The Pol II and H3K27ac ChIP-seq consequently identified a set of very transcribed muscle identity genes confirming the signal comes predominantly from muscle cells. On the 2220 identified Tead4 sites, 686 were linked with active promoters TAPI-2 web marked by Pol II and H3K27ac and enriched in muscle precise functions (Fig 9A and S12A Fig). GenesPLOS Genetics | DOI:10.1371/journal.pgen.1006600 February 8,14 /Tead4 drives myogenic differentiationFig 7. Tead-regulated genes in PMs. A. Box plots showing gene expression alterations for the duration of differentiation of PMs transfected with siTead1/4. B. Examples of genes deregulated in siTead1/4 cells in comparison with siControl. C. GSEA analyses of Tead1/4 regulated genes. One of the most important categories within the up- and down-regulated genes sets are shown. doi:ten.1371/journal.pgen.1006600.gPLOS Genetics | DOI:ten.1371/journal.pgen.1006600 February 8,15 /Tead4 drives myogenic differentiationFig eight. Very transcribed muscle cell identity genes. A. Localisation of Pol II peaks in muscle relative for the TSS. B. Study density analyses of Pol II and H3K27ac at Ensembl annotated genes distinguishing these with higher transcribing Pol II density classes A and B from those with reduced or no transcribing Pol II, classes C-E. C. Results of functional enrichment ontology analyses from the genes in classes A and B displaying the enriched terms, the enrichment score (ES) along with the p-values. D. UCSC genome browser view showing Pol II and H3K27ac profiles in the Myh4 locus. doi:ten.1371/journal.pgen.1006600.gPLOS Genetics | DOI:ten.1371/journal.pgen.1006600 February 8,16 /Tead4 drives myogenic differentiationassociated with Tead4-bound web-sites showed enrichment in terms linked with muscle structural proteins (Fig 9B). Aligning the muscle Tead4 ChIP-seq towards the coordinates on the differentiated C2C12 cell peaks revealed 1558 sites with significant signal (Fig 9C). Genes related with these shared sites had been enriched in muscle structural proteins. Inside the converse comparison making use of the top rated 2200 Tead4-bound sites in muscle as reference, 341 prevalent peaks have been identified (Fig 9D). These comparisons revealed Tead4 internet sites in muscle that weren’t named amongst the 2200 high confidence websites, but even though showing reduced occupancy in muscle have been shared with differentiated C2C12 cells. Tead4 hence bound a distinct repertoire of websites in C2C12 cells and muscle and websites with high occupancy in muscle didn’t necessarily show higher occupancy in C2C12 cells and vice-versa. We next performed ChIP-seq from muscle of mice in which Tead4 was particularly inactivated in fibres using the Hsa::Cre-ERT2 driver [32]. Mice with floxed Tead4 alleles were crossed to produce Hsa::Cre-ERT2::Tead4 lox/lox animals. These mice have been injected at six weeks with tamoxifen for 4 consecutive days and 3 weeks after injection Tead4 expression was strongly reduced in the tibialis anterior and gastrocnemius muscle tissues displaying effective recombination (Fig 9E). We performed Pol II, H3K27ac and Tead4 ChIP-seq from these Tead4 musc-/- animals. Aside a tiny variety of internet sites with signal in Tead4 musc-/- animals, Tead4 binding was lost (Fig 9F) indicating that observed signal came pretty much exclusively from sites bound in muscle. Comparison of Pol II and H3K27ac profiles in wild-type and Tead4 musc-/- muscle (Fig 9G) showed only minor changes in low intensity signals and therefore that Tead4 loss did not influence international Pol II or H3K27ac distribution.