Figure 2. HIF1α, ARNT, TRIP230, Rb and Rb-associated repressor proteins occupy hypoxia responsive regulatory regions of HIF1-regulated genes in a hypoxia-dependent fashion.

(A) A schematic of the VEGF proximal promoter and EPO enhancer and the relative location of oligonucleotides used for PCR amplification. (B) The status of HIF1α, ARNT, TRIP230, and Rb at the VEGF promoter and EPO enhancer in MCF7 cells as assayed by chromatin-immuno-precipitation (ChIP) and polymerase chain reaction (PCR) and compared to amplification reactions derived from lysates precipitated with control IgG. All ChIPs were performed at least three times except where indicated. (C) HIF1α and HIF2α protein levels are dramatically enriched during hypoxia in MCF7 cells. MCF7 cells were maintained in culture in either 20% or 1% O₂ for 24 h. Nuclear extracts were analyzed by immuno-blot and membranes were probed with affinity-purified antibodies to HIF1α and α-tubulin. Sequential ChIP of the proximal VEGF promoter and EPO enhancer using either anti-HIF1α (D) or anti-ARNT (E) affinity purified antibodies followed by immuno-precipitation with anti-TRIP230 and anti-Rb antibodies. (F) ChIP of the VEGF promoter after in MCF7 cells after transfection with either scrambled siRNA or siRb1 and immuno-precipitation with anti-TRIP230 antibody. Values are expressed as fold enrichment over control and were determined by quantitative real-time PCR. Each experimental value was corrected for input and experiments were performed twice. (G) Immuno-blot analysis of siRNA-transfected MCF7 cell lysates used in ChIP experiments. (H) ChIP of Rb-repressor complex proteins in MCF7 cells. Cells were treated as described above and chromatin complexes were isolated with affinity-purified antibodies directed to Sin3a, Sin3b, and HDACs 1–3.