Novel enhancers conferring compensatory transcriptional regulation of Nkx2-5 in heart development.

Cell type-specific expression of the developmental gene is conferred by distinct enhancer elements. Current knowledge about mechanisms in transcriptional regulation and its specific roles in multistage heart morphogenesis is limited. We comprehensively interrogate enhancers U1 and U2 in controlling transcription during heart development. Serial genomic deletions in mice reveal U1 and U2 function redundantly to confer expression at early stages, but U2 instead of U1 supports its expression at later stages. Combined deletions markedly reduce dosage as early as E7.5, despite being largely reinstated two days later, displaying heart malformations with precocious differentiation of cardiac progenitors. Cutting-edge low-input chromatin immunoprecipitation sequencing (ChIP-seq) confirmed that not only genomic NKX2-5 occupancy but also its regulated enhancer landscape is mostly disturbed in the double-deletion mouse hearts. Together, we propose a model that the temporal and partially compensatory regulatory function of two enhancers dictates a transcription factor (TF)'s dosage and specificity during development.