Control of Developmental Regulators by
Polycomb in Human Embryonic
Tong Ihn Lee,1,8Richard G. Jenner,1,8Laurie A. Boyer,1,8Matthew G. Guenther,1,8Stuart S. Levine,1,8
Roshan M. Kumar,1Brett Chevalier,1Sarah E. Johnstone,1,2Megan F. Cole,1,2Kyo-ichi Isono,3
Haruhiko Koseki,3Takuya Fuchikami,4Kuniya Abe,4Heather L. Murray,1Jacob P. Zucker,6Bingbing Yuan,1
George W. Bell,1Elizabeth Herbolsheimer,1Nancy M. Hannett,1Kaiming Sun,1Duncan T. Odom,1
Arie P. Otte,5Thomas L. Volkert,1David P. Bartel,1,2Douglas A. Melton,6David K. Gifford,1,7
Rudolf Jaenisch,1,2and Richard A. Young1,2,*
1Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3Developmental Genetics Group, RIKEN Center for Allergy and Immunology, 1-7-22, Suehiro, Tsurumi-ku, Yokohama,
Kanagawa 230-0045, Japan
4Technology and Development Team for Mammalian Cellular Dynamics, BioResource Center, RIKEN Tsukuba Institute,
3-1-1, Koyadai, Tsukuba, Ibaraki 230-0045, Japan
5Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM Amsterdam, The Netherlands
6HowardHughesMedicalInstitute, Department ofMolecular andCellularBiology,HarvardUniversity,Cambridge, MA 02138,USA
7MIT CSAIL, 32 Vassar Street, Cambridge, MA 02139, USA
8These authors contributed equally to this work.
Polycomb group proteins are essential for early
development in metazoans, but their contribu-
tions to human development are not well under-
stood. We have mapped the Polycomb Repres-
sive Complex 2 (PRC2) subunit SUZ12 across
man embryonic stem (ES) cells. We found that
SUZ12 is distributed across large portions of
over two hundred genes encoding key develop-
mental regulators. These genes are occupied by
transcriptionally repressed, and contain some of
the most highly conserved noncoding elements
are preferentially activated during ES cell differ-
entiation and that the ES cell regulators OCT4,
SOX2, and NANOG cooccupy a significant sub-
set of these genes. These results indicate that
PRC2 occupies a special set of developmental
genes in ES cells that must be repressed to
maintain pluripotency and that are poised for
activation during ES cell differentiation.
Embryonic stem (ES) cells are a unique self-renewing cell
mesodermal germ layers during embryogenesis. Human
entiated state but selectively induced to differentiate into
many specialized cell types, are thought to hold great
promise for regenerative medicine (Thomson et al., 1998;
Reubinoff et al., 2000; Mayhall et al., 2004; Pera and
must allow these cells to maintain a pluripotent state but
also allow for differentiation into more specialized states
and further understanding early development.
Among regulators of development, the Polycomb group
proteins (PcG) are of special interest. These regulators
were first described in Drosophila, where they repress
the homeotic genes controlling segment identity in the de-
veloping embryo (Lewis, 1978; Denell and Frederick,
1998; Kennison, 2004). The initial repression of these
genes is carried out by DNA binding transcriptional re-
pressors, and PcG proteins modify chromatin to maintain
these genes in a repressed state (Duncan, 1986; Bender
et al., 1987; Strutt et al., 1997; Horard et al., 2000; Hodg-
son et al., 2001; Mulholland et al., 2003).
The PcG proteins form multiple Polycomb Repressive
Complexes (PRCs), the components of which are con-
served from Drosophila to humans (Franke et al., 1992;
Shao et al., 1999; Birve et al., 2001; Tie et al., 2001; Cao
et al., 2002; Czermin et al., 2002; Kuzmichev et al., 2002;
Levine etal.,2002).ThePRCs arebroughtto thesiteof ini-
tial repression and act through epigenetic modification of
chromatin structure to promote gene silencing (Pirrotta,
Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc. 301
1998; Levine et al., 2004; Lund and van Lohuizen, 2004;
Ringrose and Paro, 2004). PRC2 catalyzes histone H3
lysine-27 (H3K27) methylation, and this enzymatic activity
is required for PRC2-mediated gene silencing (Cao et al.,
2002; Czermin et al., 2002; Kuzmichev et al., 2002; Muller
et al., 2002; Kirmizis et al., 2004). H3K27 methylation is
itates oligomerization, condensation of chromatin struc-
ture, and inhibition of chromatin remodeling activity in
order to maintain silencing (Shao et al., 1999; Francis
et al., 2001; Cao et al., 2002; Czermin et al., 2002).
Components of PRC2 are essential for the earliest
stages of vertebrate development (Faust et al., 1998;
O’Carroll et al., 2001; Pasini et al., 2004). PRC2 and its re-
lated complexes, PRC3 and PRC4, contain the core com-
ponents EZH2, SUZ12, and EED (Kuzmichev et al., 2004;
Kuzmichev et al., 2005). EZH2 is a H3K27 methyltransfer-
ase, and SUZ12 (Suppressor of zeste 12) is required for
this activity (Cao and Zhang, 2004; Pasini et al., 2004).
ES cell lines cannot be established from Ezh2-deficient
blastocysts (O’Carroll et al., 2001), suggesting that PRC2
is involved in regulating pluripotency and self-renewal.
Although the PRCs are known to repress individual HOX
genes (van der Lugt et al., 1996; Akasaka et al., 2001;
Wang et al., 2002; Cao and Zhang, 2004), it is not clear
how these important PcG regulators contribute to early
development in vertebrates.
Because the nature of PRC2 target genes in ES cells
might reveal why PRC2 is essential for early embryonic
development, pluripotency, and self-renewal, we have
mapped the sites occupied by the SUZ12 subunit
throughout the genome in human ES cells. This ge-
markable cadre of genes encoding key regulators of de-
velopmental processes that are repressed in ES cells.
The genes occupied by PRC2 contain nucleosomes that
are trimethylated at histone H3 lysine-27 (H3K27me3),
a modification catalyzed by PRC2 and associated with
the repressed chromatin state. Both PRC2 and nucleo-
somes with histone H3K27me3 occupy surprisingly large
genomic domains around these developmental regulators
and are frequently associated with highly conserved non-
coding sequence elements previously identified by com-
parative genomic methods. The transcription factors
OCT4, SOX2, and NANOG, which are also key regulators
of ES cell pluripotency and self-renewal, occupy a signifi-
cant subset of these genes. Thus, the model of epigenetic
regulation of homeotic genes extends to a large set of de-
velopmental regulators whose repression in ES cells ap-
pears to be key to pluripotency. We suggest that PRC2
functions in ES cells to repress developmental genes
that are preferentially activated during differentiation.
