Molecular Cell

ArticleContext-Dependent Wiring of Sox2 Regulatory
Networks for Self-Renewalof Embryonic and Trophoblast Stem Cells
Kenjiro Adachi,1,9,10,* Itoshi Nikaido,2,9,11 Hiroshi Ohta,3,12 Satoshi Ohtsuka,1 Hiroki Ura,1 Mitsutaka Kadota,5Teruhiko Wakayama,3,6 Hiroki R. Ueda,2,4 and Hitoshi Niwa1,7,8,*
1Laboratory for Pluripotent Stem Cell Studies
2Functional Genomics Unit
3Laboratory for Genome Reprogramming
4Laboratory for Systems Biology
5Genome Resource and Analysis Unit
RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 6500047, Japan
6Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi 4008510, Japan
7Laboratory for Development and Regenerative Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chuo-ku,
Kobe 6500017, Japan
8JST, CREST, Sanbancho, Chiyoda-ku, Tokyo, 1020075, Japan
9These authors contributed equally to this work
10Present address: Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany
11Present address: Bioinformatics Research Unit, Advanced Center for Computing and Communication, RIKEN, Wako,
Saitama 3510198, Japan
12Present address: Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku,
Kyoto 6068501, Japan*Correspondence: kenjiro.adachi@mpi-muenster.mpg.de (K.A.), niwa@cdb.riken.jp (H.N.)
http://dx.doi.org/10.1016/j.molcel.2013.09.002

SUMMARY

Sox2 is a transcription factor required for the maintenance of pluripotency. It also plays an essential rolein different types of multipotent stem cells, raising
the possibility that Sox2 governs the common
stemness phenotype. Here we show that Sox2 isa critical downstream target of fibroblast growth factor (FGF) signaling, which mediates self-renewal oftrophoblast stem cells (TSCs). Sustained expressionof Sox2 together with Esrrb or Tfap2c can replace
FGF dependency. By comparing genome-wide binding sites of Sox2 in embryonic stem cells (ESCs) and
TSCs combined with inducible knockout systems,
we found that, despite the common role in safeguarding the stem cell state, Sox2 regulates distinct
sets of genes with unique functions in these twodifferent yet developmentally related types ofstem cells. Our findings provide insights into thefunctional versatility of transcription factors during
embryogenesis, during which they can be recursively utilized in a variable manner within discrete
network structures.

INTRODUCTION
The transcriptional output of a given cell type is controlled byunique combinations of transcription factors under the control

of extrinsic signals that can modulate the expression and activityof transcription factors, forming a gene regulatory network that
dictates a specific cellular phenotype. Tissue-specific transcription factors play deterministic roles in cell-type specification,
which is manifested as lineage reprogramming by forced expression of such transcription factors (Graf and Enver, 2009; Zhou
and Melton, 2008). Sox2 is one such transcription factor required
for the maintenance of pluripotent stem cells in vivo (Avilion et al.,2003) and in vitro (Masui et al., 2007) and for the induction ofpluripotency (Takahashi and Yamanaka, 2006). However, it isalso preferentially expressed in neural, retinal, and trophoblaststem cells (TSCs) (Avilion et al., 2003; Pevny and Nicolis,2010), suggesting a possible role for Sox2 in governing a common stemness phenotype.
In embryonic stem cells (ESCs), Sox2 forms a heterodimer
with Oct3/4 (also known as Pou5f1) on DNA with the OCT-SOX
composite motifs, and these factors cooperatively activate
pluripotency-related target genes such as Nanog, Fgf4, Utf1,
Lefty1, and Fbxo15, as well as their own expression (Nakatake
et al., 2006, and references therein). Oct3/4-knockout ESCs
are differentiated along the trophoblast lineage in a highly homogeneous manner (Niwa et al., 2000). In contrast, the loss of Sox2
causes differentiation of ESCs accompanied by upregulation ofmarkers for trophoblast and embryonic germ layers, although
artificial maintenance of Oct3/4 from the transgene can sustain
self-renewal and pluripotency of Sox2-null ESCs (Masui et al.,2007), suggesting that the unique function of Sox2 may be
to maintain Oct3/4 expression. These two core transcription
factors, along with Nanog, form an interconnected and hierarchical network downstream of the leukemia inhibitory factor (LIF)Stat3 and LIF-phosphatidylinositol 3-kinase (PI3K) signaling

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pathways, which are connected together via signal-responsive
transcription factors, such as Klf4 and Tbx3 (Niwa et al., 2009).
However, the expression of core transcription factors, including
Sox2, is also maintained by shielding from ERK signaling evenin the absence of LIF, so long as the GSK3 activity is simultaneously blocked (Ying et al., 2008), indicating that the ERK
signaling, mainly activated by autocrine fibroblast growth factor(FGF) 4, negatively regulates the transcriptional network forpluripotency in ESCs.
The role of Sox2 as a crucial safeguard of stem cell state in
ESCs could be phenotypically conserved in other types ofstem cells. In neural stem cells and retinal progenitor cells,
Sox2 can cooperate with other tissue-specific homeodomaintranscription factors, such as Brn2 (also known as Pou3f2) andPax6, respectively, to activate specific target genes (Inoueet al., 2007; Tanaka et al., 2004) and plays an essential role instem cell maintenance (Favaro et al., 2009; Taranova et al.,2006). In TSCs, Sox2 is expressed predominantly in undifferentiated cells (Avilion et al., 2003). Although a previous study on
Sox2-knockout mice revealed its critical role in the trophoblast
lineage in vivo (Avilion et al., 2003), the mode of Sox2 function,
as well as its possible partners, is unknown. TSCs are derived
from embryos at a similar developmental stage as ESCs but
originate from a distinct lineage and show completely different
characteristics from ESCs. Self-renewal of TSCs is absolutely
dependent on FGF-ERK signaling that supposedly activatesthe expression of TSC-specific transcription factors such as
Cdx2 and Eomes (Kunath et al., 2004; Tanaka et al., 1998), whichis in contrast to the detrimental effect of ERK activation in
ESCs. Therefore, it is intriguing to examine the extent to which
Sox2 functions are conserved within discrete transcriptional
networks in these well-characterized and developmentally
similar stem cells.
Here, we performed a comprehensive analysis of Sox2
functions in ESCs and TSCs. First, we analyzed functions oftranscription factors that are activated downstream of FGF
signaling in TSCs and revealed the pivotal role of Sox2 in
FGF-dependent self-renewal of TSCs. Then we compared thetarget genes regulated by Sox2 in ESCs and TSCs usingtranscriptome and chromatin immunoprecipitation analyses
and found that Sox2 regulates quite different sets of genes
in ESCs and TSCs. Finally, we identified Tfap2c as one of
the TSC-specific Sox2 partners partly responsible for the
recruitment of Sox2 to TSC-specific target genes. These data
indicated that the function of Sox2 is highly flexible in thecontext of transcriptional networks controlled by different
extrinsic signals.
RESULTSFGF-ERK Signaling Converges on a Subset
of Transcription Factors in TSCs
We have previously shown that trophoblast stem (TS)-like cells
can be induced by forced repression of Oct3/4 in ESCs, which
results in gradual but homogenous downregulation of ESC
markers, like Nanog, and concomitant upregulation of TSC
markers, like Cdx2, when exogenous FGF4 is supplied (Figure 1A
and see Figure S1A available online; Niwa et al., 2000, 2005).

After subcloning, these TS-like cells (hereafter called ZHBTc4TSCs) were stably maintained as TSCs with a global expression
profile similar to that of embryo-derived EGFP-TS3.5 TSCs
(eTSCs) (Tanaka et al., 1998; Figure 3A). This in vitro systemallowed us to compare transcriptional networks in two distinct
types of stem cells with identical genotypes. We first investigated the effect of FGF4 on the expression of key transcription
factors in TSCs, which are less well understood than the ESC
transcription factors regulated by LIF. Increased expression
was found for transcription factors previously implicated in
TSC maintenance, including Cdx2 (Chawengsaksophak et al.,
1997; Strumpf et al., 2005), Eomes (Russ et al., 2000; Strumpf
et al., 2005), Elf5 (Donnison et al., 2005), Esrrb (Luo et al.,1997), and Sox2 (Avilion et al., 2003) (Figure 1B). In contrast,
the expression of differentiated trophoblast markers, such as
Ascl2 (also known as Mash2) and Cdkn1c (also known as
p57Kip2), was repressed by FGF4 (Figure S1B). Other possible
regulators of trophoblast development, such as Ets2, Tfap2c,
and Tead4, did not appear to be activated by FGF4 (Figure S1B).
The FGF responsiveness was confirmed by the rapid downregulation in ZHBTc4-TSCs and eTSCs after withdrawal of FGF4 ortreatment with the FGF receptor inhibitor PD173074 (Figures
1C and S1C). Conversely, when TSCs were stimulated with
FGF4 after starvation, the transcription factors were upregulated
in 12 hr (Figure 1D). This effect was abrogated in the presence ofthe MEK inhibitor PD0325901, but not by the PI3K inhibitor
LY294002 (Figure 1D), indicating that it was primarily mediatedby the FGF-ERK signaling pathway. Consistent with this, thesetranscription factors showed largely overlapping expression
with phosphorylated ERK in the extraembryonic ectodermof postimplantation embryos (E5.5), although the expressionof Esrrb and Sox2 was even more highly restricted, and that ofEomes and Elf5 was extended (Figure 1E). It should be notedthat the response of Esrrb and Sox2 to FGF is TSC specific, as
the expression of these genes in ESCs did not depend on FGF
signaling (Figure 1F). Instead, their expression was maintainedby either activation of LIF signaling or intrinsic transcriptional
circuitry without the counteracting FGF signal (Figure 1F; Niwa
et al., 2009; Ying et al., 2008). Indeed, around the time of
implantation (E3.5–E4.75), they are expressed exclusively inthe epiblast without detectable phosphorylated ERK (Figure 1G;
data not shown). These results also suggest that TSCs, with
their FGF/ERK dependency, correspond to a population oftrophoblasts in postimplantation, but not in preimplantation,embryos.
Sox2 Is a Critical Downstream Target of FGF Signalingthat Mediates Self-Renewal of TSCs
To evaluate the functional roles of these FGF-responsive
transcription factors in self-renewal downstream of FGF-ERK
signaling, we used a gain-of-function approach using eTSCs to
test whether the requirement for FGF4 can be replaced by artificial maintenance of these transcription factors from transgene
expression. Although sustained expression of a single factor
alone did not confer independence from FGF4, combined
expression of Sox2 and Esrrb enabled prolonged self-renewal
in the absence of FGF4 (Figures S2A and S2B). These results
were confirmed using ZHBTc4-derived TS-like cells, although

