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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell. Author manuscript; available in PMC Oct 28, 2012.
Published in final edited form as:
PMCID: PMC3212730
NIHMSID: NIHMS331001

Master Transcription Factors Determine Cell-Type-Specific Responses to TGF-β Signaling

SUMMARY

Transforming growth factor beta (TGF-β) signaling, mediated through the transcription factors Smad2 and Smad3 (Smad2/3), directs different responses in different cell types. Here we report that Smad3 co-occupies the genome with cell-type-specific master transcription factors. Thus, Smad3 occupies the genome with Oct4 in embryonic stem (ES) cells, Myod1 in myotubes, and PU.1 in pro-B cells. We find that these master transcription factors are required for Smad3 occupancy and that TGF-β signaling largely affects the genes bound by the master transcription factors. Furthermore, we show that induction of Myod1 in non-muscle cells is sufficient to re-direct Smad3 to Myod1 sites. We conclude that cell-type-specific master transcription factors determine the genes bound by Smad2/3 and are thus responsible for orchestrating the cell-type-specific effects of TGF-β signaling.

INTRODUCTION

Signaling pathways allow cells to respond to their environment and frequently act by regulating gene expression. The terminal components of these pathways tend to occupy the genes they regulate (Darnell et al., 1994; Jarriault et al., 1995; Kim et al., 1997; Molenaar et al., 1996; Pokholok et al., 2006; Sen and Baltimore, 1986) and modulate gene expression through activities that include recruitment of co-activators and chromatin remodeling machinery, modification of transcription factors, and activation of transcription (Clevers, 2006; Guasconi and Puri, 2009; Kopan and Ilagan, 2009; Massague et al., 2005; Natoli, 2009; O’Shea et al., 2002). In this way external signals produce transcriptional responses that allow cells to respond to cues from their environment.

Signaling pathways are required throughout development and play essential roles in numerous disease processes. It is notable that any one signaling pathway can direct very different responses in different cell types (Clevers, 2006; Guasconi and Puri, 2009; Kopan and Ilagan, 2009; Massague et al., 2005; O’Shea et al., 2002). How an extracellular signal produces cell-type-specific responses remains poorly understood, but these diverse responses govern nearly every aspect of cell physiology from growth to differentiation and death.

TGF-β signaling regulates processes that include stem cell maintenance, cell proliferation, differentiation, and apoptosis (Massague et al., 2005; Ross and Hill, 2008). Activation of the TGF-β receptor by TGF-β, Activin or Nodal leads to phosphorylation of the transcription factors Smad2 and Smad3 (Smad2/3). Once phosphorylated, these transcription factors accumulate in the nucleus in association with Smad4 (Massague et al., 2005; Ross and Hill, 2008). Smad3 and the less common isoform of Smad2 can both bind DNA directly through interaction with the Smad binding element (SBE) (Dennler et al., 1998; Shi et al., 1998; Zawel et al., 1998). However, due to the low affinity of this interaction, Smad transcription factors must interact with additional transcription factors in order to form stable complexes with DNA (Massague et al., 2005; Ross and Hill, 2008; Shi et al., 1998).

We mapped genome-wide binding of Smad3 in ES cells, myotubes and pro-B cells and found that a small set of cell-type-specific master transcription factors direct Smad3 to cell-type-specific binding sites and determine cell-type-specific responses to TGF-β signaling. These results are surprising as previous work has suggested that many different transcription factors in a cell are each responsible for directing Smad3 binding to a small number of sites, and it is the sum of these interactions that determines the cell-type-specific response to TGF-β signaling (Massague and Gomis, 2006; Massague et al., 2005; Seoane et al., 2004). Furthermore, we find that master transcription factors help direct Smad3 binding by establishing open chromatin that contains SBEs, allowing Smad3 to bind DNA and form a physical complex with the master transcription factors.