RESULTS AND DISCUSSION
Mapping Genome Occupancy in ES Cells
We mapped the location of both RNA polymerase II and
the SUZ12 subunit of PRC2 genome-wide in human ES
cells (Figure 1). The initiating form of RNA polymerase II
was mapped to test the accuracy of the method and pro-
vide a reference for comparison with sites occupied by
PRC2. The SUZ12 subunit of PRC2 is critical for the func-
tion of the complex and was selected for these genome-
wide experiments. Human ES cells (H9, NIH code WA09)
were analyzed by immunohistochemistry for characteris-
tic stem cell markers, tested for their ability to generate
cell types from all three germ layers upon differentiation
into embryoid bodies, and shown to form teratomas in
immunocompromised mice (Supplemental Data; Figures
DNA sequences bound by RNA polymerase II were
identified in replicatechromatin-immunoprecipitation
(ChIP) experiments using DNA microarrays that contain
over 4.6 million unique 60-mer oligonucleotide probes
spanning the entire nonrepeat portion of the human ge-
nome (Figure 1 and Supplemental Data). To obtain a prob-
abilistic assessment of binding events, an algorithm was
implemented that incorporates information from multiple
probes representing contiguous regions of the genome,
and threshold criteria were established to identify a data-
set with minimal false positives and false negatives. RNA
polymerase II was associated with the promoters of
7,106 of the approximately 22,500 annotated human
genes, indicating that one-third of protein-coding genes
are prepared to be transcribed in ES cells. Three lines of
evidence suggest this dataset is of high quality. Most of
the RNA polymerase II sites (87%) occurred at promoters
of known or predicted genes. Transcripts were detected
for 88% of the genes bound by RNA polymerase II in pre-
dent analysis using gene-specific PCR (Supplemental
Data) indicated that the frequency of false positives was
approximately 4% and the frequency of false negatives
was approximately 30% in this dataset. A detailed analy-
sis of the RNA polymerase II dataset, including binding to
miRNA genes, can be found in Supplemental Data (Tables
S1–S6 and Figures S4 and S5).
The sites occupied by SUZ12 were then mapped
ing the same approach described for RNA polymerase II
(Figure 1C). SUZ12 was associated with the promoters
of 1,893 of the approximately 22,500 annotated human
genes, indicating that ?8% of protein-coding genes are
occupied by SUZ12 in ES cells (Supplemental Data;
Tables S7 and S8). Independent site-specific analysis in-
dicated that the frequency of false positives was approx-
imately 3% and the frequency of false negatives was
approximately 27% in this dataset.
Comparison of the genes occupied by SUZ12 with
those occupied by RNA polymerase II revealed that the
two sets were largely exclusive (Figure 1D; Supplemental
Data; Table S8). There were, however, genes where
SUZ12 and RNA polymerase II cooccupied promoters.
At these genes, PRC complexes may fail to block assem-
bly of the preinitiation complex (Dellino et al., 2004), con-
sistent with the observation that Polycomb group proteins
302 Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc.
Figure 1. Genome-Wide ChIP-Chip in Human Embryonic Stem Cells
(A) DNA segments bound by the initiation form of RNA polymerase II or SUZ12 were isolated using chromatin-immunoprecipitation (ChIP) and iden-
tified with DNA microarrays containing over 4.6 million unique 60-mer oligonucleotide probes spanning the entire nonrepeat portion of the human
genome. ES cell growth and quality control, the antibodies, ChIP protocol, DNA microarray probe design, and data analysis methods are described
in detail in Supplemental Data.
(B) Examples of RNA polymerase II ChIP signals from genome-wide ChIP-Chip. The plots show unprocessed enrichment ratios (blue) for all probes
within a genomic region (ChIP versus whole genomic DNA). Chromosomal positions are from NCBI build 35 of the human genome. Genes are shown
to scale below plots (exons are represented by vertical bars). The start and direction of transcription are noted by arrows.
(C) Examples of SUZ12 ChIP signals from genome-wide ChIP-Chip. The plots show unprocessed enrichment ratios (green) for all probes within a
genomic region (ChIP versus whole genomic DNA). Chromosomal positions, genes, and notations are as described in (B).
(D) Chart showing percentage of all annotated genes bound by RNA polymerase II (blue), SUZ12 (green), both (yellow), or neither (gray).
(E)Distribution ofthedistance between bound probes andtheclosesttranscriptionstartsitesfromRefSeq, Ensembl,MGC, UCSCKnownGenesand
H-Invdatabasesfor SUZ12 (green line), andRNApolymerase II(blue line).The number of boundprobes isgiven as thepercentageoftotalprobes and
is calculated for 400bp intervalsfrom the start site. The null-distribution of the distance betweenall probes and theclosest transcriptionare shown as
a black line.
Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc. 303
can associate with components of the general transcrip-
tion apparatus (Breiling et al., 2001; Saurin et al., 2001).
The vast majority of SUZ12 bound sites were found at
gene promoters (Figure 1E). Ninety-five percent of the
SUZ12 bound regions were found within 1 kb of known or
predicted transcription start sites (Supplemental Data and
Table S7). This suggests that SUZ12 functions in human
ES cells primarily at promoters rather than at distal regula-
recent discovery of a mechanistic link between PcG pro-
teins and DNA methyltransferoses (Vire et al., 2006).
Global Transcriptional Repression by PRC2
PRC2 is composed of three core subunits, SUZ12, EED,
and EZH2, and has been shown to mediate histone
H3K27 methylation at specific genes in vivo. To confirm
that SUZ12 is associated with active PRC2 at target
genes, we used chromatin immunoprecipitation with anti-
bodies against EED and the histone H3K27me3 mark and
analyzed the results with promoter microarrays. We found
that EED and the histone H3K27me3 mark cooccurred
with SUZ12 at most genes using a high-confidence bind-
ing threshold (Figure 2). The false negative rates of thresh-
olded data can lead to an underestimate of the similarity
between different datasets. Plotting raw enrichment ratios
for genes associated with SUZ12, EED, or H3K27me3
demonstrates that SUZ12 binding represents PRC2 bind-
ing at almost all target genes (Figure S6).
