ETV2 primes hematoendothelial gene enhancers prior to hematoendothelial fate commitment.

Mechanisms underlying distinct specification, commitment, and differentiation phases of cell fate determination remain undefined due to difficulties capturing these processes. Here, we interrogate the activity of ETV2, a transcription factor necessary and sufficient for hematoendothelial differentiation, within isolated fate intermediates. We observe transcriptional upregulation of Etv2 and opening of ETV2-binding sites, indicating new ETV2 binding, in a common cardiac-hematoendothelial progenitor population. Accessible ETV2-binding sites are active at the Etv2 locus but not at other hematoendothelial regulator genes. Hematoendothelial commitment coincides with the activation of a small repertoire of previously accessible ETV2-binding sites at hematoendothelial regulators. Hematoendothelial differentiation accompanies activation of a large repertoire of new ETV2-binding sites and upregulation of hematopoietic and endothelial gene regulatory networks. This work distinguishes specification, commitment, and sublineage differentiation phases of ETV2-dependent transcription and suggests that the shift from ETV2 binding to ETV2-bound enhancer activation, not ETV2 binding to target enhancers, drives hematoendothelial fate commitment.