While endothelial cells (ECs) are pivotal in vascular development, the chromatin dynamics governing human EC specification remain elusive due to limited access to embryonic models. Here, we reconstruct the epigenomic roadmap of EC differentiation using human pluripotent stem cells (hPSCs). Temporal chromatin accessibility analysis revealed six dynamic clusters, with Cluster 5 (enriched in endothelial progenitors/ECs) exhibiting concordant chromatin opening and transcriptional activation. Key developmental transcriptional factors (ETV2, ETS1, ERG) showed stage-coordinated binding site accessibility during endothelial commitment.

ChIP-seq analysis of key histone marks showed that the broad H3K4me3 domains labeled stage-specific progenitor cell identity genes correlated with high expression of these genes. Through integrative analysis of the genome-wide association of H3K4me3, H3K27ac, H3K27me3, and accessible chromatin, we classified and characterized three types of cis-regulatory elements (CREs) that correlated with the EC fate specification and differentiation stage.

Finally, by integrating the epigenomic dataset and single-cell RNA-seq data, we identified BCL6B as an endothelial lineage-specific transcription factor regulating both differentiation commitment and arterial-venous bifurcation. Mechanistically, BCL6B functions as a transcriptional repressor. Knocking out BCL6B upregulated Notch signaling genes while inducing BCL6B overexpression downregulated NOTCH1/4 and HEY1/2 to influence arterial-venous EC fate bias.

In summary, our study profiled the dynamic epigenomic landscape, annotated regulatory DNA elements, and identified and validated BCL6B as a new transcription factor for EC differentiation and arterial-venous bifurcation. Our findings provide a valuable resource to study the epigenetic regulation of EC development.
DOI:https://doi.org/10.1093/procel/pwaf039