cate that PRC2-mediated H3K27 methylation represses
gene expression, but it has not been established if it
acts as a repressor genome-wide. If genes occupied by
SUZ12 are repressed by PRC2, then transcripts from
these genes should generally be present at lower levels
in ES cells than in differentiated cell types. To test this pre-
diction, we compared the expression levels of PRC2-oc-
cupied genesin four different ES celllines withtheexpres-
sion level of these genes in 79 differentiated human cell
and tissue types (Sato et al., 2003; Abeyta et al., 2004;
Su et al., 2004). We found that PRC2 occupied genes
cell types (Figure 2C). A small fraction of the genes occu-
pied by PRC2 were relatively overexpressed in ES cells
(Figure 2C); these tended to show less extensive SUZ12
occupancy and were more likely to be cooccupied by
Figure 2. SUZ12 Is Associated with EED,
histone H3K27me3 Modification, and
Transcriptional Repression in ES Cells
(A) Venn diagram showing the overlap of
genes bound by SUZ12 at high-confidence,
genes bound by EED at high-confidence,
and genes trimethylated at H3K27 at high-
confidence. The data are from promoter micro-
arrays that contain probes tiling ?8 kb and
+2 kb around transcription start. 72% of the
genes bound by SUZ12 at high-confidence are
also bound by EED at high-confidence; others
are bound by EED at lower confidence
(B) SUZ12 (top), EED (middle), and H3K27me3
(bottom) occupancy at NEUROD1. The plots
show unprocessed enrichment ratios for all
versus whole genomic DNA, EED ChIP versus
whole genomic DNA, and H3K27me3 ChIP
versus total H3 ChIP). Chromosomal positions
are from NCBI build 35 of the human genome.
are represented by vertical bars). The start and
direction of transcription are noted by arrows.
(C) Relative expression levels of 604 genes oc-
cupied by PRC2 and trimethylated at H3K27 in
ES cells. Comparisons were made across four
ES cell lines and 79 differentiated cell types.
Each row corresponds to a single gene that is
bound by SUZ12, associated with EED and
H3K27me3, and for which Affymetrix expres-
sion data are available. Each column corre-
sponds to a single expression microarray. ES
cells are in the following order: H1, H9, HSF6,
HSF1. For each gene, expression is shown relative to the average expression level of that gene across all samples, with shades of red indicating
function, and genes are ranked according the significance of their relative level of gene expression in ES cells.
304 Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc.
RNA polymerase II (Supplemental Data). These results are
consistent with the model that PRC2-mediated histone
H3K27 methylation promotes gene silencing at the major-
ity of its target genes throughout the genome in ES cells.
Key Developmental Regulators Are Targets of PRC2
Examination of the targets of SUZ12 revealed that they
were remarkably enriched for genes that control develop-
ment and transcription (Figure 3) and that SUZ12 tended
to occupy large domains at these genes (Figure 4). Al-
though only 8% of all annotated genes were occupied
by SUZ12, ?50% of those encoding transcription factors
associated with developmental processes were occupied
by SUZ12. By comparison, RNA polymerase II preferen-
tially occupied genes involved in a broad spectrum of
cell proliferation functions such as nucleic acid metabo-
lism, protein synthesis, and cell cycle (Figure 3A and
examples in Figure 1B; Supplemental Data; Table S10).
It was striking that SUZ12 occupied many families of
genes that control development and transcription (Figures
3B and S7 and Table S11). These included 39 of 40 of the
homeotic genes found in the HOX clusters and the major-
ity of homeodomain genes. SUZ12 bound homeodomain
genes included almost all members of the DLX, IRX,
LHX, and PAX gene families, which regulate early devel-
opmental steps in neurogenesis, hematopoiesis, axial
patterning, tissue patterning, organogenesis, and cell-
fate specification. SUZ12 also occupied promoters for
large subsets of the FOX, SOX, and TBX gene families.
The forkhead family of FOX genes is involved in axial pat-
terning and tissue development from all three germ layers
(Lehmann et al., 2003). Mutations in members of the SOX
gene family alter cell-fate specification and differentiation
and are linked to several developmental diseases
(Schepers et al., 2002). The TBX family of genes regulates
a wide variety of developmental processes such as gas-
trulation, early pattern formation, organogenesis, and
limb formation (Showell et al., 2004). Thus, the genes pref-
erentially bound by SUZ12 have functions that, when ex-
pressed, promote differentiation. This is likely to explain,
at least in part, why PRC2 is essential for early develop-
ment and ES cell pluripotency.
A remarkable feature of PRC2 binding at most genes
encoding developmental regulators was the extensive
span over which the regulator occupied the locus (Figures
4, S8, and S9). For the majority (72%) of bound sites
Figure 3. Cellular Functions of Genes
Occupied by SUZ12
(A) Genesbound bySUZ12 or RNA polymerase
II were compared to biological process gene
ontology categories; highly represented cate-
gories are shown. Ontology terms are shown
on the y axis; p-values for the significance of
enrichment are graphed along the x axis
(SUZ12 in green, RNA polymerase II in blue).
(B) Selected examples of developmental tran-
scription factor families bound by SUZ12.
SUZ12 is represented by the green oval; indi-
vidual transcription factors are represented by
circles and grouped by family as indicated. Ex-
amples of transcription factors with defined
roles in development are labeled. Transcription
factor families include homeobox protein
(HOX), basic helix-loop-helix domain contain-
ing, class B (BHLHB), HOX cofactors (MEIS/
EVX), distal-less homeobox (DLX), Forkhead
box (FOX), NEUROD, GATA binding protein
(RUNX), paired box and paired-like (PAX), LIM
homeobox (LHX),sine oculis homeobox homo-
log (SIX), NK transcription factor related (NKX),
SRY box (SOX), POU domain containing, clas-
ses 3 and 4 (POU), early B-cell factor (EBF),
atonal homolog (ATOH), hairy and enhancer of
split protein (HES), myogenic basic domain
(MYO), T-box (TBX), caudal type homeobox
(CDX), and iroquois homeobox protein (IRX).
Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc. 305
across the genome, SUZ12 occupied a small region of the
ase II (Figure 1). For the remaining bound regions, SUZ12
occupancy encompassed large domains spanning 2–35
kb and extending from the promoter into the gene. A large
portion of genes encoding developmental regulators
(72%) exhibited these extended regions of SUZ12 bind-
uous genes. For instance, SUZ12 binding extended ?100
kb across the entire HOXA, HOXB, HOXC, and HOXD
clusters but did not bind to adjacent genomic sequences,
trast, clusters of unrelated genes, such as the interleukin
1-b cluster, were not similarly bound by SUZ12. Thus,
genes encoding developmental regulators showed an un-
usual tendency to be occupied by PRC2 over much or all
of their transcribed regions.
PRC2 and Highly Conserved Elements
Previous studies have noted that many highly conserved
noncoding elements of vertebrate genomes are associ-
ated with genes encoding developmental regulators (Be-
jerano et al., 2004; Siepel et al., 2005; Woolfe et al., 2005).
we investigated the possibility that SUZ12 bound regions
are associated with these highly conserved elements. In-
spection of individual genes suggested that SUZ12 occu-
pancy was associated with regions of sequence conser-
vation (Figure 5A). Eight percent of the approximately
Figure 4. SUZ12 Occupies Large Por-
tions of Genes Encoding Transcription
Factors with Roles in Development
(A) The fraction of SUZ12 target genes associ-
ated with different sizes of binding domains.
