N undifferentiated and differentiated cells indicated that occupancy of 1495 web-sites was lost in the course of differentiation, whereas extra than 6700 web-sites had been gained and 1434 web-sites have been occupied below each circumstances (Fig 4E). For example, Tead4 constitutively occupied web-sites upstream of the Ctgf and Ccnd1 genes, whereas occupancy of web sites in the Acta1 locus is seen only in the course of STF-118804 site differentiation (S2A Fig). Websites particularly occupied in undifferentiated and differentiated cells showed enrichment in AP1 motifs inside the undifferentiated state, but enrichment of Myod1, Myog, Tcf3 and Tcf12 that cooperates with Myod1 to market myogenic differentiation [24] in differentiated cells (Fig 4F). To assess co-localisation amongst Tead4 and transcription variables whose motifs have been enriched at its binding web-sites, we compared the Tead4 profiles with those of Jun and Srf in C2C12 cells and Runx1 in principal myoblasts [25] [26]. Around 35 of websites bound by Tead4 in undifferentiated and differentiated cells overlapped with those bound by Jun (S2B Fig). In contrast, there was small overlap using the web-sites preferentially bound in the differentiated state, where the frequency of co-occurring AP1 motifs was also decreased. Similarly, a robust binding overlap among Tead4, Runx1 and Srf was seen involving websites occupied in undifferentiated and differentiated cells. A equivalent analysis of Tead1 identified 1400 bound web sites in undifferentiated cells, enriched in MCAT, AP1 and Runx motifs (S3A and S3B Fig). Nevertheless, in agreement with its absence from the differentiated cell nucleus, Tead1 occupancy was strongly lowered inside the differentiated state with only 274 detected web pages (S3C Fig). Even at web pages occupied in each situations, Tead1 occupancy was decreased inside the differentiated state (S3D Fig). Therefore, transition towards the differentiated state involved a switch from Tead1 and Tead4 occupancy to predominantly Tead4 occupancy. Within the undifferentiated state, Tead1 and Tead4 co-occupy additional than 900 web pages (S3E Fig). A set of web pages showed preferential occupancy by Tead1, but only handful of websites showed exclusive Tead1 occupancy. Hence regardless of the fact that these two proteins bind identical sequences and that Tead1 occupancy was globally decrease than Tead4, a set of web-sites was preferentially occupied by Tead1. As Tead4 regulated Tead1 expression throughout differentiation, we examined Tead4 occupancy in the Tead1 locus. Two constitutive Tead1/Tead4 occupied web pages were observed, 1 upstream with the promoter of your longest isoform and also a second upstream of an alternative promoter for any shorter isoform (S4A Fig). In the course of differentiation Tead1 occupancy diminished, but Tead4 occupancy was maintained suggesting that Tead4 directly regulates Tead1. Integration of Tead1/4 ChIP-seq information with public information on histone modifications in undifferentiated and differentiated C2C12 cells revealed the presence of H3K27ac, a mark of active promoters and enhancers, in the Tead1 promoter in undifferentiated cells overlapping with all the Tead1/4 occupied web-sites. Interestingly, upon differentiation, H3K27ac increased in the Tead1/4 occupied web pages and new regions marked by H3K27ac appeared upstream of and overlapping together with the alternative promoter. Moreover, integration with public ChIP-seq information indicated Myod1 and Myog [27] binding in the H3K27ac-enriched regions. These observations suggest that Tead4 cooperates with Myog and Myod1 to activate Tead1 expression throughout differentiation via constitutive and inducible enhancer elements.