RESULTS

SMAD3 co-occupies the genome with OCT4 in human and murine ES cells

We first investigated whether SMAD3 is directed to DNA sites co-occupied by the master transcription factor OCT4 in human (h) ES cells, where activation of SMAD2/3 is required to maintain hES cell identity (Beattie et al., 2005; James et al., 2005; Vallier et al., 2005; Vallier et al., 2009; Xu et al., 2008). ChIP-seq was performed to determine the genome-wide targets of SMAD3 and OCT4 in hES cells. If SMAD3 is directed to DNA targets by the ES cell-specific master transcription factor OCT4, we would expect to observe that SMAD3 and OCT4 bind to the same sets of genes across the genome. Analysis of the gene targets showed that SMAD3 and OCT4 were predominantly associated with active genes and had a similar distribution to each other across active, silent and bivalent genes (Fig 1A).

Figure 1
SMAD3 and OCT4 co-occupy the genome in ES cells

If SMAD3 is directed to target genes by OCT4, then SMAD3 should co-occupy DNA sites with OCT4, whereas if SMAD3 is directed to target genes by many different transcription factors, the global binding of SMAD3 and OCT4 should not be coincident. Examination of SMAD3 and OCT4 binding at individual hES cell genes revealed that SMAD3 occupies sites with OCT4; for example the two transcription factors bind the same sites at POU5F1 and LEFTY1 (Fig 1B). Furthermore, this binding pattern occurs throughout the genome (Fig 1C, Table S1). Indeed, over 80% of the 1000 highest-confidence SMAD3-bound sites are co-occupied by OCT4 (p < 1e-290, Table S1, S2).

It is possible that SMAD3 co-occupies the genome with many different factors, only one of which is OCT4. If this were true, then many different DNA binding motifs would be present at sites bound by SMAD3. However, if SMAD3 binding is most highly associated with OCT4, the OCT4 motif should be most enriched at SMAD3 sites. De novo motif discovery was performed (Bailey and Elkan, 1994) on sites bound by SMAD3 and sites bound by OCT4. The most enriched motif identifiable at sites bound by each factor was indeed the OCT4 motif (Fig 1D, Fig S1). The canonical SBE was not found by de novo motif discovery at sites bound by SMAD3 (Fig 1D, bottom), which likely reflects the difficulty in determining enrichment of a four-nucleotide motif. However, if SMAD3 and OCT4 co-occupy DNA, then SBEs should be enriched at SMAD3 and OCT4 sites. We scanned the DNA sequence around the sites bound by each factor and found that SBEs are enriched at sites bound by SMAD3 and sites bound by OCT4 (Fig 1E). These results are consistent with the model that in hES cells, SMAD3 predominantly occupies sites with OCT4 throughout the genome.

While human and murine (m) ES cells respond differently to TGF-β signaling (James et al., 2005), both cell types require the master transcription factor Oct4 to maintain cell identity (Chambers and Smith, 2004), so we investigated whether Smad3 also co-occupies sites with Oct4 in mES cells. Global analysis of Smad3 and Oct4 binding in mES cells revealed that these transcription factors occupy the same classes of genes as each other and as their counterparts in hES cells (Fig 1F). Further analysis of Oct4 and Smad3 binding revealed that the two transcription factors bind to the same sites at individual genes (Fig 1G) and throughout the genome (Fig 1H, p <1e-300). Furthermore, the Oct4 DNA motif was the most enriched motif at sites bound by Smad3 and sites bound by Oct4 (Fig 1I) and these sites were also enriched for SBEs (Fig 1J). These results show that Smad3 co-occupies the genome with the master transcription factor Oct4 in both human and murine ES cells (Fig 1K).

