epigenetic functions, including H3K4me1 (H3 lysine 4 mono-
methylation), H3K4me3, H3K9me3, H3K27me3, H3K36me3,
H3K9ac (lysine 9 acetylation), and H3K27ac. Procedures were
optimized for different tissue preparations and to accommodate
for limiting samples (Experimental Procedures). We also incor-
et al., 2011). The resource contains over 300 chromatin state
maps that significantly expand coverage of the human epige-
nome (Table S1 available online). All data sets were publically
released upon verification at www.roadmapepigenomics.org
and are also available at http://www.broadinstitute.org/pubs/
We applied automated methods to characterize the chromatin
landscapes and relate them to underlying cellular phenotypes.
First, we clustered the profiles based on pair-wise correlations
(Figure 1B). The modifications organize into separate clusters,
reflecting their associations to distinct genomic features. Modifi-
cations associated with promoter (H3K4me3, H3K9ac), tran-
script (H3K36me3), and distal element (H3K4me1, H3K27ac)
activity correlate positively with one another but show vary-
ing degrees of exclusivity with repressive marks (H3K27me3,
Next, we used principal component analysis (PCA) to measure
and visualize differences between cell types. Treating each his-
tone modification separately, we computed weighted combina-
tions of enrichment levels in genomic windows (PC1, PC2, and
PC3) that capture a large proportion of the variation between
cell types (Figure S1; Experimental Procedures). The PCA shows
a striking capacity to segregate cells and tissues based on
fundamental characteristics as seen in 3D projections of PC
coordinates (Figure 1C). This is particularly evident for modifica-
tions associated with regulatory activity (H3K4me1, H3K27ac),
which distinguish five groups of phenotypically related tissue
and cell types (see Patterns and Determinants of Regulatory
Activity and Potential in the Human Genome below). PC projec-
tions for several modifications are notable for stark translations
of certain cellular groups along PC1, indicative of major differ-
ences in chromatin state. In particular, 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
(see Developmental Specification Is Associated with Progres-
sive Chromatin Restriction). Furthermore, a clear separation of
cultured cells in the H3K9me3 projection signifies a distinct
reorganization induced by in vitro culture (see Culture Environ-
ments Trigger Macroscale Chromatin State Changes). The PCA
also provides a general tool for comparing newly characterized
cell types against representative chromatin state maps in this
Patterns and Determinants of Regulatory Activity and
Potential in the Human Genome
Enhancers and other distal regulatory elements are critical for
context-specific gene regulation but have yet to be systemati-
cally charted in primary human cells and tissues. Such elements
are associated with characteristic chromatin marks, including
H3K4me1 and H3K27ac, which facilitate their identification
(Bulger and Groudine, 2011; Hawkins et al., 2010b).
We annotated candidate regulatory elements by calling
H3K4me1 peaks in 30 cell types. After filtering out peaks that
overlap a transcription start site (TSS), we identified an average
of ?94,000 distal sites per cell type. Integrating all sites marked
by H3K4me1 in at least two cell types reveals ?377,000 putative
distal regulatory elements, with a median size of 1.2 kb. The
elements are highly tissue-specific, with 56% marked in three
or fewer cell types. Clustering on H3K4me1 patterns revealed
23 major clusters of elements with related cell-type specificities
(Figure 2A). The biological relevance of individual clusters is sup-
ported by the identities of proximal genes, which are expressed
at higher levels in the corresponding cell types and enriched for
related functional annotations (Table S2; Experimental Proce-
dures). Roughly half of all H3K4me1 sites also carry H3K27ac
Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc. 643
in at least a subset of cell types and thus represent candidate
enhancers (Figure 2B). Notably, nearly half of the candidate
enhancers are specific to in vivo tissues, blood lineages, or brain
To identify underlying sequence determinants, we scanned
the candidate enhancers for TF consensus motifs. We identified
significantly enriched motifs for each of the 23 clusters of
commonly regulated elements (Figure 2A, Table S3; Experi-
mental Procedures). The corresponding motif instances tend to
be highly conserved and to coincide with dips in the chromatin
profiles indicative of TF interactions (He et al., 2010). A sampling
of predicted TF-motif interactions were also verified using TF-
binding data (Figure S2; ENCODE Project Consortium, 2012).
