Identification of endothelial gene regulatory elements during cardiopharyngeal lineage differentiation [RNA-seq]
Endothelial cells (EC) differentiate from multiple sources during embryonic development, including the cardiopharyngeal mesoderm, which gives rise to multiple tissues including the cardiac and branchiomeric muscle. Here, we have used a cardiogenic mesoderm cell differentiation model that also activates an endothelial transcription program. Comparing our chromatin remodeling data with publicly available single-cell RNA-seq data from mouse embryos, we derived a list of over 100 putative regulatory elements activated in EC marker genes. We applied a machine-learning strategy trained with validate enhancers to predict the probability of these sequences to function as enhancers. The computational assay returned over 50% of these sequences to be likely enhancers, some of which previously reported. In addition, using genetic and epigenetic perturbations of some of these elements, we established their requirement for gene expression during endothelial differentiation. Finally, we used the integration of multiple data sources and computational tools to search for transcriptional factor binding motifs related to endothelial cell fate specification. We found motifs of GATA, ETS, and FOS/AP-1 factors as the most significantly enriched in these sequences. Overall, our approach has been efficient in the identification of novel EC regulatory sequences with high likelihood to be enhancers, and validated a subset of them. Motif analyses revealed that the core EC transcription factors GATA/ETS/FOS is a likely driver of EC differentiation in cardiopharyngeal mesoderm. Overall design: In this study we investigated the gene expression and the chromatin accessibility profile in the early phases of multipotent cardiac progenitors' differentiation into mature endothelial cells (ECs), starting from mouse embryonic stem cells (mESCs). Differentiating mESCs were collected at day2 and day4 and two biological replicates for each condition were  used for ATAC-seq and RNA-seq assay.