Mapping the chromatin occupancy of transcription factors (TFs) is a key step in deciphering developmental transcriptional programs. Here we use biotinylated knockin alleles of seven key cardiac TFs (GATA4, NKX2-5, MEF2A, MEF2C, SRF, TBX5, TEAD1) to sensitively and reproducibly map their genome-wide occupancy in the fetal and adult mouse heart. These maps show that TF occupancy is dynamic between developmental stages and that multiple TFs often collaboratively occupy the same chromatin region through indirect cooperativity. Multi-TF regions exhibit features of functional regulatory elements, including evolutionary conservation, chromatin accessibility, and activity in transcriptional enhancer assays. H3K27ac, a feature of many enhancers, incompletely overlaps multi-TF regions, and multi-TF regions lacking H3K27ac retain conservation and enhancer activity. TEAD1 is a core component of the cardiac transcriptional network, co-occupying cardiac regulatory regions and controlling cardiomyocyte-specific gene functions. Our study provides a resource for deciphering the cardiac transcriptional regulatory network and gaining insights into the molecular mechanisms governing heart development.

A genetic etiology is identified for one third of congenital heart disease (CHD) patients, including 8% attributable to coding de novo variants (DNVs). To assess the contribution of noncoding DNVs to CHD, we compared genome sequences from 749 CHD probands and their parents with 1,611 unaffected trios. Neural network prediction of noncoding DNV transcriptional impact identified a burden of DNVs in CHD ( n = 2,238 DNVs) compared to controls ( n = 4,177; P = 8.7 × 10 −4 ). Independent analyses of enhancers showed excess DNVs in associated genes (27 genes vs. 3.7 expected, P = 1 × 10 −5 ). We observed significant overlap between these transcription-based approaches (OR = 2.5, 95% CI 1.1–5.0, P = 5.4 × 10 −3 ). CHD DNVs altered transcription levels in five of 31 enhancers assayed. Finally, we observed DNV burden in RNA-binding protein regulatory sites (OR = 1.13, 95% CI 1.1–1.2, P = 8.8 × 10 −5 ). Our findings demonstrate an enrichment of potentially disruptive regulatory noncoding DNVs in a fraction of CHD at least as high as observed for damaging coding DNVs.

SignificanceBirth defects are the leading cause of infant mortality in the United States and Europe, with cardiac defects being the most prevalent. Here we define the requirement and mechanism of action of CHD4, the catalytic core component of the nucleosome remodeling and deacetylase (NuRD) complex, in embryonic heart development. CHD4 is essential from fly to human and mutations in CHD4 are causative to congenital heart disease, including atrial and ventricular septal defects. By generating a cardiac conditional null allele of CHD4, temporal transcriptional profiling, and systems-level analysis of CHD4 target genes and in utero echocardiography, we define molecular, biochemical, anatomical, and physiological mechanisms for CHD4 and the NuRD complex in repressing inappropriate expression of the skeletal and smooth muscle programs in the developing heart.

Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD). However, underlying CHD gene regulatory network (GRN) imbalances are unknown.Here, we define transcriptional consequences of reduced dosage of the CHD-linked transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5dependent pathways-including lineage decisions and genes associated with cardiomyocyte function and CHD genetics-in discrete subpopulations of cardiomyocytes. GRN analysis identified vulnerable nodes enriched for CHD genes, indicating that cardiac network stability is sensitive to TBX5 dosage. A GRN-predicted genetic interaction between Tbx5 and Mef2c was validated in mouse, manifesting as ventricular septation defects. These results demonstrate exquisite sensitivity to TBX5 dosage by diverse transcriptional responses in heterogeneous subsets of iPSC-derived cardiomyocytes. This predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.