The data implicate known and potentially novel roles for specific
TFs as regulators of cell-type-specific distal elements and gene
expression programs. To highlight one example, brain- and
neural-stem-cell-specific elements are enriched for NF1, RFX,
SOX2, SOX10, and E-box motifs (Figure 2A; clusters 18–20). In
several cases, a given TF motif is enriched in multiple unrelated
clusters and thus implicated under distinct cellular contexts. A
case in point is SOX2, a multifunctional TF with roles in pluripo-
pluripotent- (cluster 9) and neural-specific (cluster 20) distal
elements, suggesting that specificity among these clusters
may involve proximal sequence signals. Indeed, 25% of SOX2
motifs in ‘‘pluripotent’’ elements coincide with OCT4 motifs,
whereas a majority of SOX2 motifs in ‘‘neural’’ elements instead
coincide with PAX2 motifs. Further complexity is evident at the
level of enhancer usage as many loci contain multiple elements
whose activity patterns vary even between cell types in which
nearby genes are active (Figure 2C). These and other examples
suggest a prominent role for combinatorial TF activities and
complex distal element patterning in directing gene expression
programs in human cells (Bulger and Groudine, 2011).
In addition to lineage-specific TFs, the motif enrichments
implicate signaling and environmental response pathways acti-
vated in specific contexts. Clusters of distal elements broadly
associated with primary cells cultured in serum are enriched
for motifs recognized by AP-1 (clusters 12–14), a classical inte-
Primary cells and tissues (in vivo)
Inferior temporal lobe
Rectal smooth muscle
Stomach smooth muscle
stem cells and
Figure 1. Chromatin State Maps for In Vivo Tissues, Stem Cells, and Primary Culture Models
(A) Over 300 chromatin state maps were generated for human tissues, stem cells, and cultured primary cells. In the schematic, tissues and cells with related
phenotypes are grouped and color coded.
(B) Cross-correlation map generated by clustering ?200 histone modification profiles based on pair-wise correlations. Heat indicates degree of positive (red) or
(C) Projection plots show PCA coordinates for each tissue and cell type (colored as in ‘‘A’’). The data indicate that coherent variations in the chromatin landscape
distinguish cells from different developmental stages, lineages, and growth environments.
See also Figure S1 and Table S1.
644 Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc.
and Karin, 1991). A related cluster is also enriched for the p53
motif (cluster 13), consistent with documented activation of
p53 senescence programs in these primary cell models (Rhein-
wald et al., 2002).
The large number and diverse activity patterns of distal
elements prompted us to examine their global distributions.
We calculated the proportion of the genome that lies within
50 kb of an H3K4me1+ element in each cell type. This proportion
Figure 2. Genome-Wide Annotation of Tissue-Specific Distal Regulatory Elements
(A) Heatmap depicts H3K4me1 signals (blue) for ?400,000 distal elements (rows) across cell types (columns) arranged by phenotypic groups as in Figure 1A.
Clusters of distal elements with similar cell-type specificities (horizontal lines) are enriched for the indicated TF motifs (right). The data emphasize the importance
of tissue diversity for understanding distal regulatory elements, a large fraction of which is specific to in vivo tissues.
(B) Heatmap indicates the total number of H3K4me1 sites (top) and fraction of H3K4me1 sites that are also enriched for H3K27ac (bottom). Values represent
averages for each cell group.
(C) H3K36me3, H3K4me3, and H3K4me1 signal tracks are shown for the EBF1 and HLF/MMD loci in the indicated cell types. Red triangles indicate cell-type-
specific H3K4me1 sites. Although EBF1 is expressed in neurons and CD19+ B cells, distal H3K4me1 patterns vary markedly between these cell types. Similarly,
HLF and MMD are expressed in liver, muscle, and CD34+ progenitors, despite stark differences in distal element patterning.
(D) Bar graphs show the proportions of genome within 50 kb of an H3K4me1 site (top) or within an H3K36me3 interval (bottom). Values represent averages for
each cell group. The prevalence and distribution of H3K4me1 sites suggest that pluripotent cells have more accessible chromatin, but H3K36me3 patterns
suggest that total gene activity is similar to other cells.
See also Figure S2 and Tables S2 and S3.
Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc. 645
remains relatively constant at ?50% across the differentiated
tissues and cell types. However, H3K4me1 sites are more
prevalent and dispersed in pluripotent cells, such that a full
85% of the genome is within 50 kb of a site (Figure 2D). This
pattern does not appear to reflect increased gene activity as
the proportion of genome marked by H3K36me3 in pluripotent
cells (?17%) is similar to the average for differentiated cells
(?23%). Moreover, the proportion of elements with concomitant
H3K27ac is notably lower for H3K4me1 sites in pluripotent stem
cells (Figure 2B), indicating that many may represent ‘‘poised’’
enhancers or other sites of accessible chromatin. Thus, much
of the genome in pluripotent cells appears to be coincident or
proximal to accessible chromatin.
