Cardiology Division (G.G., R.M., H.R., C.J., R.S.W., P.K.R.A., A.R., M.B.S., V.V.), University of California, San Francisco.
Cardiovascular Research Institute (T.S., R.S.W., B.L.B., V.V.), University of California, San Francisco.
Circ Res. 2020 Dec 4;127(12):1502-1518. doi: 10.1161/CIRCRESAHA.120.317145. Epub 2020 Oct 12.
Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate 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 -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.
To define the epigenetic profile of PCs using comparative ATAC-seq (assay for transposase-accessible chromatin with sequencing) and to identify novel enhancers involved in SAN gene regulation, development, and function.
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 locus that was active specifically in the cardiac inflow at embryonic day 8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations.
(1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) -regulation of specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human enhancer may regulate human SAN function.
窦房结（SAN）中的心脏起搏器细胞（PCs）具有独特的基因表达程序，使其能够自动发射冲动并启动心跳。尽管已经确定了关键的 SAN 转录因子，包括 Isl1（胰岛 1）、Tbx3（T 盒转录因子 3）和 Shox2（短体同源盒蛋白 2），但控制 PC 特异性基因表达的调节结构尚不清楚，也尚未确定用于 SAN 基因调控的离散增强子。
使用比较 ATAC-seq（带有测序的转座酶可及染色质分析）来定义 PCs 的表观基因组，并鉴定参与 SAN 基因调控、发育和功能的新增强子。
我们使用分选的新生鼠 SAN 中的 ATAC-seq 比较了 PC 和右心房心肌细胞中可及染色质的区域。代表候选 SAN 调节元件的 PC 丰富的转座酶可及染色质峰位于已建立的 SAN 基因附近，并富含独特的 TF（转录因子）结合位点集。在使用转基因小鼠实验验证的几个新的 SAN 增强子中，我们鉴定出一个位于 基因座的 2.9kb 调节元件，该元件在胚胎第 8.5 天和整个后期 SAN 发育和成熟过程中仅在心脏流入道中具有活性。从小鼠基因组中删除该增强子导致 SAN 发育不良和窦性心律失常。该小鼠 SAN 增强子还在发育中的斑马鱼心脏中引导报告基因活性到流入道，证明其上游调控网络具有深度保守性。最后，人类基因组中与小鼠增强子同源的区域附近发生的单核苷酸多态性与人类群体中的静息心率存在显著关联。
（1）PCs 具有与基因表达谱相关的独特可及染色质区域，并包含新的 SAN 增强子；（2） -特异性在 SAN 中的调控取决于一个保守的 SAN 增强子，该增强子调节 PC 发育和 SAN 功能；（3）相应的人类 增强子可能调节人类 SAN 功能。
