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Cell Stem Cell. Oct 5, 2012; 11(4): 491–504.
PMCID: PMC3465555

Esrrb Is a Pivotal Target of the Gsk3/Tcf3 Axis Regulating Embryonic Stem Cell Self-Renewal

Summary

Inhibition of glycogen synthase kinase-3 (Gsk3) supports mouse embryonic stem cells (ESCs) by modulating Tcf3, but the critical targets downstream of Tcf3 are unclear. We analyzed the intersection between genome localization and transcriptome data sets to identify genes repressed by Tcf3. Among these, manipulations of Esrrb gave distinctive phenotypes in functional assays. Knockdown and knockout eliminated response to Gsk3 inhibition, causing extinction of pluripotency markers and loss of colony forming capability. Conversely, forced expression phenocopied Gsk3 inhibition or Tcf3 deletion by suppressing differentiation and sustaining self-renewal. Thus the nuclear receptor Esrrb is necessary and sufficient to mediate self-renewal downstream of Gsk3 inhibition. Leukaemia inhibitory factor (LIF) regulates ESCs through Stat3, independently of Gsk3 inhibition. Consistent with parallel operation, ESCs in LIF accommodated Esrrb deletion and remained pluripotent. These findings highlight a key role for Esrrb in regulating the naive pluripotent state and illustrate compensation among the core pluripotency factors.

Highlights

Esrrb is the principal target of Tcf3 repression in the pluripotency network ► Esrrb is essential for self-renewal downstream of Gsk3 inhibition ► Esrrb potently suppresses differentiation and sustains ESC self-renewal ► Esrrb is a core pluripotency factor but can be compensated by LIF/Stat3

Introduction

Since the original derivation of mouse embryonic stem cells (ESCs) in 1981 (Evans and Kaufman, 1981; Martin, 1981), culture conditions for sustaining pluripotency ex vivo have been progressively refined. Following the demonstration that the cytokine leukemia inhibitory factor (LIF) could replace feeder cells (Smith et al., 1988; Williams et al., 1988) and that ESC differentiation is suppressed by inhibition of mitogen activated protein kinase (Erk) signaling (Burdon et al., 1999; Kunath et al., 2007), further addition of an inhibitor of glycogen synthase kinase-3 (Gsk3) has enabled robust ESC propagation in well-defined conditions (Ying et al., 2008). When cultured using the two inhibitors (2i), ESCs display rather uniform marker expression (Wray et al., 2010) and exhibit distinctive gene expression and epigenetic features (Marks et al., 2012). A practical consequence is that it has become facile to establish ESCs from different strains of mice and also rats (Blair et al., 2011). It is noteworthy that, while the triple combination of 2i/LIF appears optimal, mouse ESCs can be propagated by providing any two of these three components (Wray et al., 2011; Wray et al., 2010), implying complementary inputs to a flexible gene regulatory circuit. However, understanding how intracellular signaling pathways engage with the core transcription factor circuitry to maintain or extinguish pluripotency remains fragmentary (Chen et al., 2008; Jaenisch and Young, 2008; Nichols and Smith, 2012; Niwa et al., 2009).

Gsk3 is a negative regulator of many different proteins (Doble and Woodgett, 2003), including transcription factors such as cMyc (Singh and Dalton, 2009). Nonetheless, the effect of Gsk3 inhibition ESC self-renewal is mediated primarily via β-catenin because ESCs lacking β-catenin do not respond productively to Gsk3 inhibitors (Lyashenko et al., 2011; Wray et al., 2011). Consistent with action through intracellular β-catenin, mutation of Apc or expression of stabilized β-catenin variants can reduce ESC differentiation (Kielman et al., 2002; Sato et al., 2004). Furthermore, Wnt3a can partially substitute for Gsk3 inhibition and support ESC propagation in conjunction with LIF (ten Berge et al., 2011; Hao et al., 2006; Ogawa et al., 2006; Yi et al., 2011).

It has been suggested that β-catenin might interact directly with Oct4 to promote pluripotent gene expression (Kelly et al., 2011). On the other hand, genetic evidence is incontrovertible that a definitive β-catenin partner, Tcf3 (also known as Tcf7l1), is a major negative regulator of ESC self-renewal (Guo et al., 2011; Pereira et al., 2006). Indeed, ablation of Tcf3 phenocopies deletion or inhibition of Gsk3 (Wray et al., 2011). Genome location analyses indicate that Tcf3 binds in proximity to many core pluripotency genes (Cole et al., 2008; Marson et al., 2008; Tam et al., 2008). Although it has been proposed that β-catenin is recruited to stimulate transcription at these sites (Cole et al., 2008), this model seems inconsistent with the Tcf3 loss of function phenotype. Furthermore, available evidence indicates that Tcf3 functions in a repressor complex (Pereira et al., 2006; Sokol, 2011; Yi et al., 2011), activity of which can be abrogated by β-catenin without requirement for its transactivation domain (Wray et al., 2011).