Pluripotent Cell Chromatin Is Refractory to Polycomb-
The dramatically reduced prevalence of accessible chromatin
in differentiated cells prompted us to test for concomitant
changes in repressive chromatin. We focused on marks associ-
ated with Polycomb-repression (H3K27me3) and constitutive
heterochromatin (H3K9me3). Both modifications contribute to
stable gene repression through interactions with protein com-
plexes involved in chromatin compaction (Margueron and Rein-
berg, 2010; Simon and Kingston, 2009).
The PCA statistics (Figure 1C) indicate that, like H3K4me1,
global H3K27me3 patterns are distinct in pluripotent cells.
Indeed, we find that the changes in H3K4me1 patterns are com-
plemented by a profound reorganization of the H3K27me3 land-
scape. In embryonic (ES) and induced pluripotent stem (iPS)
cells, this surrogate of Polycomb activity is confined to peaks
at ‘‘bivalent’’ GC-rich promoters that also carry H3K4me3. This
punctate pattern contrasts with a much broader distribution of
H3K27me3 in differentiated cells and tissues (Figure 3A). We
used a customized approach to quantify coverage in each
cell type (Figures 3B and S3; Experimental Procedures). This
confirmed that H3K27me3 affects a considerably larger propor-
tion of genome in differentiated cells (?40%) than pluripotent
cells (?8%). This dramatic shift is supported by western blots
showing that acid-extracted histones from differentiated cells
have higher H3K27me3 levels than similar extracts from ES cells
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. This led us
to consider an alternate model in which the highly dynamic chro-
matin in pluripotent cells (Meshorer et al., 2006) is refractory
to the compaction associated with Polycomb repression. To
explore this, we mapped H2A.Z, a histone variant associated
with nucleosome exchange and remodeling (Talbert and Henik-
off, 2010), in representative cell types (Figures 3C, 3D, and S3).
Consistent with prior studies (Hardy et al., 2009), H2A.Z is
depleted within elongating transcripts in all cell types exam-
ined. However, the global H2A.Z distribution diverges markedly
between pluripotent and differentiated cells. In ES and iPS cells,
the variant marks promoters and distal elements, but is also
distributed throughout intergenic regions. In differentiated cells,
H2A.Z is instead confined to promoters and distal elements. The
broad H2A.Z distribution suggests that chromatin exchange is
prevalent throughout the genome in embryonic cells. Although
H2A.Z may be compatible with punctate Polycomb sites in ES
cells (Creyghton et al., 2008), dynamic chromatin is likely incom-
patible with the stable interactions required for Polycomb
spreading and compaction (Simon and Kingston, 2009). Thus,
pervasive exchange may underlie the constrained H3K27me3
distribution and the uniquely accessible chromatin landscape
in pluripotent cells.
Developmental Specification Is Associated with
Progressive Chromatin Restriction
Togaininsightinto thetimingofthedevelopmentaltransition, we
profiled H3K27me3 in a series of cell populations representing
successive stages of specification. These include (1) embryoid
bodies (EBs) isolated from differentiating ES cells at day 4, (2)
through a 3 week differentiation procedure, and (4) neurons
differentiated from these progenitors. We found that ES cell
differentiation is accompanied by progressive enrichment of
H3K27me3 across the genome, with subtle but significant
changes in EBs, and profound alterations in neural progenitors
and neurons (Figures 3E and 3F). The latter populations exhibit
a diffuse H3K27me3 distribution akin to other differentiated cell
types. This suggests that reorganization of the chromatin land-
scape begins early in development and is recapitulated by
in vitro differentiation of ES cells.
We next examined the locations and characteristics of
genomic loci that gain H3K27me3 in the differentiated popula-
tions. We focused on a set of ?3,000 loci (100 kb size) with
variable activity across the phenotypic groups. We scored
each locus for (1) distal element H3K4me1 levels, (2) promoter
H3K4me3 levels, (3) transcript H3K36me3 levels, and (4) overall
H3K27me3 levels within each phenotypic group, and clustered
them accordingly (Figure 4A; Experimental Procedures). The re-
sulting cluster diagram conveys the variable patterning of these
loci. Promoter, transcript, and distal element activities are highly
concordant within a given locus but correlate negatively with the
atic view of how Polycomb-repressed chromatin is engaged in
specific lineages to maintain silencing of gene loci with functions
in alternate lineages.