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Figure 1. Integration of FGF-ERK Signaling into the Transcription Factor Network in TSCs
(A) Trophoblast differentiation of ZHBTc4 ESCs after treatment with Tet to repress Oct3/4 expression. The cells formed TSC-like colonies on MEF feeders only inthe presence of FGF4. Scale bar, 200 mm.
(B) Transcription factors positively regulated by FGF4 during the trophoblast differentiation. Expression levels relative to ESCs cultured without MEFs are shownin logarithmic scale.
(C) Downregulation of the TSC transcription factors by the inhibition of FGF signaling. ZHBTc4-TSCs were deprived of FGF4 or treated with the FGF receptor
inhibitor PD173074 (PD17) for 24 hr before harvest. Spry4, a direct target of the FGF signaling, served as a control.
(D) Induction of the TSC transcription factors by the FGF-ERK signaling pathway. ZHBTc4-TSCs were starved in 1% FBS for 20 hr and restimulated with FGF4 for
12 hr. The FGF receptor inhibitor PD173074 (PD17), the MEK inhibitor PD0325901 (PD03), or the PI3K inhibitor LY294002 (LY) was added 30 min prior to restimulation. Expression levels relative to unstarved control are shown.
(E) Expression of FGF-responsive transcription factors and phosphorylated ERK in postimplantation embryos. Embryos were immunostained for TSC transcription factors or dually phosphorylated ERK (dpERK) and analyzed with a confocal laser scanning microscope. Two different antibodies against Sox2 (rabbit
and goat) yielded similar results. Nonspecific binding of anti-mouse IgG to endogenous maternal immunoglobulins concentrated in the cytoplasm of vacuolated
visceral endoderm was observed (asterisks). Note that, besides their expression in the trophoblast lineage (brackets), Eomes and Sox2 were also detected in thevisceral endoderm and epiblast, respectively (arrowheads). Scale bars, 50 mm.
(F) Response to FGF or LIF in ESCs. EB5 ESCs were starved in 1% FBS for 20 hr and restimulated with FGF4 or LIF for 12 hr. The FGF receptor inhibitor PD173074(PD17) was added 30 min prior to restimulation. Note that autocrine FGFs are enough to mask the effect of exogenously added FGF4 on, for instance, Spry4expression. Socs3, a direct target of the LIF signaling, served as a control.
(G) Expression of FGF-responsive transcription factors and phosphorylated ERK in preimplantation embryos. Note that Esrrb and Sox2 were not detected in thepolar trophectoderm (arrowheads). The expression of Elf5, Esrrb, Sox2, and dpERK in the trophoblast lineage was observed only after E4.0, E4.75, E5.25, andE5.0, respectively (data not shown). Scale bars, 50 mm.
(B–D and F) Error bars indicate standard deviation (SD) of three replicates.
See also Figure S1.

Sox2 alone slightly promoted self-renewal (Figures S2C and
S2D). We next assessed the multipotency of the transgenic
TSCs by chimera formation assay. The stable transfection ofan expression vector for Sox2 and Esrrb, linked by a selfcleaving 2A peptide sequence and flanked by loxP sites, enabled

the self-renewal of eTSCs at a clonal density in the absence of
FGF4 (Figure 2A). These cells maintained Cdx2 and Eomes
expression even in the presence of PD173074, confirming their
independence from FGF signaling (Figure 2B). After the culture
without FGF4 for 4 weeks, the cells were transiently transfected

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with Cre recombinase to excise the floxed transgene in thepresence of FGF4, followed by negative selection against thymidine kinase with gancyclovir. The resulting transgene-deleted
clones restored FGF dependency and readily differentiated after
withdrawal of FGF4 (Figures 2C and S2E). When the reverted
TSCs were injected into blastocysts, they contributed to all of
the placental cell lineages in chimeras at the same efficiency
as the parental line maintained in the presence of FGF4 (Figures
2D, 2E, and S2F), demonstrating that the expression of Sox2and Esrrb is minimally sufficient to maintain the multipotencyof TSCs in the absence of FGF4.
Induced Knockout of Sox2 in ESCs and TSCs
Causes Differentiation along Their Respective
Developmental Pathways
The results described above indicate that Sox2 plays a central
role in regulating self-renewal of TSCs downstream of FGF
signaling. The function of Sox2 in TSCs stands in contrast tothat in ESCs, in which Sox2 acts as a core transcription factor
downstream of LIF signaling to govern pluripotency (Niwaet al., 2009). In ESCs, Sox2 prevents trophoblast differentiation
mainly by maintaining Oct3/4 expression or repressing Eomes
expression and restricts the specification of the primary germlayers as well (Ivanova et al., 2006; Masui et al., 2007).
To compare the functional role of Sox2 in ESCs and TSCs in
more detail, we performed whole-genome expression analysis
after induced knockout of Sox2 in each cell type. For this, we firstestablished a stable TSC line by transient induction of Cdx2
(Niwa et al., 2005) in the Tet-inducible Sox2-knockout ESC
line, 2TS22C (Masui et al., 2007). Global gene expression profilesof ZHBTc4- and 2TS22C-derived TSCs, together with embryoderived EGFP-TS3.5, revealed that these TSCs are clustered
together and separated from the ESCs along the primary principal component (PC1) axis (Figure 3A). Upon knockout of
Sox2 in TSCs, expression of TSC markers, especially Esrrb,
gradually decreased even in the presence of FGF4, concomitantwith upregulation of differentiated trophoblast markers (Figures
3B and S3A). These TSCs gradually lost their self-renewal
capacity, manifesting flat morphological changes (Figure 3C).
Global expression analysis identified 3,440 and 1,066 genesthat were affected by Sox2 knockout in ESCs and TSCs,
respectively (Figures 3D, 3E, and S3B–S3F; Table S1). The
smaller number of affected genes in TSCs, especially upregulated genes, could represent the restricted developmental
capacity of TSCs compared to ESCs. Only a few genes werecommonly downregulated in ESCs and TSCs, while larger
numbers of genes, especially genes involved in placental development, were upregulated in both cell types; this makes sense,
considering Sox2’s role in preventing trophoblast commitment
in ESCs (Masui et al., 2007). Downregulation of pluripotency
markers and concomitant upregulation of mesodermal andendodermal markers, but not neuroectodermal markers, wasobserved exclusively in ESCs (Figures 3F and 3G). In contrast,
genes involved in cell-cycle regulation, especially in mitotic
checkpoint control, were downregulated only in TSCs (Figure 3G); this likely reflects the endoreduplication cycle, characteristic of differentiating trophoblast giant cells (Ullah et al.,2008). Gene ontology (GO) analysis using DAVID (Huang da

et al., 2009) showed that GO terms related to placental function,
like blood vessel development and negative regulation of immune system processes, are overrepresented in genes upregulated in TSCs and, to a lesser extent, in genes upregulated
in ESCs (Figures 3G, S3G, and S3H; Table S2). Gene set
enrichment analysis (GSEA; Subramanian et al., 2005) using
published microarray data (Ralston et al., 2010; Schulz et al.,2009) further revealed that genes affected by Sox2 knockout
are preferentially enriched in genes whose expression is similarly
regulated during normal differentiation of the respective cell
types (Figures 3H, 3I, and S3I). These results indicate thatSox2 regulates unique sets of genes to maintain stemness
and prevent precocious differentiation along the possible developmental pathways in each stem cell type.
Sox2 Occupies Distinct Sets of Genes Implicatedin Later Differentiation in ESCs and TSCs
To better understand the mechanisms underlying the versatility in transcriptional regulation by Sox2, we next analyzed
genome-wide binding sites of Sox2 during the differentiation of
ZHBTc4 ESCs into TS-like cells by chromatin immunoprecipitation, followed by high-throughput sequencing (ChIP-seq) (Figures 4A–4C and S4A–S4F; Table S3). During the differentiation,
Sox2 binding sites changed dynamically. For instance, aconserved region 4 within a distal enhancer of Pou5f1 was preferentially occupied in ESCs (Figure 4A), while the Cdx2 locus was
specifically occupied in differentiating TSCs (Figure 4B). Persistent binding was less frequently observed, as exemplified at the
Col4a1 locus (Figure 4C). As a whole, the numbers of peaks(FDR < 0.05) that overlapped with those of other samples werequite limited (Figure S4G). K-means clustering of normalized
ChIP-seq signals around the merged peak regions over thecourse of differentiation revealed that the vast majority of Sox2
binding sites are cell-type specific (Figure 4D). About half of
the Sox2 binding sites are ESC specific (cluster 2 [c2] and c5),
while the other half are predominant in differentiating TSCs (c1,c3, and c4), which are further classified as either increased(c1), transiently increased (c3), or gained (c4) binding. The
Sox2 biding sites in any clusters are predominantly found at transcription start site (TSS)-distal intronic or intergenic regions(Figure 4E). To gain insights into the underlying mechanisms
for such a context-dependent Sox2 binding, we performed denovo motif analysis using MEME (Bailey et al., 2009) and foundthat different noncanonical motifs, besides the canonical SOX
motif, were enriched in the peak centers of each cluster (Figure 4F). We then mapped these motifs to the peak regions(Figure 4G). As expected, overrepresentation of the OCT-SOX
composite motif was observed only in ESC-specific clusters(c2 and c5), confirming the pivotal role of Oct3/4 as a recruiterof Sox2 to the ESC-specific target sites. Interestingly, the AP-2
binding motif was overrepresented only in the TSC-predominant
clusters (c1, c3, and c4) and found together with the canonical
SOX motif within a subset of peaks (Figure 4H), suggesting cobinding of Sox2 and AP-2 transcription factors. It also should
be noted that although the canonical SOX motif was enrichedin all clusters, it was more overrepresented in c1 and c3, where
Sox2 binding is relatively persistent. Although the ESRRB motif
was also overrepresented in Sox2 peak clusters, we could not

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find a significant difference of the motif enrichment in each peakcluster (data not shown). We also mapped these motifs to thepeak summits of original samples, which were ranked by thelog10 (q-value) for peak call (Figure 4I). The more confident
peaks in ESCs were highly enriched for the OCT-SOX motif
compared to the less confident peaks. In contrast, the more
confident peaks in differentiating TSCs were enriched for thecanonical SOX motif, suggesting a stronger dependence of
Sox2 binding on its own recognition motif. These results suggest that sequence features could, in part, explain the dynamic
behavior of Sox2 binding.
To explore the functional significance of differential Sox2
binding, GO enrichment analysis was performed using GREAT(McLean et al., 2010), considering the predominance of TSSdistal binding sites (Figure 4J; Table S4). Genes involvedin stem cell maintenance were enriched for the peaks of allclusters, but especially c5, while genes for early placental
development were enriched for c3, c4, and c5. Enrichmentof genes associated with placental functions, such as arterydevelopment, regulation of lymphocyte differentiation, andregulation of T cell activation, are observed in c1, c2, or c3.
These results suggest that contextual Sox2 binding is related
to specific developmental processes.
Next we investigated the functional consequence of Sox2
binding by comparing the ChIP-seq data with the microarraydata. The peaks of each cluster were assigned to RefSeq genes
whose TSSs are located within a 10 kb distance (Table S5).
Enrichment of these genes in genes affected by Sox2 knockoutin ESC or TSC was analyzed by GSEA (Figures 4K, 4L, and 4N).
We found that genes associated with the ESC-specific clusters(c2 and c5) are significantly enriched in genes downregulated
in Sox2-knockout ESCs, while genes near the TSC-predominant
clusters (c1, c3, and c4) are enriched in genes downregulated
in Sox2-knockout TSCs. GSEA using the microarray data of
ZHBTc4 ESCs differentiating into TSCs showed that genes
associated with the ESC-specific clusters (c2 and c5) and withthe cluster showing gains of Sox2 binding in TSCs (c4) are significantly enriched in genes downregulated and upregulated
during the trophoblast differentiation, respectively (Figures 4M
and 4N). It should be noted that when more distal bindingsites were assigned to genes, such a positive effect of Sox2
binding on expression of nearby genes was less evident (datanot shown). These results suggest that Sox2 primarily contributes to activation of neighboring genes in both ESCs andTSCs. Nevertheless, this does not exclude a role of distal Sox2
binding in transcriptional regulation and a role of Sox2 binding
in priming gene expression without significant consequences(see Discussion). Taken together, self-renewal of ESCs andTSCs is regulated by a context-dependent role of Sox2.
We also performed ChIP-seq analysis of Esrrb using thesame chromatin samples (Figures S4A–S4F). In stark contrast
to Sox2, Esrrb bindings were less dynamically regulated during
the trophoblast differentiation (Figure S4G). K-means clusteringof normalized ChIP-seq signals around the merged peakregions identified ESC-specific binding peaks (c5) as a majorcluster (Figure S4I). The remaining peaks were classified as
relatively persistent clusters, with a TSC-predominant cluster
not apparent. The Esrrb biding sites, especially of c2 and c3,
were frequently found around the TSS (Figure S4J), consistent
with the suggested role of Esrrb in collaboration with basal transcription machinery (van den Berg et al., 2010). The peaks of allclusters were enriched for the canonical ESRRB recognition
motif (a motif for the nuclear receptor superfamily), and strong
binding sites were associated with the occurrence of the ESRRB
motifs (Figures S4L and S4M). These results suggest a common
mechanism for Esrrb in regulating stemness in ESCs and TSCs.
Although a minor fraction of Sox2 peak regions was also boundby Esrrb, we found only a slight enrichment of genes related to
specific biological processes near these regions (Figures S4N–
S4P), suggesting that Sox2 and Esrrb might play complementaryroles via different sets of target genes.
Tfap2c Is a Cooperative Factor of Sox2 in TSCs
The motif analysis described above suggests a possible role ofAP-2 transcription factors in cooperating with Sox2 in TSCs.
Among the five members of the AP-2 family of transcription
factors, Tfap2c (also known as Tcfap2c or AP-2g) is most abundantly expressed in TSCs (Figure 5A), and its expression isrestricted to the trophoblast lineages in vitro and in vivo (Figures
5B, 5C, and S1B). The forced expression of Tfap2c rapidly
suppressed Oct3/4 expression and induced trophoblast differentiation in ESCs (Figures S5A and S5B), confirming previous
reports (Berg et al., 2011; Kuckenberg et al., 2010). When Tfap2c
expression was induced in ESCs, the enhanced recruitment of