Tcf3 has previously been proposed to act via repression of Nanog (Pereira et al., 2006). However, Nanog null ESCs retain responsiveness to Gsk3 inhibition (Silva et al., 2009), and knockdown of Nanog in Tcf3 null ESCs does not prevent upregulated expression of other pluripotency genes (Yi et al., 2011). It is therefore unclear whether Tcf3 functions as a general repressor of pluripotency genes or acts via a selective target or subset of targets. To resolve this issue, we integrated available high resolution genome location data with transcriptome profiles from Tcf3 perturbation studies. This enabled identification of a small number of transcription factor candidates that were subjected to loss and gain of function analyses.

Results

Genome-Scale Identification of Candidate Genes Directly Regulated by Tcf3

Genome-wide mapping of transcription factor binding events is achievable by chromatin immunoprecipitation and deep sequencing (ChIP-seq). Integrated analysis of multiple transcription factor ChIP-seq studies (Hannah et al., 2011), has the potential to provide new mechanistic insights (Ouyang et al., 2009; Wilson et al., 2010). Given the dominant role of transcriptional regulation in ESC function (Jaenisch and Young, 2008; Nichols and Smith, 2012; Niwa, 2007; Smith, 2009; Young, 2011) we generated a compendium of transcription factor ChIP-seq analyses (see Figures S1A and S1B and Table S1 available online and http://bioinformatics.cscr.cam.ac.uk/ES_Cell_ChIP-seq_compendium.html). We used the ChIP-seq compendium to explore candidate target genes for the Tcf3 transcriptional repressor. We focused on those Tcf3 ChIP-seq targets also bound by other pluripotency factors. As previously indicated from promoter-based ChIP-on-chip analysis (Cole et al., 2008), extensive colocalization of Tcf3 with core pluripotency transcription factors Oct4 and Nanog was evident in the genome-wide ChIP-seq data sets (Figure 1B).

Figure 1
Identification of Tcf3 Direct Targets in Mouse ESCs

We then analyzed published gene expression profiles for ESCs in which Tcf3 has been inactivated, either by gene targeting (Yi et al., 2011) or by RNAi (Cole et al., 2008). Tcf3 null and Tcf3 knockdown ESCs show similarly enhanced self-renewal so we reasoned that genes differentially expressed in both studies are likely to underlie this phenotype. Tcf3 appears to function in ESCs primarily, if not exclusively, as a transcriptional repressor (Pereira et al., 2006; Wray et al., 2011; Yi et al., 2011). We therefore focused on genes with increased expression after Tcf3 inactivation and found 379 genes upregulated in Tcf3 null cells and 1,972 in Tcf3 knockdown cells (Figure 1C). Of these, 120 genes were common to both groups and therefore were considered the most likely specific responders.

We intersected the list of Tcf3 target genes from the ChIP-seq compendium with the 120 upregulated genes and found a statistically significant enrichment (p value = 0.0034), with 50 genes identified as candidates for direct regulation, being both bound by Tcf3 and derepressed after Tcf3 ablation. The same analysis for genes downregulated following Tcf3 inactivation did not show significant enrichment for Tcf3 binding (p value = 0.57, Figure S1C), indicating indirect regulation. To survey possible biological roles of Tcf3 direct targets, we performed Gene Ontology analysis and found an overrepresentation of developmental processes such as cell differentiation, anatomical structure development, cell morphogenesis, and embryonic development (Figure S1D). This is in line with the role of Tcf3 as a repressor of pluripotency (Wray et al., 2011) and mediator of axial patterning (Merrill et al., 2004).

For functional validation, we focused on genes associated with cell differentiation and development and among them selected transcription factors. This gave a short list of nine genes (in bold in Figure S1E). We used gene expression analysis to test responsiveness to the Gsk3 inhibitor Chiron99021 (CH). Four genes showed very low expression with or without CH (data not shown). The other five transcription factor genes exhibited significantly increased expression in response to CH (Figure 2D, orange columns). Esrrb, Klf2, Nanog, Nr0b1, and Tcfcp2l1 thus emerge as candidate mediators of Gsk3/Tcf3-mediated ESC self-renewal. Figure 2E shows gene tracks of Tcf3, Nanog, and Oct3/4 binding at these five gene loci.

Figure 2
Esrrb Is Required to Mediate Self-Renewal Downstream of GSK3

We also analyzed Tcf3 null cells and found an increased level of expression of the five genes, similar to that in wild-type (WT) cells treated with CH (Figure 2D, blue columns). Importantly, when Tcf3 null cells were treated with CH, we did not observe any further increase in expression (Figure S1F). These data indicate that CH promotes the transcription of Esrrb, Klf2, Nanog, Nr0b1, and Tcfcp2l1 through Tcf3 inhibition without requirement for canonical activation through other Tcf/Lef family members (Yi et al., 2011).

