tion to the input DNA controls in each treatment. Phospho-Smad2 enriched regions detected by the PCR primers are indicated in the genomic coordinates above each graph. Error bars show s.e.m. for n = 12 replicates. (TIF) Table S1 64.2% of Microarray Targets Differentially Expressed by .1.5-fold are also Targets of Differential Phospho-Smad2 Binding. Table showing 104 out of the 162 microarray target genes from Figure 3A where expression is induced and/or repressed by .1.5-fold during stimulation or inhibition of Nodal/Activin signaling in ES cells. These targets also exhibit pSmad2 binding at +/250 kb from the 59/39UTRs and within the genes themselves. Only ChIP-Seq peaks that satisfy a size cutoff of .12 sequencing tags in at least 1 condition are considered significantly above background sequencing levels and counted for each gene during Activin (ACT), DMSO vehicle control and SB inhibitor treatments. Gene IDs of the target genes, their chromosomal locations (Chr) and their orientation on the sense (+)/antisense (2) strands are as indicated. Gene names highlighted in yellow are for Activin/SB co- regulated targets, red for Activin specific targets and green for SB responsive genes only. The number of sequencing tags within the largest ChIP-Seq peaks for each gene and under each condition are as shown. For genes that contain multiple pSmad2 binding sites, the largest peaks counted may not occur at the same genomic location. Changes in the level of overall pSmad2 binding during Activin treatment compared to SB (ACT vs SB), Activin compared to DMSO control (ACT vs DMSO) and SB compared to DMSO (SB vs DMSO) are shown as (+) for increased binding and (–) for decreased binding by .1.5-fold. The overall level of pSmad2 binding for each gene is calculated as the sum of all peak heights (enrichment counts) proximal to the gene and under each condition. A value of 0 denotes no significant change in binding at a 1.5-fold cutoff and indicates no detectable peaks for statistical analysis. (DOC) Table S2 There Are Dynamic Changes in Phospho-Smad2 Binding Motifs During Graded Nodal/Activin Signaling. Moti- fEnrich program identification of co-motifs enriched within #5000 bp of pSmad2 binding peaks. Co-motifs are ranked based References 1. Ashe HL, Briscoe J (2006) The interpretation of morphogen gradients. Development 133: 385–394. 2. Gurdon JB, Bourillot PY (2001) Morphogen gradient interpretation. Nature 413: 797–803. 3. Tabata T, Takei Y (2004) Morphogens, their identification and regulation. Development 131: 703–712. 4. Green JB, Smith JC (1990) Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate. Nature 347: 391–394. 5. Gurdon JB, Harger P, Mitchell A, Lemaire P (1994) Activin signalling and response to a morphogen gradient. Nature 371: 487–492. 6. 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Tremblay KD, Hoodless PA, Bikoff EK, Robertson EJ (2000) Formation of the definitive endoderm in mouse is a Smad2-dependent process. Development 127: 3079–3090. Nodal/Activin Signaling in ES Cell Fate Decisions on frequency of occurrence from highest to lowest. Matching of co-motifs with associated transcription factors was performed using the TRANSFAC PWM database with p-value thresholds of 1e-05. Transcription factor names in black are present across all 3 signaling conditions with Activin, DMSO and SB treatments. Activin enriched transcription partners binding on the co-motifs in proximity to pSmad2 sites are highlighted in red, DMSO enriched factors in blue and SB in green. The intervals of significant enrichment for each co-motif are indicated in base pairs (bp). (DOC) Table S3 Primers Used for Quantification of ChIP DNA and Marker/Gene Expression Levels by Real-Time PCR. List of forward/reverse primer pairs used in SYBR Green real-time PCR reactions for the quantification of ChIP-DNA (ChIP-qPCR) and Marker/Gene expression. For tiling ChIP-qPCR analysis of pSmad2 binding on Pitx2, Lefty2 and Smad7, primers for each gene are numbered sequentially according to the 59–39 order of their respective amplified regions shown in Figure S1. (DOC) Text S1 Supporting Experimental Procedures and Supporting References. Description of methods used for motif identification and statistical analysis of ChIP-Seq data and immunostaining of the protein markers of differentiation in cells with the associated supplementary references. (DOC) Acknowledgments We are grateful to R. Soong, M. C. Wu, and L. H. Abdul Hadi for their assistance with ChIP-Seq. We would also like to thank C. W. Chang for the MotifEnrich program and W. K. Sung, N. D. Clarke, and H. Kato for scientific discussion. Author Contributions Conceived and designed the experiments: KLL SKL BL. 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PLoS Genetics | www.plosgenetics.org 18 June 2011 | Volume 7 | Issue 6 | e1002130