FIG. 5.

CBP and SRC-1 cooperatively enhance HIF-1α-mediated transcriptional activation. (A) COS7 cells were transiently cotransfected with pCMV4/HIF-1α and 0.75 to 1.5 μg of SRC-1 and/or 0.75 to 1.5 μg of CBP expression plasmids together with a hypoxia-responsive reporter gene (pT81/HRE-luc) and subsequently exposed to 21 or 1% O₂. Luciferase values are presented as relative luciferase activity as described in the legend to Fig. 1. The results of three independent experiments performed in duplicate ± SE are shown. (B) Cooperative activation by CBP and SRC-1 of GAL4–HIF-1α fusion proteins. COS7 cells were cotransfected with GAL4–HIF-1α fusion constructs together with a GAL4-responsive reporter plasmid in the absence or presence of CBP and/or SRC-1, as indicated (in micrograms). Cells were exposed to 21 or 1% O₂ before harvest. After normalization for transfection efficiency using alkaline phosphatase activity, reporter gene activities are expressed as relative to that of GAL4 in normoxia. The results of two independent experiments performed in duplicate ± SE are shown. (C) p300 protein stimulates the activity of the two minimal transactivation domains of HIF-1α in a hypoxia-dependent manner. (Right) Schematic representation of full-length p300 (pCMVβ-p300-HA) and p300Δ (pCMVβ-p300Δ1254-1376). (Left) The two minimal transactivation domains of HIF-1α were fused to GAL4 DBD and transfected into COS7 cells together with a GAL4-responsive reporter plasmid in the absence or presence of p300, p300Δ, and/or SRC-1 expression vectors, as indicated. Cells were exposed to 21 or 1% O₂ before harvest. Normalized reporter gene activities are expressed as relative to that of GAL4 in normoxia. The results of a representative experiment performed in duplicate are shown.