Genetic dissection of cell type–specific congenital heart disease variants reveals ETS1 control of vascular smooth muscle cell fate

Numerous congenital heart disease (CHD) risk variants lie in cis-regulatory elements, yet the impact of variants in repressed and bivalent chromatin on cell type–specific heart development is unclear. Here, we generated a single-cell multiomic atlas of human cardiac differentiation by jointly profiling H3K27ac, H3K27me3, H3K4me3, and transcriptomes in individual cells, classifying cis-regulatory elements into six chromatin states, enabling prioritization of 610 CHD-associated noncoding variants enriched in lineage- and state-specific regulatory elements, including repressive and bivalent regions. CRISPR knockin of selected variants perturbed nearby gene expression and impaired cardiac differentiation. Dissection of a Polycomb-marked variant near ETS1 (ETS proto-oncogene 1) revealed local H3K27me3 loss, H3K27ac gain, and ETS1 up-regulation, which redirects differentiation toward second-heart-field progenitors with epithelial characteristics at early stages and, ultimately, highly migratory ETS1 ⁺ vascular smooth muscle cells. Together, our work establishes a single-cell chromatin-state framework for interpreting noncoding CHD risk variants and uncovers an ETS1-driven program linking Polycomb-deregulated noncoding variation to aberrant vascular smooth muscle fate commitment during human cardiogenesis.