Smad2/3 has been shown to interact with many different transcription factors (Massague et al., 2005; Ross and Hill, 2008), and the analysis described thus far does not eliminate the possibility that Smad3 co-occupies sites with many different transcription factors other than Oct4 in ES cells. To determine if Smad3 co-occupies sites with other characterized transcription factors, we analyzed ChIP-seq data for six different factors in mES cells. Examination of binding data at individual genes such as that encoding Max (Fig 2A) revealed that Smad3 co-occupied sites with Oct4 as well as Sox2 and Nanog, which are key ES cell transcription factors that occupy the genome together (Boyer et al., 2005; Chen et al., 2008; Marson et al., 2008). In contrast, Smad3 did not occupy sites bound by Ronin, Zfx, or c-Myc, despite previous evidence that c-Myc can interact with Smad2/3 in other cell types (Chen et al., 2008; Dejosez et al., 2010; Feng et al., 2002). Genome-wide analysis confirmed that Smad3 tended to co-occupy sites with Oct4, Sox2 and Nanog but rarely co-occupied sites with Ronin, Zfx, or c-Myc (Fig 2B, FigS2A, Table S1). We further quantified the preference for Smad3 to co-occupy sites with these factors by calculating the percent of Smad3 sites co-occupied by each factor (Fig 2C) and determining the distance from each Smad3 site to the nearest binding site for each transcription factor (Fig 2D). Over 90% of the top 1000 Smad3 sites were co-occupied by Oct4, Sox2 and Nanog individually while only a small fraction of Smad3 sites were co-occupied by Ronin, Zfx, or c-Myc (Fig 2C). Furthermore, the majority of sites occupied by Ronin, Zfx or c-Myc are located far from sites occupied by Smad3 (Fig 2D). Thus in mES cells, Smad3 tends to co-occupy the genome specifically at sites bound by Oct4, Sox2 and Nanog.

Figure 2
Smad3 co-occupies the genome with the ES cell core master transcription factors

Oct4 recruits Smad3

If Oct4 directs Smad3 to sites in ES cells then we would expect Oct4 and Smad3 to co-occupy the same sites at the same time. We performed ChIP for Smad3 in mES cells, followed by Western analysis to detect Oct4, which revealed that Smad3 and Oct4 were contained in the same cross-linked complex (Fig 3A). In addition, co-immunoprecipitation (Co-IP) experiments showed that Smad3 and Oct4 form a physical complex that is dependent on TGF-β signaling (Fig 3B). We then performed ChIP for Oct4 followed by re-ChIP for Smad3 (ChIP-re-ChIP) to determine if Smad3 and Oct4 occupied binding sites in mES cells simultaneously (Fig 3C). The results showed that Oct4 and Smad3 did temporally co-occupy DNA sites. We then asked if Oct4 and Smad3 co-occupy the Lefty1 enhancer by electrophoretic mobility shift analysis (EMSA); antibodies against both Smad3 and Oct4 caused a supershift of the complex suggesting that both Oct4 and Smad3 were part of the complex bound to the Lefty1 enhancer (Fig 3D).

Figure 3
Oct4 recruits Smad3

To determine if Oct4 is required for recruiting Smad3 to DNA sites bound by Oct4 in ES cells, we inhibited Oct4 expression in ZHBTc4 mES cells with doxycycline (dox) treatment for 24 hr (Niwa et al., 2000). We found that loss of Oct4 did not affect the levels of Smad3 protein (Fig 3E). However, loss of Oct4 expression did result in a dramatic reduction in Smad3 occupancy at key genes normally co-occupied by Oct4 and Smad3 (Fig 3F). Furthermore, loss of Oct4 resulted in reduced responsiveness to TGF-β signaling at genes normally co-occupied by Oct4 and Smad3 (Fig 3G).

Oct4 might recruit Smad3 through a direct interaction and/or by inducing a more open chromatin state to make Smad binding elements (SBEs) available for Smad3 binding. Our evidence suggests that Oct4 and Smad3 are associated in some fashion (Fig 3A–F), although analysis of the positions of Oct4 and Smad3 binding peaks indicates that these two proteins do not interact in a unique and direct manner such as that observed for Oct4 and Sox2 (Fig 3H). Analysis of nucleosome occupancy at sites bound by Oct4 revealed that these sites are relatively depleted of nucleosomes (Fig3I). These results are consistent with the possibility that Oct4 recruits Smad3 to adjacent SBEs that are available due to nucleosome depletion.

