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Additional Supporting Information may be found in the online version of this article.

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STEM_666_sm_suppfigure1.tif3175KSupporting Figure 1. Chromatin immunoprecipitation performed in the presence of additional protein cross linkers and combined with deep sequencing reveal that Activin/Nodal signalling controls the core pluripotency transcriptional network in hESCs. (A) Lack of efficiency of conventional ChIP approach to detect the interactions of Smad2/3 proteins with genomic DNA in hESCs. Three separate ChIP-QPCR experiments were performed on hESCs to detect the presence of Smad2/3 on the promoters of Smad7 and Lefty, two known genes of Activin/Nodal signaling. Results were normalised against a control region (Smad7 3UTR) and are expressed as +/- standard derivation from three technical replicates. (B) Combination of diverse chemical cross linkers improves ChIP efficiency. ChIP was performed in the presence of different combination of cross linkers (Dimethyl dithiobispropionimidate or DTBP; Disuccinimidyl tartarate or DST, Ethylene glycol bis succinimidylsuccinate, or EGS) as described in the Supplementary Methods. Importantly, these experiments were performed simultaneously on the same batch of cells to avoid experimental variations. The immunoprecipitated DNA was then amplified using Q-PCR with specific primers to detect enrichment in the denoted genomic regions (Supplementary Table 7). Results were normalised against a control region (Smad7 UTR) and are expressed as +/- standard derivation from three replicates. (C) FACS analyses showing that hESCs grown in CDM+Activin+FGF2 express homogenously the pluripotency marker Nanog. hESCs immuno-stained with secondary antibody alone were used as negative control to define the gate for statistical analyses. (D) Representative view of Smad2/3 binding sites in genomic regions containing known Activin/Nodal target genes. Y axis shows the number of sequence tags per 200 bp window. Red Arrows point to statistically significant enriched genomic regions while blue arrow point to previously characterised binding sites. (E) Validation of Smad2/3 binding sites in hESCs. ChIP-QPCR were performed on hESCs grown in the presence or in the absence of Actvin/Nodal signalling to confirm the specific presence of Smad2/3 on the genomic regions identified using ChIP-XL-Seq. Results were normalised against control region (Smad73UR) and are expressed as +/- standard deviation. (F) Schematic representation of transcription factor binding motifs inferred from genomic regions bound by Smad2/3 in hESCs. Statistical analyses performed as described in supplementary materials. (G) Gene Ontology analysis of genes bound by Smad2/3 which are down or up regulated in the absence of Activin/Nodal signalling in hESCs. The Y axis shows the GO term and the X axis shows the p value for the significance of enrichment for the top 30 GO terms.
STEM_666_sm_suppfigure2.tif3175KSupporting Figure 2. Activin/Nodal signalling pathway controls a broad part of the transcriptional network characterising endoderm cells. (A) FACS analysis showing that endoderm cells generated from hESCs express homogenously Sox17. DE cells immuno-stained with secondary antibody alone were used as negative control to define the gate for statistical analyses. (B) Gene Ontology analysis of genes bound by Smad2/3 which are down or up regulated in the absence of Activin/Nodal signalling in endoderm cells. The Y axis shows the GO term and the X axis shows the p value for the significance of enrichment for the top 30 GO terms. (C) Validation of Smad2/3 binding sites in endoderm cells. ChIP-XL-QPCR was performed on endoderm cells to confirm the presence of Smad2/3 on the genomic regions identified using ChIP-XL-Seq. Results were normalised against control region (Smad73UTR) and are expressed as +/- standard deviation from three experiments. (D) Smad2/3 binding regions in endoderm cells are evolutionary conserved. The average conservation (phastCons, scaled 0-1) of the genomic regions bound by Smad2/3 was determined as described in Materials and Methods. Matched non-specific genomic regions were used as negative control (Red lines). (E) Relative position of the genomic regions bound by Smad2/3 in endoderm to the Transcriptional Start Site (TSS). (F) Expression of pluripotency markers is down regulated upon endoderm differentiation. hESCs (H9) were grown for 3 days in culture conditions inductive for endoderm differentiation (CDM + Activin + FGF2 + BMP4 +Ly) (Day1, Day2 and Day3). RNAs were extracted every day, and then Q-PCR analyses were performed to detect the genes denoted. Data represent the mean of three independent experiments and error bars indicate standard deviation.
STEM_666_sm_suppfigure3.tif3175KSupporting Figure 3. Nanog and Smad2/3 cooperate to maintain the transcriptional network characterizing pluripotency in hESCs. (A) Q-PCR analyses showing the absence of Nanog transcripts in hESCs stably expressing ShRNA targeting Nanog (ShNanog-hESCs). hESCs (H9) grown in CDM + Activin + FGF2 were used as positive control (hESCs) while hESCs grown for 7 days in the presence of SB431542 were used as negative control (hESCs + SB). (B) Q-PCR analyses showing the expression of Nanog in hESCs stably transfected with an expression vector for Nanog grown in CDM + Activin + FGF2 (OE Nanog) or grown for 7 days in the presence of SB431542 (OE Nanog + SB). hESCs (H9) grown in similar culture conditions were used as control (hESCs and hESCs +SB). (C) ChIP analyses showing that Smad2/3 does not bind candidate target genes in hESCs overexpressing Nanog grown in the presence of SB431542 for 24 hours.
STEM_666_sm_supptable1.XLSX5463KSupporting Table 1. Smad2/3 binding sites and associated genes in hESCs.
STEM_666_sm_supptable2.XLSX897KSupporting Table 2. Smad2/3 target genes up and down regulated in the absence of Activin/Nodal signalling in hESCs.
STEM_666_sm_supptable3.XLSX4400KSupporting Table 3. Smad2/3 binding sites and associated genes in Definitive Endoderm.
STEM_666_sm_supptable4.XLSX1443KSupporting Table 4. Smad2/3 target genes up and down regulated in the absence of Activin/Nodal signalling in Definitive endoderm.
STEM_666_sm_supptable5.XLSX315KSupporting Table 5. Candidate target genes bound in hESC and in Definitive Endoderm.
STEM_666_sm_supptable6.XLSX5198KSupporting Table 6. Genes bound by Smad2/3 and Nanog in hESCs
STEM_666_sm_supptable7.XLS32KSupporting Table 7. List of primers for ChIP-Q-PCR and luciferase assays.
STEM_666_sm_suppinformation.doc77KSupporting Information

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