Chromatin restriction can also proceed beyond this initial
developmental reorganization, under certain contexts. In par-
ticular, the brain sections exhibit uniquely high H3K27me3
coverage over intergenic regions, relative to annotated genes
(Figure 4B). Expansion of the Polycomb-repressed state is
accompanied by a dramatic restriction of accessible chromatin,
such that ?70% of H3K4me1 sites in brain reside within tran-
scriptional units (Figures 4B and 4C). To test the generality of
this finding, we examined H3K4me1 profiles for assorted mouse
tissues (Shen et al., 2012). Out of 19 cell and tissue types exam-
ined, we found that cerebellum and cortex have the highest
proportions of H3K4me1 sites within genic regions (Figure S3).
The brain sections are unique among tissues in the resource
in that they comprise specialized, terminally differentiated cell
types—primarily neurons and glial cells. We reasoned that the
restrictive chromatin environment in these cells might obstruct
access to intergenic sequences and thus favor recognition of
646 Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc.
Figure 3. Global Chromatin Restriction during Developmental Specification
(A) H3K27me3 signal tracks for a 20 Mb region of chromosome 16 are shown for pluripotent (top) and differentiated (bottom) cell types.
(B) Two-dimensional box plot compares the fraction of genome within 50 kb of an H3K4me1 site (y axis) and the proportion of genome enriched for H3K27me3
(x axis). For each phenotypic cell group, squares and midpoints indicate 25th, 50th, and 75thpercentiles, whereas the crosses designate minimum and maximum
values. Specification is accompanied by marked restriction of accessible chromatin and increased prevalence of the Polycomb-repressed state.
(C) H3K27me3, H3K36me3, and H2A.Z signal tracks for a 7 Mb region of chromosome 15 are shown for ES cells and endothelial cells.
(D) Bar plot contrasts normalized intergenic H2A.Z and H3K27me3 signal medians in ES and differentiated cells (see Figure S3). These normalized signals are
asensitive indicator of chromatin state transitions. H2A.Z is distributedbroadly inpluripotent cells, indicativeof genome-wide remodeling, but is confinedtosites
of regulatory activity in differentiated cells.
(E) Box plots indicate normalized intergenic H3K27me3 signals for cells in each phenotypic group. Boxes indicate 25th, 50th, and 75thpercentiles, whereas
whiskers indicate minimum and maximum values. Corresponding values for ES cell derivatives are indicated at right.
(F) H3K27me3 profiles for ES cell derivatives are projected onto PC space as in Figure 1C. The arrow emphasizes the developmental progression.
See also Figure S3.
Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc. 647
functional elements within introns. In support of this possibility,
we find that the genes with a high density of conserved noncod-
ing sequences in their introns are expressed at higher levels in
brain and enriched for functional annotations related to neuronal
physiology (Figure 4D; Experimental Procedures).
In summary, our data suggest that developmental specifica-
tion is accompanied by a striking transition from a permissive
with pervasive Polycomb repression. Chromatin restriction may
alsoproceed significantly furtherincertain specializedcells,with
brain sections in particular exhibiting severe sequestration of
intergenic sequences and evidence for preferential utilization of
regulatory elements within introns.
Relating Macroscale Chromatin Features to Nuclear
The coarse partitioning of the genome between loci of coordi-
nated gene regulatory activity and large Polycomb-repressed
regions prompted us to investigate macroscale patterns of
histone modification. Focusing on ?3,000 1 Mb intervals, we
−25K TSSTES 25K
Intergenic / Intragenic
Intergenic / Intragenic
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. Rows correspond to clusters of loci with similar cell-type specificities.
Heatmap depicts relative levels of H3K4me1 over distal elements (light blue), H3K4me3 over promoters (green), H3K36me3 over gene bodies (dark blue), and
element activities are largely concordant within a locus, but are exclusive with H3K27me3.
(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).Brainsectionsarenotableforahighprevalenceofrepressivechromatinthroughout intergenicregions andarelative confinementofH3K4me1sites
(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). Brain
sections display higher H3K4me1 signals in gene bodies, even when TSS proximal regions (±5 kb) are masked (gray).
(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). The genes are ordered according to the density of conserved elements within their introns. The top quintile of genes (n = 1760) is
strongly enriched for functional annotations related to brain physiology, including axon guidance (p < 10?13) and synapse (p < 10?9), and exhibit higher RNA
expression in brain (heatmap at right; red indicates high expression; blue indicates low expression). These data suggest that a highly restrictive chromatin
structure in specialized brain cells favors access to intronic regions and may influence the function of sequence elements that mediate corresponding regulatory
See also Figure S3.
648 Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc.
quantified relative coverage by each modification and clustered
the intervals accordingly. We found that most intervals are domi-
nated by a single coherent chromatin state, allowing us to segre-
gate them into 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).
Several lines of evidence suggest that the macroscale chro-
matin patterns reflect chromosomal and nuclear architecture.
