conditions. ChIP samples from each condition were sequenced to a similar depth of 10-13 million tags. Interestingly the number and magnitude of pSmad2 binding events did not correspond to the 2- fold up- or downregulation of pSmad2 in ES cells under Activin and SB treatments. In fact the greatest number of binding peaks (7423) occurred in the control DMSO condition that maintains self-renewal and pluripotency of ES cells (Figure 4A). When homeostatic Nodal/Activin signaling was perturbed by Activin and SB treatments, the number of binding events decreased to 5094 and 4859 respectively suggesting that any change in the levels of endogenous pSmad2 from the ES cell undifferentiated condition also caused a dynamic change in pSmad2 binding across the ES cell genome. The lower numbers may also be reflective of the transition where pSmad2 is dissociating from former target genes and establishing the recruitment of new genes. This was further supported by the percentage of overlapping peaks that were common to the 3 treatments being relatively small at 10.3% with a significantly larger number of unique peaks appearing in specific treatments (37.25% in DMSO, 20.44% in SB and 19.5% in Activin out of 12979 total peaks in the union). A previous study has profiled Smad2/3 binding sites using promoter arrays in human keratinocytes [31]. However, reporter assays on Nodal/Activin responsive target genes such as Lefty1/2, Nodal [36,51] and Pitx2 [35] suggest that Smad2/3 may also regulate DNA elements in the introns rather than at the promoter region. Consistent with the reporter assay studies, our ChIP-Seq data showed that the majority of pSmad2 binding (Figure 4B) occurs in introns (,30%) with only a minority of sites in the proximal promoters (,10%). Furthermore, there was a significant shift in pSmad2 binding from the distal 59 and 39 regions towards the promoters of genes in the SB treatment compared to DMSO and Activin (Figure 4B). Examination of binding specifically in the promoter region showed a clear preference for pSmad2 to associate in the +/2600 bp proximal region of transcriptional start sites (TSS) with a steady decrease in binding further away from the TSS (Figure 4C). In addition, the increase in number of pSmad2 binding peaks during low signaling with the SB treatment can be confirmed in the promoter region both up- and downstream of the TSS. In conclusion, pSmad2 binding, similar to the changes in gene expression identified by microarrays, also demonstrates binding to distinct subsets of genomic locations at different signaling levels. Phospho-Smad2 Binding Is Positively Associated with Transcriptional Activity and Its Binding Preference for Specific DNA Motifs Changes with Signaling Levels We next examined the relationship governing the degree of pSmad2 binding and the level of transcription across the genome (Figure 5A). In all 3 conditions, a clear trend emerges suggesting that more pSmad2 binding drives higher levels of gene expression. However, the possibility that pSmad2 is not driving expression but preferentially associates with more transcriptionally active genes and open chromatin cannot be excluded. To distinguish between the 2 possibilities, we examined the trend between pSmad2 binding events and differential gene expression from the micro- array analysis in the 3 signaling conditions. Indeed, a significant majority (64.2%) of microarray target genes had pSmad2 binding within +/250 kb and all displayed .1.5 fold change in binding in each signaling condition or had different number of binding events or changed the location of pSmad2 binding (Table S1) suggesting that the pattern of gene expression was indeed dynamically driven by pSmad2-DNA interactions. To account for how pSmad2 is able to switch binding locations during differential Nodal/Activin signaling, we examined its Nodal/Activin Signaling in ES Cell Fate Decisions preference for specific DNA motifs under each condition. It is known that Smad2/3 are able to bind directly the basic CAGA motif and at the same time they possess a number of partner transcription factors that modulate the specificity and strength of binding. Here we see that there is strong pSmad2 association with the basic CAGA SBE specifically in the Activin treatment (Figure 5B). This was also confirmed when we examined the strong CAGAC canonical SBE as defined by the TRANSFAC PWM database which also appears with high frequency at the center of pSmad2 ChIP-seq peaks in the Activin treatment and not in DMSO, SB or the random mouse genome sequence control (Figure 5C). This suggests that both CAGA and CAGAC displayed graded pSmad2 binding that varied with the signaling level and were preferentially bound in the Activin condition. To compare the contribution of CAGA against non-CAGA sequences towards pSmad2 binding, the top 10 de novo motifs in each condition were identified using the Weeder program (Figure 5D). Motifs that occurred with significant frequency but were not enriched in the center of pSmad2 ChIP-Seq peaks were excluded to remove the influence of comotifs around the peaks. A number of non-CAGA motifs that occurred with similar or greater frequency than CAGA were isolated. Interestingly, these de novo motifs also showed a graded effect on pSmad2 association similar to the CAGA SBE. Other non-CAGA motifs were preferentially bound by pSmad2 only in the DMSO and SB condition and depleted during the high signaling Activin condition. This suggested that while CAGA binding was significant, binding to non-CAGA sequences accounted for the majority of pSmad2 association within the ES cell genome suggesting that this was primarily mediated by transcriptional co-partners. Indeed, when the top consensus motifs in the center of all ChIP-seq peaks in each signaling condition and in the combined dataset were studied (Figure 5E), there was a strong enrichment for motifs belonging to transcription partners such as E2f and Ap1 instead of Smad binding CAGA boxes. To confirm the association of the putative transcriptional cofactors and establish their identity, we expanded the analysis to TRANSFAC co-motifs occurring within +/2 1 kb range of pSmad2 binding sites (Table S2). A large number of known pSmad2 transcription partners such as Ap1, Sp1 and E2f are indeed associated within the vicinity of pSmad2 peaks regardless of the level of Nodal/Activin signaling. However, there were additional co-motifs bound by transcription factors such as Oct4, Stat3 and p53 that only appear prominently in Activin treatments and Hes1, Lrf and Plzf appearing in SB. This is supportive of an exchange of transcription partners in association with pSmad2 that was governed by the level of Nodal/Activin signaling which was likely to be responsible for the change in specificity of pSmad2 transcriptional complexes for target gene subsets and their level of expression. Furthermore, while pSmad2 does bind to its own CAGA sequence, transcriptional copartners played a greater role both in binding affinity and specificity of pSmad2 protein complexes for the ES cell genome. Graded, Low Signaling Dominant, High Signaling Dominant, and Multimodal Models of Binding Occur for Phospho-Smad2 Target Genes To investigate the different models of pSmad2 binding during differential Nodal/Activin signaling, we examined the ChIP-Seq profiles including those of the transcriptionally regulated micro- array targets and identified at least 4 types of pSmad2 binding. The first model is that of ‘‘graded’’ target genes that follow closely the changes in Nodal/Activin signaling with increased binding and transcription during high signaling, have moderate response in endogenous baseline signaling and showed a loss of binding with PLoS Genetics | www.plosgenetics.org 8 June 2011 | Volume 7 | Issue 6 | e1002130