Figure 4. Graded expression of putative target genes of the Activin A/TGFβ signaling pathway in day 4 mESCs.

(a) Schematic representation of the experimental setup. Mouse ESCs were differentiated in serum free conditions and different dosages of Activin A and SB-431542 were introduced to create a graded activation of the Activin A/TGFβ signaling pathway. Cells were harvested at day 4 for sequencing library generation. Differential gene expression analysis identified ~15–20% of expressed transcripts as differentially regulated in each sample in comparison with untreated controls (see Supplementary Methods online). (b) Regulation of Activin A pathway components in response to SB-431542 and Activin A. (c) Putative TGFβ target genes in differentiating mESCs at day 4. The heat map corresponds to fold changes observed for transcripts in comparison to untreated control. Putative target genes were classified as transcripts that followed opposite trends of regulation upon treatment with Activin A and SB. Fifty transcripts were successively up-regulated while 23 transcripts followed graded down-regulation with increasing dosages of Activin A. The majority of the TGFβ target genes (marked with *) had FoxH1 transcription factor binding sites separated by 30–200 bp (also called ASE) in 10 Kb upstream and downstream of the transcription start site. Known TGFβ target genes are highlighted in bold. Low copy number transcripts (RPKM < 3 in AA3 sample) are displayed in red font.