Methylation status of CpG sites in the NOTCH4 promoter region regulates NOTCH4 expression in patients with tetralogy of Fallot

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.

CpG site hypomethylation at ETS1‑binding region regulates DLK1 expression in Chinese patients with Tetralogy of Fallot

“…Aberrant methylation of promoter CpG islands is known to contribute to the phenotypic expression of CHDs, including TOF and VSD ( 3 ). Our previous studies identified that the altered expression of some genes involved in cardiac development, such as Notch4 and COUP transcription factor 2 in TOF, may be associated with the abnormal methylation of their promoters ( 26 , 27 ). In the current study, no significant difference was observed in the integral methylation level of DLK1 promoter between the patients with TOF and controls.…”

“…In the current study, no significant difference was observed in the integral methylation level of DLK1 promoter between the patients with TOF and controls. However, previous studies have demonstrated that the methylation of certain CpG sites can affect gene expression ( 26 , 28 , 29 ). Therefore, the methylation status of each CpG site in the DLK1_R region was analyzed in the present study.…”

“…The Ethics Committee of the Children's Hospital of Fudan University [Shanghai, China; approval no. 2015( 26 )] and Soochow University (Suzhou, China) approved the collection of cardiac tissues. Written informed consent of all study participants was obtained from their parents or relatives, and the study was registered in the Chinese Clinical Trial Registry (registration no.…”

Genome-Wide Analysis of DNA Methylation in Buccal Cells of Children Conceived through IVF and ICSI

“…Intriguingly, we found DMR in ZFPM2 which is a gene highly related to Tetralogy of Fallot (TOF) in the DisGeNET database and ZFPM2 promoter hypermethylation has been found in TOF patients [ 67 ]. Moreover, HAND1 and NOTCH4 , in which we found DMR, display abnormal methylation patterns of their promoter regions in TOF patients [ 68 , 69 ]. Interestingly, TOF has been shown to be more prevalent in the ART-conceived population (adjusted OR 2.4, 95% CI 1.5–3.7) [ 70 ] even if an up-to-date meta-analysis did not find any increased risk of this congenital heart defect [ 71 ].…”

Uncovering a Key Role of ETS1 on Vascular Abnormality in Glioblastoma

“…Vascular abnormality is associated with a distinct gene signature in ECs (10,28). ETS1 could control the expression of several genes driving vascular abnormality, including MCAM (29), ANGPT2 (30), SOX4 (31), VEGFA/VEGFR2 (25,32), ITGA1 (33), NOTCH4 (34,35). However, the direct effects of ETS1 on function of tumor ECs remain poorly defined.…”