Figure 2. FGF-Independent Self-Renewal of TSCs by Sustained Expression of Sox2 and Esrrb(A) Scheme for assessment of developmental capacity of FGF-independent TSCs with sustained expression of Sox2 and Esrrb. A piggyBac vector carrying afloxed transgene cassette for bicistronic expression of Sox2 and Esrrb was transfected into the eTSC line EGFP-TS3.5 (Tanaka et al., 1998). PB, terminal repeatsof the piggyBac transposon; 2A, a 2A peptide from porcine teschovirus-1; pacDtk, a fusion gene composed of a puromycin N-acetyltransferase and a truncatedversion of HSV-1 thymidine kinase; GANC, gancyclovir. The letters in brackets refer to the panels in this figure corresponding to each step.
(B) Immunostaining of the Sox2/Esrrb transgenic TSCs (clone 628-1) grown in the absence of FGF4 with or without PD173074. Without FG4, transgenic cells
were positive for the TSC markers Cdx2 and Eomes, although some spontaneously differentiated cells (asterisks) were negative or only weakly positive. Note thatthe parental EGFP-TS3.5 line constitutively expresses EGFP, but the fluorescence intensity was low and almost negligible compared to the immunofluorescencesignals. Scale bar, 200 mm.
(C) Restoration of FGF dependency in reverted TSCs. The self-renewal of TSCs after Cre-mediated excision of the transgenes (clone 628-1c2) became FGF
dependent. Note that the reverted clone showed higher EGFP expression than the parental line. Scale bar, 200 mm.
(D) E13.5 chimeric embryos and placentas obtained by blastocyst injection of the parental (EGFP-TS3.5, upper) or reverted (clone 628-1c2, lower) TSCs
maintained in the presence of FGF4. Scale bars, 4 mm.
(E) Transverse section of placentas from the chimeras (clone 628-1c2). The differentiated progeny of the injected TSCs were observed in the spongiotrophoblast(sp) and labyrinth (la) layers, as well as in the interstitial spaces of the maternal decidua (de). Scale bar, 1 mm.
See also Figure S2.

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Figure 3. Microarray Analysis of Genes Affected by Sox2 Knockout in ESCs and TSCs
(A) Principal component analysis (PCA) of global gene expression profiles. The first two PCs explain 37% and 24%, respectively, of the total variance.
(B) Changes in gene expression after Tet-induced Sox2 knockout in TSCs. Error bars indicate SD of three replicates.
(C) Photomicrographs of Tet-inducible Sox2-knockout ESC line 2TS22C and its derivative TSC line cultured in the absence or presence of Tet for 4 days.
Scale bar, 200 mm.
(D) MA plots showing the log2 ratio and average log2 intensity of control and Sox2-knockout ESCs or TSCs. Whole-genome expression analysis was performed
4 days after Sox2 knockout. Blue lines indicate log2 ratio cutoff of 0.25. Blue, red, or green dots indicate selected genes affected only in ESCs, in TSCs, or in both,respectively.
(E) The number of genes affected by Sox2 knockout in ESCs and TSCs. Cutoffs for q-value and log2 ratio were set to 0.1 and 0.25, respectively. The numbersof genes upregulated or downregulated are shown in red or blue, respectively. The remaining genes in the intersection were affected in an opposite mannerin ESCs and TSCs. Selected genes downregulated or upregulated in common are shown. Genes in bold were also considered significant when more stringent
cutoff values (q-value < 0.05) were used (Figure S3F).
(F) Changes in expression levels of lineage markers. Note that some markers for trophoblast lineage (asterisks) are also expressed in embryonic tissues.
(G) GO analysis of genes affected by Sox2 knockout using DAVID. The log10 (p value) for selected GO terms in the Biological Process category(GOTERM_BP_FAT) is shown in a heatmap.
(H and I) GSEA of changes in expression of genes affected by Sox2 knockout during the differentiation of ESCs (H) and TSCs (I). Genes are ordered according totheir expression levels in differentiating ESCs (embryoid bodies) relative to undifferentiated ESCs (H) or levels in differentiating TSCs relative to undifferentiatedTSCs (I). Normalized enrichment scores (NES) and FDR are shown.
See also Figure S3.

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Figure 4. ChIP-Seq Analysis of Sox2 Occupancy during Differentiation of ESCs into TSCs
(A–C) Normalized Sox2 ChIP-seq signals at the Pou5f1 (A), Cdx2 (B), and Col4a1 (C) loci during the differentiation of ZHBTc4 ESCs into TS-like cells. ChIP-seq
signals were normalized as levels per million mapped reads. The chromatin of Sox2 KO ESCs was used as a negative control. The Cdx2 region analyzed by
ChIP-PCR in Figure 5 is shown (an arrowhead).
(D) A heatmap of the ChIP-seq signals around the merged peak regions. ChIP-seq peaks of each sample (q-value < 0.05) were merged, and normalized ChIP-seq
signals over ± 1 kb regions around the merged peak centers were clustered with the k-means algorithm into five clusters.
(E) Annotation of ChIP-seq clusters with genomic features. The merged peak centers of each cluster were annotated with genomic features using CEAS
(Shin et al., 2009). Annotation of the whole genome is shown as a control.
(legend continued on next page)

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Sox2 was observed at the Tfap2c binding sites, including theCdx2 locus, as early as one day after induction, even with sustained expression of Oct3/4 by the transgene (Figures 5D and
S5C). Conversely, knockdown of Tfap2c significantly decreased
TSC-specific recruitment of Sox2 (Figures 5E and S5E). We
confirmed that overexpression or knockdown of Tfap2c did notincrease or decrease Sox2 protein levels, respectively (Figure S5F), which excludes the possibility that the observedchanges in Sox2 binding are simply due to the modulation of
Sox2 protein levels. To elucidate the mechanistic basis of cooperative binding, we performed coimmunoprecipitation experiments of endogenous Sox2 and Tfap2c proteins in TSCs.
Tfap2c was coimmunoprecipitated with Sox2 (Figure 5F, left)
and, reciprocally, Sox2 was coimmunoprecipitated with Tfap2c(Figure 5F, right), indicating that these proteins physicallyinteract with each other. Importantly, overexpression of Tfap2c
together with Sox2 also enabled the self-renewal of TSCs
without FGF4 (Figures 5G and 5H), whereas knockdown ofTfap2c prevented the formation of undifferentiated TSC colonies(Figure S5D). Taken together, these results suggest that Tfap2c
is a crucial cooperative factor for Sox2 to maintain self-renewalof TSCs.
To gain more global insights into the cooperativity between
Sox2 and Tfap2c, we performed ChIP-seq analysis of thesefactors, along with Cdx2, in ZHBTc4-TSCs. We also analyzed
the Sox2 binding sites in eTSCs, confirming a large overlap
with those in ZHBTc4-TSCs (Figure S6I). As expected, a numberof previously identified TSC-predominant Sox2 binding sites,
including the Esrrb, Eomes, and Atrx/Magt1 loci, were co-occupied by Sox2 and Tfap2c in ZHBTc4-TSCs (Figures 6A–6C and
S6A–S6H). A significant number of Sox2 peaks overlappedwith Tfap2c peaks, while Cdx2 peaks that overlapped with
Sox2 or Tfap2c peaks were limited, irrespective of the relatively
large numbers of peaks (Figure 6D). Interestingly, Tfap2c peaks
were significantly enriched at proximal promoters of known
genes, while Cdx2 binding sites were found exclusively at
TSS-distal regions (Figure 6E). De novo motif discovery identified
the canonical recognition motifs for each factor as primary
motifs (Figure 6F). The motifs for AP-1 and AP-2, and TEAD
and AP-1 transcription factors were also enriched in Sox2 andTfap2c peaks, respectively. Motif mapping into the ranked peaks
suggests that Tfap2c and Cdx2 bindings primarily depend on
their recognition sequences, while the presence of canonical

