Figure 5

TET1 served as a co-activator to activate hypoxia downstream target and TET1 interacted with HIF-1α through a specific domain together with mapping of the transactivation domain in TET1. (a) Synergistic activation of the Twist1-driven reporter construct by co-transfecting HIF-1α, TET1 (wild-type or point mutant), and CBP. N: normoxia; H: hypoxia. The asterisk (*) indicates statistical significance (P <0.05) between experimental and control transfections. pXP2-Twist1 promoter-driven luciferase construct alone was used as the control transfection. Error bars indicate standard deviations (s.d.) of triplicate luciferase activity. (b) Co-immunoprecipitation assays showed the interaction between HIF-1α and TET1. The expression levels of transfected proteins (WCE: whole cell extracts) were shown on the right panel. (c) Mapping of the domain in HIF-1α that interacted with TET1. The HIF-1α-175-305 truncation mutant interacted with TET1 using the co-immunoprecipitation assays. (d) Yeast one hybrid assays showed that the transactivation activity of TET1 is mapped to the region of a.a. 451-674. The HIF-1α (a.a. 1-826) was used as a positive control. The yeasts transformed with different plasmids can survive in the Sc-Ura medium, whereas only the yeasts containing plasmids that can activate the HIS3 gene can survive in the selection medium of Sc-Ura-His. (e) Co-immunoprecipitation experiments showed that the anti-TET1 antibody pulled down both endogenous OGT and HIF-1α in H1299 cells.