20 pmol siRNA

Nuclear receptor-binding SET domain-containing protein 1 (Nsd1) acts as a histone lysine methyltransferase, and its role in oxidative stress-related abnormal embryonic heart development remains poorly understood. In the present study, H2O2 decreased the expression of Nsd1 and NK2 transcription factor related locus 5 (Nkx2.5). We further focused on Nkx2.5 modulating the transcription of Nsd1 in response to H2O2. Luciferase activity analysis indicated that a regulatory region from - 646 to - 282 is essential for the basal transcriptional activity, in which, an a Nkx2.5-binding element (NKE) was identified at - 412/- 406 of the Nsd1 promoter by electrophoresis mobility shift assay and a chromatin immunoprecipitation assay. H2O2 obviously reduced the p646-luc promoter activity, and the depletion of Nkx2.5 expression weakened H2O2 inhibition on the p646-luc promoter. The overexpression of Nkx2.5 increase Nsd1 p646-luc promoter activity, but did not affected p646-luc-mut. Furthermore, overexpression and depletion of Nkx2.5 led to the increase and decrease of Nsd1 protein and mRNA levels. These data indicated that H2O2-induced Nsd1 suppression resulted from the decrease of Nkx2.5 expression through the NKE element.