Long-Range Enhancer-Promoter Interactions Revealed by Hi-C in Cardiac Development

Cardiomyocyte differentiation requires the coordinated activation of cardiac structural and transcription factor genes, many of which are controlled by enhancers located tens to hundreds of kilobases from their target promoters, yet how these long-range enhancer-promoter interactions are established during the differentiation programme itself has not been comprehensively mapped at high resolution in a human cardiomyocyte differentiation system. Here we performed in situ Hi-C, H3K27ac and GATA4 ChIP-seq, and ATAC-seq at four stages of directed cardiomyocyte differentiation from human induced pluripotent stem cells (mesoderm, cardiac progenitor, immature cardiomyocyte and mature cardiomyocyte), and used CRISPR interference to test the functional contribution of newly formed enhancer-promoter loops at key cardiac loci. Hi-C contact maps identified 6,847 enhancer-promoter loops that were absent or weak at the pluripotent stage and progressively strengthened across the differentiation time course, with loop formation preceding, by one differentiation stage on average, the acquisition of H3K27ac at the corresponding enhancer and transcriptional induction of the target gene. GATA4 ChIP-seq showed that 71% of stage-specific loop anchors were occupied by GATA4 at or before the stage at which the loop first strengthened, and GATA4 occupancy preceded H3K27ac deposition at these anchors, consistent with a pioneering role for GATA4 in nucleating loop formation ahead of enhancer activation. CRISPR interference targeting individual loop anchors at the TBX5 and NKX2-5 loci reduced target gene induction at the corresponding differentiation stage without measurably altering induction of neighbouring genes outside the affected loop, confirming that these interactions are functionally required rather than incidental consequences of activation. These results establish a temporal hierarchy in which GATA4-dependent loop formation generally precedes enhancer activation and transcriptional induction during human cardiomyocyte differentiation, and demonstrate the functional necessity of individual stage-specific loops at core cardiac transcription factor loci.