STAT3 or USF2 Contributes to HIF Target Gene Specificity

<div><p>The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein.</p> </div

STAT3 and USF2 are enriched on the promoters of HIF1 or HIF2 targets respectively.

<p>Chromatin immunoprecipitation was performed in chromatin lysates from normoxic RCC4 (<b>A</b>) or hypoxic Hep3B cells (<b>C</b>). Antibodies against STAT3 or USF2 were used to co-precipitate STAT3- and USF2-associated genomic DNA. Q-PCR was used to detect the promoters of HIF1 target genes, <i>CA9</i> and <i>PGK1</i>, the enhancer or promoter of HIF2 target genes, <i>EPO</i> and <i>PAI1</i>, or the HIF1/HIF2 common target, <i>VEGF</i>. Results were normalized to input samples as % of input. The relative fold binding was calculated by dividing each of the % input values by the % input value of STAT3 associated with CA9 promoter here in Figure 3A. Similar calculations were made for Figure 3C and ChIP/Re-ChIP in Figure 9. <b>B</b>) The relative expression levels of <i>CA9</i>, <i>PAI1</i> and <i>VEGF</i> mRNAs in normoxic RCC4 cells. <b>D</b>) The fold of induction of HIF1 target genes <i>CA9</i> and <i>PGK1</i>, HIF2 target genes <i>PAI1</i> and <i>EPO</i> in hypoxic Hep3B cells.</p

STAT3 increases the binding kinetics of HIF1α protein on its target gene promoters of CA9, PGK1 and VEGF in hypoxic RCC4T cells.

<p>Detection of HIF1α or STAT3 on the promoters of HIF1 target genes, <i>CA9</i> (<b>A</b>), <i>PGK1</i> (<b>B</b>) and <i>VEGF</i> (<b>C</b>) in RCC4T cells with or without STAT3 inhibitor S3I-201, cultured under normoxia or hypoxia for 1, 3, 8 and 16 hours. Results were expressed as % input DNA for easy appreciation of binding changes.</p

USF2 functions alone or with HIF2α to activate endogenous HIF2 target genes in normoxic Hep3B cells.

<p><b>A</b>) Western blot analysis of Flag-tagged USF2, HIF1αTM and HIF2αTM to monitor the expression of these plasmids in transfected Hep3B cells. The starts * indicate several HIF2α protein bands expressed from the vector. <b>B</b>) mRNA levels of HIF1 target genes, <i>PGK1</i>, <i>GLUT1</i>, <i>LDHA</i> and <i>CA9</i>, in normoxic Hep3B cells in response to transient transfection of the indicated plasmids. <b>C</b>) mRNA levels of HIF2 target genes, <i>EPO</i>, <i>PAI1</i>, <i>OCT4</i> and <i>PLAC8</i>, in normoxic Hep3B cells in response to transient transfection of the indicated plasmids.</p

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.</p

N-TADs of HIF1α or HIF2α is required for its functional interaction with STAT3 or USF2 to activate HIF1 or HIF2 target gene promoter.

<p><b>A</b>) Schematic presentation of HIF1αDPA (Double Proline to Alanine), HIF2αDPA and HIF1α/HIF2α hybrid constructs. <b>B</b>) Western blot analysis of HIF1α, HIF2α, HIF hybrids and STAT3 to monitor the protein expression during CA9/Luc reporter gene assays. <b>C</b>) Activation of HIF1 target gene reporter, CA9/Luc, by the indicated plasmids. HIS is an empty vector that contains a histidine tag only. <b>D</b>) Western blot analysis of HIF1α, HIF2α, HIF hybrids and USF2 to monitor the protein expression during PAI1/Luc reporter gene assays. <b>E</b>) Activation of HIF2 target gene reporter, PAI1/Luc, by the indicated plasmids.</p

The HIF2α/USF2 physical interaction requires the C-TAD and N-TAD of the HIF2α protein.

<p><b>A</b>) WB detection of USF2, HIF1α and HIF2α in RCC4 cell lysate (input), in precipitated materials by the protein A/protein G beads and pre-immuno serum (beads) or in precipitated materials by USF2 antibody and protein A/protein G beads (USF2-IP). <b>B</b>) Schematic presentation of Flag-tagged full-length (HIF2α), N-terminal (HIF2α-N) or C-terminal (HIF2α-C) halves of HIF2αTM or HIF2α deletion of N-TAD (HIF2α∆NTAD), IH (HIF2α∆IH) or C-TAD (HIF2α∆CTAD). <b>C</b>) Anti-Flag or anti-HA WB detection of Flag-tagged HIF2α or HA-tagged USF2 protein in cell lysates (Lysates) or in anti-Flag beads precipitated materials (IP). The red starts indicated the positions of HIF2αFL, HIF2α-N and HIF2α-C proteins. The Δ labeled band in USF2+HIF2-C lane was consistently observed, likely expressed from downstream ATG of HIF2α cDNA. <b>D</b>) Anti-Flag or anti-HA WB detection of Flag-tagged HIF2α or HA-tagged USF2 protein in cell lysates (Lysates) or in anti-Flag beads precipitated materials (IP). The background signals in USF2-HA only lane was deducted from the “anti-HA IP’ signal to calculate the % IP.</p

Model for HIF target gene specificity.

<p>Although HIF1α/ARNT and HIF2α/ARNT lack binding specificity, HIF1α/ARNT and HIF2α/ARNT activate unique target genes. Other transcription factors such as STAT3 and USF2 are required for HIF1 or HIF2 target gene activation, these HIF1 or HIF2 specific trascription partners contribute to HIF target gene specificity by mechanisms including: 1) A specific/preferential binding of STAT3 or USF2 to the promoters of HIF1 or HIF2 target genes. 2) Specific physical and functional interaction of HIF1α with STAT3, or HIF2α with USF2. and 3) HIF1α/ARNT specific transcription partners such as STAT3 or HIF2α/ARNT specific transcription partners such as USF2 are required for the formation of the functional transcription complexes on the promoters of HIF1 or HIF2 target genes respectively.</p

The role of HIF and USF2 binding sites on HIF target gene specificity.

<p><b>A</b>) Schematic presentation of the <i>CA9</i> promoters, a HIF1 target gene. Construct 1 was generated by inserting 2 copies of -191 HBS of PAI1 promoter, a HIF2 target gene near the -13 HBS of CA9 promoter. Constructs 2 and 3 were generated by inserting -684 and -565 USF2 binding sites of PAI1 promoter near the -13 HBS (construct 2) or near -1001 of CA9 promoter (construct 3). <b>B</b>) Schematic presentation of a shorter version of <i>CA9</i> promoters (-506/+25). Construct 4 was generated by replacing the original -13 HBS with -191 HBS of PAI1 promoter. Constructs 5 and 6 were generated by replacing the STAT3 binding sites at -499 and -464 of CA9 promoter with -191 HBS (construct 5) or -684 and -565 USF2 binding sites (construct 6) from the PAI1 promoter. Construct 7 was made by replaced -13 HBS and -499 and -464 STAT3 binding sites in the CA9 promoter with HBS and USF2 binding sites from PAI1 promoter. <b>C</b>) Fold of induction of CA9/Luc reporters (-1096/+25) activated by the indicated plasmids. <b>D</b>) Fold of induction of CA9/Luc reporters (-506/+25) activated by the indicated plasmids. The same activators used in Figure 1 were used for experiments here.</p