ChIP-Seq Chromatin Immunoprecipitation (ChIP) using a certified ChIP- grade rabbit polyclonal anti-Smad2 (phospho S465+S467) anti- body (ab16509, Abcam) was carried out in ES cells under chemically defined high, medium or low Nodal/Activin signaling conditions according to the Agilent Mammalian ChIP-on-chip protocol v9.1 up to the ChIP DNA purification step. Adapter ligation, library amplification and size selection in the 200–300 bp range were performed according to the Illumina ChIP Sample Prep protocol (#11257047, Rev. A). Massively parallel sequencing was carried out for ChIP samples in all 3 signaling conditions with their respective input DNA controls on the Genome Analyzer (Illumina) up to a sequencing depth of at least 106106 tags pass filter. The ChIP-Seq data was deposited in NCBI GEO with accession number GSE23581. Details of the ChIP-Seq, motif and statistical analysis can be found in the Text S1. Luciferase Assays The 503 bp fragment of the mouse Oct4 promoter region corresponding to chr17:35640683–35641185 was cloned into the pGL4.23[luc2/minP] Firefly luciferase reporter construct (Pro- mega) to generate pGL4.23 Oct4. This construct was point mutated by oligo cloning into unique StuI and NsiI sites to produce pGL4.23 m4 Oct4 (CAGAC mutated to CATGC) and pGL4.23 m345 Oct4 (TCTGGGCAGACGGCAGA mutated to TATGGG- CATGCGGCATA). The constructs were transfected into mouse ES cells in an 80:1 ratio with the pGL4.75[hRluc/CMV] Renilla luciferase co-transfection control (Promega) using Lipofectamine 2000 (Invitrogen). A control transfection was included with an 80:1 ratio of the empty pGL4.23 vector to pGL4.75 Renilla control. Immediately after lipofections, the ES cells were pretreated with FBS media without LIF and with 10 mM SB for 6 hours. Subsequently the cells were split into replicates and plated in FBS media without LIF containing 25 ng/ml Activin, 1/5000 DMSO vehicle or 10 mM SB, which induces high, medium or low Nodal/Activin signaling respectively for 8 hours. The cells were washed once in PBS and lysed in 1XPassive Lysis Buffer and luciferase assays were performed using the Dual-Luciferase Reporter Assay System on the GloMax 96 Microplate Luminom- eter with Dual Injectors (Promega). Supporting Information Figure S1 There Is Graded pSmad2 Binding to Pitx2 and Lefty2 but Not the Smad7 Proximal Promoter. Real-time PCR quantifi- cation of pSmad2 ChIP enrichments using tiling primers across the intronic enhancer in Pitx2 (A) and the promoter regions of Lefty2 (B) and Smad7 (C). Y-axis shows fold enrichment over the non-binding control region in the Sox2 locus after normalizing to the input DNA for each condition while the x-axis represents genomic distance in base pairs (bp) from the transcriptional start site (TSS) of each gene. Upstream and downstream distances are denoted as negative and positive coordinates respectively. Trends show the level of pSmad2 ChIP enrichment for the indicated genomic regions obtained from ES cells treated with Activin (red), DMSO carrier control (blue) and SB (red) in chemically defined KSR media. Horizontal bars show coverage of each PCR primer, vertical error bars show s.e.m. for n = 3 replicates. (TIF) Figure S2 Activin/SB Treatments of ES Cells Promote Exit from Self-Renewal towards Mesendoderm and Trophectoderm Differentiation. Immunofluorescence detection of the markers of pluripotency and mesendoderm or trophectoderm differentiation in ES cells treated for 6 days with Activin, DMSO control and SB. Nodal/Activin Signaling in ES Cell Fate Decisions Phase contrast bright field (BF) images are in the top panels followed by Hoechst 33342 nuclear staining in blue. Alexa Fluor 546 staining for Lim1, Oct4 and Hand1 are in red for Activin, DMSO and SB treated cells respectively. Alexa Fluor 488 staining for Mixl, SSEA-1 and placental Cadherin (P-cad) are in green while overlays of all fluorescent channels (Merge) are shown in the bottom panels. Scale bar shows distance under a 20X objective lens. (TIF) Figure S3 Pitx2, the Lefty1/2 Hotspot, and Smad7 Contain Multiple pSmad2 Regulatory Sites in Their Genetic Loci. UCSC Genome Browser representation of the genomic loci for Pitx2 (A) on chromosome 3:128,894,461–128,931,307, Lefty1/2, Pycr2 and Tmem63a (B) on chromosome 1:182,813,583–182,908,336 and Smad7 (C) on chromosome 18:75,513,703–75,561,010. Genomic coordinates are shown on the x-axis while raw pSmad2 ChIP-Seq tag counts are presented on the y-axis in the top panel followed by relative enrichments normalized to the respective input DNA controls of Activin (red), DMSO (blue) and SB (green) treated ES cells in the bottom panel. The RefSeq genetic structures of the indicated genes and their isoforms if any are indicated below the ChIP-Seq panels. Scale bar shows genomic distance in kilo base pairs (kb) while pSmad2 enrichment peaks corresponding to the real-time PCR results in Figure S1 are indicated with asterisks (*). (TIF) Figure S4 Phospho-Smad2 Binding to Majority of Sites and Target Genes Varies by .1.25-Fold during Graded Signaling. Pie charts showing patterns and statistics of pSmad2 binding to sites and target genes that vary by at least 1.25-fold during Activin, DMSO and SB treatments. Colored segments display the indicated models of pSmad2 binding in the 3 signaling conditions. Proportional segment sizes define the relative contribution of each type of binding behavior for ChIP-Seq peaks (A) and target genes within +/250 kb of the peaks (B) out of the total for each. Values indicate the number of peaks (A) or genes (B) in each regulatory model with percentages of the total in parentheses. (TIF) Figure S5 The Mesendodermal Genes Mixl, Nodal, and Fgf8 Are Targets of Differential pSmad2 Binding. Binding profile of pSmad2 ChIP-Seq enrichments on Mixl (A), Nodal (B) and Fgf8 (C) as visualized on the UCSC Genome Browser at the coordinates chr1:182,571,609–182,632,748, chr10:60,873,730– 60,895,009 and chr19–45:805,804–45,822,014 respectively. Changes in the level of pSmad2 ChIP-Seq peak enrichments (raw peaks top panel, normalized peaks bottom panel) are indicated for Activin (red), DMSO (blue) and SB (green) treated ES cells on the y-axis. X-axis shows genomic location and genetic features of the indicated genes are below the Genome Browser panels. PSmad2 binding peaks showing significant differential binding with Nodal/Activin signaling are marked with asterisks (*). Scale bar shows genomic distance in kilo base pairs (kb). (TIF) Figure S6 Validation of 15 Genes as Direct Targets of Nodal/ Activin Signaling via Differential Phospho-Smad2 Binding. Real- time PCR validation of pSmad2 ChIP-Seq enrichment on original unamplified ChIP DNA from ES cells treated with Activin (ACT), DMSO and SB in KSR media for 18 hours. Indicated genes were selected based on transcriptional regulation under the same conditions in the microarray analysis of Figure 3A and real-time PCR gene expression detection in Figure 7D. Vertical scale shows fold-enrichment over the non-binding control region in the Pfkm locus on chromosome 15:97,934,552–97,934,626 after normaliza- PLoS Genetics | www.plosgenetics.org 17 June 2011 | Volume 7 | Issue 6 | e1002130