Smad3 co-occupies the genome with master transcription factors in various cell types

We next investigated whether Smad3 co-occupies the genome of additional cell types with the master transcription factors of those cell types. We performed ChIP-seq analysis for Myod1 in myotubes (Davis et al., 1987; Tapscott, 2005) and PU.1 in pro-B cells (DeKoter and Singh, 2000; Nutt and Kee, 2007) and found that sites bound by Oct4, Myod1 and PU.1 were largely unique (Fig 4A, Table S1). We also performed ChIP-seq analysis of Smad3 binding in myotubes and pro-B cells and found that Smad3 also tends to bind unique sites in these different cell types (Fig 4B).

Figure 4
Smad3 co-occupies DNA with cell-type-specific master transcription factors

If Smad3 is recruited to DNA sites by master transcription factors, then we would expect Smad3 sites in each cell type to be occupied by the master transcription factor found in that cell type. We initially analyzed the top 1000 bound sites for Smad3 in mES cells, myotubes and pro-B cells and asked if those sites were occupied by master transcription factors (Fig 4C, S3A). Indeed, Oct4 occupied sites with Smad3 in mES cells but did not occupy sites bound by Smad3 in myotubes or pro-B cells. Myod1 occupied sites with Smad3 in myotubes, and PU.1 occupied sites with Smad3 in pro-B cells. We next analyzed all Smad3 sites in each cell type, which confirmed that Smad3 tends to co-occupy sites with the cell-type-specific master transcription factors (Fig S3B).

The cell-type-specific association of master transcription factors and Smad3 was striking at individual genes (Fig 4D). For example, Smad3 and Oct4 co-occupied sites at the gene encoding Sox2 in mES cells, but these sites were not occupied by Smad3 or Myod1 in myotubes or Smad3 or PU.1 in pro-B cells. The gene encoding Adora1 was uniquely co-occupied by Smad3 and Myod1 in myotubes, and the gene encoding Vpreb2 was uniquely occupied by Smad3 and PU.1 in proB cells. Genome-wide analysis confirmed that Smad3 occupied unique sites with Oct4 in mES cells, Myod1 in myotubes and PU.1 in pro-B cells (Fig 4E). In addition, motif discovery revealed enrichment of the Myod1 motif in Smad3-bound regions of myotubes and the PU.1 motif in Smad3-bound regions of pro-B cells (Fig 4F), further supporting the conclusion that Smad3 co-occupies the genome with cell-type-specific master transcription factors.

We next investigated whether interactions could be detected between Smad3 and the master transcription factors of each cell type and whether Smad3 binding depends on Myod1 in myotubes and PU.1 in pro-B cells. We found that Smad3 immunoprecipitated with Oct4 in mES cells, Myod1 in myotubes and PU.1 in pro-B cells (Fig 4G). We next asked if knockdown of Myod1 affected binding of Smad3 in myotubes. Myoblasts were transfected with Myod1 siRNA, and a 52% knockdown of Myod1 was observed after 48 hr of myotube differentiation. The reduction in Myod1 was associated with an intermediate level of myotube differentiation (data not shown) and resulted in decreased Smad3 occupancy at sites normally co-occupied with Myod1 (Fig S3C). Similarly, deletion of PU.1 and the functionally redundant protein spib (DeKoter et al., 2002) in pro-B cells resulted in decreased Smad3 occupancy at sites co-occupied by PU.1 (Fig S3D). Together these results suggest that master transcription factors are required for wild-type levels of Smad3 recruitment.

If Smad3 binds DNA at sites with cell-type-specific master transcription factors, then SBEs should be enriched at these sites. We scanned Oct4, Myod1 and PU.1 sites and found that these sites were each enriched for SBEs (Fig 4H). This result indicates that Smad3 binds a unique subset of SBEs in each cell type, and that this subset is associated with sites bound by the cell-type-specific master transcription factors.

