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The nuclear receptor subfamily 2 group F member 2 (NR2F2) gene encodes a ligand-inducible transcription factor involved in angiogenesis and heart development. This study aimed to elucidate the molecular mechanism of epigenetic regulation of NR2F2 in tetralogy of Fallot (TOF) development. In the present study, immunohistochemical staining showed that NR2F2 protein expression was significantly higher in the right ventricular outflow tract (RVOT) tissues of TOF cases compared with controls. The methylation status of the CpG island shore (CGIS) of the NR2F2 gene was decreased in TOF cases, and the CpG site 3 in the CGIS region of NR2F2 promoter was a differential methylation site. Furthermore, the methylation level of the CpG site 3 and the NR2F2 protein expression were significantly negatively correlated in TOF patients. In vitro functional analysis revealed that RXRα could upregulate the NR2F2 gene by directly binding to the CGIS in the NR2F2 promoter, while hypomethylation of the NR2F2 promoter via treatment with 5-azacytidine influenced the affinity of RXRα to its binding sites, as shown by ChIP-qPCR. These findings suggest that promoter hypomethylation activates NR2F2 by enhancing RXRα binding to NR2F2 CGIS in the development of TOF.
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