FIG. 4.

Definition of HIF-1α structures which are regulated by the CBP and SRC-1 coactivators. (A) Different subregions of HIF-1α were fused to the GAL4 DBD and transfected into COS7 cells together with a GAL4-responsive reporter plasmid in the absence or presence of 1.5 μg of either CBP or SRC-1 expression plasmid. Cells were exposed to 21 or 1% O₂ for 36 h before harvest. N-TAD and C-TAD, N- and C-terminal transactivation domains. (B) The C-terminal transactivation domain of Arnt mediates transcriptional activation by CBP and SRC-1. COS7 cells were cotransfected with the indicated GAL4-Arnt fusion proteins and 1.5 μg of CBP or SRC-1 expression vectors together with a GAL4-responsive reporter plasmid. Six hours after transfection, cells were exposed to either 21 or 1% O₂ for 36 h before harvest and reporter gene assays. (C) HIF-1α prevents Arnt from functionally interacting with CBP and SRC-1. GAL4/Arnt 128-774 and a deletion mutant (GAL4/Arnt 128-603) were transfected into COS7 cells together with 0.2 μg of hHIF-1α expression vector and 1.5 μg of CBP and SRC-1 expression vectors, as indicated. Luciferase activity was measured following 36 h of exposure to either 21 or 1% O₂. Normalized reporter gene activities are expressed relative to that of nonfusion GAL4 in normoxia. The results of three independent experiments performed in duplicate ± SE are shown.