Finally, we investigated whether nucleosome depletion occurs at sites bound by cell-type-specific master transcription factors and Smad3, as increased accessibility of SBEs in these regions may contribute to Smad3 binding. Genome-wide ChIP-seq analysis of histone H3 occupancy revealed that master transcription factors occupied cell-type-specific regions that were relatively depleted of nucleosomes (Fig S3E). In addition, Smad3 and the master transcription factors co-occupied unique nucleosome-depleted regions in each cell type (Fig 4I). For example, in mES cells Oct4 and Smad3 co-occupied sites that were relatively depleted of nucleosomes (Fig 4I, left, blue), while sites occupied by Smad3 and Myod1 in myotubes (purple), or Smad3 and PU.1 in pro-B cells (green) were not associated with significant nucleosome depletion in mES cells. Nucleosome depletion was unchanged when TGF-β signaling was inhibited (Fig S3F) suggesting that Smad3 did not affect nucleosome occupancy but was directed to sites that were already depleted of nucleosomes. Thus, Smad3 appears to bind unique SBEs in nucleosome-depleted regions adjacent to master transcription factors in each cell type (Fig 4J).

Smad3 can occupy different cell-type-specific enhancers at the same gene

Master transcription factors can bind cell-type-specific enhancers (Heintzman et al., 2009). Thus, we would expect that Smad3 could occupy different enhancers of the same gene in different cell types. We analyzed the intersection of genes bound by Smad3 in mES cells, myotubes and pro-B cells. While only 1% of the Smad3 binding sites were occupied in more than one cell type (Fig 4B), 13% of genes bound by Smad3 were occupied by Smad3 in more than one cell type (Fig 5A). Analysis of individual genes showed that Smad3 could occupy the same gene in different cell types but usually did so by co-occupying cell-type-specific enhancers with the cell-type-specific master transcription factor (Fig 5B). For example, Id3 was occupied by Oct4 in mES cells and PU.1 in pro-B cells. However, Oct4 and PU.1 occupied Id3 at different sites, and Smad3 co-occupied sites with the cell-type-specific master regulators. Thus, for the small fraction of genes that appear to be targeted by TGF-β signaling in multiple cell types, these genes tend to be bound by Smad3 at different enhancers (Fig 5C).

Figure 5
Smad3 can bind the same gene at different sites in different cell types

TGF-β signaling regulates genes bound by master transcription factors

If Smad3 is directed to sites occupied by Oct4 then genes bound by Oct4 should be modulated by TGF-β signaling. Genome wide expression analysis was performed on mES cells under standard conditions and after treatment with the TGF-β inhibitor SB431542 for 24 hr (James et al., 2005; Ross et al., 2006; Vallier et al., 2005; Xu et al., 2008) to identify genes that are modulated by TGF-β signaling. We found that genes bound by Oct4, Sox2, Nanog, or Smad3 were affected by a block in TGF-β signaling (Fig 6A, B, Table S3, S4). As a control we also analyzed genes bound by Zfx, a transcription factor that binds a similar number of genes to Oct4 (Table S1, S3) but does not co-occupy sites with Smad3. In contrast to genes bound by Oct4, genes bound by Zfx were not affected by TGF-β signaling (Fig 6B, bottom).

Figure 6
TGF-β signaling regulates genes bound by master transcription factors

To determine if key ES cell genes bound by Smad3 and Oct4 are direct targets of TGF-β signaling, we next asked if activation of TGF-β signaling resulted in rapid recruitment of transcriptional co-activators (Ross et al., 2006). mES cells were treated with SB431542 for 24 hr before they were washed and re-treated with fresh SB431542 or Activin to activate the TGF-β pathway. ChIP was performed for the histone acetyltransferase p300 one hr after activation and showed that TGF-β signaling led to rapid recruitment of p300 to genes co-occupied by Oct4 and Smad3 (Fig 6C).

If master transcription factors direct Smad3 to different targets in different cell types we would expect that the genes regulated by TGF-β signaling are different in different cell types. Genome wide expression analysis was performed on myotubes and pro-B cells to identify genes that changed in expression after treatment with TGF-β for 12 hr. Expression changes in myotubes and pro-B cells were also compared to changes in mES cells after inhibition of TGF-β signaling for 24 hr. As expected, a largely unique set of genes was found to be affected in each cell type (Fig 6D, left). We next asked if the set of genes bound by Smad3 or the master transcription factors in each cell type were associated with the set of genes affected by TGF-β signaling in each cell type. In each case we found that only genes bound by the cell-type-specific master transcription factor or cell-type-specific Smad3 were affected by TGF-β signaling (Fig 6D, right), indicating that TGF-β signaling regulates genes bound by cell-type-specific master transcription factors.

