Abstract:
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.
摘要:
法洛四联症(TOF)是紫红色先天性心脏病(CHD)的最常见形式。尽管在TOF患者中已经证明了整个基因组的甲基化水平较低,关于特定基因的DNA甲基化变化及其与TOF发育的关系知之甚少。NOTCH4是Notch信号通路的介质,在正常心脏发育中起重要作用。然而,NOTCH4基因的表观遗传调控在TOF发病中的作用尚不清楚.考虑到NOTCH4在TOF患者中的低突变频率和低表达,我们假设NOTCH4基因的异常DNA甲基化改变可能影响其表达并负责TOF的发育。在这项研究中,我们测量了NOTCH4的启动子甲基化状态,并对其调控机制进行了探讨,这可能与TOF疾病有关。此外,我们对NOTCH4启动子甲基化状态进行了测定,以进一步了解可能在TOF发生中起作用的表观遗传机制.免疫组织化学分析用于检测TOF患者右室流出道心肌组织中NOTCH4的表达。与健康对照相比,TOF患者NOTCH4表达显著降低(P=0.0055).此外,亚硫酸氢盐测序表明,患者NOTCH4启动子中CpG位点2的甲基化水平明显高于对照组(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转录因子可以通过与靶基因的特定DNA序列结合作为转录激活因子,如DLL4和NOTCH4,在正常心脏发育中起重要作用。双荧光素酶报告基因和电泳迁移率变化分析表明,ETS1转录因子可以与NOTCH4启动子区结合。然而,ETS1与NOTCH4启动子的结合被推定的ETS1结合位点的甲基化所取消.这些结果表明,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。由于ETS1的结合受损。