The chromatin states align with chromosome banding patterns,
with active (1) and inactive states (2–4), respectively, enriched in
light and dark bands. All three inactive states are also enriched
for contacts with the nuclear lamina (Guelen et al., 2008). We
also examined the relationship between the macroscale chro-
matin domains and chromosomal interactions (Lieberman-Aiden
et al., 2009). The interaction maps indicate that the genome can
be partitioned into two compartments such that contacts within
ments are depleted. We found that the chromatin state-based
classification scheme mirrors the HiC compartmentalization,
with active states coinciding with one compartment and repres-
sive states with the other (Figure 5). In fact, the chromatin states
enable prediction of HiC compartments with an overall accuracy
of 83%. These correspondences suggest that macroscale chro-
matin patterns are reflective of chromosomal and nuclear archi-
Culture Environments Trigger Macroscale Chromatin
Although the macroscale chromatin patternsare each recovered
in all of the differentiated cell types, one configuration—the pure
H3K9me3 state—varies markedly in its prevalence. It is rare
in brain sections and other in vivo models but ?50-fold more
prevalent in cultured primary cells (Figure S4). Visual inspec-
tion of the profiles confirms expansive regions of modest but
contiguous H3K9me3 enrichment that are particularly pro-
nounced in cultured cells (Figure 6A). We called H3K9me3-
enriched intervals in each cell type and merged overlapping
intervals to collate a set of 296 domains (median size 1.4 Mb).
We then calculated normalized H3K9me3 signals for each
domain in each cell type and clustered the domains accordingly
Two clusters comprise constitutive H3K9me3 domains (Fig-
ure 6B, clusters VI, VII; Table S4). The corresponding loci include
olfactory receptor, zinc finger, and protocadherin gene clusters
and several imprinted loci, which have previously been associ-
ated with H3K9me3 (Magklara et al., 2011; O’Geen et al.,
2007). The remaining clusters comprise domains with variable
enrichments across the cell types. These domains tend to be
near telomeres (cluster I), AT rich (cluster II–IV), gene poor (clus-
ters I–IV), and in contact with the nuclear lamina. Their H3K9me3
signals are most pronounced in in vitro cultured cells, such as
endothelial cells (HUVEC), keratinocytes (NHEK) and fibroblasts
(NHLF) (Figure 6B; clusters I–IV). Preferential association of vari-
able H3K9me3 domains with the culture environment is further
supported by a direct comparison of surgically resected skeletal
muscle against cultured skeletal muscle cells (Figure S4).
Although they are representative of different lineages, the cell
types with pronounced H3K9me3 domains are all grown as
adherent cultures in the presence of serum or other potent
growth stimuli. By contrast, hematopoietic cells grown in sus-
pension and stem cells grown in defined media without serum
lack the variable H3K9me3 domains. Growth stimuli have
previously been linked to global chromatin changes. Trans-
forming growth factor b (TGF-b)-mediated epithelial-to-mesen-
marks and a reduction in the lamina-associated modification,
H3K9me2 (Guelen et al., 2008; McDonald et al., 2011). Although
we find that the genome-wide patterns of H3K9me3 and
H3K9me2 are distinct (Figure S4), we nonetheless considered
whether the culture-induced H3K9me3 domains might relate
to EMT-like nuclear architecture changes. In support of this
possibility, we found that inhibition of TGF-b signaling in WI-38
fibroblasts leads to a reduction in H3K9me3 domain signals
(Figure S4). Furthermore, EMT and serum-exposure both lead
to subtle increases in the median expression of genes under-
lying H3K9me3 domains (Figure 6E). Finally, we note that the
H3K9me3 domains occupy the same inactive compartment as
the nuclear lamina in the chromatin interaction data (Figures 5
and 6B). These findings are suggestive of a model in which
growth stimuli in culture media promote alterations to nuclear
architecture and lamina contacts that render formerly inert loci
susceptible to H3K9me3 modification.
In addition to having related growth environments, the
affected models are all nontransformed, primary cells that
will undergo senescence-related growth arrest (Figure S4).