Esrrb Is Critical for Self-Renewal Downstream of the Gsk3/Tcf3 Axis

Gsk3 deletion or inhibition is sufficient to maintain ESC self-renewal in bulk culture for several passages, albeit with ongoing differentiation (Doble et al., 2007; Wray et al., 2010; Ying et al., 2008). This condition provides a stringent assay for downstream gene requirement. To obtain a quantitative readout, we used Rex1GFPd2 cells, in which a destabilized GFP protein is expressed from the Rex1 (Zfp42) locus (Wray et al., 2011). Rex1 is expressed specifically in ground state pluripotent cells and is downregulated within 24 hr at the onset of differentiation (Toyooka et al., 2008; Wray et al., 2010). Consequently the fraction of undifferentiated ESCs in a population can be quantified by flow cytometric analysis for GFP (Wray et al., 2011). We knocked down the five transcription factor genes in Rex1GFPd2 cells by small interfering RNA (siRNA) transfection (Figure 2A). Two different siRNAs were used for each gene (Figure S2A). Cells were maintained in the presence of CH and evaluated by flow cytometry (Figure 2B). ESCs treated with control siRNA are mostly Rex1GFP-positive with a small shoulder of negative cells. This profile is unaltered by siRNA against Nr0b1 while Nanog knockdown induces only a small increase in the negative fraction. The effect of Klf2 and Tcfcp2l1 siRNAs is rather more pronounced, but in both cases the majority of cells remain GFP-positive. In contrast, knockdown of Esrrb caused a dramatic shift from Rex1GFP-positive to -negative (Figures 2B and 2C). This is not a selective elimination effect because viable cell numbers determined by live cell gating were similar in all conditions. To exclude nonspecific effects of siRNAs on GFP expression, we confirmed a reduction in Rex1 transcript (Figure 2D). Further qPCR analyses showed a marked downregulation in other pluripotency markers and a concomitant increase in the early differentiation marker Fgf5 upon treatment with siEsrrb.

To address whether decreased expression of Rex1 and pluripotency genes following Esrrb knockdown is associated with compromised self-renewal, we tested ability to form undifferentiated colonies 48 hr after siRNA transfection. Cells were plated at single cell density in 2i+LIF and after 5 days stained for alkaline phosphatase (AP). AP activity is a classical marker of undifferentiated ESCs but does not reliably discriminate them from postimplantation epiblast stem cells (EpiSCs) (Bao et al., 2009). However, EpiSCs differentiate or die in 2i (Guo et al., 2009), therefore in this condition the marker is reliably indicative of ESC status. Cells transfected with a control siRNA (siControl) or siRNA targeting eGFP (siGFP) gave rise to numerous AP-positive colonies, while knockdown of Nr0b1, Nanog, Klf2, or Tcfcp2l1 caused up to 2-fold reduction (Figure 2E). Knockdown of Esrrb had a much stronger effect, however, almost entirely eliminating AP-positive colonies (Figure 2E, green columns).

Previously, we reported that CH promotes self-renewal by relieving Tcf3 repression (Wray et al., 2011). The expression of each of the five candidate genes is elevated in Tcf3 null cells (Figure 1D). We therefore examined their importance for the ability of ESCs lacking Tcf3 to self-renew efficiently without requirement for CH. We knocked down the genes in Tcf3 null cells and tested ability to form colonies in the presence of the Mek inhibitor PD0325901 (PD) alone. Null cells transfected with either siControl or siGFP readily generated AP-positive colonies (Figure 2F, blue columns). Klf2 and Tcfcp2l1 siRNA treatment resulted in slightly fewer colonies, whereas knockdown of Esrrb or Nanog almost eliminated colony formation (Figure 2F, green and purple columns). We also used titrated siRNA to partially reduce Esrrb expression to levels close to those in WT ESCs (Figures S2B and S2C). This resulted in restoration of colony formation to similar numbers as for WT ESCs (Figure S2D). Taken together, these results suggest that Esrrb is necessary to mediate self-renewal downstream of Gsk3 inhibition and Tcf3 derepression.

Esrrb Expression Reproduces the Effect of Gsk3 Inhibition on Self-Renewal

We then examined whether expression of Esrrb or the other factors may be sufficient to substitute for Gsk3 inhibition. Rex1GFPd2 cells maintained in 2i were transfected with Piggybac expression vectors (Guo et al., 2009). Following hygromycin selection for two passages in 2i, the response to inhibitor withdrawal was tested in colony forming assays and in bulk culture (Figures 3A and 3B). PB-vector and PB-Tcfcp2l1 transfectants yielded negligible colonies when plated at clonal density in N2B27 alone. PB-Nr0b1, PB-Nanog, and PB-Klf2 produced some colonies but many fewer than in the presence of CH. PB-Esrrb, however, generated undifferentiated colonies in comparable number and size to those obtained in the presence of CH. The absence of any significant additive effect when Esrrb is overexpressed in the presence of CH (Figure 3A) suggests that Esrrb is capable of fully recapitulating the effect of Gsk3 inhibition on ESC clonogenicity.

