Hierarchical enhancers 1 confer the temporal and partially compensatory transcriptional regulation of Nkx2-5 during heart development

We comprehensively interrogate enhancers U1 and U2 in controlling Nkx2-5 transcription during heart development. Serial genomic deletions in mice reveal a functional hierarchy of U1 and U2 as evidenced by stage-dependent phenotypic variations. Specifically, U1 and U2 function redundantly to confer Nkx2-5 expression at early stages, but U2 instead of U1 supported its expression at later stages of heart chamber formation and maturation. Combined deletions markedly reduce Nkx2-5 dosage as early as E7.5, despite being largely reinstated two days later, displaying heart malformations with precocious differentiation of cardiac progenitors. Together, we propose a model that the temporal and partially compensatory regulatory function of two enhancers dictates a TF?s dosage and specificity during development. 1) H3K27ac ChIP-seq and ATAC-seq data on E16.5 ventricles from Control and U2KO mice. DKO mice were generated by deleting both U1 and U2 locus and U2KO mice were generated by deleting only U2 locus using CRISPR-Cas9 genome editing. 2) Single cells of outflow tract (OFT) or heart tube (HT) were derived for scRNA-seq from DKO and Control mice at E8.25. 3) H3K27ac ChIP-seq data on E9.5 hearts from Control and DKO mice. 4) NKX2-5 ChIP-seq data on E9.5 hearts from DKO mice. 5) Hi-C with WT and U1U2-DKO E8.5 mouse hearts.