Cellular senescence is also associated with global architecture
changes—specifically, the formation of ‘‘senescence-associ-
ated heterochromatin foci (SAHFs),’’ which are DAPI-dense
Open HiC compartment
Close HiC compartment
Nuclear lamina contact
Figure 5. Macroscale Chromatin Features and Nuclear Architecture
Nonoverlapping 1 Mb genomic windows (n = 2,725) were clustered by their
coverage by four histone modifications. Roughly half of the windows clustered
into four main states: (I) ‘‘active’’ loci with high H3K36me3 and H3K4me1, (II)
‘‘Polycomb-repressed’’ loci with high H3K27me3, (III) heterochromatic loci
with high H3K9me3, and (IV) ‘‘null’’ loci devoid of histone modification. Top:
coverage for each interval (data points) and average coverage for each cluster
(horizontal lines) by the indicated modification. Bottom: intervals that occupy
active (top) or inactive (middle) nuclear compartments and show enrichment
for nuclear lamina contacts (bottom). The data relate macroscale modification
patterns to genome compartmentalization and nuclear architecture.
Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc. 649
nuclear structures that stain for H3K9me3 and related chromatin
markers (Adams, 2007). Moreover, oncogene-induced senes-
cence is dependent on the H3K9 methyltransferase Suv39h1
(Braig et al., 2005). We tested whether Suv39h1 mediates the
culture-specific domains by profiling H3K9me3 in fibroblasts
after shRNA knockdown. We observed markedly reduced
H3K9me3 in the culture-specific domains, indicating that
Suv39h1 is required for their maintenance (Figures 6C and
6D). We suggest that context-specific induction of H3K9me3
domains by Suv39h1 in cells subjected to growth stimuli in
nonphysiologic environments may underlie their vulnerability to
senescence-associated chromatin changes (see Discussion).
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). The domains are clustered into seven
groups based on their cell-type specificities. GC content, gene density, and nuclear lamina contact enrichment are plotted for each cluster (right). Black arrows
(left) indicate domains that coincide with loci found to have aberrant DNA methylation patterns in iPS cells (Lister et al., 2011).
(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 culture-specific domains are shown for lung fibroblasts after 4 or 10 days of Suv39h1 knockdown.
(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-b-mediated EMT.
See also Figure S4 and Table S4.
650 Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc.
In contrast to the primary cell models, ES and iPS cells lack
the variable H3K9me3 domains (Figure 6B). Because iPS cells
are typically derived from cultured fibroblasts, this implies that
chromatin in these regions is repaired during cellular reprogram-
ming. However, evidence suggests that the domains may be
repaired inefficiently and thus present an impediment to reprog-
ramming. Specifically, recent studies have highlighted more
than 20 ‘‘hot spots’’ of aberrant epigenomic reprogramming
that frequently exhibit aberrant DNA methylation patterns in
iPS cells, relative to ES cells (Lister et al., 2011; Ruiz et al.,
2012). Remarkably, we find that essentially all of these hot spots
overlap regions with H3K9me3 domains in differentiated cells
(Figure 6B). Recent work has also shown that suppression of
Suv39h1 enhances reprogramming (Onder et al., 2012). Thus,
although H3K9me3 patterns are largely reset in iPS cells, the
culture-specific domains may present a barrier to reprogram-
ming and potentially retain a memory of architectural aberrations
in the preprogrammed donor cell.
Discerning the principles by which a single genome can give
rise to a multiplicity of cellular states remains a critical goal.
Model organism studies have presented paradigms by which
interactions among TFs, epigenetic regulators, and genome
sequence elements mediate lineage-specific gene expression
and dynamic responses to environmental stimuli. Yet our under-
standing of these components and their functions in humans has
lagged, with limited existing knowledge derived largely from
in vitro models.
This study aimed to characterize systematically the sequence
genome function in native contexts. We mapped chromatin
blood lineages, brain sections, gastrointestinal tissues, adipose,
liver, and muscle, and compared them to pluripotent and differ-
entiating stem cells and other in vitro counterparts. We used
the maps to locate and classify regulatory sequences and to
characterize large-scale epigenetic states and their dynamics
across a breadth of cellular phenotypes.
The distal element annotations extend prior studies of in vitro
cells with a broad survey of in vivo tissues. Integration of
genome-wide profiles for H3K4me1 and H3K27ac across 30
cell and tissue types yielded nearly 400,000 putative distal
elements, roughly half of which have enhancer-like chromatin
patterns. The elements show exquisite tissue specificity, with
most showing activity in just a few cell types. By analyzing their
tissue specificities and the underlying DNA sequences, we
predict upstream factors that drive genome regulatory programs
in specific cellular contexts. These include master regulator TFs
that dictate specific lineages, as well as regulators such as AP1
whose activity patterns appear to reflect increased mitogenic
signaling triggered by the culture environment. The large propor-
tion of candidate enhancers specific to in vivo tissues should be
valuable for human genetics given their potential to facilitate the
identification and interpretation of causal sequence variants
from genome-wide association studies (Ernst et al., 2011; Gaul-
ton et al., 2010; ENCODE Project Consortium, 2012).
