Figure 10

HIF2α binds to birc5a and birc5b promoter. (a) HIF2α binds to HRE as confirmed by EMSA. COS-1 cells were transfected with zHif2A-HA and zARNT1A-HA as indicated. Nuclear extracts were collected at 48 h post-transfection followed by gel mobility shift assays by using the HRE oligonucleotides as a probe. The asterisks in lanes 3, 4 and 6 indicate the specific HIF2–HRE complex formed. NE, nuclear extract; COS-1, nuclear extract of COS-1 cells without transfection as a control; Hif2A, nuclear extract of COS-1 cells transfected with pcDNA3-zHif2A-HA; Hif2A+zARNT1A, nuclear extract of COS-1 cells co-transfected with pcDNA3-zHif2A-HA and pcDNA3-zARNT1A-HA. (b) HIF2α binds to the birc5a and birc5b promoters in brain tissues under physiological conditions. A chromatin immunoprecipitation (ChIP) assay was performed with polyclonal antibody against anti-HIF2α or preimmune serum as control. Chromatin was prepared from adult brain tissue and then immunoprecipitated using polyclonal antibody against anti-HIF2α or preimmune serum (IgG) as control. The immunoprecipitates were subjected to PCR using specific primers spanning the HREs in the birc5a and birc5b promoters (shown in panel c). The module 5 of birc5a and modules 2 and 6 of birc5b were significantly enriched by the HIF2α antibody. The rest modules failed to be amplified. A portion of chromatin solution (2.4%) was used as input. The primers used in ChIP assay were listed in Table 1. (c) Schematic representation of the birc5a (NM_194397), birc5b (NM_145195), human BIRC5 (NM_001012271) and mouse Birc5 (NM_001012273) promoters. The putative binding sites for HIF2, including HRE1 (ACGTG, black bars) and HRE2 (GCGTG, gray bars), are indicated. The position of the DNA modules analyzed in ChIP-PCR assays is indicated by the boxes. The amplified fragments in ChIP assays (panel b), including the module 5 in birc5a promoter and the modules 2 and 6 in birc5b promoter, are indicated by black boxes