SOX motif is less predictive for Sox2 bindings in vivo, further
emphasizing the contextual binding of Sox2 (Figure 6G). Finally,
we found TSC-predominant Sox2 peak clusters (c1, c3, and c4),
but not ESC-specific clusters (c2 and c5), shown in Figure 4D
were significantly overlapped with the Sox2 and Tfap2c peaksin TSCs (Figure 6H). Taken together, these results suggest a
global cooperativity of Sox2 and Tfap2c in regulating transcriptional network unique to TSCs.
DISCUSSION
Tissue-specific transcription factors are primarily responsible fordirecting transcriptional programs that confer unique cellular
phenotypes. However, it is frequently found that the same transcription factors are recurrently expressed in different cell types
at different stages during development. Sox2 is one such factor
whose expression is dynamically regulated: inner cell mass at
blastocyst stage; primitive ectoderm and extraembryonic ectoderm at egg-cylinder stage; neuroectoderm around gastrulation;and central nervous system, sensory placodes, and foregut
endoderm thereafter (Collignon et al., 1996; Wood and Episkopou, 1999). In addition, Sox2 is also preferentially expressed inputative stem cell populations of various adult tissues (Arnold
et al., 2011). Studies of germline or conditional knockout mice
revealed that Sox2 is definitely required for the development
and maintenance of these tissues (Arnold et al., 2011; Avilion
et al., 2003; Favaro et al., 2009; Que et al., 2009; Taranova
et al., 2006). Is such a pleiotropic role of Sox2 based on its common functions in different cell types or different modes of action
in different contexts? Here, we have shown the different modesof Sox2 action in ESCs and TSCs underpinning the recursive
deployment of Sox2 in different cell types during embryonicdevelopment.
First we found that Sox2 is both necessary and sufficient, if
combined with Esrrb or Tfap2c, to mediate the effect of FGF
on self-renewal of TSCs. Although some transcription factors
are considered to be involved in this process, no factors havebeen identified that can replace FGF dependency. Although
Sox2-deficient mice die shortly after implantation due to defective epiblast development (Avilion et al., 2003), chimeric analysis
using Sox2-null blastocyst injected with wild-type ESCs suggests a critical role of Sox2 in the trophoblast lineage aroundE7.5, when Erk2 (also known as Mapk1)- or Frs2-deficient mice(F) Motifs found by MEME to be overrepresented in 600 randomly selected ± 100 bp regions around the merged peak centers of each cluster with E-value scores(from c1 to c5, top to bottom).
(G) Occurrence of the motifs in peaks of each cluster. The motifs were mapped to the ± 200 bp regions around the merged peak centers of each cluster, and thepercentages of peaks containing at least one sequence for a given motif are shown.
(H) Co-occurrence of the motifs in peak centers of each cluster (from c1 to c5, top to bottom).
(I) Occurrence of the motifs in the ranked Sox2 peaks during differentiation of ESCs into TSCs. The peak summits of each sample were ranked by the log10(q-value) for peak call. The cumulative percentages of peaks (summits ± 200 bp) containing at least one sequence for a given motif are plotted against ranks ofpeaks. The ESRRB motif is a motif discovered in the Esrrb ChIP-seq data (Figure S4K).
(J) GREAT ontology enrichments for the peaks of each cluster. The binomial raw p values for selected GO Biological Process and Mouse Phenotype are shown ina heatmap.
(K–N) GSEA of changes in expression of genes associated with the peaks of each cluster after Sox2 knockout in ESCs (K), after Sox2 knockout in TSCs (L), andduring the trophoblast differentiation of ESCs induced by Oct3/4 knockout (M). The ChIP-seq peaks of each cluster were assigned to RefSeq genes whose TSSs
are located within 10 kb of the peak centers. The NES for gene sets with a FDR < 0.01 is shown in a heatmap (N). The core enrichment genes that contributed mostto the enrichment results are listed in Table S5.
See also Figure S4.

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Distinct Sox2 Networks in ESCs and TSCs

A

B

F

C

G

D

E

H

Figure 5. Tfap2c-Dependent Recruitment of Sox2 in TSCs
(A) Expression of AP-2 family members during the differentiation of ZHBTc4 ESCs into TS-like cells. Log2-transformed expression values from the Affymetrix
Exon Array data are shown. Note that AP-2 transcription factors, except Tfap2c, are expressed at negligible levels.
(B) Immunostaining for Tfap2c in ZHBTc4 ESCs and TSCs. Scale bar, 100 mm.
(C) Immunostaining for Tfap2c in postimplantation embryos (E5.5). Scale bar, 50 mm.
(D) Recruitment of Sox2 after induced expression of Tfap2c or knockout of Oct3/4. Oct3/4 expression was regulated by the Tet-off transgene, and expression ofTfap2c was induced using an ecdysone receptor (EcR)-based system in the same cell line. Relative occupancies to control regions (Ctrl-1 and Ctrl-2) are shown.
(E) Abrogation of Sox2 recruitment by knockdown of Tfap2c. ESCs stably expressing miR-155-based shRNA against Tfap2c were induced to differentiate into
TS-like cells for 2 days by Tet-induced repression of Oct3/4.
(F) Coimmunoprecipitation of Tfap2c with Sox2 in eTSCs. The cell lysates were prepared from EGFP-TS3.5 treated with or without formaldehyde (FA) for 10 min
and immunoprecipitated with anti-Sox2 (left) or anti-Tfap2c (right) antibodies. The whole-cell lysate (input) and immunoprecipitates (IP) were analyzed byimmunoblotting (IB).
(G) Immunostaining of the Sox2/Tfap2c transgenic TSCs cultured in the absence of FGF4 for more than five passages. Scale bar, 200 mm.
(H) Maintained expression of TSC markers in Sox2/Tfap2c-transfected TSCs grown in the absence of FGF4. The parental EGFP-TS3.5 line was cultured in thepresence or absence of FGF4 for 3 days.
(D–E and H) Error bars indicate SD of three replicates.
See also Figure S5.

also result in lethality (Gotoh et al., 2005; Hadari et al., 2001;
Saba-El-Leil et al., 2003). The function of Esrrb is also essential
but auxiliary, since, unlike Sox2, Esrrb alone did not affect selfrenewal of TSCs in the absence of FGF4 (Figure S2C). It should
be noted that, as exemplified by the FGF-ERK dependency,
extraembryonic ectoderm in postimplantation embryos isphenotypically and molecularly distinct from the trophectoderm
in preimplantation embryos, in which Tead4 and Cdx2 play
central roles. Thus, Sox2 is deployed for FGF-dependent selfrenewal of TSCs after implantation (Figures 1E and 1G).
In two types of stem cells, Sox2 can connect different extrinsic
signals, LIF-STAT and FGF-ERK, to different sets of transcription
factors, enabling cell-type-specific responses to environmental
stimuli (Figure 6I). We found that one of the mechanisms forsuch context-dependent functions of Sox2, with intrinsic lowaffinity DNA-binding activity (Kamachi et al., 2000), is attributable
to the cell-type-specific transcription factors that assist in its

recruitment or binding to the target sites: Oct3/4 in ESCs andTfap2c in TSCs. Mechanistically, Sox2 physically interacts withTfap2c in TSCs, which may facilitate the efficient binding of
Sox2 to the targets co-occupied with Tfap2c. The cooperativerole of Sox2 and Tfap2c in vivo is supported by the evidence
that Tfap2c-knockout embryos die between E7.5 and E8.5 due
to a defect in trophoblast-derived tissues (Auman et al., 2002;
Werling and Schorle, 2002), as is the case in Sox2-null embryos
rescued by wild-type ESCs. Interestingly, such cooperative
action of Sox2 and Tfap2c in TSCs was found on Cdx2, whichis the most important regulator of trophoblast development. It
should be noted that although Sox2 activates Cdx2 in TSCs, it
is expressed in a highly reciprocal manner to Cdx2 from E3.0
to E5.5, in which Sox2 begins to be expressed in trophoblast
lineages as well (Figures 1E and 1G; data not shown). Althoughepigenetic constraints imposed on the chromatin might beinvolved in different accessibility of target genes in different

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Distinct Sox2 Networks in ESCs and TSCs

A

D

B

E

C

F

I

H
G

Figure 6. Global Cooperativity of Sox2 and Tfap2c in TSCs
(A–C) Normalized Sox2, Tfap2c, and Cdx2 ChIP-seq signals at the Esrrb (A), Eomes (B), and Atrx/Magt1 (C) loci in ZHBTc4-TSCs. The Esrrb region analyzed by
ChIP-PCR in Figure 5 is shown (an arrowhead).
(D) A Venn diagram showing the number of overlapping peaks in each data set. Peak regions were defined as ± 100 bp regions around peak summits.
(E) Annotation of ChIP-seq peaks with genomic features using CEAS.
(F) Motifs found by MEME to be overrepresented in the top 1,000 ranked peaks (summits ± 100 bp).
(G) Occurrence of the motifs in the ranked peaks. The cumulative percentage of peaks (summits ± 200 bp) containing at least one sequence for a given motif areplotted against ranks of peaks.
(H) A heatmap representing the fractions of overlapping peaks of Sox2 ChIP-seq clusters found in Figure 4D with Sox2, Tcfap2c, and Cdx2 peaks in TSCs. The
numbers of overlapping peaks are shown in the grid.
(I) Self-renewal networks regulated by transcription factors and extrinsic signals in ESCs (Niwa et al., 2009) and TSCs. Context-dependent role of Sox2 is in part
mediated by the lineage-specific and mutually antagonistic transcription factors Oct3/4 and Tfap2c.

cellular contexts, its contribution would be minimal, since theepigenetic state of ESCs is considered as a tabula rasa (Murakami et al., 2011; Niwa, 2007); this was confirmed by the
dynamic change of the Sox2 binding sites immediately after
elimination of Oct3/4.
However, even in a different mode of action with different
partners, Sox2 plays a conceptually common role in ESCs andTSCs: the maintenance of self-renewal. This role is achievedby the activation of genes involved in maintaining the undifferentiated states and, albeit less commonly, restricts the expressionof genes promoting differentiation. Furthermore, although alarge fraction of the Sox2-bound genes whose functions are
related to later developmental processes were not significantly
affected by Sox2 knockout, Sox2 may prime these genes for
subsequent activation (Wegner, 2011). A flexible mode ofregulation by Sox2 fits well with the idea that Sox2 is one ofthe putative stemness genes that is predominantly expressedand serves as a molecular hub to respond to diverse environmental cues for stem cell maintenance in different types ofstem cells. This is in sharp contrast to the case of Esrrb, asshown here, and Myc, which regulates a common set of genes
involved in cell proliferation and metabolism in wide variety ofstem or progenitor cells (Kim et al., 2010). The versatile functionsof Sox2 provide a clear example of how tissue-specific transcription factors have maximum flexibility to play divergent roles indifferent networks. Considering that the sequence specificities
of individual transcription factors are generally limited, and only
a few of the potential recognition sites are bound in vivo, thecontext-dependent redeployment of these transcription factors
to drive tissue-specific transcriptional programs, as shown
here, may be prevailing. Such mechanisms should contributeto maximizing the complexity and diversity of cellular functions
with a minimal set of transcription factors. Reorganization oftranscription factor networks might occur extensively during

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Distinct Sox2 Networks in ESCs and TSCs

embryogenesis, in which networks transit from one state toanother along the developmental trajectory. This strategy also
should be beneficial in evolution, since it allows establishmentof novel networks, such as those for placental formation, only
from existing transcription factors in the genome. This idea is
supported by the evidence that very few transcription factors
are expressed only in the sole cell type, and their combinatorialusage is quite flexible. We expect that the concept found in
this study will emphasize the significance of the entire transcription factor network for the understanding of refined strategies in
evolution and development.
EXPERIMENTAL PROCEDURES
Cell Culture and Cell Lines
ESCs were maintained in GMEM supplemented with 10% FBS, 1 3 nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and
1,000 U/ml LIF on gelatin-coated dishes. TSCs were induced and maintainedin GMEM/10% FBS supplemented with 50 ng/ml human FGF4, 2 mg/ml
heparin, 10 ng/ml human Activin A, and 2 mM L-glutamine on mitomycin C
treated mouse embryonic fibroblasts (MEFs). Tet-inducible Oct3/4-knockout
ESC line ZHBTc4 and its derivative (4S2IP14) carrying an IRES-pac cassetteknocked in downstream of the Sox2 coding region for selection of Sox2positive cells were maintained as described previously (Niwa et al., 2000,
2005). Tet-inducible Sox2-knockout ESC line 2TS22C and its TSC derivative(22CEROH2) carrying a 4-hydroxytamoxifen (4-OHT)-inducible Cdx2-ERTM
transgene and a hygromycin-thymidine kinase fusion gene targeted into thePou5f1 locus were maintained as described previously (Masui et al., 2007;
Niwa et al., 2005). ESC-derived TSC lines were established from ZHBTc4 in
TSC medium supplemented with Tet on MEF feeders followed by subcloning,or from 22CEROH2 treated with 4-OHT followed by culture without 4-OHT
and with GANC to eliminate undifferentiated ESCs. Inducers and inhibitors
were used at the following concentrations: Tet, 1 mg/ml; doxycycline (Dox),
1 mg/ml; 4-OHT, 200 ng/ml; GANC, 1 mM; PD173074, 100 nM; PD0325901,
1 mM; LY294002, 20 mM.
ChIP
ChIP assays were performed using the ChIP-IT Express Enzymatic Kit (Active
Motif) following the manufacturer’s instructions. Briefly, cells were crosslinked
with 1% formaldehyde for 10 min at room temperature, and chromatin was
enzymatically sheared into fragments with an average size of 300–500 bp.
The chromatin fragments were immunoprecipitated with the antibodies
against Sox2 (raised by us), Esrrb (H6705, Perseus Proteomics), Tfap2c(sc-8977, Santa Cruz Biotechnology), and Cdx2 (CDX2-88, BioGenex). Precipitated DNA was analyzed by real-time PCR. For each experiment, the percentage of input was determined and normalized to the value obtained at the 28S
gene and intergenic spacer of ribosomal DNA, which gave reliable amplification due to their multiple copies in a genome. All primers are listed in Table S6.

