SRP174411 PRJNA511670 GSE124337 Hand2 selectively reorganizes chromatin accessibility to induce pacemaker-like transcriptional reprogramming (RNA-seq) Gata4, Mef2c, Hand2, and Tbx5 (GHMT) can reprogram transduced fibroblasts into induced pacemaker-like myocytes (iPMs), but the underlying mechanisms remain obscure. Here we explored the role of Hand2 in iPM formation by using a combination of transcriptome, genome, and biochemical assays. We found many shared transcriptional signatures between iPMs and endogenous sinoatrial node (SAN), yet key regulatory networks remain missing. We demonstrate that Hand2 augments chromatin accessibility at loci involved in sarcomere organization, electrical coupling, and membrane depolarization. Focusing on an established cardiac Hand2 cistrome, we observe selective reorganization of chromatin accessibility to promote pacemaker-specific gene expression. Moreover, we identify a novel Hand2 cardiac subtype diversity (CSD) domain through biochemical analysis of the N-terminus. By integrating our RNA-seq and ATAC-seq datasets, we highlight desmosome organization as a hallmark feature of iPM formation. Collectively, our results illuminate Hand2-dependent mechanisms that may guide future efforts to rationally improve iPM formation. Overall design: GHMT driven Hcn4-GFP pacemaker cells, GMT-iCMs, and control samples were harvested after 14 days of induction for ATAC and RNAseq library preparation. GSE124337 pubmed 31116981 parent_bioproject PRJNA511667