Figure 3
Esrrb Recapitulates the Effect of GSK3 Inhibition on ESC Self-Renewal

In bulk culture in the presence of PD alone, PB-vector and PB-Tcfcp2l1 cells collapsed within two passages. Other transfectants could be maintained longer but progressively lost Rex1GFP expression (Figure 3B) and completely differentiated or died by five passages. By contrast, PB-Esrrb cells could easily be expanded in PD for more than 12 passages. Their Rex1GFP profile showed only a small shoulder of GFP low cells and the cells retained undifferentiated morphology (Figures 3B and 3C). PB-Esrrb cells in these conditions expressed Oct4, Sox2, and Nanog at a level similar to control cells in 2i (Figure 3D, orange columns). They showed reduced Klf4 and Klf5 but increased Klf2.

We then tested whether expression of Esrrb could confer clonal self-renewal in the presence of serum, which is normally dependent on LIF. Colonies were scored as undifferentiated, mixed, or differentiated according to the AP staining pattern because serum supports the survival and proliferation of differentiated cells. PB-vector cells formed undifferentiated colonies only in the presence of LIF, whereas PB-Esrrb cells were equally capable of generating AP-positive colonies without LIF (Figure 3E), similarly to PB-Nanog cells that serve as a positive control. Strikingly, the combination of Esrrb overexpression and LIF caused a further increase in the number of undifferentiated colonies, indicating that Esrrb is in a parallel pathway to LIF/Stat3. We also used this assay to examine whether Esrrb could rescue the differentiation phenotype induced by overexpression of Tcf3. Indeed the generation of undifferentiated colonies is diminished by transfection with PB-Tcf3 alone and regained by cotransfection with PB-Esrrb (Figure 3E). Consistent with the colony assay, Tcf3 transfectants showed reduced expresson of Nanog that was restored by Esrrb (Figure S3A).

Esrrb-expressing cells formed undifferentiated AP-positive colonies when LIF or Wnt signaling were blocked by a Jak inhibitor or DKK respectively (Figures S3B and S3C). Furthermore, neither the LIF target Socs3 nor the Wnt target Axin2 showed elevated expression (Figures S3D and S3E), confirming that Esrrb does not act by stimulating these pathways. We also cocultured GFP labeled Esrrb transfectants with unlabelled WT cells. After three passages in serum without LIF, the entire population was GFP-positive (Figure S3F), demonstrating that Esrrb acts cell autonomously.

In unscreened PB-Esrrb cells, the expression of Esrrb is typically up to 6- to 8-fold higher than endogenous levels. This creates the possibility of neomorphic effects. We therefore transfected Rex1GFPd2 ESCs with an alternative PB-Esrrb vector allowing capture of low expressors using G418 (Figure 3F). We screened for level of Esrrb expression and identified a pool in which Esrrb messenger RNA (mRNA) and protein are constitutively expressed at levels comparable to those found in control vector transfectants in 2i (Figures 3G and 3H). These PB-Esrrb-neo cells recapitulate the phenotype of Esrrb overexpressing cells: in the absence of CH or LIF, they can be cultured for multiple passages, they express pluripotency markers (Figures 3G and 3I), and they self-renew at clonal density (Figure 3J). We conclude that constitutive expression of Esrrb at endogenous levels is sufficient to sustain ESC self-renewal.

Because constitutive expression of Esrrb blocks differentiation, transgene excision (Chambers et al., 2003; Niwa et al., 2009) was necessary to determine whether ESCs maintained by Esrrb retain pluripotency. After transfection with an excisable vector, cells were clonally selected and expanded in serum without LIF for 1 month. They were then transiently transfected with a Cre expression vector followed by subcloning of DsRed-positive cells in the presence of LIF (Figure 3K). These subclones no longer expressed the Esrrb transgene and reacquired dependency on LIF (bottom panels of Figure 3L and Figure S3H). Reverted cells were injected into blastocysts and gave rise to chimeric embryos in which DsRed-expressing cells contributed widely (Figure 3M).

Collectively, these results indicate that constitutive expression of Esrrb can replace Gsk3 inhibition and furthermore maintain self-renewal and pluripotency independently of LIF/Stat3.

Esrrb Is Dispensable for Self-Renewal in the Presence of LIF

Although Esrrb appears essential for ESC propagation downstream of Gsk3 inhibition, the forced expression studies indicate that it does not lie downstream of LIF/Stat3. Therefore Esrrb might be dispensable in the presence of LIF, just as Stat3 can be deleted when Gsk3 is inhibited (Ying et al., 2008). We tested this hypothesis by knocking down Esrrb in Rex1GFPd2 cells cultured in the presence of LIF and PD (Figure 4A). Under these conditions we found that Esrrb siRNA had a modest effect on the naive pluripotency marker Rex1 (Figures 4B and 4C) and caused only a partial reduction in the ability to form undifferentiated colonies (Figure 4D). These findings are in stark contrast with the near elimination of self-renewal by Esrrb siRNA in the absence of LIF (Figure 2).

