Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor (HIF), a heterodimer of HIF-␣ and the aryl hydrocarbon receptor nuclear translocator subunits. The HIF-1␣ and HIF-2␣ subunits are structurally similar in their DNA binding and dimerization domains but differ in their transactivation domains, implying they may have unique target genes. Previous studies using Hif-1␣ ؊/؊ embryonic stem and mouse embryonic fibroblast cells show that loss of HIF-1␣ eliminates all oxygen-regulated transcriptional responses analyzed, suggesting that HIF-2␣ is dispensable for hypoxic gene regulation. In contrast, HIF-2␣ has been shown to regulate some hypoxia-inducible genes in transient transfection assays and during embryonic development in the lung and other tissues. To address this discrepancy, and to identify specific HIF-2␣ target genes, we used DNA microarray analysis to evaluate hypoxic gene induction in cells expressing HIF-2␣ but not HIF-1␣. In addition, we engineered HEK293 cells to express stabilized forms of HIF-1␣ or HIF-2␣ via a tetracycline-regulated promoter. In this first comparative study of HIF-1␣ and HIF-2␣ target genes, we demonstrate that HIF-2␣ does regulate a variety of broadly expressed hypoxia-inducible genes, suggesting that its function is not restricted, as initially thought, to endothelial cell-specific gene expression. Importantly, HIF-1␣ (and not HIF-2␣) stimulates glycolytic gene expression in both types of cells, clearly showing for the first time that HIF-1␣ and HIF-2␣ have unique targets.Oxygen (O 2 ), the final electron acceptor during oxidative phosphorylation, is absolutely required for invertebrate and vertebrate life. The immediate response to O 2 deprivation (hypoxia) is a defense phase, which suppresses ATP consumption by arresting protein translation and ion channel activity, two major ATP sinks during normoxia. During a rescue phase, in spite of a general reduction in RNA synthesis, transcription of some genes increases dramatically under low O 2 (21, 34). These hypoxia-responsive genes are involved in glucose transport, glycolysis, erythropoiesis, angiogenesis, vasodilation, and respiratory rate, and together they function to minimize the effects caused by low O 2 at cellular, tissue and systemic levels (93, 106).The activation of many O 2 -regulated genes is mediated by hypoxia-inducible factor (HIF), a heterodimer consisting of HIF-1␣ and HIF-1␤ (also called the aryl hydrocarbon receptor nuclear translocator [ARNT]) in most cells (52,104,105). Both HIF-1␣ and ARNT belong to the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of transcription factors, which share several conserved structural domains, including a bHLH region for DNA binding and two PAS domains for target gene specificity and dimerization (102). Although ARNT is absolutely required for HIF activity (63, 110), HIF function is primarily regulated by HIF-1␣ protein stability (37,46,84). Under normoxia, HIF-1␣ is ubiquitinated through interaction with the von Hippel-Lindau tumor suppr...

HIF-2alpha promotes von Hippel-Lindau (VHL)-deficient renal clear cell carcinoma (RCC) tumorigenesis, while HIF-1alpha inhibits RCC growth. As HIF-1alpha antagonizes c-Myc function, we hypothesized that HIF-2alpha might enhance c-Myc activity. We demonstrate here that HIF-2alpha promotes cell-cycle progression in hypoxic RCCs and multiple other cell lines. This correlates with enhanced c-Myc promoter binding, transcriptional effects on both activated and repressed target genes, and interactions with Sp1, Miz1, and Max. Finally, HIF-2alpha augments c-Myc transformation of primary mouse embryo fibroblasts (MEFs). Enhanced c-Myc activity likely contributes to HIF-2alpha-mediated neoplastic progression following loss of the VHL tumor suppressor and influences the behavior of hypoxic tumor cells.

The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic "knock-in" strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1␣ with HIF-2␣ results in expanded expression of HIF-2␣-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2␣, but not HIF-1␣, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2␣ KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2␣ severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2␣-specific target gene and indicate that HIF-2␣ can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2␣'s tumor promoting activity.[Keywords: HIF; hypoxia; HIF-2␣; Oct-4; VEGF; TGF-␣; stem cells; cancer] Supplemental material is available at http://www.genesdev.org.

