Pioneer factor ETV2 safeguards endothelial cell specification by recruiting the repressor REST to restrict alternative lineage commitment.

Mechanisms of cell fate specification are central to developmental biology and regenerative medicine. ETV2 is a master regulator for the endothelial cell (EC) lineage specification. Here we study mechanisms by which ETV2 overexpression in human induced pluripotent stem-cell-derived mesodermal progenitors efficiently specifies ECs. We used CUT&RUN, scRNA-seq and scATAC-seq to characterize the molecular features of EC differentiation mediated by ETV2. We defined the scope of ETV2 pioneering activity and identified its direct downstream target genes. Induced ETV2 expression both directed specification of endothelial progenitors and suppressed acquisition of alternative fates. Functional screening and candidate validation revealed cofactors essential for efficient EC specification, including the transcriptional activator GABPA. Notably, the transcriptional repressor REST was also necessary for efficient EC specification. ETV2 recruited REST to repress non-EC lineage genes. Our study provides an unparalleled molecular analysis of EC specification at single-cell resolution and highlights the important role of pioneer factors to recruit repressors that suppress commitment to alternative lineages.