Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer

Abstract

Rationale: The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3, ISL1 and SHOX2. Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. Objective: Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. Methods and Results: We performed ATAC-seq on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative regulatory DNA elements specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse orthologue of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. Conclusions: We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3 and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3. exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.

A gene desert required for regulatory control of pleiotropicShox2expression and embryonic survival

“…Consistent with our observations in embryonic hearts of S2 GD Δ/Δ embryos ( Fig. 4A, 5A, B ), Van Eif et al confirm the embryonic lethality phenotype in their embryos lacking the 250kb region and show that the lethality is likely a result of a hypoplastic SAN (and venous valves) due to loss of Shox2 protein in the SV 49 . In addition, through our targeted exploration we now define a 1.5kb element located within this 250kb window and driving transcriptional activity specifically in the Shox2 domain of the SV ( Fig.…”

“…The cumulative removal of enhancers via deletion of the gene desert further allowed functional assessment of fundamental cis -regulatory activities in other tissues. Most notably, in absence of the gene desert, we observed a depletion of Shox2 transcripts in the sinus venosus (or inflow tract), comprising the SAN pacemaker population and most likely cause of the observed embryonic lethality phenotype 22,49 . Furthermore, our results demonstrate that craniofacial Shox2 expression and in particular Shox2 transcripts in the mandibular and nasal processes critically depend on the presence of the gene desert.…”

“…Here we demonstrate an essential regulatory requirement of the gene desert for embryonic viability at mid-gestation by maintaining Shox2 transcription in the cardiac sinus venosus (SV) encompassing the SAN pacemaker myocardium. In a very recent, independently published study, van Eif et al report complementary observations at the same locus 49 . In this study, they performed ATAC-seq on SAN-like pacemaker cells differentiated from human pluripotent embryonic stem cells (hESC) and Hcn4 + SAN cells of newborn mice to delineate the cis -regulatory modules controlling the expression of TFs promoting cardiac pacemaker cell fate, such as TBX3, ISL1 and SHOX2 .…”

“…In this study, they performed ATAC-seq on SAN-like pacemaker cells differentiated from human pluripotent embryonic stem cells (hESC) and Hcn4 + SAN cells of newborn mice to delineate the cis -regulatory modules controlling the expression of TFs promoting cardiac pacemaker cell fate, such as TBX3, ISL1 and SHOX2 . While the authors initially focused on human cis -regulatory landscapes near these genes, they used CRISPR/Cas9 deletions to investigate the function of homologous SAN-specific accessible chromatin regions in the Shox2 and Tbx3 loci in mouse embryos 49 . In particular, within the 582kb gene desert domain deleted here, their study narrows the critical space down to a ∼250kb region.…”

“…6C, E ), potentially acting as critical enhancer controlling Shox2 in SAN pacemaker cells. Further enhancer deletion analyses will uncover whether Shox2 transcription in the SAN is controlled by a single cis -regulatory unit or is shielded by multiple enhancers as it could be the case in human embryos 49 .…”