Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that initiates the heartbeat. Although critical SAN transcription factors, including Isl1 (Islet-1), Tbx3 (T-box transcription factor 3), and Shox2 (short-stature homeobox protein 2), have been identified, the cis-regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified. This study aimed to define PCs’ epigenetic profile using comparative ATAC-seq and to identify novel enhancers involved in SAN gene function, regulation, and development.

We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched assay for transposase-accessible chromatin peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF (transcription factor) binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at embryonic day 8.5 throughout later SAN development and maturation.

In conclusion, PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, cis-regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and a corresponding human ISL1 enhancer may regulate human SAN function.