AUTHOR CONTRIBUTIONS
K.A. and H.N. designed the study; K.A. performed experiments and analyzed
the data; K.A., I.N., and H.R.U. performed bioinformatic analysis; H.O. and
T.W. performed blastocyst injection; S.O. performed coimmunoprecipitation
experiments; K.A., H.U., and M.K. performed ChIP-seq experiments; andK.A. and H.N. interpreted the data and wrote the paper.
ACKNOWLEDGMENTS
We thank A. Smith and G. Guo (University of Cambridge, Cambridge) for
piggyBac transposon vectors, S. Tanaka (University of Tokyo, Tokyo) andS. Kuraku (Genome Resource and Analysis Unit, RIKEN, Kobe) for the EGFPTS3.5 line, the RIKEN Omics Science Center (Yokohama) for high-throughput
sequencing analysis, K. Uno (Functional Genomics Unit, CDB, RIKEN, Kobe)
for microarray analysis, H. Schöler (Max Planck Institute for Molecular Biomedicine, Münster) for generous support, and Y. Nakai-Futatsugi and D. Sipp (CDB,
RIKEN, Kobe) for their critical reading of the manuscript. This work was supported by a RIKEN research grant and by the Cell Innovation Program from
the Ministry of Education, Culture, Sports, Science and Technology (MEXT)
to H.N. and H.R.U. It was also supported by the CREST Program, from theJapan Science and Technology Agency (JST) to H.N.
Received: March 1, 2013
Revised: July 8, 2013
Accepted: August 29, 2013
Published: October 10, 2013
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Molecular Cell, Volume 52

Supplemental Information
Context-Dependent Wiring of Sox2 Regulatory
Networks for Self-Renewalof Embryonic and Trophoblast Stem Cells
Kenjiro Adachi, Itoshi Nikaido, Hiroshi Ohta, Satoshi Ohtsuka, Hiroki Ura,
Mitsutaka Kadota, Teruhiko Wakayama, Hiroki R. Ueda, and Hitoshi Niwa

Figure S1
A

Cdx2 (rabbit)

Oct3/4

HoechstNanog

Cdx2 (mouse)

Hoechst

Eomes

Sox2 (goat)

Hoechst

Sox2 (rabbit)

Esrrb

Hoechst

Day 0

Day 1

Day 2

Day 3

Day 4

Day 0

Day 1

Day 2

Day 3

Day 4

Day 4
(differentiated)

Figure S1
Bi) ESC-associated genes
Nanog

Fgf4

Esrrb

-FGF4

Cdx2

Sox2

1

100

0.1

10

0.01
0.001
0

1

2

3

4

Foxd3

Klf2

1

10

0.1

1

0.01

0.1

0.001

0.01

0

1

2

3

4

Myc

0

Sall4

1

2

Klf4

3

Tbx3

Klf5

4

Bmp4

Spry4

10

+FGF4
Elf5

Fgfr2

Hand1

Tead4

Ets2

Gata2

Gata3

Tfap2cTpbpa

Cdkn1c

Pparg

1
0.1

0

1

2

3

4

Ascl2
100
10
1
0.1

0

1

2

3

4

Plf

Syncytin A

100

1000

10

100

1

10

0.01

0.1

1

0

1

2

3

4

0

1

2

3

4

1

0

1

2

3

4

Days after Oct3/4 KO

Expression levels
relative to +FGF4 0h

10

1

+FGF4
-FGF4 24h
+PD17 24h

0.1

0.01

Spry4 Cdx2 Eomes Elf5

Esrrb

Sox2 Tfap2c

Figure S1. Screening for FGF4-Responsive Transcription Factors Expressed in Trophoblasts, Related to
Figure 1(A) Immunostaining for ESC and TSC markers after treatment of ZHBTc4 with Tet to repress Oct3/4 expression.
The cells were cultured in the presence of FGF4 on MEF feeders. Oct3/4 expression from the Tet-off transgene
was rapidly repressed upon Tet treatment, while Nanog expression was gradually decreased. Note that expression
of

-FGF4

Eomes

0.1

Days after Oct3/4 KO

Cii) TSC-associated genes

Expression levels relative to ESC w/o MEF

Expression levels relative to ESC w/o MEF

Pou5f1 (CDS)+FGF4

Sox2 and Esrrb was maintained only in the undifferentiated TS-like cells, but not in the spontaneously

differentiated trophoblast-like cells (arrowheads). Eomes is expressed, albeit at a low level, in ZHBTc4 ESCs andupregulated during the differentiation into TS-like cells (see also Figure S1B). Scale bars, 200 µm.
(B) FGF4-responsiveness of ESC- and TSC-associated genes during the differentiation of ZHBTc4 ESCs into TSlike cells. Kinetics of gene expression was analyzed by real-time PCR during the trophoblast differentiation in thepresence or absence of FGF4. The expression of Sox2 and Esrrb was rapidly downregulated without exogenous
FGF4. The persistent activation of TSC markers, Cdx2, Eomes and Elf5, was also dependent on FGF4. In contrast,
markers of differentiated trophoblast such as Cdkn1c (trophoblast giant cells), Ascl2 (spongiotrophoblasts) andsyncytin A (syncytiotrophoblasts) were reduced by the addition of FGF4. Only genes positively regulated by FGF4
are shown in Figure 1B.
(C) Downregulation of the TSC transcription factors by the inhibition of FGF signaling in the eTSC line EGFP-TS3.5.
The cells were deprived of FGF4 or treated with the FGF receptor inhibitor PD173074 (PD17) for 24 h before
harvest.
(B and C) Error bars indicate standard deviation (SD) of three replicates.

Figure S2
ACD

Without FGF4

Transgene

Without FGF4

Cdx2

±

Cdx2-ERTM

－

Eomes

－

Eomes-ERTM

－

Elf5

－

Elf5-ERTM

－

Esrrb

－

Esrrb

－

Sox2

－

Sox2

±

Tfap2c－Sox2+Cdx2ERTM±

Cdx2+Esrrb±

Sox2+Esrrb＋

Cdx2+Sox2±

Esrrb+Sox2＋

Transgene

Cdx2

Hoechst

Sox2 (Tg)

Esrrb (Tg)

HoechstTfap2cElf5

Hoechst

Cdx2

Sox2

Esrrb

Tfap2c

Cdx2

Eomes

Sox2

Esrrb

Esrrb+Sox2

Cdx2+Sox2

Cdx2+Esrrb

Cdx2

B

Eomes

-FGF4

+FGF4

E

F
Cell line

Recovered/Transferred

Chimeric (% recovered)

Highly contributed (% recovered)

EGFP-TS3.5

22/50

6 (27.3)

3 (13.6)

628-1c1

28/50

7 (25.0)

4 (14.3)

628-1c2

32/53

6 (18.8)

6 (18.8)

Figure S2
Figure S2. FGF-Independent Self-Renewal of Transgenic TSCs, Related to Figure 2(A) Gain-of-function screen for transcription factors that support FGF-independent self-renewal of eTSCs. The
constitutive expression vectors were stably transfected into embryo-derived EGFP-TS3.5 line using piggyBac
transposon system. After selection, the cells were cultured in the absence of FGF4. The self-renewal capacity was
examined by serial passaging. Combined expression of Sox2 and Esrrb enabled the self-renewal of TSCs.
Although Cdx2 also promoted proliferation, the transgenic cells tended to lose undifferentiated morphology andexpression of other TSC transcription factors (see Figure S2B).
(B) Immunostaining of the transgenic TSCs cultured without FGF4 for more than five passages. Scale bar, 200 µm.
(C) Gain-of-function screen for transcription factors that support FGF-independent self-renewal of ZHBTc4-derived
TS-like cells. The constitutive or 4-OHT-inducible expression vectors for these factors were stably transfected into
ZHBTc4 ESCs by electroporation. The resulting clones were then induced to differentiate into trophoblast by theaddition of Tet (and 4-OHT) in the absence of FGF4. Note that Sox2 alone promoted short-term proliferation (lessthan two passages).
(D) Immunostaining of the ZHBTc4-derived transgenic TSCs grown in the absence of FGF4. Scale bar, 200 µm.
(E) Immunostaining of the transgene-deleted eTSCs (clone 628-1c2) cultured in the presence or absence of FGF4
for four days. Scale bar, 200 µm.
(F) Contribution of the transgene-deleted eTSCs to the chimeric placentas. The two transgene-excised TSC clones(628-1c1 and 628-1c2) and the parental EGFP-TS3.5 line were injected into host blastocysts. The embryos andplacentas were recovered at E13.5 and the contribution of TSCs was assessed by EGFP fluorescence.

