窦房结(SAN)起搏器心肌细胞的发育和功能,心脏的主要起搏器,由保守的转录因子网络严格控制,包括TBX3(T-box转录因子3),ISL1(ISLLIMhomeobox1),和SHOX2(身材矮小的Homeobox2)。然而,在SAN起搏器细胞中控制靶基因表达的调节DNA元件(RE)仍未定义。
鉴定人类SAN样起搏器细胞的调控景观和可能参与起搏器细胞基因调控的SAN特异性RE的功能评估。
我们对人类多能干细胞衍生的SAN样起搏器细胞和心室样细胞进行了测序,对转座酶可接近的染色质进行了测定,并鉴定了数千个对任一人类细胞类型具有特异性的推定RE。我们验证了SHOX2和TBX3基因座中的起搏器细胞特异性元件。CRISPR介导的小鼠非编码区的纯合缺失与SHOX2基因座中的候选起搏器特异性REs导致发育中的SAN和胚胎致死性选择性丧失Shox2表达。在靠近MED13L的区域中,在TBX3上游发现了高达1Mbp的推定起搏器特异性RE,该区域具有与运动后心率恢复相关的变异。在小鼠中删除直系同源区域,导致SAN和(心脏)神经节中Tbx3表达的选择性丧失以及新生儿致死率。Tbx3的表达在包括房室传导系统在内的其他组织中得以维持,肺,还有肝脏.杂合成年小鼠在起搏后显示增加的SAN恢复时间。携带相关变体的人类RE强烈驱动转基因小鼠胚胎SAN中的表达。
我们提供了一个全基因组的候选人类起搏器特异性RE集合,包括SHOX2,TBX3和ISL1的基因座,并确定了影响运动后心率恢复的人类遗传变异与具有高度保守功能的变异RE之间的联系,驱动TBX3的SAN表达式。
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 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined.
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.
We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs 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 ortholog 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.
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.