Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor (HIF), a heterodimer of HIF-α and the aryl hydrocarbon receptor nuclear translocator subunits. The HIF-1α and HIF-2α 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-1α^−/− embryonic stem and mouse embryonic fibroblast cells show that loss of HIF-1α eliminates all oxygen-regulated transcriptional responses analyzed, suggesting that HIF-2α is dispensable for hypoxic gene regulation. In contrast, HIF-2α 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-2α target genes, we used DNA microarray analysis to evaluate hypoxic gene induction in cells expressing HIF-2α but not HIF-1α. In addition, we engineered HEK293 cells to express stabilized forms of HIF-1α or HIF-2α via a tetracycline-regulated promoter. In this first comparative study of HIF-1α and HIF-2α target genes, we demonstrate that HIF-2α 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-1α (and not HIF-2α) stimulates glycolytic gene expression in both types of cells, clearly showing for the first time that HIF-1α and HIF-2α have unique targets.

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-1α with HIF-2α results in expanded expression of HIF-2α-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2α, but not HIF-1α, binds to theOct-4promoter and inducesOct-4expression and transcriptional activity, thereby contributing to impaired development in homozygousHif-2αKI/KIembryos, 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-2α severely reduces the number of embryonic primordial germ cells, which requireOct-4expression for survival and/or maintenance. These results identifyOct-4as a HIF-2α-specific target gene and indicate that HIF-2α can regulate stem cell function and/or differentiation through activation ofOct-4, which in turn contributes to HIF-2α's tumor promoting activity.

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-α proteins. HIF-1α and HIF-2α exhibit high sequence homology but have different mRNA expression patterns; HIF-1α is expressed ubiquitously whereas HIF-2α 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-α isoforms. Global deletion of Hif-2 α results in distinct phenotypes depending on the mouse strain used for the mutation, clearly demonstrating an important role for HIF-2α in mouse development. The function of HIF-2α in adult life, however, remains incompletely understood. In this study, we describe the generation of a conditional murine Hif-2 α allele and the effect of its acute postnatal ablation. Under very stringent conditions, we ablate Hif-2 α after birth and compare the effect of acute global deletion of Hif-2 α and Hif-1 α. Our results demonstrate that HIF-2α plays a critical role in adult erythropoiesis, with acute deletion leading to anemia. Furthermore, although HIF-1α 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-2α is the critical α isoform regulating Epo under physiologic and stress conditions in adults.

The basic helix-loop-helix-Per-ARNT-Sim–proteins hypoxia-inducible factor (HIF)-1α and HIF-2α 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-2α is unable to activate multiple endogenous HIF-1α–specific target genes (e.g., glycolytic enzymes), HIF-2α 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-1α and HIF-2α 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-2α with the analogous region of HIF-1α is sufficient to convert HIF-2α into a protein with HIF-1α 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-2α to activate specific target genes such as Cited-2, EPO, and PAI-1. These results demonstrate that the HIF-α TADs, particularly the N-TADs, confer HIF target gene specificity, by interacting with additional transcriptional cofactors.

Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factors (HIFs), HIF-1alpha and HIF-2alpha. The HIF-1alpha and HIF-2alpha subunits are structurally similar in their DNA binding and dimerization domains but differ in their transactivation domains, implying they may have unique target genes and require distinct transcriptional cofactors. Our previous results demonstrated that HIF-1alpha and HIF-2alpha regulate distinct target genes. Here, we report that HIF-2alpha is not transcriptionally active in embryonic stem (ES) cells, as well as possible inhibition by a HIF-2alpha-specific transcriptional repressor. Using DNA microarray analysis of hypoxia-inducible genes in wild-type (WT), Hif-1alpha(-)(/)(-), and Hif-2alpha(-)(/)(-) ES cells, we show that HIF-1alpha induces a large number of both confirmed and novel hypoxia-inducible genes, while HIF-2alpha does not activate any of its previously described targets. We further demonstrate that inhibition of HIF-2alpha function occurs at the level of transcription cofactor recruitment to endogenous target gene promoters. Overexpression of WT and, notably, a DNA-binding-defective HIF-2alpha mutant restores endogenous HIF-2alpha protein activity, suggesting that ES cells express a HIF-2alpha-specific corepressor that can be titrated by overexpressed HIF-2alpha protein. HIF-2alpha repression may explain why patients with mutations in the VHL tumor suppressor gene display cancerous lesions in specific tissue types.