Figure S3
A

Cdh2

Cdx2

Elf5

Eomes

Esrrb

Fli1

Foxh1

Grhl3

Hoxc9

Mycl1

Col4a1Col4a2

Dkk1

Fos

Fzd5

Gata2

Pparg

Ppargc1b

Vdr

Wnt4

Bmp4
10

1
0.1

1

0.01
0.001

0

1

2

3

0.1

4

0

Spry1

1

2

3

4

Spry4

10

10

1

1

0.1

0

1

2

3

0.1

4

0

1

2

3

4

-Tet

Days after Sox2 KO

Bmp4

Eomes

Esrrb

Fgf4

Klf4

Nanog

Pou5f1

Cdx2

Col4a1Col4a2

Dkk1

Elf5

q<0.1, log>0.5

Wnt4

Vdr

Spry4

Spry1

Pparg

Mycl1

Hoxc9

Grhl3

Gata2

Fzd5

Foxh1

E
10

Fos

Klf4 Nanog Pou5f1

Fli1

Fgf4

Esrrb

Sox2 Bmp4 Eomes Esrrb(CDS)

q<0.05, log>0.5

Eomes

Expression levels
relative to -Tet

q<0.05, log>0.25

C
Expression levels
relative to -Tet

+Tet

q<0.1, log>0.25

1

0.1

q<0.1, log>0.25

1

q<0.05, log>0.25
q<0.1, log>0.5
0.1

F

Cdh2

D
10

Bmp4

B

Ppargc1b

Expression levels relative to -Tet (Day 0)

Sox2 (CDS)
10

Sox2
(CDS)

Bmp4

Cdx2

Eomes

ESC

Esrrb

Hoxc9

q<0.05, log>0.5

Pparg

ESC

ESC

TSC
2998
1267
1731

442
92
222

UniProt_TISSUE

G

TSC
1992
734
1258

153
32
84

q<0.05, log>0.25

220
115
105

TSC
944
413
531

103
25
65

q<0.1, log>0.5

188
103
85

TSC
777
300
477

50
15
29

q<0.05, log>0.5

Brain
Embryonic stem cell
Macrophage
AmnionLiver
Mammary gland
Trophoblast stem cell
Cerebellum
Mammary tumor
Egg
Embryonic germ cell
Pituitary gland
Blastocyst
Embryonic eye
Hypothalamus
Mast cell
Brain cortex
Heart
Dendritic cell
Colon
Bone marrow
Neural Stem Cell
Skin
SpleenAdipose tissue
Osteoblast
Ovary and uterus
TongueInner ear
Ectoplacental cone
Cecum
Limb
ThymusEye
KidneyAdult pancreatic islet
Pituitary
Hippocampus
RetinaSpinal ganglion
Thyroid
EyeballLung
Medulla oblongata
Placenta
Placenta and extra embryonic tissueq<0.1, log>0.25

624
359
265

ESC

ESC down_q<0.1 log<0.25
ESC down_q<0.05 log<0.25
ESC down_q<0.1 log<0.5
ESC down_q<0.05 log<0.5
ESC up_q<0.1 log<0.25
ESC up_q<0.05 log<0.25
ESC up_q<0.1 log<0.5
ESC up_q<0.05 log<0.5
TSC down_q<0.1 log<0.25>3.0

TSC down_q<0.05 log<0.25

TSC down_q<0.05 log<0.5
TSC up_q<0.1 log<0.25
TSC up_q<0.05 log<0.25-log10(p-value)

TSC down_q<0.1 log<0.5
2.0

1.0

TSC up_q<0.1 log<0.5
TSC up_q<0.05 log<0.5

0.0

107
60
47

I

TSC dif. vs undif.
-log10(NES)TSC up_q<0.05 log<0.5

TSC up_q<0.1 log<0.5

TSC up_q<0.05 log<0.25

TSC up_q<0.1 log<0.25

TSC down_q<0.05 log<0.5

TSC down_q<0.1 log<0.5

TSC down_q<0.05 log<0.25

TSC down_q<0.1 log<0.25

ESC up_q<0.05 log<0.5

ESC up_q<0.1 log<0.5

ESC up_q<0.05 log<0.25

ESC up_q<0.1 log<0.25

0.0

ESC down_q<0.05 log<0.5

1.0

M phase of mitotic cell cycle
formation of primary germ layer
endoderm development
Wnt receptor signaling pathway
mesenchymal cell development
mesoderm development
forebrain development
kidney development
branching morphogenesis of a tube
neural tube development
skeletal system development
gland development
heart development
actin cytoskeleton organization
regulation of small GTPase mediatedcell motion
epithelium development
blood vessel development
placenta development
regulation of angiogenesis
cell adhesion
neuron differentiation
positive regulation of cell prolife
hindbrain development
lung development
extracellular matrix organization
activation of NF-kappaB-inducing ki
female pregnancy
adaptive immune response
cytokine-mediated signaling pathway
muscle organ development
genitalia development
regulation of BMP signaling pathway
epithelial to mesenchymal transitio
eye development
sensory organ development
salivary gland development
mammary gland development
gut development
ectoderm development
positive regulation of angiogenesis
transforming growth factor beta rec
negative regulation of immune syste
positive regulation of MAP kinase atrophectodermal cell differentiatio
cellular response to insulin stimul
steroid metabolic process
apoptosis
response to insulin stimulus
ear development
liver development
embryonic limb morphogenesis
response to DNA damage stimulus
DNA replication
chromosome segregation
positive regulation of cytokine pro
negative regulation of MAP kinase apositive regulation of apoptosis
regulation of cell-substrate adhesi
eye photoreceptor cell development
regulation of Notch signaling pathw
fibroblast growth factor receptor s
gamete generation
response to retinoic acidepithelial cell proliferation
stem cell maintenancestem cell differentiation>3.0

ESC down_q<0.1 log<0.5

2.0

GOTERM_BP_FAT

H

ESC down_q<0.05 log<0.25

ESC down_q<0.1 log<0.25-log10(p-value)

Figure S3

ESC down_q<0.05 log<0.25
ESC down_q<0.1 log<0.25

ESC down_q<0.05 log<0.5
ESC down_q<0.1 log<0.5

ESC up_q<0.05 log<0.25
ESC up_q<0.1 log<0.25

ESC up_q<0.05 log<0.5
ESC up_q<0.1 log<0.5

TSC down_q<0.05 log<0.25
TSC down_q<0.1 log<0.25

TSC down_q<0.05 log<0.5
TSC down_q<0.1 log<0.5

TSC up_q<0.05 log<0.25
TSC up_q<0.1 log<0.25

TSC up_q<0.05 log<0.5
TSC up_q<0.1 log<0.5

3.0

0.0 or FDR >0.01

ESC dif. vs undif.

-3.0

Figure S3. Microarray Analysis of Sox2 Knockout ESCs and TSCs, Related to Figure 3(A) Changes in gene expression after Tet-induced Sox2 knockout in TSCs.

(B and C) Changes in gene expression four days after Sox2 knockout in 2TS22C-ESCs (B) and -TSCs (C)analyzed by RT-PCR.

(D and E) Genes affected by Sox2 knockout in ESCs (D) and TSCs (E) identified by microarray with different q-

value and log2 ratio cutoffs (colored in yellow). Note that the sensitivity to identify genes found to be affected by

RT-PCR was maximized with the less stringent cutoff.

(F) The number of genes affected by Sox2 knockout in ESCs and TSCs. Use of the relaxed and stringent cutoffs

resulted in identification of similar proportions of affected genes in each group.

(G and H) Tissue Expression (G) and GO (H) enrichment analysis of genes affected by Sox2 knockout. The-log10(p-value) for UniProt report for expressed tissues (UniProt_TISSUE) and GO terms in the Biological Process

category (GOTERM_BP_FAT) are shown in a heatmap. Note that use of the less stringent cutoffs showed better

sensitivity with reasonable specificity.

(I) GSEA analysis of changes in expression of genes affected by Sox2 knockout during the differentiation of ESCs

that contributed most to the enrichment results are listed in the Table S2.

and TSCs. The -log10(NES) for gene sets with a FDR <0.01 are shown in a heatmap. The core enrichment genes(A-C) Error bars indicate standard deviation (SD) of three replicates.

Figure S4A

1 kb

Pou5f1

Tcf19

α-Sox2

Oct3/4 KO D0
Oct3/4 KO D1
Oct3/4 KO D2
Oct3/4 KO D3
Oct3/4 KO D4
Sox2 KO

α-Esrrb

CR4 CR2

Oct3/4 KO D0
Oct3/4 KO D1
Oct3/4 KO D2
Oct3/4 KO D3
Oct3/4 KO D4

3

3

3

3

1 kb

1 kb

3

668

921

518

403

358

213

Sox2 D2 465 760 1179 697

601

355

243

234

221

126

Sox2 D4

Sox2 D1 1063 2699 758 825

Sox2 D3

185

Sox2 D2

297

Sox2 D1

Esrrb D4

2

Esrrb D3

3

Esrrb D2

3

Esrrb D1

3

319

1 kb

Col4a2F6449 2045 439

Col4a1

5 kb

Esrrb

Nr0b1

Sox2 D0 5299 1063 464 563

Sox2 D0

E

SRR2

Sox2

Esrrb D0

α-Sox2α-Esrrb

G

C

3

SRR1

Oct3/4 KO D0
Oct3/4 KO D1
Oct3/4 KO D2
Oct3/4 KO D3
Oct3/4 KO D4

1 kb

Cdx2

10

D
Oct3/4 KO D0
Oct3/4 KO D1
Oct3/4 KO D2
Oct3/4 KO D3
Oct3/4 KO D4
Sox2 KOB

Sox2 D3 562 825 697 2076 912 494 287 328 355 194
223

270

299

157

Esrrb D0 2091 955 366 509

372 19826 2560 2227 2413 1584

Esrrb D1 440 522 244 287

222 2538 3915 1765 1616 1048

Esrrb D2 320 404 236 328

270 2195 1761 3703 1938 1247

Esrrb D3 300 360 223 359

302 2375 1615 1946 4463 1442

Esrrb D4 186 213 127 195

158 1559 1044 1245 1432 3538

0Fraction of overlapping peaks

Sox2 D4 448 669 600 912 1807 363

00.0-log10(p-value) >10

c1 c2 c3 c4 c5 MGI Expression-log10(p-value) >10

c1 c2 c3 c4 c5 Mouse Phenotype
abnormal visceral yolk sac morphology
abnormal trophoblast layer morphology
abnormal intestine physiology
abnormal forebrain development
abnormal mesoderm development
abnormal heart development
abnormal rostral-caudal axis patterningabnormal embryonic epiblast morphology
exencephaly
abnormal neural fold morphology
abnormal placenta labyrinth morphology
abnormal limbs/digits/tail development
abnormal artery development
abnormal hepatobiliary system development
abnormal ectoderm development
liver hypoplasia
decreased hematopoietic stem cell number
abnormal digestive system development
abnormal keratinocyte morphology
abnormal liver development
abnormal embryonic hematopoiesis
absent allantois
abnormal forelimb zeugopod morphology
anophthalmia
absent mature B cells
absent somites
abnormal anterior visceral endoderm morphology
abnormal neuroendocrine cell morphology
abnormal extraembryonic ectoderm morphology
abnormal extraembryonic endoderm formation
placental labyrinth hypoplasia
abnormal notochord morphology
abnormal blastocyst morphology
abnormal vasculogenesis
abnormal olfactory system morphology
abnormal telencephalon development
failure of chorioallantoic fusion
microcephaly>8c1 c2 c3 c4 c5 GO Biological Process
1.0

H
0

-log10(p-value)