Genes are grouped into four categories ac-
cording to their function: Signaling, Adhesion/
migration, Transcription, and Other.
(B) Examples of SUZ12 (green) and RNA poly-
merase II (blue) binding at the genes encoding
developmental regulators TBX5 and PAX6.
The plots show unprocessed enrichment ratios
for all probes within a genomic region (ChIP
versus whole genomic DNA). Genes are shown
to scale below plots (exons are represented by
vertical bars). The start and direction of tran-
scription are noted by arrows.
(C) Binding profiles of SUZ12 (green) and RNA
polymerase II (blue) across ?500 kb regions
encompassing HOX clusters A–D. Unpro-
cessed enrichment ratios for all probes within
a genomic region are shown (ChIP versus
whole genomic DNA). Approximate HOX clus-
ter region sizes are indicated within black bars.
306 Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc.
described by Woolfe and colleagues (Woolfe et al., 2005)
were found to beassociated withthe SUZ12bound devel-
opmental regulators (p-value 10?14). Using entries from
the PhastCons database of conserved elements (Siepel
et al., 2005), we found that SUZ12 occupancy of highly
conserved elements was highly significant (using highly
conserved elements with a LoD conservation score of
100 or better, the p-value for significances was less than
10?85). Since PRC2 has not been shown to directly bind
DNA sequences, we expect that specific DNA binding
proteins occupy the highly conserved DNA sequences
and may associate with PRC2, which spreads and oc-
cupies adjacent chromatin. Thus, the peaks of SUZ12
occupancy might not be expected to precisely colocate
with the highly conserved elements, even if these ele-
ments are associated with PRC2 recruitment.
Remarkably, the degree of the association between
considering sequences with an increasing degree of con-
servation (Figure 5B). By comparison, RNA polymerase II
showed no such enrichment. These results suggest that
the subset of highly conserved noncoding elements at
genes encoding developmental regulators may be associ-
ated with PcG-mediated silencing of these regulators.
Signaling Genes Are among PRC2 Targets
The targets of SUZ12 were also enriched for genes that
encode components of signaling pathways (Figure 3A
and Table S12). There is evidence that transforming
growth factor-b (TGFb), bone morphogenic protein (BMP),
wingless-type MMTV integration site (Wnt), and fibro-
blast growth factor (FGF) signaling pathways, which
Figure 5. SUZ12 Binding Is Associated
with Highly Conserved Regions
(A) SUZ12 occupancy (green) and conserved
elements are shown at NKX2-2 and adjacent
genomic regions. The plots show unprocessed
enrichment ratios for all probes within this ge-
nomic region (SUZ12 ChIP versus whole geno-
mic DNA). Conserved elements (red) with LoD
scores > 160 derived from the PhastCons pro-
gram (Siepel et al., 2005) are shown to scale
plots (exons are represented by vertical bars).
A higher resolution view is also shown below.
(B) Enrichment of conserved noncoding ele-
ments within SUZ12 (green) and RNA polymer-
ase II (blue) bound regions. The maximum non-
exonic PhastCons conservation score was
ison, the same parameter was determined us-
ing a randomized set of genomic regions with
the same size distribution. The graph displays
the ratio of the number of bound regions with
that score versus the number of randomized
genomic regions with that score.
Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc. 307
Figure 6. Preferential Activation of PRC2 Target Genes during ES Cell Differentiation
(A) Fold enrichment in the number of genes induced or repressed during ES cell differentiation. The change in gene expression is given as the log(2)
transformed ratio of the signals in differentiated H1 cells versus pluripotent H1 cells and is binned into six groups. The upper limit of each bin is in-
dicated on the x axis. The two lines show genes transcriptionally inactive in ES cells (absence of RNA polymerase II) and bound by SUZ12 (green) and
genes transcriptionally inactive in ES cells and repressed by other means (blue). In both cases, fold enrichment is calculated against the total pop-
ulation of genes and normalized for the number of genes present in each group.
(B) Expression changes of genes encoding developmental regulators during ES cell differentiation. Expression ratio (differentiated/pluripotent) is rep-
resented by color, with shades of red indicating upregulation and shades of green downregulation according to the scale shown above. Genes are
orderedaccording tochange ingene expression, withgenesexhibitinghigherexpressioninpluripotent EScells totheleftandgenesexhibitinghigher
expression in differentiated cells to the right. Genes bound by SUZ12 in undifferentiated ES cells are indicated by blue lines in the lower panel.
transformed ratio of the signals in Suz12-deficient cells versus wild-type ES cells. The two lines show genes transcriptionally inactive in human ES
cells (absence of RNA polymerase II) and bound by SUZ12 (green) and genes transcriptionally inactive in human ES cells and repressed by other
means (blue). In both cases, fold enrichment is calculated against the total population of genes.
(D) Gene expression ratios (log base 2) of Suz12 target genes in differentiated human H1 ES cells relative to pluripotent H1 ES cells (x axis) and in
Suz12-deficient mouse cells relative to wild-type mouse ES cells (y axis). Upper right quadrant: genes upregulated during human ES cell differenti-
ation and in Suz12-deficient mouse cells; lower right: genes upregulated during ES cell differentiation and downregulated in Suz12-deficient cells;
lower left: genes downregulated during ES cell differentiation and in Suz12-deficient cells; upper left: genes downregulated during ES cell differen-
tiation and upregulated in Suz12-deficient cells.
(E) SUZ12 binding profiles across the gene encoding muscle regulator MYOD1 in H9 human ES cells (green) and primary human skeletal myotubes
(gray). The plots show unprocessed enrichment ratios for all probes within a genomic region (ChIP versus whole genomic DNA). Genes are shown to
scale below plots (exons are represented by vertical bars). The start and direction of transcription are noted by arrows.
308 Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc.
are required for gastrulation and lineage differentiation in
the embryo, are also essential for self-renewal and dif-
ferentiation of ES cells in culture (Loebel et al., 2003; Mo-
lofsky et al., 2004). SUZ12 generally occupied the pro-
moters of multiple components of these pathways, but it
occupied larger domains withina groupof signaling genes
that contained highly conserved elements. This group
contained members of the Wnt family (WNT1, WNT2,
WNT6) as well as components of the TGFb superfamily
(BMP2, GDF6). Recent studies have shown that Wnt sig-
naling plays a role in pluripotency and self-renewal in
both mouse and human ES cells (Sato et al., 2004), and
our results suggest that it is important to maintain specific
family members in a repressed state in ES cells.
