miR-30 Regulates Mitochondrial Fission through Targeting p53 and the Dynamin-Related Protein-1 Pathway
Figure 5
p53 binds to and activates Drp1 promoter.
(A) The potential p53 binding sites in the Drp1 promoter region. Drp1 promoter region contains two potential p53 DNA binding sites (indicated as BS1 and BS2). The mutations were introduced to BS1 and BS2 (the converted nucleotides are underlined), respectively. (B) ChIP analysis of in vivo endogenous p53 binding to Drp1 promoter upon hydrogen peroxide treatment. 6 h after treatment cells were harvested for the ChIP analysis. Chromatin-bound DNA was immunoprecipitated with the anti-p53 antibody. Anti-actin antibody was used as a negative control. Immunoprecipitated DNA was analyzed by PCR using a primer combination that encompassed p53 BS1 (upper panel) or BS2 (lower panel). (C) p53 activates Drp1 promoter activity. Cardiomyocytes were infected with Adp53 or Adβ-gal at a moi of 50. 24 h after infection cells were transfected with the constructs of the empty vector (pGL-4), Drp1 wild-type promoter, or the mutated Drp1 promoters (m-BS1 and m-BS2), respectively. Firefly luciferase activities were normalized to Renilla luciferase activities. Data are expressed as the relative luciferase activity. (D) Hydrogen peroxide activates Drp1 promoter activity. Cardiomyocytes were infected with Adp53RNAi or Adp53-S-RNAi at a moi of 50. 24 h after infection cells were transfected with the constructs. 24 h after transfection cells were exposed to hydrogen peroxide. Data are expressed as the mean±SEM of three independent experiments.