Genetic Dissection of a Super Enhancer Controlling the Nppa-Nppb Cluster in the Heart

Rationale: ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide), encoded by the clustered genesNppa andNppb , are important prognostic, diagnostic, and therapeutic proteins in cardiac disease. The spatiotemporal expression pattern and stress-induction of theNppa andNppb are tightly regulated, possibly involving their coregulation by an evolutionary conserved enhancer cluster.Objective: To explore the physiological functions of the enhancer cluster and elucidate the genomic mechanism underlyingNppa-Nppb coregulation in vivo.Methods and Results: By analyzing epigenetic data we uncovered an enhancer cluster with super enhancer characteristics upstream ofNppb . Using CRISPR/Cas9 genome editing, the enhancer cluster or parts thereof,Nppb and flanking regions or the entire genomic block spanningNppa-Nppb , respectively, were deleted from the mouse genome. The impact on gene regulation and phenotype of the respective mouse lines was investigated by transcriptomic, epigenomic, and phenotypic analyses. The enhancer cluster was essential for prenatal and postnatal ventricular expression ofNppa andNppb but not of any other gene. Enhancer cluster–deficient mice showed enlarged hearts before and after birth, similar toNppa-Nppb compound knockout mice we generated. Analysis of the other deletion alleles indicated the enhancer cluster engages the promoters ofNppa andNppb in a competitive rather than a cooperative mode, resulting in increasedNppa expression whenNppb and flanking sequences were deleted. The enhancer cluster maintained its active epigenetic state and selectivity when its target genes are absent. In enhancer cluster–deficient animals,Nppa was induced but remained low in the postmyocardial infarction border zone and in the hypertrophic ventricle, involving regulatory sequences proximal toNppa .Conclusions: Coordinated ventricular expression ofNppa andNppb is controlled in a competitive manner by a shared super enhancer, which is also required to augment stress-induced expression and to prevent premature hypertrophy.