Activation of PRC2 Target Genes
PRC2 is associated with an important set of developmen-
tal regulators that must be silent in ES cells but activated
during differentiation. This observation suggests that
PRC2 ultimately functions to repress occupied genes in
ES cells and that these genes may be especially poised
for transcriptional activation during ES cell differentiation.
We reasoned that if this model is correct, genes bound by
SUZ12 should be preferentially activated upon ES cell dif-
ferentiation orin cellsthatlack SUZ12.Furthermore, in dif-
ferentiated cells, SUZ12 might continue to be observed at
silent genes but must be removed from genes whose ex-
pression is essential for that cell type.
We first examined gene expression in ES cells stimu-
lated to undergo differentiation (Sato et al., 2003). We
found that genes occupied by SUZ12 were more likely
to be activated during ES cell differentiation than genes
that were not occupied by SUZ12 (Figure 6A; Supplemen-
tal Data; Table S13), indicating that SUZ12-occupied
genes show preferential activation during differentiation
under these conditions. Thirty-six percent of genes bound
sion during ES cell differentiation, whereas only 16% of
genes not bound by SUZ12 showed such an increase.
This effect was particularly striking at the set of develop-
mental regulators (Figure 6B). SUZ12 occupied most
(83%) of the developmental regulators that were induced
more than 10-fold during ES cell differentiation.
We next examined the expression of SUZ12 target
genes in Suz12-deficient cell lines derived from homozy-
gous mutant blastocysts (Supplemental Data). We rea-
soned that genes bound by SUZ12 in human ES cells
have orthologs in mice that should be upregulated in
Suz12-deficient mouse cells, although we expected the
overlap in these sets of genes to be imperfect because
of potential differences between human and mouse ES
cells, the possible repression of PRC2 target genes by ad-
ditional mechanisms, and pleiotropic effects of the Suz12
knockout on genes downstream of Suz12-target genes.
Differences in gene expression between Suz12 homozy-
gous mutant cells and wild-type ES cells were measured
using gene expression microarrays and the human SUZ12
binding data mapped to orthologous mouse genes using
HomoloGene (www.ncbi.nlm.nih.gov/HomoloGene). We
found that a significant portion of mouse genes whose
counterparts were bound by SUZ12 in human ES cells
were upregulated in Suz12-deficient mouse cells (70 of
346 genes, p = 6 ? 10?4); these genes are listed in Table
S14. Orthologs of genes occupied by SUZ12 in human
ES cells were more likely to be activated and less likely
logsofgenesnotoccupied bySUZ12(Figure6C). Further-
more, we found that orthologs of Suz12 target genes that
were induced upon human ES cell differentiation were
generally also induced upon loss of Suz12 in mouse cells
(Figure 6D). Genes that were activated during ES cell dif-
ferentiation and in Suz12-deficient cells included those
encoding transcriptional regulators (GATA2, GATA3,
GATA6, HAND1, MEIS2, and SOX17) signaling proteins
(WNT5A, DKK1, DKK2, EFNA1, EFNB1, EPHA4, and
EPHB3) and the cell-cycle inhibitor CDKN1A. These data
indicate that Suz12 is necessary to fully repress the genes
that are occupied by PRC2 in wild-type ES cells and have
since been confirmed with binding data and knockout
studies of a second PRC subunit in mouse (Boyer et al.,
If PRC2 functions to repress genes in ES cells that are
activated during differentiation, then in differentiated tis-
sues SUZ12 occupancy should be diminished at genes
encoding developmental regulators that have a role in
specifying the identity of that tissue, similar to results
seen with Ezh2 at specific genes in mouse (Caretti
et al., 2004). To test this, we designed an array focused
on the promoters of developmental regulators and used
ChIP-Chip to investigate SUZ12 occupancy at these
promoters in primary differentiated muscle cells. The re-
sults demonstrated that genes encoding key regulators
of muscle differentiation, including MYOD1, displayed
greatly diminished SUZ12 occupancy when compared to
ES cells (Figure 6E). MYOD1 is a master regulator for
muscle differentiation (Tapscott, 2005), and the gene en-
coding this transcription factor displayed no significant
SUZ12 occupancy when compared to the levels of
SUZ12 occupancy observed in ES cells. Genes encoding
other transcriptional regulators that play a central role in
muscle development, such as PAX3 and PAX7 (Brand-
Saberi, 2005), showed reduced levels of SUZ12 occu-
pancy in muscle cells relative to ES cells (Supplemental
Data and Figure S11). In contrast, other developmental
regulators important for differentiation of nonmuscle
tissues remained occupied by SUZ12 in differentiated
muscle cells (Figure 6F and Table S15). These data
(F) Suz12 binding profiles across the gene encoding LHX9 in H9 human ES cells (green) and primary human skeletal myotubes (gray). The plots show
unprocessed enrichment ratios for all probes within a genomic region (ChIP versus whole genomic DNA). Genes are shown to scale below plots
(exons are represented by vertical bars). The start and direction of transcription are noted by arrows.
Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc. 309
support a model where PRC2 binding in ES cells re-
presses key developmental regulators that are later ex-
pressed during differentiation.
Targets of PRC2 Are Shared with Key
ES Cell Regulators
The transcription factors OCT4, SOX2, and NANOG have
essential roles in early development and are required for
the propagation of undifferentiated ES cells in culture
(Nichols et al., 1998; Avilion et al., 2003; Chambers
et al., 2003; Mitsui et al., 2003). We recently reported
that these transcription factors occupied promoters for
many important developmental regulators in human ES
cells (Boyer et al., 2005). This led us to compare the set
of genes encoding developmental regulators and occu-
pied by OCT4, SOX2, and NANOG with those occupied
by PRC2 (Figure 7 and Supplemental Data). We found
that each of the three DNA binding transcription factors
occupied approximately one-third of the PRC2-occupied
genes that encode developmental transcription factors
(Figure 7A; Supplemental Data; Table S11). Remarkably,
we found that the subset of genes encoding developmen-
tal regulators that were occupied by OCT4, SOX2, and
NANOG and repressed in the regulatory circuitry high-
lighted in Boyer et al. were almost all occupied by PRC2
(Figure 7B). These included genes for transcription factors
known to be important for differentiation into extraembry-
onic, endodermal, mesodermal, and ectodermal lineages
(e.g., ESX1L, ONECUT1, HAND1, HOXB1). As expected,
active genes encoding ES cell transcription factors (e.g.,
ZIC3, STAT3, OCT4, NANOG) were occupied by OCT4,
SOX2, NANOG, and RNA polymerase II but not by PRC2
The observation that OCT4, SOX2, and NANOG are
bound to a significant subset of developmental genes oc-
velopmental regulators and stem cell pluripotency. Like
PRC2, OCT4 and NANOG have been shown to be impor-
tant for early development and ES cell identity. It is possi-
ble, therefore, that inappropriate regulation of develop-
mental regulators that are common targets of OCT4,
NANOG, and PRC2 contributes to the inability to establish
et al., 1998; O’Carroll et al., 2001; Chambers et al., 2003;
Mitsui et al., 2003).
