Fig. 1.

TiPARP is a direct target gene and a negative regulator of HIF. (A) A schematic representation of TiPARP promoter. Core sequence of hypoxia-response element (HRE) is highlighted in red. (B) HCT116 and MCF-7 cells were cultured at normoxia or 1% O₂ (hypoxia) for 16 h. Expression of TiPARP was analyzed by qRT-PCR. Data are represented as means + SD (n = 3). (C) HCT116 and MCF-7 cells were treated with 1 mM DMOG or incubated at 1% O₂ (hypoxia) for 18 h. Endogenous TiPARP and HIF-1α were analyzed by Western blot. (D) Luciferase reporter assay showing that HIF-1 binds to HRE of TiPARP. Luciferase reporter construct used in this experiment contained about 1.2-kb proximal promoter fragment of human Tiparp gene. (Top) Schematic representation of the luciferase reporter construct with WT or mutated (Mut) HRE. (Bottom) HIF-1α transactivation measured by luciferase reporter with WT or mutant HRE from TiPARP promoter. Transfection efficiencies were normalized to cotransfected Renilla-luciferase. Data are represented as means ± SD (n = 2 for the vector control and n = 4 for WT and mutant). (E) ChIP assay assessing the binding of HIF-1α to HRE in endogenous TiPARP promoter. RNA polymerase II (Pol II) was used as a positive control. (F) HIF-1 luciferase reporter activity in HEK 293T cells showing that WT TiPARP, but not inactive H532A mutant (HA), decreases HIF-1 transcriptional activity. Luciferase reporter construct used in this experiment contained three hypoxia response elements from the Pgk-1 gene. Relative luciferase activities were normalized with the cotransfected Renilla-luciferase. Data are represented as means ± SD (n = 6). (G) HIF-1 luciferase reporter activity measured in hypoxic HEK 293T cells transfected with empty vector, Flag-tagged TiPARP WT or H532A mutant. Luciferase reporter construct used in this experiment contained three HREs from the Pgk-1 gene. Data are represented as means ± SD (n = 6 for the hypoxic WT TiPARP samples and n = 3 for other samples). (H) qRT-PCR analysis of HIF-1 target gene induction in response to hypoxia, in HCT116 cells stably expressing control shRNA (Control) or shTiPARP (KD). The ratio of hypoxic to normoxic gene expression is shown. Data are represented as means + SD (n = 3). (I) HCT116 cells were treated with 1 μg/mL doxycycline for 24 h to induce the overexpression of empty vector (EV), Flag-tagged WT TiPARP, or inactive H532A mutant. Cells were then incubated at hypoxia (1% O₂) for 16 h. Hypoxic induction of HIF target genes were measured by qRT-PCR. Data are represented as means + SD (n = 6). Statistical analyses were performed using unpaired two-tailed t tests. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.