Our study also helps resolve controversy regarding how chro-
matin is reorganized during developmental specification. Prior
involves expansion of repressive chromatin domains or, alterna-
tively, is dominated by localized state changes (Hawkins et al.,
2010a; Lienert et al., 2011; Wen et al., 2009). We critically ad-
dressed this issue by comparing the distributions of multiple
histone modifications and a marker of chromatin exchange
across tissues and cells at various stages of commitment. We
implemented a statistical model to quantify differences in the
chromatin landscapes and verified inferences by measuring
global modification levels with western blots.
We conclude that specification is accompanied by a stark
transition in the epigenetic landscape from a uniquely accessible
state to increasingly restrictive configurations (Figure 7). In
embryonic cells, active and inactive loci both appear subject to
dynamic chromatin remodeling that is likely incompatible with
repressive chromatin compaction (Simon and Kingston, 2009).
Accordingly, the Polycomb mark is largely confined to poised
promoters in ES and iPS cells. Differentiated cells present an
inverse pattern, with chromatin exchange confined to loci under
active regulation and much of the remaining landscape affected
by Polycomb repression. Our findings build upon prior reports of
expanded H3K27me3 domains in differentiated cells (Hawkins
et al., 2010a; Pauler et al., 2009) and suggest a prominent role
for hyperdynamic chromatin in pluripotent cells as a hindrance
do not support prior claims that expanded H3K9me3 domains
arise upon specification, as we find that such features are
instead triggered by thecultureenvironment (see below). Further
study is needed to clarify how developmental and environmental
cues alter H3K9me2 patterns, which appear distinct from the
other repressive states.
In certain contexts, chromatin restriction can proceed well
ment. Case in point is the brain sections wherein intergenic
regions are almost entirely covered by Polycomb-repressed
chromatin and, conversely, a large majority of accessible chro-
of neurons, glia, and other highly specialized cell types that may
be permissive to the accumulation of repressive chromatin. A
corollary of the chromatin patterns is that intronic regulatory
elements may be more accessible and more readily engaged
in such cells. Indeed, we find that introns of genes with neuronal
functions have a relatively higher density of conserved noncod-
ing sequence elements, raising the provocative concept that ep-
igenomic landscapes shape the evolution of genome sequence.
Chromatin architecture changes have also been associated
with cellular responses to environmental cues, yet their influ-
ences on specific genomic loci have remained vague. Here, we
describe a set of megabase-sized H3K9me3 domains that arise
in primary cultured cells but which are rare or absent in tissues,
blood lineages, and stem cells (Figure 7). The domains likely
reflect nuclear architecture changes as they correspond to
gene-poor regions that contact the lamina and occupy the
same inactive compartment. Prior indications that TGF-b-medi-
ated EMT and cellular senescence perturb nuclear architecture
prompted us to examine whether the culture-specific domains
Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc. 651
might relate to such processes (Adams, 2007; McDonald et al.,
2011). We found that EMT modestly increases the expression
lowers their H3K9me3 signals. The domains are dependent on
Suv39h1, a histone methyltransferase that mediates onco-
gene-induced senescence (Braig et al., 2005). Cellular senes-
cence is associated with the formation of nuclear foci (SAHFs)
that stain for H3K9me3 and are thought to arise by rearrange-
ment of pre-existing regions of histone modification (Chandra
et al., 2012). We speculate that H3K9me3 domains represent
an initial response to growth stimuli and nonphysiologic environ-
ments that primes cells for senescence-associated events.
Regardless, the direct identification of genomic regions as
candidate targets of EMT and presenescence changes should
facilitate the study of processes that are fundamental to human
health, aging and cancer.
The fate of culture-specific H3K9me3 domains during cellular
reprogramming presents an interesting question. Although they
are prominent in fibroblasts, the domains appear to be erased
during reprogramming, as they are absent in iPS cells. Yet the
ylated regions found to distinguish iPS cells from ES cells, which
have not undergone reprogramming (Lister et al., 2011; Ruiz
et al., 2012). Somatic cell reprogramming is enhanced by inhibi-
tion of TGF-b signaling and suppression of the H3K9 methyl-
transferase Suv39h1 (Maherali and Hochedlinger, 2009; Onder
et al., 2012). Moreover, H3K9me3 heterochromatin domains
have recently been found to impede the initial binding of plurip-
otency TFs during this process (Soufi et al., 2012). These
correspondences raise the intriguing possibility that H3K9me3
domains present a hindrance to reprogramming and possibly
retain an epigenetic memory of presenescence changes in the
donor cell. Although further study is clearly needed to appreciate
their significance, the identification and initial characterization of
these macroscale chromatin aberrations provides a starting
point for such investigations.
roadmapepigenomics.org and are available at NCBI’s GEO database
(GSE17312, GSE19465 and GSE25249) and a dedicated website http://
setswere publicallyreleased upon verificationat www.
