Functional organization of the natriuretic peptide gene cluster in development and disease

The expression of Nppa (ANF) and Nppb (BNP) marks the chamber myocardium in the embryo, and both genes serve as accurate markers for hypertrophy and heart failure. However, despite decades of research, the mechanisms regulating Nppa and Nppb gene expression during development and disease are not well understood. Because of their organization in an evolutionary conserved cluster and their near identical expression patterns, the genes form a valuable model to study co-regulation, regulatory landscape organization and regulatory element sharing. We analyzed the in vivo chromatin conformation, epigenetic status and enhancer potential of sequences surrounding the Nppa-Nppb cluster to gain insight into its transcriptional control. We show that the regulatory landscape of the cluster is confined to a 60 kbp region centered on Nppb. The conformation of the chromatin brings both promoters and several potential regulatory elements in close proximity to each other. Remarkably, this conformation is tissue- and stage-independent. The distribution of H3K27ac and association of Pol2 at several contact sites changed after stress, revealing their potential involvement in gene regulation. To explore co-regulation of Nppa and Nppb expression, we generated double-reporter transgenic mice carrying modified bacterial artificial chromosomes. Both bioluminescent signal from Luciferase, and red fluorescence from Katushka precisely recapitulated the spatio-temporal patterns of Nppa and Nppb, respectively, during development, hypertrophy and myocardial infarction. Simultaneous analysis of two reporters in vivo revealed that Nppa and Nppb share developmental, but not stress-response enhancers responsible for their co-regulation, and that the stress response of Nppa requires the promoter of Nppb.