Institute of Paediatrics, Children's Hospital of Fudan University, Shanghai 201102, P.R. China.
Cardiovascular Centre, Children's Hospital of Fudan University, Shanghai 201102, P.R. China.
Mol Med Rep. 2020 Nov;22(5):4412-4422. doi: 10.3892/mmr.2020.11535. Epub 2020 Sep 24.
Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease (CHD). Although a lower methylation level of whole genome has been demonstrated in TOF patients, little is known regarding the DNA methylation changes in specific gene and its associations with TOF development. NOTCH4 is a mediator of the Notch signalling pathway that plays an important role in normal cardiac development. However, the role of epigenetic regulation of the NOTCH4 gene in the pathogenesis of TOF remains unclear. Considering the NOTCH4 low mutation frequency and reduced expression in the TOF patients, we hypothesized that abnormal DNA methylation change of NOTCH4 gene may influence its expression and responsible for TOF development. In this study, we measured the promoter methylation status of NOTCH4 and was measured and its regulation mechanism was explored, which may be related to TOF disease. Additionally, the promoter methylation statuses of NOTCH4 was measured in order to further understand epigenetic mechanisms that may serve a role in the development of TOF. Immunohistochemical analysis was used to examine NOTCH4 expression in right ventricular outflow tract myocardial tissues in patients with TOF. Compared with healthy controls, patients with TOF displayed significantly reduced in NOTCH4 expression (P=0.0055). Moreover, bisulphite sequencing suggested that the methylation levels of CpG site 2 in the NOTCH4 promoter was significantly higher in the patients than in the controls (P=0.0459). NOTCH4 expression was negatively associated with CpG site 2 methylation levels (r=‑0.51; P=0.01). ETS1 transcription factor can serve as transcriptional activators by binding to specific DNA sequences of target genes, such as DLL4 and NOTCH4, which serves an important role in normal heart development. Dual‑luciferase reporter and electrophoretic mobility shift assays indicated that the ETS1 transcription factor could bind to the NOTCH4 promoter region. However, binding of ETS1 to the NOTCH4 promoter was abrogated by methylation at the putative ETS1 binding sites. These findings suggested that decreased NOTCH4 expression in patients with TOF may be associated with hypermethylation of CpG site 2 in the NOTCH4 promoter region, due to impaired binding of ETS1.
法洛四联症（TOF）是最常见的发绀型先天性心脏病（CHD）。虽然已有研究表明 TOF 患者的全基因组甲基化水平较低，但关于特定基因的 DNA 甲基化变化及其与 TOF 发生发展的关系仍知之甚少。NOTCH4 是 Notch 信号通路的介体，在心脏正常发育中发挥重要作用。然而，NOTCH4 基因的表观遗传调控在 TOF 发病机制中的作用尚不清楚。鉴于 TOF 患者 NOTCH4 基因突变频率低且表达降低，我们假设 NOTCH4 基因的异常 DNA 甲基化改变可能影响其表达，并导致 TOF 的发生。在这项研究中，我们测量了 NOTCH4 的启动子甲基化状态，并探讨了其调控机制，这可能与 TOF 疾病有关。此外，还测量了 NOTCH4 的启动子甲基化状态，以进一步了解可能在 TOF 发展中起作用的表观遗传机制。免疫组织化学分析用于检测 TOF 患者右心室流出道心肌组织中 NOTCH4 的表达。与健康对照组相比，TOF 患者的 NOTCH4 表达明显降低（P=0.0055）。此外，亚硫酸氢盐测序表明，NOTCH4 启动子 CpG 位点 2 的甲基化水平在患者中明显高于对照组（P=0.0459）。NOTCH4 表达与 CpG 位点 2 甲基化水平呈负相关（r=-0.51；P=0.01）。ETS1 转录因子可通过与靶基因如 DLL4 和 NOTCH4 的特定 DNA 序列结合而作为转录激活因子，在正常心脏发育中发挥重要作用。双荧光素酶报告基因和电泳迁移率变动分析表明，ETS1 转录因子可与 NOTCH4 启动子区结合。然而，在假定的 ETS1 结合位点发生甲基化后，ETS1 与 NOTCH4 启动子的结合被阻断。这些发现表明，TOF 患者 NOTCH4 表达降低可能与 NOTCH4 启动子区 CpG 位点 2 的高甲基化有关，这是由于 ETS1 结合受损所致。