Sample Acquisition and Production Mapping
ES and iPS cell lines were cultured in serum-replacement media (Bock et al.,
2011; Boulting et al., 2011). EBs were generated by in vitro differentiation of
H9 ES cells in low attachment plates in EB differentiation media. Neural
progenitors and neurons were derived from H9 ES cells by in vitro differentia-
tion for 3 and 5 weeks, respectively (Dhara and Stice, 2008). Blood lineages
were isolated from cord or peripheral bloods. Liver, adipose, skeletal muscle,
and gastrointestinal tissues were harvested at surgical resection. Brain
sections were obtained postmortem within hours of death.
Tissue and cell preparations were subjected to ChIP-seq, as described
(Adli et al., 2010; Ku et al., 2008). Aligned reads were used to derive 25 bp
resolution density maps. Noncentered PCA was carried out on each modi-
fication separately based on the number of reads in all nonoverlapping 1 kb
windows. Detailed descriptions are presented in Extended Experimental
Distal Regulatory Element and Repressive Chromatin States
We used a scanning window approach to call H3K4me1 and H3K27ac peaks
(Guttman et al., 2010). After excluding sites within 2.5 kb of a TSS, we desig-
nated H3K4me1 sites as candidate distal elements and clustered (K-means)
them by their cell-type specificities. We quantified their predictive power for
gene activity by correlating the state of each element and the expression of
the nearest TSS. We scanned the distal element clusters for over-represented
Figure 7. Model for Large-Scale Chromatin State Transitions
Illustration depicts large-scale chromatin patterns and their relative prevalence in cells from different developmental stages or environments. Although the
cells (left), inactive regions are diffusely enriched for markers of chromatin exchange and accessibility. Indifferentiatedcells acquired in vivo(middle), inactive loci
instead tend to adopt a Polycomb-repressed chromatin state. In differentiated cells cultured in vitro (right), large domains enriched for the heterochromatin
marker H3K9me3 arise in regions associated with the nuclear lamina.
652 Cell 152, 642–654, January 31, 2013 ª2013 Elsevier Inc.
TF motifs, and validated a sampling of predicted TF-enhancer interactions
using published TF-binding profiles (ENCODE Project Consortium, 2012).
We quantified H3K27me3 and H3K36me3 distributions in each cell type by
modeling foreground and background signals, and then used a scanning
procedure to call intervals of contiguous enrichment. We clustered 2,976
by their chromatin states to elucidate context-specific chromatin regulation
and repression. We also clustered all 1 Mb windows based on their relative
coverage by each modification to characterize macroscale chromatin fea-
tures. To collate H3K9me3 domains, we masked repeat elements and used
a 100 kb window-scanning procedure to call intervals in each cell type.
Detailed descriptions are presented in Extended Experimental Procedures.
Suv39h1 Knockdown and TGF-b Inhibitor Experiments
WI-38fibroblasts weresubjectedtoSuv39h1 knockdown withtwoshRNAs,as
described (Onder et al., 2012). For TGF-b inhibition, WI-38 cells were treated
with 2 or 4 mM TGF-b RI Kinase Inhibitor II (Calbiochem 616452) for 6 days.
H3K9me3 was profiled by ChIP-seq, as described above.
Supplemental Information includes Extended Experimental Procedures, four
figures, and four tables and can be found with this article online at http://dx.
We acknowledge members of the Broad Institute’s Epigenomics Program and
Genome Sequencing and Analysis Program, and the NIH Epigenomics
Mapping Consortium for constructive comments. We thank Kevin Eggan for
ES and iPS lines; Allen Powe and Steve Stice for neural cells; Greg Lauwers
and the MGH Tissue Repository for tissue procurement; and David Flowers,
Irwin Bernstein, John Stamatoyannopoulos and Shelly Heimfeld for blood
samples. We also thank Leslie Gaffney and Lauren Solomon for assistance
with figures. This research was supported by the NIH Common Fund
(U01 ES017155); the National Human Genome Research Institute (U54
HG004570); the National Heart, Lung and Blood Institute (U01 HL100395);
the National Institute on Aging (P30 AG10161); the Howard Hughes Medical
Institute; the Starr Cancer Consortium; and the Burroughs Wellcome Fund.
Received: September 21, 2011
Revised: August 30, 2012
Accepted: December 11, 2012
Published: January 17, 2013
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