We have mapped the sites occupied by SUZ12 through-
out the genome to gain insights into how PRC2 contrib-
utes to pluripotency in human embryonic stem cells. ES
cells proliferate in an undifferentiated state yet remain
poised to respond to development cues. Genes encoding
the transcriptional regulators that promote differentiation
must therefore be repressed in ES cells but activated
Figure 7. SUZ12 Is Localized to Genes also Bound by ES Cell
(A) Transcriptional regulatory network model of developmental regula-
tors governed by OCT4, SOX2, NANOG, RNA polymerase II, and
SUZ12 inhuman ES cells. The EScell transcriptionfactors eachbound
to approximately one-third of the PRC2-occupied, developmental
transcription factor genes. Developmental regulators were selected
based on gene ontology. Regulators are represented by dark blue cir-
cles; RNA polymerase II is represented by a light blue circle; SUZ12 is
represented by a green circle; gene promoters for developmental reg-
ulators are represented by small red circles.
(B) SUZ12 occupies a set of repressed developmental regulators also
bound by OCT4, SOX2, and NANOG in human ES cells. Genes anno-
tated as bound by OCT4, SOX2, and NANOG previously and identified
as active or repressed based on expression data (Boyer et al., 2005)
were tested to see if they were bound by SUZ12 or RNA polymerase
II. Ten of eleven previously identified active genes were found to be
bound by RNA polymerase II at known promoters, while eleven of
twelve previously identified repressed genes were bound by SUZ12.
Regulators are represented by dark blue circles, RNA polymerase II
by a light blue circle, and SUZ12 by a green circle. Gene promoters
are represented by red rectangles.
310 Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc.
upon receiving signals to differentiate. We found that
PRC2 occupies large domains at genes encoding a key
set of repressed developmental regulators that are prefer-
entially activated upon cellular differentiation, thus impli-
cating this complex directly in the maintenance of the plu-
Transcription factors and chromatin regulators contrib-
ute to the transcriptional regulatory circuitry responsible
for pluripotency and self-renewal in human ES cells. Un-
derstanding this circuitry is fundamental to understanding
human development and realizing the therapeutic poten-
tial of these cells. In this context, we find it exciting that
the outlines of the core transcriptional regulatory circuitry
of human ES cells are emerging. The transcription factors
OCT4, SOX2, and NANOG are associated with actively
transcribed genes that contribute to growth and self-re-
newal (Boyer et al., 2005). These factors also occupy
genes encoding key developmental regulators that are
transcriptionally repressed, due at least in part to their as-
H3K27me3. Further study of transcription factors and
to produce a more comprehensive map of transcriptional
regulatory circuitry in ES cells and to test models that
sights into approaches by which pluripotent cells can be
stimulated to differentiate into different cell types.
Cells and Cell Culture
Human H9 ES cells (WiCell, Madison, WI) were cultured as described
(Boyer et al., 2005). Primary human skeletal muscle cells were ob-
tained from Cell Applications (San Diego, CA) and expanded and dif-
ferentiated into myotubes according to the supplier’s protocols.
Suz12 ?/? mouse cell lines were derived from blastocysts from
crosses between heterozygous Suz12 mutant animals, as described
in Supplemental Data.
Chromatin Immunoprecipitation and DNA Microarray Analysis
ChIPwascombined withDNAmicroarray analysisasdescribed (Boyer
et al., 2005). The antibodies used here were specific for hypophos-
phorylated RNA polymerase II (8WG16) (Thompson et al., 1989),
SUZ12 (Upstate, 07-379), EED (Hamer et al., 2002), H3K27me3 (Ab-
cam, AB6002), and total histone H3 (Abcam, AB1791). The design of
the oligo-based arrays, which were manufactured by Agilent Technol-
ogies, is described in detail in Supplemental Data. A whole-chip error
model was used to calculate confidence values from the enrichment
ratio and the signal intensity of each probe (probe p-value) and of
each set of three neighboring probes (probe-set p-value). Probe-sets
with significant probe-set p-values (p < 0.001) and significant individ-
ual probe p-values were judged to be bound (see Supplemental
Data for additional information). Bound regions were assigned to
genes if they were within 1 kb of the transcription start site from one
of five genomic databases; RefSeq, MGC, Ensembl, UCSC Known
Gene, or H-Inv. All microarray data is available at ArrayExpress under
the accession designation E-WMIT-7.
Gene Expression Analysis
Gene expression data were collated from H1 ES cells (Sato et al.,
2003), H9, HSF1, and HSF6 ES cells (Abeyta et al., 2004), and 79 dif-
ferentiated human cell and tissue types (Su et al., 2004) and analyzed
as described in detail in Supplemental Data. Replicate gene expres-
sion data was obtained for wild-type mouse ES cells and Suz12-
deficient cells using Agilent Mouse Development arrays and were
analyzed as described in Supplemental Data.
Supplemental Data include fifteen figures, fifteen tables, Experimental
Procedures, and References and can be found with this article online
We thank Elizabeth Jacobsen for technical assistance and Robert
Brady for help with array design. L.A.B. and H.L.M. were supported
by NRSA postdoctoral fellowships. M.G.G. is an Amgen Fellow of
LSRF. R.M.K. was supported by a fellowship from the ACS. D.T.O.
was supported by NIH award DK070813. This work was supported
by NIH grants HG002668 and GM069400. T.L., T.L.V., D.K.G., and
R.A.Y. consult for Agilent Technologies.
Received: October 25, 2005
Revised: January 20, 2006
Accepted: February 23, 2006
Published: April 20, 2006
Abeyta, M.J., Clark, A.T., Rodriguez, R.T., Bodnar, M.S., Pera, R.A.,
and Firpo, M.T. (2004). Unique gene expressionsignatures of indepen-
dently-derived human embryonic stem cell lines. Hum. Mol. Genet. 13,
Akasaka, T., van Lohuizen, M., van der Lugt, N., Mizutani-Koseki, Y.,
Kanno,M.,Taniguchi,M.,Vidal, M.,Alkema,M.,Berns, A.,andKoseki,
H. (2001). Mice doubly deficient for the Polycomb Group genes Mel18
and Bmi1 reveal synergy and requirement for maintenance but not
initiation of Hox gene expression. Development 128, 1587–1597.
Badge, R. (2003). Multipotent cell lineages in early mouse develop-
ment depend on SOX2 function. Genes Dev. 17, 126–140.
