Cotransfected p300 enhances hypoxic induction of transcription from the Epo enhancer (30). Transient transfection of Hep3B cells was performed with 1 μg of a luciferase reporter gene (E) containing the Epo enhancer upstream of the simian virus 40 promoter or one in which the HIF-1 binding site has been mutated (Em). Cells were cotransfected with 0–2 μg (Left) and 0 or 2 μg (Right) of pCMV-p300 (4) with 1 μg of pCMV-lacZ (to correct for variations in transfection efficiency). The y axis shows the ratio of luciferase expression at 1% O₂ to that at 21% O_2. Results on the Left are the mean of triplicate experiments ± 1 SD and on the Right are duplicate experimental points.

p300 binds HIF-1α (28). (A) GST fusion proteins containing the indicated portions of p300 C/H1 were constructed. ³⁵S-Labeled HIF-1α was transcribed and translated in vitro and mixed with the indicated GST fusion proteins immobilized on glutathione beads. Bead-bound proteins were visualized by SDS/PAGE and autoradiography. (B) Full-length p300 (WT) and p300 deleted within the first cysteine/histidine-rich region (C/H1) were synthesized in insect cells using the baculovirus system. ³⁵S-labeled HIF-1α was synthesized in vitro, and translation products were mixed with the indicated baculo-p300 species and immunoprecipitated with a p300 antibody [RW128 (4)] using protein-A Sepharose beads. Radiolabeled bead-bound proteins were visualized by SDS/PAGE and autoradiography. Lanes 1 in A and B each contain 20% of the input translation products analyzed in the other lanes. (C) U-2 OS cells were transfected with 10 μg of pCMVβ-HA-HIF-1α (+) or vector alone (−). Cells were labeled with [³⁵S]methionine, and cellular extracts were prepared, mixed (where indicated) with the relevant baculovirus-p300 species, and then immunoprecipitated with either anti-p300 or anti-HA antibody, as in B. Bead-bound proteins were released by boiling, and reimmunoprecipitated with antibody to the hemagglutinin (HA) epitope (12CA5). Proteins bound to beads in this second round were visualized by SDS/PAGE and autoradiography. The open arrowhead indicates HA–HIF-1α. The identity of the faster migrating band is not clear but may represent a degradation product. Standard molecular weights (kDa; Sigma) are indicated. IVT, in vitro translate; pp, precipitation; IP, immunoprecipitation.

E1A inhibits hypoxia-driven transcription (31). (A) Hep3B cells were cotransfected with 1 μg of pCMV-lacZ and 0.5 μg of Epo49-Luc (32) (diagramed below the bar graph) and brought to 16 μg of total with Bluescript DNA (Stratagene). Twenty-four hours later, cells were infected with adenovirus type 5 bearing the indicated E1A deletion mutations. The 1101 mutant deletes E1A residues 4–25, while the 1107 mutant deletes residues 111–123. E1AΔ111-123 interacts with p300/CBP but not with pRB (33), while E1AΔ4-25 does not recognize p300/CBP but does bind to pocket proteins (33). The adenoviruses also bore a mutation (dl520) rendering them unable to synthesize 13S E1A (34). (B) Hep3B cells were infected with either wild-type (wt) or with mutant adenoviruses as in A. After infection, cells were placed in either 1% or 21% O₂ for 6 hr; total cellular RNA was extracted, and Northern blots analyses using a VEGF probe were performed.

One or more members of the p300/CBP family interact(s) with HIF-1α in a DNA-bound complex (27). (A) Nuclear extracts from Hep-3B cells were analyzed by EMSA using a HIF-site-containing probe. Extracts in lanes 1–5 and 7–8 were prepared from cells treated for 5 hr with 1% O₂, and the extract in lane 6 was prepared from normoxic cells. An 100-fold excess of unlabeled wild-type Epo 3′ enhancer probe (WT, lane 5), mutant probe (mut, lane 4), probe containing a central midline element (CME, lane 3), probe containing a xenobiotic response element (XRE, lane 2), or nonspecific probe (NS: myc E2F site, lane 1) were used as competitors. HIF-1α antibody (OZ15) was tested as a potential supershifting reagent in lane 8. In both experiments, free probe was in excess and is not shown. (B) Nuclear extracts from Hep-3B cells were analyzed as in A. Extracts in lanes 2–4 were prepared from cells treated for 5 hr with 1% O₂. Extracts in lanes 1 and 5 were prepared from normoxic cells, and lane 6 contains no cellular protein. HIF-1α antibody (OZ15) and p300/CBP monoclonal antibody (AC240) were analyzed as potential supershifting reagents in lanes 3 and 4–6, respectively. Induced (I) and constitutive (C) complexes are indicated.