Nodal/Activin Signaling in ES Cell Fate Decisions positions using the same control calculations as in (B). Insert shows the logo of the Smad binding motif (TRANSFAC PWM SMAD_Q6_01) containing the CAGAC sequence. (D) Graph showing the sequence and frequency of the top 6-mer motifs in Activin, DMSO and SB. The motifs were defined by the Weeder program using sequences that occur only in the center of ChIP-Seq peaks. The frequency is expressed as fraction of +/–25 bp sequences spanning ChIP-Seq peaks containing the indicated motifs. CAGA containing 6-mers are highlighted in red. (E) 8-mer motifs defined by Weeder program in top 1000 ChIP-Seq peaks ranked by enrichments in +/250 bp intervals for Activin, DMSO, SB and combined conditions correspondingly. Transcription factors binding to sequences similar to these motifs identified by the STAMP program using TRANSFAC PWM are indicated with corresponding p-values. doi:10.1371/journal.pgen.1002130.g005 decreased mRNA levels during signaling repression. This category of pSmad2 binding comprises 23.87% of high confidence ChIP- Seq peaks corresponding to 16.28% of target genes associated within +/250 kb of these peaks (Figure S4). Radil a Rap GTPase effector that plays a role in the migration of neural crest progenitors [52] exemplified such pSmad2 binding and transcrip- tional regulation (Figure 3A, Figure 6A, and Table S1) in the first intron with normalized relative enrichments of 107 tags in Activin compared to 51 in the DMSO control and complete loss of binding indistinguishable from background sequencing levels in SB. The known target gene Pitx2 showed reproducible results with the ChIP data obtained by real-time PCR (Figure S1A) both in terms of the binding location in the intronic enhancer as well as the level of pSmad2 enrichments under graded Nodal/Activin signaling. There were normalized enrichments of up to 201 tags in Activin, 156 in DMSO control and again complete loss of binding in SB (Figure S3A). Interestingly, Pitx2 had 2 graded binding sites, one of which is in the known intronic region and a novel site in the 39 region. The graded binding in the Pitx2 locus also correlates with transcriptional consequences showing strong induction/ inhibition of Pitx2 mRNA levels from 0 to 24 hours (Figure 1A). The two inducers of the mesendoderm cell fate Mixl [53] and Nodal [54] also show evidence of graded pSmad2 binding within 50 kb of the genes (Figure S5A and S5B) suggesting that they may be directly regulated by Nodal/Activin signaling for this purpose. The binding location in the first intron of Nodal also corresponds to the intronic enhancer previously described to be important for left side expression in the early embryo via the Nodal/Activin signaling autoregulatory loop [55,56] confirming that Nodal is itself a direct target. It was also unclear if pSmad2 binding and regulation of target genes only exists in a 1-to-1 relationship or if the same binding sites were capable of regulating multiple targets in the genomic vicinity. While Lefty2 was a known direct target with pSmad2 binding in its promoter region (Figure S1B), for the first time, to our knowledge, we characterized an important pSmad2 transcriptional hotspot in the entire 100 kb Lefty1/2 locus where all the genes within this region were co-regulated by pSmad2 binding suggesting a coordinated mode of transcriptional regulation (Figure S3B). This was further confirmed in the microarray analysis (Figure 3A) demonstrating that Lefty1/2, Pycr2 and Tmem63a display the same pattern of gene expression following a graded response to Nodal/ Activin signaling. This was consistent with the real-time PCR quantification of the pSmad2 pulldown of the Lefty2 promoter (Figure S1B) that corresponds to the most upstream pSmad2 binding site in the Lefty1/2 hotspot as did a time course profiling of Lefty2 expression from 0 to 24 hours (Figure 1B). In the second model of pSmad2 binding, we describe ‘‘low signaling dominant’’ conditions that permit pSmad2 binding but less so under other signaling levels. The Id1/2/3 (Figure 6B, Figure S6, and Table S1) family of transcriptional repressors shows pSmad2 binding to these genes only in the SB treatments and not in Activin or the DMSO control. Statistically, 32.73% of pSmad2 binding sites display this mode of behavior associated with 23.44% of target genes (Figure S4). In contrast, the third model showed the opposite ‘‘high signaling dominant’’ mode of binding such as in the case of 220011C2Rik (Figure 6C and Table S1) where pSmad2 only binds strongly in the Activin condition but to a lesser degree in DMSO or SB also resulting in transcriptional consequences (Figure 3A and Figure S4). Another known component of the mesendodermal cell fate Fgf8 [57] also shows strong pSmad2 binding in the promoter region specifically during high signaling (Figure S5C). Intriguingly, findings in the chick embryo show that Fgf8 also plays important roles in left-right asymmetry where it can be induced by Activin [58] in agreement with our results. In the fourth model which accounts for the regulation of the largest proportion (33.69%) of target genes associated with pSmad2 ChIP-Seq peaks (Figure S4B), the same target gene may be regulated by ‘‘multimodal pSmad2 binding’’ events. Copz2 has two pSmad2 association sites in the intron and promoter region (Figure 6D). The promoter site only binds pSmad2 in the SB condition while the intronic enhancer shows a graded response to the signaling level. In the case of the known target gene Smad7, we have shown that the pSmad2 binding peak in the promoter region is invariant in all 3 signaling conditions (Figure S1C) which could not explain how Smad7 was differentially expressed during graded Nodal/Activin signaling (Figure 1C). In confirmation with these results, the ChIP-Seq data showed the same pSmad2 association on the Smad7 proximal region with no change in binding under all 3 signaling conditions. Surprisingly, we discovered a previously undescribed pSmad2 regulatory element in the distal Smad7 promoter region that binds pSmad2 in a graded manner (Figure S3C) and could account for why Smad7 was responsive to different Nodal/Activin signaling levels. Hence pSmad2 binding in the Smad7 proximal region may not be the dominant regulatory region for Nodal/Activin signaling but may depend instead on the dynamically changing pSmad2 distal promoter element for Smad7 regulation. Indeed the proximal promoter element may be more of a Smad3 regulated region instead of Smad2 as previously described [38]. In conclusion we demonstrate that pSmad2 dependent binding and transcription during graded Nodal/Activin signaling occurs in the ES cell genome in a graded, low or high signaling dominant, many-to-one or one-to-many multimodal manner in relation to the target genes that they regulate. Nodal/Activin Signaling Titrates the Level of Oct4 in ES Cells, Which Is a Direct Target of Phospho-Smad2 Binding in the Promoter Region The mesendodermal and trophectodermal cell fate decisions brought about by graded Nodal/Activin signaling strikingly resemble the ES cell response to a less than 2-fold up- or downregulation of the Oct4 master regulator of stemness in driving differentiation towards similar cell fates [59]. Furthermore, an important mechanism for trophectoderm differentiation depends on the Oct4 repression of Cdx2 expression and the induction of this lineage is thought to be indicative of loss of stemness [60]. We therefore hypothesized that Oct4 may be a key downstream target under Nodal/Activin control during the specification of divergent cell fate decisions and investigated how the pathway may be governing Oct4. PLoS Genetics | www.plosgenetics.org 11 June 2011 | Volume 7 | Issue 6 | e1002130