Bejerano, G., Pheasant, M., Makunin, I., Stephen, S., Kent, W.J., Mat-
tick, J.S., and Haussler, D. (2004). Ultraconserved elements in the hu-
man genome. Science 304, 1321–1325.
Bender, M., Turner, F.R., and Kaufman, T.C. (1987). A development
genetic analysis of the gene regulator of postbithorax in Drosophila
melanogaster. Dev. Biol. 119, 418–432.
Birve, A., Sengupta, A.K., Beuchle, D., Larsson, J., Kennison, J.A.,
Rasmuson-Lestander, A., and Muller, J. (2001). Su(z)12, a novel Dro-
sophila Polycomb group gene that is conserved in vertebrates and
plants. Development 128, 3371–3379.
J.P., Guenther, M.G., Kumar, R.M., Murray, H.L., Jenner, R.G., et al.
(2005). Core transcriptional regulatory circuitry in human embryonic
stem cells. Cell 122, 947–956.
Boyer, L.A., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L.A., Lee,
T.I., Levine, S.S., Wernig, M., Tajonar, A., Ray, M.K., Otte, A.P., Vidal,
M., Gifford, D.K., Young, R.A., and Jaenisch, R. (2006). Polycomb
complexes repress developmental regulators in murine embryonic
stem cells. Nature, in press.
Brand-Saberi, B. (2005). Genetic and epigenetic control of skeletal
muscle development. Ann. Anat. 187, 199–207.
Breiling, A., Turner, B.M., Bianchi, M.E., and Orlando, V. (2001). Gen-
eral transcription factors bind promoters repressed by Polycomb
group proteins. Nature 412, 651–655.
Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc. 311
Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H., Tempst,
P., Jones, R.S., and Zhang, Y. (2002). Role of histone H3 lysine 27
methylation in Polycomb-group silencing. Science 298, 1039–1043.
Cao, R., and Zhang, Y. (2004). SUZ12 is required for both the histone
methyltransferase activity and the silencing function of the EED-EZH2
complex. Mol. Cell 15, 57–67.
Caretti, G., Di Padova, M., Micales, B., Lyons, G.E., and Sartorelli, V.
(2004). The Polycomb Ezh2 methyltransferase regulates muscle
gene expression and skeletal muscle differentiation. Genes Dev. 18,
Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie,
ripotency sustaining factor inembryonic stem cells. Cell113, 643–655.
Czermin, B., Melfi, R., McCabe, D., Seitz, V., Imhof, A., and Pirrotta, V.
(2002). Drosophila enhancer of Zeste/ESC complexes have a histone
H3 methyltransferase activity that marks chromosomal Polycomb
sites. Cell 111, 185–196.
Dellino, G.I., Schwartz, Y.B., Farkas, G., McCabe, D., Elgin, S.C., and
Pirrotta, V. (2004). Polycomb silencing blocks transcription initiation.
Mol. Cell 13, 887–893.
Denell, R.E., and Frederick, R.D. (1983). Homoeosis in Drosophila:
a description of the Polycomb lethal syndrome. Dev. Biol. 97, 34–47.
Duncan, I. (1986). Control of bithorax complex functions by the seg-
mentation gene fushi tarazu of D. melanogaster. Cell 47, 297–309.
Faust, C., Lawson, K.A., Schork, N.J., Thiel, B., and Magnuson, T.
(1998). The Polycomb-group gene eed is required for normal morpho-
genetic movements during gastrulation in the mouse embryo. Devel-
opment 125, 4495–4506.
Francis, N.J., Saurin, A.J., Shao, Z., and Kingston, R.E. (2001). Recon-
Franke, A., DeCamillis, M., Zink, D., Cheng, N., Brock, H.W., and Paro,
protein complex in chromatin of Drosophila melanogaster. EMBO J.
Hamer, K.M., Sewalt, R.G., den Blaauwen, J.L., Hendrix, T., Satijn,
D.P., and Otte, A.P. (2002). A panel of monoclonal antibodies against
human polycomb group proteins. Hybrid. Hybridomics 21, 245–252.
recognition of a 70-base-pair element containing d(GA)(n) repeats me-
diates bithoraxoid polycomb group response element-dependent si-
lencing. Mol. Cell. Biol. 21, 4528–4543.
Horard, B., Tatout, C., Poux, S., and Pirrotta, V. (2000). Structure of
a polycomb response element and in vitro binding of polycomb group
complexes containing GAGA factor. Mol. Cell. Biol. 20, 3187–3197.
Kennison, J.A. (2004). Introduction to Trx-Gand Pc-G genes. Methods
Enzymol. 377, 61–70.
get genes is associated with methylation of histone H3 Lys 27. Genes
Dev. 18, 1592–1605.
Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P., and
Reinberg, D. (2002). Histone methyltransferase activity associated
with a human multiprotein complex containing the Enhancer of Zeste
protein. Genes Dev. 16, 2893–2905.
Kuzmichev, A., Jenuwein, T., Tempst, P., and Reinberg, D. (2004). Dif-
ferent EZH2-containing complexes target methylation of histone H1 or
nucleosomal histone H3. Mol. Cell 14, 183–193.
Kuzmichev, A., Margueron, R., Vaquero, A., Preissner, T.S., Scher, M.,
Kirmizis, A., Ouyang, X., Brockdorff, N., Abate-Shen, C., Farnham, P.,
and Reinberg, D. (2005). Composition and histone substrates of poly-
comb repressive group complexes change during cellular differentia-
tion. Proc. Natl. Acad. Sci. USA 102, 1859–1864.
Lehmann, O.J., Sowden, J.C., Carlsson, P., Jordan, T., and Bhatta-
charya, S.S. (2003). Fox’s in development and disease. Trends Genet.
Levine, S.S., Weiss, A., Erdjument-Bromage, H., Shao, Z., Tempst, P.,
and Kingston, R.E. (2002). The core of the polycomb repressive com-
Mol. Cell. Biol. 22, 6070–6078.
Levine, S.S., King, I.F., and Kingston, R.E. (2004). Division of labor in
polycomb group repression. Trends Biochem. Sci. 29, 478–485.
Lewis, E.B. (1978). A gene complex controlling segmentation in Dro-
sophila. Nature 276, 565–570.
eage choice and differentiation in mouse embryos and embryonic
stem cells. Dev. Biol. 264, 1–14.
Lund, A.H., and van Lohuizen, M. (2004). Polycomb complexes and si-
lencing mechanisms. Curr. Opin. Cell Biol. 16, 239–246.
Mayhall, E.A., Paffett-Lugassy, N., andZon, L.I. (2004). The clinical po-
tential of stem cells. Curr. Opin. Cell Biol. 16, 713–720.