Adaptive transcriptional responses to oxygen deprivation (hypoxia) are mediated by the hypoxia-inducible factors (HIFs), heterodimeric transcription factors composed of two basic helix-loop-helix-PAS family proteins. The transcriptional activity of HIF is determined by the hypoxic stabilization of the HIF-␣ proteins. HIF-1␣ and HIF-2␣ exhibit high sequence homology but have different mRNA expression patterns; HIF-1␣ is expressed ubiquitously whereas HIF-2␣ expression is more restricted to certain tissues, e.g., the endothelium, lung, brain, and neural crest derivatives. Germ-line deletion of either HIF subunit is embryonic lethal with unique features suggesting important roles for both HIF-␣ isoforms. Global deletion of Hif-2␣ results in distinct phenotypes depending on the mouse strain used for the mutation, clearly demonstrating an important role for HIF-2␣ in mouse development. The function of HIF-2␣ in adult life, however, remains incompletely understood. In this study, we describe the generation of a conditional murine Hif-2␣ allele and the effect of its acute postnatal ablation. Under very stringent conditions, we ablate Hif-2␣ after birth and compare the effect of acute global deletion of Hif-2␣ and Hif-1␣. Our results demonstrate that HIF-2␣ plays a critical role in adult erythropoiesis, with acute deletion leading to anemia. Furthermore, although HIF-1␣ was first purified and cloned based on its affinity for the human erythropoietin (EPO) 3 enhancer hypoxia response element (HRE) and regulates Epo expression during mouse embryogenesis, HIF-2␣ is the critical ␣ isoform regulating Epo under physiologic and stress conditions in adults.Epo ͉ hypoxia-inducible factor ͉ hypoxia ͉ red blood cell H ypoxia-inducible factors (HIFs), members of the basic helix-loop-helix (bHLH)-PAS family of transcription factors, are master regulators of oxygen (O 2 ) homeostasis and stimulate genes important for angiogenesis, erythropoiesis and glucose metabolism (1-3). HIFs are heterodimeric factors consisting of ␣ (HIF-1␣, -2␣, and -3␣) and ␤ subunits [HIF-1␤ or ARNT (arylhydrocarbon-receptor nuclear translocator)].

The basic helix-loop-helix-Per-ARNT-Sim-proteins hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha are the principal regulators of the hypoxic transcriptional response. Although highly related, they can activate distinct target genes. In this study, the protein domain and molecular mechanism important for HIF target gene specificity are determined. We demonstrate that although HIF-2alpha is unable to activate multiple endogenous HIF-1alpha-specific target genes (e.g., glycolytic enzymes), HIF-2alpha still binds to their promoters in vivo and activates reporter genes derived from such targets. In addition, comparative analysis of the N-terminal DNA binding and dimerization domains of HIF-1alpha and HIF-2alpha does not reveal any significant differences between the two proteins. Importantly, replacement of the N-terminal transactivation domain (N-TAD) (but not the DNA binding domain, dimerization domain, or C-terminal transactivation domain [C-TAD]) of HIF-2alpha with the analogous region of HIF-1alpha is sufficient to convert HIF-2alpha into a protein with HIF-1alpha functional specificity. Nevertheless, both the N-TAD and C-TAD are important for optimal HIF transcriptional activity. Additional experiments indicate that the ETS transcription factor ELK is required for HIF-2alpha to activate specific target genes such as Cited-2, EPO, and PAI-1. These results demonstrate that the HIF-alpha TADs, particularly the N-TADs, confer HIF target gene specificity, by interacting with additional transcriptional cofactors.

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Solid tumors often exhibit simultaneously inflammatory and hypoxic microenvironments. The ‘signal transducer and activator of transcription-3’ (STAT3)-mediated inflammatory response and the hypoxia-inducible factor (HIF)-mediated hypoxia response have been independently shown to promote tumorigenesis through the activation of HIF or STAT3 target genes and to be indicative of a poor prognosis in a variety of tumors. We report here for the first time that STAT3 is involved in the HIF1, but not HIF2-mediated hypoxic transcriptional response. We show that inhibiting STAT3 activity in MDA-MB-231 and RCC4 cells by a STAT3 inhibitor or STAT3 small interfering RNA significantly reduces the levels of HIF1, but not HIF2 target genes in spite of normal levels of hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α protein. Mechanistically, STAT3 activates HIF1 target genes by binding to HIF1 target gene promoters, interacting with HIF1α protein and recruiting coactivators CREB binding protein (CBP) and p300, and RNA polymerase II (Pol II) to form enhanceosome complexes that contain HIF1α, STAT3, CBP, p300 and RNA Pol II on HIF1 target gene promoters. Functionally, the effect of STAT3 knockdown on proliferation, motility and clonogenic survival of tumor cells in vitro is phenocopied by HIF1α knockdown in hypoxic cells, whereas STAT3 knockdown in normoxic cells also reduces cell proliferation, motility and clonogenic survival. This indicates that STAT3 works with HIF1 to activate HIF1 target genes and to drive HIF1-depedent tumorigenesis under hypoxic conditions, but also has HIF-independent activity in normoxic and hypoxic cells. Identifying the role of STAT3 in the hypoxia response provides further data supporting the effectiveness of STAT3 inhibitors in solid tumor treatment owing to their usefulness in inhibiting both the STAT3 and HIF1 pro-tumorigenic signaling pathways in some cancer types.

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