HIF1 or HIF2 target gene promoters/enhancers are bound by distinct HIF1α/Pol II or HIF2α/Pol II transcriptional complexes and formation of these transcriptional complexes depends on STAT3 or USF2 activity.

<p>Sonicated chromatin from normoxic RCC4, RCC4/STAT3 shRNA, RCC4/USF2 shRNA or hypoxic Hep3B cells or Hep3B/USF2 shRNA cell was subjected to anti-Pol II ChIP, the precipitated protein/DNA complexes were then subjected to a secondary ChIP using HIF1α or HIF2α antibodies. Precipitated DNA was analyzed for HIF target gene promoter and results were displayed as percent of relative fold of binding. <b>A</b>) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, <i>CA9</i> and <i>PGK1</i> (two left columns) and mRNA levels of CA9 and PGK1 in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). <b>B</b>) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, <i>PAI1</i> and <i>EPO</i> (two left columns) and mRNA levels of PAI-1 and EPO in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). <b>C</b>) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, <i>CA9</i> and <i>PGK1</i> and fold of induction of CA9 and PGK1 gene expression in hypoxic Hep3B and Hep3B/USF2 shRNA cells. <b>D</b>) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, <i>PAI1</i> and <i>EPO</i> and fold of induction of PAI1 and EPO in hypoxic Hep3B and Hep3B/USF2 shRNA cells.