Nodal/Activin Signaling in ES Cell Fate Decisions conditions in ES cells. Genomic locations of signaling regulated peaks correspond to (A) graded binding to intron of Radil in all 3 conditions, (B) discrete binding to promoter and exon of Id1 specific to SB treatment, (C) binding to the 39 region of 2210011C2Rik only in the Activin condition and (D) multimodal recruitment of pSmad2 in ChIP-Seq peaks marked with (*) where it is SB specific in the promoter region of Copz2 and graded in correlation with signaling in the intronic region. All target genes were identified to be differentially expressed under the 3 signaling conditions in the microarray analysis of Figure 3A. Top panels show raw sequencing tag enrichments in Activin/DMSO/SB on the vertical axis. Bottom panels show ChIP-Seq peak positions and intensities defined by the MACS program based on normalization to the respective input DNA sequencing controls for each condition. Horizontal scale shows genomic coordinates on the indicated chromosomes and scale bar denotes genomic distance. Structure of the indicated target genes is represented by thick solid lines for exons, thin solid lines for UTRs and continuous arrows running from 59 to 39 for introns. doi:10.1371/journal.pgen.1002130.g006 We discovered that the Oct4 locus was rich in multiple pSmad2 binding events from ChIP-Seq profiling (Figure 7A). During graded Nodal/Activin signaling in chemically defined conditions however, only a pSmad2 peak in the promoter region of Oct4 showed a similarly graded response suggesting that this was the functional Nodal/Activin signaling response element. We exam- ined the transcript levels of endogenous Oct4 expression (Figure 7D) upon inhibition of Nodal/Activin signaling with SB in serum containing media and found that it was also significantly downregulated within 24 hours. In agreement with the transcript data, Oct4 protein levels were similarly downregulated in SB treated ES cells (7E). Analysis of the 503 bp promoter region encompassing the beginning and end of the pSmad2 binding peak showed that it contained eight CAGA sites or their inversion (Figure 7B). To determine if this regulatory sequence was indeed a Nodal/Activin response element of the Oct4 promoter, we cloned this into luciferase reporter constructs and transfected ES cells subjected to the 3 signaling conditions with Activin, DMSO and SB (Figure 7C) in serum containing media. The reporter activity of the wild type Oct4 promoter construct was .100X higher than that of the empty reporter construct in the DMSO control signaling condition suggesting that the 503 bp sequence had strongly driven Oct4 promoter activity in ES cells. Crucially, the Oct4 promoter reporter displayed a specific graded response to Nodal/Activin signaling while the control empty reporter did not. To confirm that the Oct4 response to graded Nodal/Activin signaling was functionally driven by pSmad2 binding, we determined the exact SBE responsible for Oct4 inducibility (Figure 7C). Mutagenesis experiments on the Oct4 promoter region in luciferase assays revealed that the strong CAGAC consensus SBE site in the middle of the 503 bp fragment was indispensable for graded Oct4 promoter activity. Loss of this site completely abolished the promoter response to both high and low Nodal/Activin signaling. Further point mutations of two minimal CAGA SBEs flanking the CAGAC site led to no further significant effects on the Oct4 promoter. We therefore conclude that Oct4 is a direct target of pSmad2 binding and Nodal/Activin signaling regulates both its promoter activity and endogenous expression. The 503 bp Oct4 promoter response element with the essential CAGAC SBE was sufficient and independent of all other pSmad2 binding events in the Oct4 locus or other DNA regulatory elements in cis that may be mediated by Nodal/Activin signaling. The regulation of Oct4 is well known for its importance in cell fate decisions and its downregulation during loss of Nodal/Activin signaling is signifi- cant not only as an impetus for trophectoderm differentiation but also reconciles the alternative role of Nodal/Activin signaling in maintaining self-renewal and pluripotency. Discussion The molecular basis of extracellular signaling instructions governing differential cell fate decisions in the Nodal/Activin pathway has been postulated but not shown conclusively. Primarily, the transcriptional events occurring at the interface between pSmad2 signal transduction from the activated cell surface receptors to manipulation of the global stem cell transcriptome driving specific lineage programs have not been well characterised. This study provides an important insight into how quantitative signaling is translated into qualitative cell fate decisions by showing for the first time, to our knowledge, that the same transcription factor pSmad2 is able to bind and transcrip- tionally regulate different subsets of target genes in a dose- dependent manner. The specification of cell fate decisions is governed by 2 distinct events. The first requires an exit from self-renewal and maintenance of stemness programs by direct control of pSmad2 over key pluripotency factors. Previous studies have revealed that Nanog is a direct target of Smad2/3 transcription in human ES cells [61]. Here we show an additional level of control over the stem cell program by direct transcriptional regulation of the Oct4 master pluripotency gene by pSmad2. The second event requires an entry into a specific differentiation program that is in turn brought about by direct and indirect pSmad2 regulation of differentiation genes such as Gata3, Tcfap2c and Igf2 that are known to be important factors for trophectoderm cell fates. This cell fate decision is further reinforced by loss of Oct4 with inhibition of Smad2 phosphorylation as the former is known to be a potent repressor of the trophectoderm gene Cdx2 in the blastocyst [60]. The pSmad2 binding target genes driving mesendodermal differentiation include Mixl, Fgf8 and Nodal itself, while other genes such as Chst15 expressed in definitive endoderm and Fgf15 for cardiac mesoderm have also been identified as strong Nodal/Activin transcriptional targets. It is likely that over the course of long-term differentiation for 6 days, additional target genes may be recruited for the specification of both lineage decisions that may not be apparent at the 18 hours time point in this study which may be too early for endpoint differentiation. Indeed, strong regulation of Mixl and Fgf8 and to a lesser extent Nodal could be detected at 3 and 6 days (Figure 2B and data not shown) of treatment in correlation with the level of Nodal/Activin signaling. Consistent with the role of Nodal/Activin as morphogens, we found that many components of the pathway were themselves feedback targets that were directly regulated by pSmad2 binding in ChIP-Seq and/or differentially expressed in our microarray analysis. These include the negative feedback inhibitors such as Lefty1/2 and Smad7 which are already known targets of Nodal/ Activin signaling. In this study, graded pSmad2 binding could be detected in the intronic region of Tmepai (Figure S6) which sequesters Smad2/3/4 from receptor kinase activity [62]. Similarly, SnoN [63] and Ski [64] also present graded intronic binding of pSmad2 (Figure S6) and both function as transcrip- tional repressors of Smad2/3/4. There were also positive feedback components such as Nodal, its cofactor Cripto and FoxH1 the transcriptional copartner of Smad2 (Figure 8 and Figure S6) that show graded binding in the intron and promoter regions. The preponderance of the negative components in the autoregulatory loop of Nodal/Activin signaling is significant, as it suggests that the PLoS Genetics | www.plosgenetics.org 13 June 2011 | Volume 7 | Issue 6 | e1002130