Figure 4
Esrrb Is Not Required for Self-Renewal in the Presence of LIF

To substantiate these findings, we generated ESCs in which the Esrrb gene can be conditionally inactivated. Through serial gene targeting, we inserted loxP sites to flank the second exon of both alleles of Esrrb (Figure S4A). The second exon encodes the start codon and part of the DNA-binding domain. Esrrb−/− ESCs generated by Cre-mediated recombination are therefore expected to be functionally null. Tamoxifen-regulatable Cre was used to effect deletion. Homozygous deletion was confirmed in a clonally expanded population by immunostaining and qPCR (Figures 4E and 4F). These Esrrb null ESCs remained morphologically undifferentiated in serum-containing medium with LIF over multiple passages. Expression of some pluripotency-associated genes, Klf4 and Tbx3, was reduced (Figure 4F) and the null cells exhibited a rather more flattened morphology (Figure 4H), but they showed similar proliferation to WT ESCs. Interestingly, Nanog−/− ESCs also show reduced expression of Klf4 (Figure 4G). From examination of the ChIP-seq compendium, Klf4 is a likely direct target of both Esrrb and Nanog, which may explain its lower level in null cells.

When Esrrb null ESCs were cultured in serum-free conditions, they expanded in 2i+LIF and LIF+PD. They were less compact than parental cells but remained undifferentiated (Figure 4H) with no overt compromise of proliferation or viability. However, on LIF withdrawal, the null cells collapsed within one passage. Thus, in the absence of Esrrb, ESCs lose the ability to respond effectively to Gsk3 inhibition and their propagation appears strictly dependent on LIF. Clonal analysis of Esrrb−/− cells confirmed these findings, showing a dramatic reduction in the number of AP-positive colonies in the absence of LIF (Figure 4I, compare 2i+LIF to 2i and LIF+PD to CH).

We carried out gene expression analysis after 48 hr of culture either in LIF+PD or in CH (Figures 4K and 4L). In LIF+PD, Esrrbfl/fl and Esrrb−/− cells showed a comparable profile, apart from reduced Klf4 and Tbx3 in the null cells (Figure 4K). In contrast, after 48 hr in CH Esrrb−/− cells showed lower expression of all pluripotency markers (Figure 4L). These deletion findings are fully consistent with results obtained after Esrrb knockdown (Figures 2 and and4A–4D),4A–4D), confirming that Esrrb is essential for self-renewal downstream of CH, but can be compensated for by LIF stimulation. In contrast, Nanog null cells could be propagated under all conditions described above, consistent with previous observations (Silva et al., 2009) that Nanog is not required for responsiveness to Gsk3 inhibition (Figure 4J).

To explore why loss of Esrrb has more severe consequences than deletion of Nanog, we utilized the ChIP-seq compendium (Figure S1) to compare profiles of Nanog, Oct4, Sox2, Tcf3, and Esrrb. As shown in Figure 4M using the Tbx3 locus as an example, there are regions bound by Nanog, Oct4, Sox2, Tcf3 (O/S/T) together with Esrrb (see blue box), as well as regions bound only by Esrrb (see green box). Global analysis revealed that a majority of the genomic regions bound by Nanog are co-occupied by at least one of Oct4, Sox2, and Tcf3 (O/S/T), as well as Esrrb. However, only a minority of Esrrb-bound regions are occupied by any of the other four pluripotency factors (Figure 4N; Figure S4C). To investigate whether Esrrb-specific occupancy translates into Esrrb-specific candidate target genes, we mapped binding peaks to genes (see Table S1 and Experimental Procedures section) and intersected the resulting gene lists. Whereas over 90% (2,921 out of 3,230) of the predicted Nanog target genes were also targets of O/S/T and/or Esrrb, only ~55% (1,992 out of 3,647) of Esrrb candidate targets were shared with any of the other factors (Figure 4O; Figure S4D). The extensive overlap of Nanog with Oct4, Sox2, Tcf3, and/or Esrrb candidate targets may explain why Nanog deletion can be tolerated in established ESCs. Conversely, the wider occupancy of Esrrb is consistent with the observation that Esrrb function is less readily dispensable.

We then examined whether ESCs lacking Esrrb and maintained using LIF are pluripotent. Cells grafted under the kidney capsule gave rise within 6 weeks to large multidifferentiated tumors that contained neural, mesodermal, and endodermal tissues along with undifferentiated embryonal carcinoma (Figure 5A). The tumors showed no overt differences in size or differentiation from teratocarcinomas generated by Esrrbfl/fl cells. Most significantly, the embryonic identity and developmental potential of Esrrb null cells was confirmed by integration into the inner cell mass after aggregation with morulae (Figures 5B and 5C) and widespread contribution to the midgestation embryo after blastocyst injection (Figure 5D; Figure S5).

