Fig. 3: HIF-1α and p300 physically binds to the promoter of NDRG1-OT1.
A Schematic representation of firefly luciferase constructs containing the NDRG1-OT1 promoter (−2000 to −1 bp), showing both the wild-type sequence (WT) and the mutation of the HIF-1α binding sites (mut). There are two putative HREs ([A/G]CGTG) within the promoter of NDRG1-OT1. Site 1: −1773 to −1769 bp; site 2: −647 to −643 bp. B Luciferase reporter assays of NDRG1-OT1 promoter in cells treated with CoCl2, or overexpressing HIF-1α or HIF-2α. HEK293T cells were transfected with HIF-1α or HIF-2α expressing plasmids, firefly luciferase plasmids, and Renilla reporter vectors. The CoCl2 treatments were administered for 24 h. The relative firefly luciferase activity was measured and normalized to Renilla luciferase activity. C Luciferase reporter assays of NDRG1-OT1 mutant promoters in cells treated with CoCl2. HEK293T cells were transfected with indicated firefly plasmids and Renilla reporter vectors. The CoCl2 treatments were administered for another 24 h after transfection of plasmids for 24 h. D, E ChIP of HIF-1α, p300, and NDRG1-OT1 promoter in MCF-7 (D) and MDA-MB-453 (E) cells under hypoxia. Anti-HIF-1α or anti-p300 antibody was used to precipitate chromatin, and DNA was examined by primers flanking site 1 or site 2 of the NDRG1-OT1 promoter. VEGF promoter served as a positive control. Chromatin precipitated by anti-RNA polymerase II antibody was amplified by primers flanking the GAPDH promoter and served as a negative control. The relative chromatin enrichment (%) was normalized to input. All data shown are the means ± SDs (n = 3). IP immunoprecipitation. *P < 0.05.