TS4_inner cell mass
TS4_zona pellucida
TS5_inner cell mass
TS5_trophectoderm
TS12_embryo; ectoderm; neural ectoderm
TS19_limb; mesenchyme
TS28_oocyte
TS6_epiblast
TS13_primordial germ cells
TS15_hindgut
TS8_epiblast
TS14_central nervous system
TS15_urogenital system
TS9_extraembryonic component; endoderm
TS15_eye
TS15_inner ear
TS13_yolk sac
TS25_hair; root sheath
TS15_telencephalon
TS22_femur cartilage condensation
TS21_skin; dermis
TS28_neural retinal epithelium
TS8_extraembryonic component; ectoderm
TS11_ectoplacental cone
TS11_amnion
TS15_extraembryonic component
TS11_chorion
TS15_otocyst; epithelium
TS20_liver
TS26_spleen
TS23_trachea; epithelium
TS20_stomach; epithelium
TS28_glomerular capsule
TS23_oesophagus; epithelium
TS22_stomach; mesentery
TS15_forelimb bud
TS13_gut
TS22_optic nerve
TS22_respiratory tract
TS15_trunk mesenchyme; paraxial mesenchyme
TS22_gonad

embryonic placenta development
actin filament organization
gamete generationneural tube development
lung development
stem cell maintenancestem cell development
stem cell differentiation
mesoderm development
trophectodermal cell differentiation
blastocyst formation
dorsal/ventral pattern formation
epidermis development
anterior/posterior axis specification
regulation of glial cell differentiation
liver development
kidney development
skin development
regulation of cell-cell adhesion
hair follicle development
pancreas development
regulation of astrocyte differentiation
keratinocyte differentiation
regulation of reproductive process
JAK-STAT cascade
regulation of Notch signaling pathway
embryonic camera-type eye development
myeloid leukocyte differentiationembryonic skeletal system morphogenesis
cardiac cell differentiation
regulation of lymphocyte differentiation
thymus development
regulation of S phase of mitotic cell cycle
ectodermal placode formationartery development
TGF beta receptor signaling pathway
negative regulation of MAP kinase activity
Ras protein signal transduction
regulation of JUN kinase activity
erythrocyte homeostasis
regulation of T cell activation
response to retinoic acidnegative regulation of BMP signaling pathway
diencephalon development
hindgut morphogenesis
neural crest cell development
digestive tract development
regulation of skeletal muscle tissue development
inner ear development
auditory receptor cell fate commitment
regulation of epithelial cell differentiation
negative regulation of myoblast differentiation
mesenchyme development
hindbrain development
pituitary gland development
regulation of oligodendrocyte differentiation
canonical Wnt receptor signaling pathway
positive regulation of leukocyte migration
T-helper 1 type immune response
forebrain neuron differentiation
negative regulation of retinoic acid receptor signaling pathway
progesterone receptor signaling pathway
regulation of histone H3-K9 methylation
negative regulation of calcium ion-dependent exocytosis

Figure S4
I

K

J
D1

D2

D3

L

D4
851 2090

1
2

3280

3

% peaks with motifs

1

0

ESRRB / 1.3e-534

20

40

60

80 100

ESRRB

D0

2
ESRRB / 1.3e-041

1 2 3 4 5
3
4148

4

ESRRB / 3.6e-091

4
ESRRB / 6.3e-407

5
ESRRB / 6.3e-069

GenomeM% peaks with motifs

14420

5

D0

D1

D2

D3

D4

100

Promoter (<1 kb)
Promoter (1-2 kb)
Promoter (2-3 kb)
5‘ UTR
3’ UTR
Coding exon
IntronIntergenic

80

SOX
OCT-SOX
AP-2
ESRRB

60
40
20
0

1

19826 1

3915

1

3703

1

4463

1

3538

Ranked peaks-1 kb 0 +1 kb

N Sox2
D0

Sox2
D1

Sox2
D2

Sox2
D3

Sox2
D4

Esrrb
D0

Esrrb
D1

Esrrb
D2

Esrrb
D3

O Sox2 w/ Esrrb Sox2 w/o Esrrb

Esrrb
D4

Genome112Promoter (<1 kb)
Promoter (1-2 kb)
Promoter (2-3 kb)
5‘ UTR
3’ UTR
Coding exon
IntronIntergenic

2

3

3

4

5

P
Sox2 allSox2 w/ Esrrb

c1 c2 c3 c4 c5 c1 c2 c3 c4 c5Sox2 w/ Esrrb

0-log10(p-value)

Sox2 all>8

Sox2 w/ Esrrb

c1 c2 c3 c4 c5 c1 c2 c3 c4 c5

Mouse Phenotype
abnormal visceral yolk sac morphology
abnormal ectoderm development
abnormal forebrain development
abnormal embryonic epiblast morphology
abnormal mesoderm development
abnormal placenta labyrinth morphology
abnormal trophoblast layer morphology
failure of chorioallantoic fusion
abnormal blastocyst morphology
abnormal artery development
abnormal embryonic hematopoiesis
abnormal keratinocyte morphology

5
Sox2 w/o Esrrb
0-1 kb 0 +1 kb

GO Biological Process
stem cell maintenancestem cell differentiation
epidermis development
embryonic placenta development
actin filament organization
mesoderm development
regulation of S phase of mitotic cell cycle
artery development
regulation of lymphocyte differentiation
negative regulation of MAP kinase activity
regulation of T cell activation
hindbrain development4-log10(p-value) >10

Figure S4
Figure S4. ChIP-Seq Analysis of Sox2 and Esrrb Binding Sites in Differentiating TSCs, Related to Figure 4(A-F) Normalized Sox2 and Esrrb ChIP-seq signals at the Pou5f1 (A), Cdx2 (B), Col4a1 (C), Sox2 (D), Nr0b1 (E),and Esrrb (F) loci during the differentiation of ZHBTc4 ESCs into TS-like cells. ChIP-seq signals were normalized
as levels per million mapped reads.
(G) A heatmap representing the fraction of overlapping peaks in each data set. Peak calling was performed using
MACS2 with a q-value cutoff of 0.05. Peak regions were defined as ±100-bp regions around peak summits. The
fractions of peaks that overlap at least 1 bp with those of other samples are shown in a heatmap. The numbers ofoverlapping peaks are shown in the grid. The larger numbers of Sox2 and Esrrb peaks in ESCs may be in part due
to their higher expression levels (Figure 1B and Figure S1A).
(H) GREAT ontology enrichments for the peaks of each Sox2 cluster. Only selected GO terms are shown in Figure
4J.
(I) A heatmap of the Esrrb ChIP-seq signals around the merged peak regions. ChIP-seq peaks of each sample
were merged and normalized ChIP-seq signals over ±1-kb regions around the merged peak centers wereclustered with the k-means algorithm into 5 clusters.
(J) Annotation of ChIP-seq clusters with genomic features. The merged peak centers of each cluster wereannotated with genomic features using CEAS.
(K) Motifs found by MEME to be overrepresented in 600 randomly selected ±100-bp regions around the merged
peak centers of each cluster (from c1 to c5, top to bottom).
(L) Occurrence of the ESRRB motif in peaks of each cluster. The motif was mapped to the ±200-bp regions
around the merged peak centers of each cluster and the percentages of peaks containing at least one sequence
for the motif are shown.
(M) Occurrence of the motifs in the ranked Esrrb peaks during differentiation of ESCs into TSCs. The peak summitsof each sample were ranked by the -log10(q-value) for peak call. The cumulative percentage of peaks (summit±200 bp) containing at least one sequence for a given motif are plotted against ranks of peaks. The SOX, OCTSOX, and AP-2 motif are motifs discovered in the Sox2 ChIP-seq data (Figure 4F).
(N) A heatmap of the Sox2 and Esrrb ChIP-seq signals around the merged peak regions of Sox2. Each Sox2 peakcluster shown in Figure 4D was further split with the k-means algorithm into 3 subclusters only based on Esrrb
ChIP-seq signals. The first and second subclusters of each major cluster showing strong and moderate Esrrb
binding, respectively, were considered as co-occupied with Esrrb.
(O) Annotation of Sox2 subclusters with genomic features.
(P) GREAT ontology enrichments for the peaks of each Sox2 cluster co-occupied with Esrrb. For comparison, theenrichments for all the Sox2 peaks of each cluster (Figure 4J) are also shown.

Figure S5+Ec+Ec +FGF4/MEF

B

D

4S2IP14

shTfap2c_1

Tfap2c/Venus

Nanog

HoechstTfap2c/Venus

Oct3/4 (Tg)

HoechstTfap2c/Venus

Nanog

Hoechst+Ec D1+Ec D2
Cdx2

HoechstTfap2cHoechstshTfap2c_1

4S2IP14Venus (shRNA)F
Tfap2c/Venus

Untreated

Untreated

Tfap2c/Venus

shTfap2c_4

shTfap2c_2Untreated+Dox D1+Dox D2

Untreated+Ec D2+Dox D2+Ec D1+Dox D1

Untreated

Hoechst

Untreated

Oct3/4

shTfap2c_3

EC
Tfap2c/Venus

shTfap2c_2

shTfap2c_3

Untreated

shTfap2c
_4

A

Cdx2

-

HoechstEc Tet

-

Tet

-

Tet

-

Tet

-

Tet

Sox2

Tubulin+Ec D2+Dox D2+Ec D1+Dox D1

Tfap2cFigure S5. Trophoblast Differentiation Induced by Tfap2c in ESCs, Related to Figure 5(A) Morphological alterations of ZHBTc4:Sox2-IRES-pac (4S2IP14)-derived 14RheoTfap2c ESCs four days after
induction of Tfap2c. Expression of the transgene encoding Tfap2c (NM_009335) tagged with IRES-Venus was
induced by the addition of an ecdysteroid agonist (Ec) in the absence or presence of FGF4/MEFs. Scale bar, 200µm.
(B) Immunostaining for Oct3/4, Nanog and Cdx2 after induction of Tfap2c in EB5 ESCs using a Tet-on system.
Preferential suppression of Oct3/4 expression by Tfap2c was observed. Scale bars, 100 µm.
(C) Immunostaining for Oct3/4, Nanog and Cdx2 after induction of Tfap2c with Ec in 4S2IP14 ESCs, in which
Oct3/4 is constitutively expressed from the Tet-off transgene. Scale bars, 100 µm.
(D) Photomicrographs of 4S2IP14 ESCs stably expressing miR-155-based shRNA against Tfap2c three days after
induction of trophoblast differentiation by Oct3/4 knockout. Scale bar, 200 µm.
(E) Immunostaining of 4S2IP14 ESCs stably expressing miR-155-based shRNA against Tfap2c two days after
induction of trophoblast differentiation by Oct3/4 knockout. Expression of Venus, which is encoded by the sametranscript as miR-155-shRNA, was detected using anti-GFP antibody (GF090R, Nacalai Tesque). Note that nuclear
staining of Tfap2c was weakly detected in the shTfap2c_1- and _2- expressing cells, but not in the shTfap2c_3and _4- expressing cells. Scale bar, 100 µm.
(F) Western blot analysis two days after Tfap2c induction (Ec) or Oct3/4 knockout (Tet) in 14RheoTfap2c ESCs
(left), and two days after Oct3/4 knockout (Tet) in Tfap2c knockdown 4S2IP14 ESCs (right).