Figure 5
Esrrb Null Cells Are Pluripotent

Direct Regulation of Esrrb Expression by the Gsk3/β-Catenin/Tcf3 Axis

In keeping with several other core pluripotency factors, Esrrb is expressed in a mosaic fashion in ESCs cultured in serum. When CH is added to the cultures, however, Esrrb immunostaining becomes more uniform and increased in intensity (Figures 6A and 6B). To confirm binding of Tcf3 to the Esrrb gene, we carried out single point ChIP assays at five regions detected in previous ChIP studies (Cole et al., 2008; Marson et al., 2008). Marked enrichment was observed in WT ESCs compared with Tcf3 null cells (Figure 6C). As noted above, Tcf3 null ESCs show elevated expression of Esrrb that is not further induced by CH alone. We repeated this analysis in the presence of LIF and PD and obtained similar results (Figure 6D). β-catenin null ESCs also fail to upregulate Esrrb in response to CH, consistent with their inability to self-renew without LIF (Wray et al., 2011). These data confirm that both Tcf3 and β-catenin are necessary for Esrrb induction by Gsk3 inhibition. We found that Wnt3a can induce Esrrb, although to lower levels than CH (Figure S6), likely reflecting the operation of negative feedback in the canonical Wnt pathway that can be short-circuited by Gsk3 inhibition. Finally, we investigated the possibility that induction of Esrrb may involve Nanog. Nanog is modestly upregulated by both CH and Tcf3 ablation (Figure 1D) and it has been shown to bind to the Esrrb promoter region (Chen et al., 2008). We found that Esrrb is fully induced by CH in Nanog null ESCs (Figure 6E). Steady state expression is lower in the absence of Nanog, however (see also Figure 4G). We conclude that Nanog plays a role in maintenance of Esrrb expression level, but that induction by Gsk3 inhibition is mediated directly by abrogation of Tcf3 repression (Figure 6F).

Figure 6
Mechanism of Esrrb Regulation by Tcf3

Discussion

Several mechanisms have been proposed through which inhibition of Gsk3 may stabilize the naive state of mouse ESCs (Cole et al., 2008; Kelly et al., 2011; Sokol, 2011; Wray et al., 2011; Yi et al., 2011). The present analyses clarify this debate by identifying Esrrb as a direct functional target of Tcf3 that is derepressed downstream of Gsk3 inhibition. Perturbation studies demonstrate that Esrrb activity is both necessary and sufficient for the response to Gsk3 inhibition. Notably, Esrrb exhibits markedly more potent capacity than other Tcf3 targets to suppress differentiation, sustain propagation, and maintain key pluripotency genes. While recognizing that Tcf3 also regulates other key genes in the pluripotency circuitry, these findings pinpoint Esrrb as the main effector through which the Gsk3/β-catenin/Tcf3 axis modulates ESC self-renewal.

We generated a compendium of publicly available ChIP-seq data for 38 transcriptional regulators in mouse ESCs (Figures S1A and S1B). This integrated resource with a unified data structure enables streamlined cross-referencing of individual data sets from different laboratories. Distinct clusters of binding profiles can be discerned that appear to partition the ESC transcriptional program into several major subcompartments. The compendium thus provides a powerful analytical tool with the potential to fuel new hypotheses on the transcriptional control of ESC function. Tcf3 binds at a large number of sites, including many genes implicated in ESC biology (Cole et al., 2008; Marson et al., 2008; Tam et al., 2008). We used the compendium resource to examine in greater detail genes bound by both Tcf3 and core pluripotency factors and generate a refined list of candidate targets, which included Esrrb.

Esrrb is an orphan nuclear receptor related to the estrogen receptor (Luo et al., 1997). A potential role in ESCs was previously suggested from results of an RNAi screen by Ivanova and colleagues who found that knockdown of Esrrb reduced ESC self-renewal efficiency and promoted differentiation (Ivanova et al., 2006). Subsequently, Esrrb was reported to replace Klf4 in fibroblast reprogramming, albeit with reduced efficiency (Feng et al., 2009). Through protein interaction studies, Esrrb was found to bind to Oct4 and cooperate in transcriptional regulation of Nanog (van den Berg et al., 2008). Esrrb is also proposed to interact with Nanog and thereby play a reciprocal role in regulation of Oct4 expression (Zhang et al., 2008). These authors also reported that overexpression of Esrrb can support formation of AP colonies in serum in the absence of LIF (Zhang et al., 2008). However, the identity of the colonies was not characterized further and neither self-renewal nor retention of pluripotency were investigated. Nor is there any evidence that Esrrb is induced by LIF. In the embryo, deletion of Esrrb causes midgestation lethality due to placental defects (Luo et al., 1997), but mutant embryos develop through implantation and gastrulation. Furthermore, tetraploid complementation rescued embryo development at least to midgestation (Luo et al., 1997), confirming that there is no defect in the ICM or epiblast. Hence the functional significance of Esrrb in ESCs and its position in the regulatory hierarchy have been uncertain.

Here, through loss-of-function studies by siRNA knockdown and definitive gene deletion via homologous recombination, we established that Esrrb is specifically required for the self-renewal effect of Gsk3 inhibition. Furthermore, Esrrb gain-of-function can replace Gsk3 inhibition and support long-term ESC propagation in the presence of Mek inhibition alone. Constitutive expression of Esrrb can also confer clonogenic LIF-independent self-renewal in serum. Importantly, ESCs propagated under the direction of Esrrb remain pluripotent and capable of colonizing chimeras when the transgene is removed.