Induction of Myod1 redirects Smad3 binding in mES cells

If master transcription factors direct Smad3 to their sites of occupancy, then induction of a master transcription factor that is not normally expressed in a specific cell type should direct Smad3 to the unique sites occupied by the induced master transcription factor. To test this idea, we induced expression of Myod1 in mES cells (Fig 7A) (Nishiyama et al., 2009). ChIP-seq was performed for Smad3 in mES cells that were maintained for 5 days in mES cell culture conditions with and without induction of Myod1. Despite expression of Myod1, mES cells maintained expression of Oct4 (Fig 7B). However, expression of Myod1 was sufficient to direct a fraction of Smad3 to sites occupied by Myod1 (Fig 7C). Inspection of ChIP-seq profiles showed that Smad3 continued to occupy sites with Oct4 in the setting of Myod1 expression (Fig 7D), while also occupying new sites with Myod1 (Fig 7E).

Figure 7
Myod1 expression redirects Smad3 occupancy in mES cells

DISCUSSION

Transcription factors bind specific DNA sequences and regulate gene expression (Ptashne, 1988), and master transcription factors are required for establishment, maintenance and even reprogramming of cell identity (Takahashi and Yamanaka, 2006; Feng et al., 2008; Lassar et al., 1986; Seale et al., 2008; Zhou et al., 2008; Graf and Enver, 2009). Here we report that master transcription factors also are responsible for directing the gene targets of TGF-β signaling and thus determine the cell-type-specific effects of TGF-β signaling.

This conclusion is supported by the finding that Smad3 co-occupies the genome with Oct4 in ES cells, Myod1 in myotubes and PU.1 in pro-B cells. In addition, Smad3 interacts with these master transcription factors and binds accessible DNA sites adjacent to those bound by master transcription factors. Furthermore, the expression of genes bound by master transcription factors is modulated by TGF-β signaling. Finally, expression of Myod1 in non-muscle cells can re-direct Smad3 to the new sites occupied by Myod1.

If many different transcription factors can interact with Smad2/3 (Chen et al., 2002; Chen et al., 1996; Chen et al., 1997; Germain et al., 2000; Liu et al., 1997; Massague et al., 2005; Seoane, 2004), why do master transcription factors have such a profound effect in determining the genes regulated by TGF-β signaling in each cell type? The answer may lie in the relative concentration of different transcription factors, as master transcription factors tend to be expressed at high levels compared to other transcription factors (Young, 2011). Thus, the abundance of cell-type-specific master transcription factors may allow them to dominate the competition for interactions with Smad2/3. Recent work in mES cells has also suggested that the Wnt, LIF and BMP pathways target sites bound by mES cell master transcription factors (Chen et al., 2008; Cole et al., 2008; Young, 2011), and our findings now suggest that cell-type-specific master transcription factors may operate in many different cell types to determine the gene targets of signaling pathways.

TGF-β signaling, through activation of Smad2 and Smad3, plays an essential role in normal development and tissue homeostasis as well as in human diseases from cancer to autoimmunity to cirrhosis (Friedman, 2008; Li and Flavell, 2008; Massague et al., 2005; Padua and Massague, 2009; Wandzioch and Zaret, 2009). It is therefore critical to understand how activation of Smad2/3 can lead to such diverse cellular responses. Our findings reveal that the cell-type-specific effects of TGF-β signaling are determined in large part by the interaction of Smad2/3 proteins with master transcription factors that specify and maintain cell identity. It is through this mechanism that TGF-β signaling is tailored to modulate genes that are most relevant to cell identity, which may explain why aberrations in this pathway can have such profound effects in a range of human diseases.

