Figure 9

MED15 deficiency inhibits the TAD function of HIF1α and the binding of HIF1α to HRE.A, schematic diagram of Gal4/UAS reporting system. The DNA-binding domain of Gal4 (Gal4-DBD) and transcriptional activation domain of HIF1α (HIF1α-TAD) are fused and expressed. B, luciferase reporter assays in WT or MED15 KO (KO #19) HCT116 cells. Cells were cotransfected with GalO or GalA, pSV40-Renilla, and reporter plasmid (pGal4-UAS-Elb-FLuc), followed by being exposed to 21% O₂ or 1% O₂ for 24 h. Data are represented as means ± SD. n = 3 biologically independent extracts. ∗∗∗∗p < 0.0001 by two-way ANOVA with Tukey's multiple comparisons test. C–F, Cut&Run analysis in WT or MED15 KO (KO #19) HCT116 cells. Cells were exposed to 1% O₂ for 24 h. Cut&Run assays were performed with antibody against IgG or HIF1α followed by qPCR analysis of HRE enrichment multiples of HIF target genes CA9 (C), PKM (D), VEGFA (E), and CPT1A (F). Data are represented as means ± SD. n = 3 biologically independent extracts. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-way ANOVA with Tukey's multiple comparisons test. CA9, carbonic anhydrase 9; CPT1A, carnitine O-palmitoyltransferase 1; E1b, a TATA box from the promoter region of adenovirus E1b gene; Gal4-UAS, Gal4 binding DNA sequence; HIF, hypoxia-inducible factor; MED15, Mediator complex subunit 15; ns, not significant; PKM, pyruvate kinase; VEGFA, vascular endothelial growth factor A.