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takaha-
shi, K., Maruyama, M., Maeda, M., and Yamanaka, S. (2003). The ho-
meoprotein Nanog is required for maintenance of pluripotency in
mouse epiblast and ES cells. Cell 113, 631–642.
Molofsky, A.V., Pardal, R., and Morrison, S.J. (2004). Diverse mecha-
nisms regulate stem cell self-renewal. Curr. Opin. Cell Biol. 16, 700–
Mulholland, N.M., King, I.F., and Kingston, R.E. (2003). Regulation of
Polycomb group complexes by the sequence-specific DNA binding
proteins Zeste and GAGA. Genes Dev. 17, 2741–2746.
Muller, J., Hart, C.M., Francis, N.J., Vargas, M.L., Sengupta, A., Wild,
Histone methyltransferase activity of a Drosophila Polycomb group re-
pressor complex. Cell 111, 197–208.
Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius,
D., Chambers, I., Scholer, H., and Smith, A. (1998). Formation of pluri-
potent stem cells in the mammalian embryo depends on the POU tran-
scription factor Oct4. Cell 95, 379–391.
O’Carroll, D., Erhardt, S., Pagani, M., Barton, S.C., Surani, M.A., and
Jenuwein, T. (2001). The polycomb-group gene Ezh2 is required for
early mouse development. Mol. Cell. Biol. 21, 4330–4336.
Orlando, V.,andParo, R.(1995). Chromatinmultiprotein complexesin-
volved in the maintenance of transcription patterns. Curr. Opin. Genet.
Dev. 5, 174–179.
Pasini, D., Bracken, A.P., Jensen, M.R., Denchi, E.L., and Helin, K.
(2004). Suz12 is essential for mouse development and for EZH2 his-
tone methyltransferase activity. EMBO J. 23, 4061–4071.
Pera, M.F., and Trounson, A.O. (2004). Human embryonic stem cells:
prospects for development. Development 131, 5515–5525.
Pirrotta, V. (1998). Polycombing the genome: PcG, trxG, and chroma-
tin silencing. Cell 93, 333–336.
Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A.
(2000). Embryonic stemcelllinesfrom human blastocysts: somatic dif-
ferentiation in vitro. Nat. Biotechnol. 18, 399–404.
Ringrose, L., and Paro, R. (2004). Epigenetic regulation of cellular
memory by the Polycomb and Trithorax group proteins. Annu. Rev.
Genet. 38, 413–443.
Sato, N., Sanjuan, I.M., Heke, M., Uchida, M., Naef, F., and Brivanlou,
A.H. (2003). Molecular signature of human embryonic stem cells and
its comparison with the mouse. Dev. Biol. 260, 404–413.
Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., and Brivanlou, A.H.
(2004). Maintenance of pluripotency in human and mouse embryonic
stem cells through activation of Wnt signaling by a pharmacological
GSK-3-specific inhibitor. Nat. Med. 10, 55–63.
312 Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc.
Saurin, A.J., Shao, Z., Erdjument-Bromage, H., Tempst, P., and King- Download full-text
ston, R.E. (2001). A Drosophila Polycomb group complex includes
Zeste and dTAFII proteins. Nature 412, 655–660.
Schepers, G.E., Teasdale, R.D., andKoopman, P. (2002). Twentypairs
of sox: extent, homology, and nomenclature of the mouse and human
sox transcription factor gene families. Dev. Cell 3, 167–170.
Shao, Z., Raible, F., Mollaaghababa, R., Guyon, J.R., Wu, C.T.,
Bender, W., and Kingston, R.E. (1999). Stabilization of chromatin
structure by PRC1, a Polycomb complex. Cell 98, 37–46.
Showell, C., Binder, O., and Conlon, F.L. (2004). T-box genes in early
embryogenesis. Dev. Dyn. 229, 201–218.
Siepel, A., Bejerano, G., Pedersen, J.S., Hinrichs, A.S., Hou, M.,
Rosenbloom, K., Clawson, H., Spieth, J., Hillier, L.W., Richards, S.,
et al. (2005). Evolutionarily conserved elements in vertebrate, insect,
worm, and yeast genomes. Genome Res. 15, 1034–1050.
Simon, J., Chiang, A., and Bender, W. (1992). Ten different Polycomb
group genes are required for spatial control of the abdA and AbdB
homeotic products. Development 114, 493–505.
Strutt, H., Cavalli, G., and Paro, R. (1997). Co-localization of Polycomb
protein and GAGA factor on regulatory elements responsible for the
maintenance of homeotic gene expression. EMBO J. 16, 3621–3632.
Su, A.I., Wiltshire, T., Batalov, S., Lapp, H., Ching, K.A., Block, D.,
Zhang, J., Soden, R., Hayakawa, M., Kreiman, G., et al. (2004). A
gene atlas of the mouse and human protein-encoding transcriptomes.
Proc. Natl. Acad. Sci. USA 101, 6062–6067.
Tapscott, S.J. (2005). The circuitry of a master switch: Myod and the
regulation of skeletal muscle gene transcription. Development 132,
Thompson, N.E., Steinberg, T.H., Aronson, D.B., and Burgess, R.R.
(1989). Inhibition of in vivo and in vitro transcription by monoclonal
antibodies prepared against wheat germ RNA polymerase II that react
with the heptapeptide repeat of eukaryotic RNA polymerase II. J. Biol.
Chem. 264, 11511–11520.
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A.,
Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). Embryonic
stem cell lines derived from human blastocysts. Science 282, 1145–
The Drosophila Polycomb Group proteins ESC and E(Z) are present in
a complex containing the histone-binding protein p55 and the histone
deacetylase RPD3. Development 128, 275–286.
van der Lugt, N.M., Alkema, M., Berns, A., and Deschamps, J. (1996).
expression. Mech. Dev. 58, 153–164.
Vire, E., Brenner, C., Deplus, R., Blanchon, L., Fraga, M., Didelot, C.,
Morey, L., Van Eynde, A., Bernard, D., Vanderwinden, J.M., Bollen,
M., Esteller, M., Di Croce, L., de Launoit, Y., and Fuks, F. (2006). The
Polycomb group protein EZH2 directly controls DNA methylation.
Nature 439, 871–874.
Wang, J., Mager, J., Schnedier, E., and Magnuson, T. (2002). The
mouse PcG gene eed is required for Hox gene repression and extra-
embryonic development. Mamm. Genome 13, 493–503.
Woolfe, A., Goodson, M., Goode, D.K., Snell, P., McEwen, G.K., Va-
vouri, T., Smith, S.F., North, P., Callaway, H., Kelly, K., et al. (2005).
Highly conserved non-coding sequences are associated with verte-
brate development. PLoS Biol. 3, e7.
Cell 125, 301–313, April 21, 2006 ª2006 Elsevier Inc. 313