Mouse ESC self-renewal is highly responsive to LIF, acting through Jak kinase and the downstream transcription factor Stat3 (Matsuda et al., 1999; Niwa et al., 1998). Neither LIF nor Gsk3 inhibition alone can fully suppress ESC differentiation, but the combination of both together is very effective (Wray et al., 2010). Furthermore, Stat3 null ESCs can be derived and maintained using 2i and are functionally nonresponsive to LIF (Ying et al., 2008), whereas β-catenin null ESCs self-renew in the presence of LIF but do not respond to Gsk3 inhibition (Lyashenko et al., 2011; Wray et al., 2011). These observations indicate that Gsk3 inhibition/Tcf3 derepression supports self-renewal in parallel with LIF/Stat3 and inputs independently into the core pluripotency network (Figure 6F). Indeed, although Esrrb expression can act dominantly to confer LIF independence, addition of LIF further augments clonogenic capacity. Consistent with parallel pathways, the requirement for Esrrb is not absolute. ESCs lacking Esrrb remain undifferentiated if cultured in the presence of LIF with either PD or serum. Thus the need for Esrrb, just as for Gsk3 inhibition or β-catenin (Lyashenko et al., 2011; Wray et al., 2011), is conditional on whether ESCs receive other pro- and antidifferentiation stimuli, in particular LIF. Parallel compensatory capacity could explain why the pluripotent lineage in the early embryo can develop without Esrrb (Luo et al., 1997).

When Esrrb knockdown or knockout ESCs are cultured without LIF, they downregulate pluripotency genes and exit self-renewal. We therefore conclude that Esrrb is a component of the core transcription factor network that underpins pluripotency. Esrrb appears to be highly interconnected with other pluripotency factors by both protein interactions and transcriptional cross-regulation (van den Berg et al., 2008; van den Berg et al., 2010; Zhang et al., 2008). Interestingly, examination of the ChIP-seq compendium reveals that although Esrrb binds at many of the same genes as other core pluripotency factors (Nanog, Oct4, Sox2, Tcf3), it often occupies different sites. In addition, Esrrb is located at a large group of genes not bound by the other factors. These observations are consistent with the functional evidence that Esrrb makes a distinctive contribution to ESC self-renewal. Indeed, we find that Esrrb binding is detected at ~70% of genes (8,149 out of 12,051) that show significant expression by RNA-seq (>0.5 RPKM) in ground state ESCs (Marks et al., 2012). This suggests that it may play a generalized role in ESC transcription, particularly when considered with evidence that Esrrb interacts physically with components of the basal transcriptional machinery (van den Berg et al., 2010).

In summary, these findings place Esrrb on a par with Nanog, Klf4, Klf2, and Tbx3 in the pantheon of intrinsic pluripotency factors that together with Oct4 and Sox2 establish and sustain naive ESCs (Figure 6F). Specifically, Esrrb is a direct target of Tcf3 repression and the principal factor mediating the self-renewal response to Gsk3 inhibition and stabilization of β-catenin. Esrrb acts independently of LIF/Stat3, conferring flexibility and robustness to naive ESC self-renewal. Future work will explore the mutual compensation and plasticity in the pluripotency gene regulatory network that allows LIF stimulation to accommodate deletion of Esrrb. It will also be of interest to examine epiblast development in blastocysts doubly deficient for Esrrb and Stat3. Finally, we note that Esrrb is not significantly expressed in mouse postimplantation epiblast stem cells (EpiSCs), nor in currently available human pluripotent stem cells, neither of which show a self-renewal response to Gsk3 inhibition.

Experimental Procedures

Generation of ChIP-seq Compendium

Processed data files (density maps, wig format; peak coordinates, bed format) were acquired from the NCBI Gene Expression Omnibus (GEO) and Short Sequence Read Archive (SRA). They were displayed in the UCSC Genome Browser allowing a visual inspection of the ChIP-seq data and peak calling quality. Where the published peak coordinates appeared to over- or underpredict, or were unavailable, creation of density maps and peak calling was performed de novo using the mapped reads as described (Hannah et al., 2011). The specific peak parameters used are indicated in Table S1.

A binary peak matrix was generated as described (Hannah et al., 2011), analyzed by unsupervised hierarchical clustering using Pearson correlation coefficients, and displayed using the heatmap function in R (Figure S1A).

Lists of candidate target genes for all factors have been generated by peak-to-gene mapping using the same uniform parameters for all studies, which may be different from the parameters used in the individual original studies. The requirements used are as follows: if a TF peak is within 100 bp of the TSS, it is associated with that gene alone; all other peaks can be associated with up to two genes, by examining 50 kb of flanking sequence on either side of the peak.

The lists of candidate targets have been analyzed by unsupervised hierarchical clustering using Pearson correlation coefficients, and displayed using the heatmap function in R (Figure S1B). All raw and processed data files used for this study are available for download from http://bioinformatics.cscr.cam.ac.uk/ES_Cell_ChIP-seq_compendium.html. The gene tracks have been generated by loading the density maps of the indicated factors into the UCSC genome browser as custom tracks.

