CpG site hypomethylation at ETS1-binding region regulates DLK1 expression in Chinese patients with Tetralogy of Fallot
CpG site hypomethylation at ETS1-binding region regulates DLK1 expression in Chinese patients with Tetralogy of Fallot
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ETS1α结合区的 CpG 位点低甲基化调节中国法洛四联症患者的 DLK1 表达
DOI：
10.3892/mmr.2022.12609
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发表时间：
2022-01
影响因子：
3.4
通讯作者：
Guoying Huang
中科院分区：
文献类型：
--
作者：
Guixiang Tian;Lili He;Ruoyi Gu;Jingwei Sun;Weicheng Chen;Yanyan Qian;Xiaojing Ma;Weili Yan;Zhenshan Zhao;Ziqing Xu;Meijiao Suo;Wei Sheng;Guoying Huang
法洛四联症（TOF）是最常见的紫绀型先天性心脏畸形，约占10%的病例。虽然TOF的发病机制是复杂的，在很大程度上是未知的，表观遗传学起着巨大的作用，特别是DNA甲基化。蛋白δ样非经典Notch配体1（DLK1）基因编码Notch信号通路的非经典配体，其参与心脏发育。然而，DLK1在TOF发病机制中的表观遗传机制尚未阐明。因此，本研究旨在阐明其具体机制。本研究采用免疫组织化学方法检测DLK1蛋白表达，亚硫酸氢盐测序PCR检测DLK1启动子甲基化状态。采用双荧光素酶报告基因检测转录因子ETS 1对DLK1基因表达的影响。电泳迁移率变动分析和染色质免疫沉淀分析，在体内和体外，被用来验证ETS1转录因子的DLK1启动子的结合，以及DLK1启动子的甲基化状态对这种结合亲和力的影响。法洛四联症（TOF）患者右室流出道DLK 1的表达明显低于对照组（P<0.001）。TOF患者DLK1_R区域CpG 10、11位点甲基化水平明显低于对照组（P<0.01）。DLK1_R的甲基化水平和CpG位点11的甲基化状态均与DLK1蛋白表达呈正相关。发现ETS1通过结合到CpG位点11来抑制DLK1的转录活性，并且这种亲和力可以受到DLK1启动子的甲基化水平的影响。这些结果表明DLK1启动子的低甲基化可以增加ETS1转录因子的结合亲和力，从而抑制DLK1基因的转录活性，并有助于TOF的发展。
Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart malformation accounting for ~10% of cases. Although the pathogenesis of TOF is complex and largely unknown, epigenetics plays a huge role, specifically DNA methylation. The protein δ like non-canonical Notch ligand 1 (DLK1) gene encodes a non-canonical ligand of the Notch signaling pathway, which is involved in heart development. However, the epigenetic mechanism of DLK1 in the pathogenesis of TOF is yet to be elucidated. Therefore, the present study aimed to clarify its specific mechanism. In this study, immunohistochemistry was used to detect the protein expression of DLK1 and the methylation status of the DLK1 promoter was measured via bisulfite sequencing PCR. Dual-luciferase reporter assays were performed to examine the influence of transcription factor ETS proto-oncogene 1 (ETS1) on DLK1 gene expression. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay, both in vivo and in vitro, were used to verify the binding of the ETS1 transcription factor to the DLK1 promoter as well as the influence of methylation status of DLK1 promoter on this binding affinity. The expression of DLK1 in the right ventricular outflow tract was significantly lower in patients with Tetralogy of Fallot (TOF) than that in controls (P<0.001). Moreover, the methylation level of CpG site 10 and CpG site 11 in the DLK1_R region was significantly decreased in TOF cases compared with controls (P<0.01). The integral methylation levels of DLK1_R and the methylation status of the CpG site 11 were both positively associated with DLK1 protein expression in TOF cases. ETS1 was found to inhibit DLK1 transcriptional activity by binding to the CpG site 11 and this affinity could be influenced by the methylation level of the DLK1 promoter. These findings demonstrated that the hypomethylation of the DLK1 promoter could increase the binding affinity of ETS1 transcription factor, which in turn inhibited DLK1 gene transcriptional activity and contributed to the development of TOF.
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影响因子：
3.7
作者：
Serra-Juhé C;Cuscó I;Homs A;Flores R;Torán N;Pérez-Jurado LA
通讯作者：
Pérez-Jurado LA
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影响因子：
3.4
作者：
Zhu Y;Ye M;Xu H;Gu R;Ma X;Chen M;Li X;Sheng W;Huang G
通讯作者：
Huang G
影响因子：
8.8
作者：
Finn, Johanna;Sottoriva, Kilian;Liu, Yuru
通讯作者：
Liu, Yuru
法洛四联症
DOI：
10.1017/cbo9780511581656.023
发表时间：
2009
期刊：
影响因子：
--
作者：
R. Langford
通讯作者：
R. Langford
DOI：
10.1007/978-1-4471-4999-6_18-2
发表时间：
2020-12
期刊：
Pediatric and Congenital Cardiology, Cardiac Surgery and Intensive Care
影响因子：
--
作者：
Jennifer S. Nelson;E. Bove;J. Hirsch-Romano
通讯作者：
Jennifer S. Nelson;E. Bove;J. Hirsch-Romano