-

TetFigure S6

α-Sox2ZHBTc4-TSC

A

1 kb

2

Oct3/4 KO D0
Oct3/4 KO D1
Oct3/4 KO D2
Oct3/4 KO D3
Oct3/4 KO D4
Sox2 KO3

5 kb

Gm14492

α-Sox2ZHBTc4-TSC

Lsp1
5

Oct3/4 KO D0
Oct3/4 KO D1
Oct3/4 KO D2
Oct3/4 KO D3
Oct3/4 KO D4
Sox2 KO

2

2

3

2

3

3

3

3

E

3

5 kb

4

5 kb

3

3

3

3

3

6

4

H

5 kb

F

3

Gm10782

5 kb

Id2

Hs6st1

Gm17746

6

α-Sox23
α-Tfap2c
3
α-Cdx2
Oct3/4 KO D0
Oct3/4 KO D1
Oct3/4 KO D2
Oct3/4 KO D3
Oct3/4 KO D4
Sox2 KO

5 kb

Mark2

Cxcl14Nctc1

α-Sox25
α-Tfap2c
5α-Cdx2

C

Hoxc11 Hoxc10 Hoxc9 Hoxc8 Hoxc6 Hoxc5 Hoxc4

Krt18

Mrp123
Tnnt3

G
ZHBTc4-TSC

5 kb

Krt8

α-Sox22
α-Tfap2c2α-Cdx2D

α-Sox2

B

Cdx2

6
6

3

4

I
ZHBTc4-TSC

7568

6611

eTSC

6725

Figure S6. ChIP-Seq Analysis of Sox2, Tfap2c, and Cdx2 Binding Sites in TSCs, Related to Figure 6(A-H) Normalized ChIP-seq signals at the Cdx2 (A), Krt8/Krt18 (B), Hoxc11-Hoxc4 (C), Tnnt3 (D), Mark2 (E),
Cxcl14/Gm10782 (F), Hs6st1 (G), and Id2/Gm17746 (H) loci in ZHBTc4-TSCs and during the differentiation of
ZHBTc4 ESCs into TS-like cells. The regions analyzed by ChIP-PCR in Figure 5 are shown (arrowheads).
(I) A venn diagram showing the number of overlapping peaks of Sox2 in ZHBTc4-TSCs and eTSCs. Peak regions
were defined as ±100-bp regions around peak summits.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Generation of Stable Cell Lines with Constitutive and Inducible Expression or Knockdown
To obtain stable transfectants, expression constructs driven by the CAG promoter (Niwa et al.,1991) were introduced into ESCs and TSCs by electroporation or using the piggyBac transposon
system (Guo et al., 2009), followed by selection, subcloning, and screening for transgene
expression. For inducible expression using a Tet-on system, the piggyBac transposon vectors forthe expression of rtTA2S-M2 and a Tet-responsive transgene followed by an IRES-Venus cassette
were co-transfected and the selected clones were treated with 1 µg/ml Dox. For inducible
expression using an EcR-based system, the piggyBac transposon vectors for the expression of
GAL4-EcR, VP16-RXR (from the RheoSwitch Inducible Expression System, New England
Biolabs) and a GAL4-responsive transgene followed by an IRES-Venus cassette wereco-transfected and the selected clones were treated with a non-steroidal ecdysteroid agonist,
diacylhydrazine (GenoStat Ligand, 500 nM, Millipore). For stable knockdown, miR-155-based
shRNA expression vectors containing the stem-loop sequence derived from the miR-155
precursor followed by an IRES-Venus cassette under the control of the CAG promoter wereconstructed. The following target sequences were selected using BLOCK-iTTM miR RNAi Select(Invitrogen): shTfap2c_1, 1223-1243; shTfap2c_2, 979-999; shTfap2c_3, 352-372; shTfap2c_4,
678-698 (nucleotide positions respective to the translation initiation site of NM_009335).

RT-PCR Analysis
Total RNA was isolated using QuickGene RNA cultured cell HC kit S (Fuji Film). First-strand cDNA
was synthesized with ReverTra Ace reverse transcriptase (TOYOBO). Real-time PCR was
performed using SYBR Green Realtime PCR Master Mix (TOYOBO). Expression levels werenormalized to Gapdh and presented as fold change over control samples. Each experiment was
performed in technical triplicate. Error bars indicate SD of three replicates. Similar results wereobtained in at least two independent experiments. All primers are listed in the Table S6.

Immunofluorescence Analysis
Immunostaining was performed using the following primary antibodies with dilutions: mousemonoclonal anti-Cdx2 (CDX2-88, BioGenex), 1:1,000; rabbit monoclonal anti-Cdx2 (3977, Cell
Signaling Technology), 1:200; rabbit anti-Eomes (AB9618, Millipore), 1:1000; mouse monoclonalanti-Esrrb (H6705, Perseus Proteomics), 1:1,000; rabbit anti-Sox2 [raised by ourselves (Masui etal., 2007)], 1:1,000; goat anti-Sox2 (GT15098, Neuromics), 1:1000; rabbit anti-Tfap2c (sc-8977,
Santa Cruz), 1:250; goat anti-Elf5 (sc-9645, Santa Cruz), 1:100; rabbit monoclonalanti-phosphorylated ERK (4370, Cell Signaling Technology), 1:100; mouse monoclonalanti-Oct3/4 (sc-5279, Santa Cruz), 1:1,000; rabbit anti-Nanog (A300-397A, Bethyl Laboratories),
1:1000; rat monoclonal anti-Nanog (14-5761, eBioscience), 1:250.

Production of TSC Chimeras
Two reverted TSC clones and parental EGFP-TS3.5 line were injected into E3.5 ICR blastocysts(10-20 TSCs/blastocyst) as described previously (Oda et al., 2009). Injected blastocysts weretransferred into the uteri of 2.5 days post-coitum (dpc) pseudopregnant ICR female mice andanalyzed at 13.5 dpc. For histological analysis, placentas were fixed with 4% paraformaldehyde
for overnight at 4°C, infiltrated with sucrose, embedded in OCT compound and frozen at -80°C.
Cross sections were prepared and analyzed for EGFP expression by fluorescent microscopy.
They were subsequently stained with hematoxylin-eosin and micrographs of the same field weretaken.

Microarray Analysis
Total RNA was extracted from ZHBTc4 ESCs treated with Tet, ZHBTc4-TSCs, EGFP-TS3.5 TSCs,
or 2TS22C-ESCs and -TSCs treated with Tet cultured in ESC or TSC media on MEF feeders. For
each dataset, the experiments were done in triplicate. cDNA synthesis and cRNA labelingreactions were performed using the Affymetrix GeneChip WT Terminal Labeling Kit and the
Ambion WT Expression Kit according to the manufacturer’s protocols. Affymetrix high-density
oligonucleotide arrays for Mus musculus (GeneChip Mouse Exon 1.0 ST Array) were hybridized,
stained, and washed according to the Expression Analysis Technical Manual (Affymetrix). The
expression values were summarized and normalized by the robust multiarray analysis andquantile normalization method using by Expression Console software (Affymetrix). The microarraydata are available at the GEO (GSE28455). For classification of the 11 cell types, PCA was
performed using the prcomp function in R (http://www.r-project.org/). For identification of genes
affected by Sox2 knockout, the less stringent cutoff (q-value <0.1, log2 ratio > 0.25) was applied tomaximize sensitivity while maintaining reasonable specificity. For functional annotation of the genesets, DAVID (Huang da et al., 2009) was used. Heatmaps showing the enrichment were generatedusing Cluster 3.0 and Java Treeview. GSEA (Subramanian et al., 2005) was performed to assessenrichment of the gene sets. FDR was estimated by gene set permutation tests. Previously
published microarray data for CGR8 ESCs undergoing differentiation through embryoid bodyformation (Schulz et al., 2009) and TS3.5 eTSCs differentiating into mature trophoblast cell types
upon withdrawal of FGF4 and MEF-conditioned medium (Ralston et al., 2010) were obtained fromthe EBI ArrayExpress (E-TABM-672) and the GEO (GSE12985), respectively. Enrichment wasdetermined by comparing the expression levels in the undifferentiated ESCs or TSCs with those inthe rest of the samples.

ChIP-Seq and Data Analysis
Chromatin was prepared from ZHBTc4 ESCs cultured in TSC medium supplemented with Tet for 0
to 4 days on MEFs, or ZHBTc4- and EGFP-TS3.5 TSCs cultured in TSC medium on MEFs.
Sequencing libraries were prepared according to the Illumina protocols and sequenced on the
Genome Analyzer IIx or HiSeq 1500 (Illumina) as single-end 50-bp reads. Sequence reads werealigned to the mouse reference genome (mm9) using Bowtie2 (Langmead and Salzberg, 2012)
with default parameters. Peak Calling was performed using MACS2 (Zhang et al., 2008) with input
DNA as a control and with a q-value cutoff of 0.05. The signals were normalized by sequencing
depth of million mapped reads with the --SPMR option. The peaks that overlap with the Sox2
peaks in Sox2 knockout ESCs (2TS22C +Tet) were removed. To calculate the number ofoverlapping peaks, ±100-bp regions around peak summits were considered to avoid a bias toward
longer peak regions. K-means clustering and visualization of ChIP-seq signals around the peakregions were performed using the heatmapr script from the Cistrome package (Liu et al., 2011).
Annotation of peak regions with genomic features around the RefSeq genes were performed using
CEAS (Shin et al., 2009). De novo motif discovery was performed using the MEME Suite (Bailey etal., 2009). The sequences of ±100-bp regions around the 600 randomly selected merged peak
centers or around the top 1,000 ranked peak summits that were masked for repetitive elements
using RepeatMasker were obtained from the UCSC Genome Browser. The overrepresented
motifs identified by MEME were then searched against the JASPAR CORE and UniPROBE
databases using TOMTOM. MAST was used for mapping of the motifs to ±200-bp regions aroundeach peak center/summit with a p-value cutoff of 1e-4. Functional annotation of genes associatedwith peak regions was performed using GREAT (McLean et al., 2010) to integrate distal binding
events, with the default association rule (5+1 kb basal, up to 1 Mb extension) and the whole
mouse genome as background. For GSEA, each peak center was assigned to RefSeq genes
whose TSSs are located within a 10-kb distance using the peak2gene script from the CEAS
package. Enrichment was determined by comparing the expression levels in Sox2 knockout ESCs
or TSCs with those in their respective controls, or the levels in ZHBTc4 ESCs differentiating to
TSCs (+Tet D1-D4) with those in the control (D0). The ChIP-Seq data are available at the GEO(GSE28455).

Co-Immunoprecipitation Analysis
Co-immunoprecipitation was performed using Dynabeads Co-Immunoprecipitation Kit (Invitrogen)following the manufacturer's instructions. Briefly, EGFP-TS3.5 cells were treated with or without 1%

formaldehyde for 10 min, and lysed in buffer containing 100 mM NaCl, protease and phosphataseinhibitor cocktails. The whole cell lysate was immunoprecipitated with anti-Sox2 (raised byourselves) or anti-Tfap2c antibodies covalently coupled to beads. The immunoprecipitatedsamples were analyzed by Western blot with anti-Sox2 (GT15098, Neuromics) and anti-Tfap2c
antibodies.

SUPPLEMENTAL REFERENCES
Guo, G., Yang, J., Nichols, J., Hall, J.S., Eyres, I., Mansfield, W., and Smith, A. (2009). Klf4 reverts
developmentally programmed restriction of ground state pluripotency. Development 136, 1063-1069.
Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9,357-359.
Liu, T., Ortiz, J.A., Taing, L., Meyer, C.A., Lee, B., Zhang, Y., Shin, H., Wong, S.S., Ma, J., Lei, Y., et al.
(2011). Cistrome: an integrative platform for transcriptional regulation studies. Genome Biol 12, R83.
Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants
with a novel eukaryotic vector. Gene 108, 193-199.
Oda, M., Tanaka, S., Yamazaki, Y., Ohta, H., Iwatani, M., Suzuki, M., Ohgane, J., Hattori, N.,
Yanagimachi, R., Wakayama, T., et al. (2009). Establishment of trophoblast stem cell lines from somatic
cell nuclear-transferred embryos. Proc Natl Acad Sci U S A 106, 16293-16297.
Zhang, Y., Liu, T., Meyer, C.A., Eeckhoute, J., Johnson, D.S., Bernstein, B.E., Nusbaum, C., Myers,
R.M., Brown, M., Li, W., et al. (2008). Model-based analysis of ChIP-Seq (MACS). Genome Biol 9,
R137.