The ChIP-seq data used for peaks intersection, target gene intersection, and gene tracks (in Figures 1 and and4)4) are as follows: Esrrb, GSE11431 (GSM288355); Nanog, GSE11724 (GSM307140 and GSM307141); Oct4, GSE11724 (GSM307137); Sox2, GSE11724 (GSM307138 and GSM307139); Tcf3, GSE11724 (GSM307142 and GSM307143).

Embryonic Stem Cell Culture

ESCs were cultured without feeders on plastic coated with 0.1% gelatine (Sigma, cat. G1890) and replated every 3 days at a split ratio of 1 in 10 following dissociation with Accutase (PAA, cat. L11-007). Cells were cultured either in the GMEM (Sigma, cat. G5154) supplemented with 10% FCS (Sigma, cat. F7524), 100 μM 2-mercaptoethanol (Sigma, cat. M7522), 1× MEM nonessential amino acids (Invitrogen, cat. 1140-036), 2 mM L-glutamine, 1 mM sodium pyruvate (both from Invitrogen), and 100 units/ml LIF, or in the serum-free media N2B27 (NDiff N2B27 base medium, Stem Cell Sciences Ltd, cat. SCS-SF- NB-02) supplemented, as indicated, with small-molecule inhibitors PD (1 μM, PD0325901) and CH (3 μM, CHIR99021) and LIF prepared in-house. Colony forming assays were carried out by plating 600 ESCs per well on plates coated with laminin (Sigma, cat. L2020). Plates were fixed and stained for AP (Sigma, cat. 86R-1KT) according to the manufacturer’s protocol. Plates were scanned using a CellCelector (Aviso) and scored manually.

Gene Expression Analysis by Quantitative PCR with Reverse Transcription

Total RNA was isolated using the RNeasy Kit (QIAGEN) and complementary DNA (cDNA) was made from 500 ng using SuperScriptIII (Invitrogen) and oligo-dT primers. For real-time PCR, we used TaqMan Fast Universal Master Mix and TaqMan probes (Applied Biosystems) or the Universal Probe Library (UPL, Roche) system. In Figures 4F, 4J, and 4K, we used SYBR green system. Primers and UPL probe numbers are detailed in Table S2. Technical replicates were carried out for all quantitative PCR reactions. An endogenous control (Gapdh, glyceraldehyde-3-phosphate dehydrogenase, Applied Biosystems 4352339E) was used to normalize expression.

RNAi Experiments

siRNAs were transfected at a final concentration of 40 nM using Dharmafect 1 (Dharmacon, cat. T-2001-01), following the protocol for reverse transfection. For a 12-well plate (4cm2), we used 2 μl of transfection reagent, 2 μl of 20 microM siRNA solution, and 30,000 ESCs in 1 ml of N2B27 medium. The medium was changed after overnight incubation; 48 hr after transfection, the cells were analyzed as indicated. See Table S3 for sequences of the siRNAs used.

Flow Cytometry

After treatment with Accutase, live ESCs were resuspended in PBS with 3% FCS and ToPro-3 (Invitrogen) was added at a concentration of 0.05 nM to detect dead cells. Flow cytometry analyses were performed using a Dako Cytomation CyAn ADP high-performance cytometer with Summit software.

Essrb Gene Targeting

LoxP sites were inserted to flank exon2 of Esrrb using a promoter-trap vector containing an FRT flanked SA-IRES-βgeo selection cassette (Figure S4). Subsequent transfection with the FLPe expression vector removed this cassette to generate Esrrbfl/+ ESCs. Targeting was confirmed by genomic PCR. This strategy was repeated for the second allele to create homozygous Esrrbfl/fl ESCs. To enable efficient conditional deletion, we stably transfected cells with an expression vector for the tamoxifen-inducible form of Cre (MerCreMer). Null cells were obtained by treatment with 200ng/ml of Tamoxifen for 3 days and expanded from single cells in medium containing serum and LIF.

Mouse studies were carried out in a designated facility under licenses granted by the UK Home Office.

Acknowledgments

We thank Jason Wray for advice and discussion. We are grateful to Brad Merrill for generously providing Tcf3 null ESCs. Tüzer Kalkan, Jörg Betschinger, and Ge Guo provided reagents. Bill Mansfield, Charles Dumeau, Peter Humphreys, Rachael Walker, and Margaret McLeish provided specialist technical support. G.M. and T.S. performed and interpreted ESC experiments; E.T., A.J., and R.H. generated the ChIP-seq compendium; S.O. produced chimaeras; and B.G., H.N., and A.S. supervised the study and wrote the paper with G.M. This study was funded by the Medical Research Council of the UK, the Wellcome Trust, and RIKEN, and was facilitated by a Biotechnology and Biological Sciences Research Council Japan Partnering Award to A.S. and H.N. G.M. is a Human Frontier Science Program fellow, A.J. is an EMBO long term fellow, R.H. is supported by the NIHR Cambridge Biomedical Research Campus, and A.S. is a Medical Research Council Professor.

Supplemental Information

Document S1. Figures S1–S6, Tables S1–S4, and Supplemental Experimental Procedures:
Document S2. Table S5:

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