EXPERIMENTAL PROCEDURES

Cell culture

hES (BGO3) cells were maintained as previously described (Ludwig et al., 2006) using mTESR1 media (Stem Cell Technologies), which contains TGF-β. mES cells were cultured as previously described (Marson et al., 2008). mES cells were maintained on murine embryonic fibroblast (MEF) feeder cells and then passaged 2x off feeders prior to analysis. Exogenous Activin was not added to mES cell cultures unless stated. When indicated, mES cells were treated with SB431542 (10 μM) for 24 hr to inhibit TGF-β signaling. ZHBTc4 mES cells were treated with dox for 24 hr to repress Oct4 expression as previously described (Niwa et al., 2000). Myod1 was induced in ES cells by dox withdrawal as previously described (Nishiyama et al., 2009). Analysis of Myod1 and PU.1 binding was performed under standard culture conditions and analysis of Smad3 binding was performed after treatment with TGF-β in myotubes and pro-B cells. See Extended Experimental Procedures for additional details.

Chromatin Immunprecipitation (ChIP)

ChIP and ChIP coupled with massively parallel sequencing (ChIP-seq) was performed as previously described (Marson et al., 2008). Analysis of H3 occupancy was performed by normalizing the average H3 density across all sites co-occupied by the indicated transcription factors. Antibodies and additional information can be found in the Extended Experimental Procedures.

ChIP-seq Analysis

Analysis methods were derived from previously published methods (Marson et al., 2008). Briefly, the number of extended reads were calculated in bins across the genome and bins that contained statistically significant ChIP-seq enrichment were identified by comparison to a Poissonian background model. A p-value cutoff of 1e-9 was used for all datasets except for c-Myc and Ronin where a cutoff of 1e-5 was used in order to analyze comparable numbers of bound sites. Refer to the Extended Experimental Procedures for details.

Mircroarray Analysis

Analysis was performed using Agilent Whole-Mouse Genome Microarrays (Agilent, G4122F) as previously described (Cole et al., 2008). A gene was determined to be significantly affected by TGF-β signaling if its expression level changed by at least 1.5 fold with a p-value less than or equal to 0.05. Refer to the Extended Experimental Procedures for details.

Co-immunoprecipitation (Co-IP)

Co-IP experiments using nuclear extracts were performed as previously described (Kagey et al., 2010). Refer to the Extended Experimental Procedures for details.

Electrophoretic mobility shift (EMSA)

A 40 bp sequence containing adjacent Smad and Oct4 binding sites in the murine Lefty1 enhancer was labeled with [γ-32P]-ATP and incubated with nuclear extract from mES cells treated with Activin for 1 hr. Competitor DNA was used at 100-fold excess to labeled DNA. Supershift was performed by incubating the assembled complex with antibodies against Smad3 and Oct4. Refer to Extended Experimental Procedures for additional details.

Previously published ChIP-seq Datasets used in this study

The following previously published datasets were used: Oct4, Nanog, Sox2 in mES cells (Marson et al., 2008), c-Myc and Zfx in mES cells (Chen et al., 2008) and Ronin in mES cells (Dejosez et al., 2010).

Supplementary Material

04

05

Acknowledgments

We are grateful to M. S. H. Ko for the gift of ES[MC1R(20)]:tetMyod1 cells, D. Wotton for the gift of Smad2/3 antibody and H. Singh for providing the 38B9 cells with permission from N. Rosenberg. We thank T. Lee and L. Lawton for helpful discussions, J. Love, J-A. Kwon, V. Dhanapal, S. Gupta and T. Volkert for Illumina sequencing and microarray preparation, G. Frampton, and B. Yuan for help with computational analysis, and T. DiCesare for help with graphics. We also thank A. Blais for assistance with siRNA transfection. This work was supported by fellowships from the American Gastroenterological Association and by NIH grant DK090122 (A.C.M), the Canadian Institutes of Health Research (S.B.) and by NIH grant HG002668 (R.A.Y.).

Footnotes

ACCESSION NUMBERS

The ChIP-seq and microarray data are deposited in GEO under accession numbers GSE21621 and GSE23830.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures, three figures and four tables.

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