Hypoxia induces a group of physiologically important genes such as erythropoietin and vascular endothelial growth factor. These genes are transcriptionally upregulated by hypoxia-inducible factor 1 (HIF-1), a global regulator that belongs to the basic helix-loop-helix PAS family. Although HIF-1 is a heterodimer composed of ␣ and ␤ subunits, its activity is primarily determined by hypoxiainduced stabilization of HIF-1␣, which is otherwise rapidly degraded in oxygenated cells. We report the identification of an oxygen-dependent degradation (ODD) domain within HIF-1␣ that controls its degradation by the ubiquitinproteasome pathway. The ODD domain consists of Ϸ200 amino acid residues, located in the central region of HIF-1␣. Because portions of the domain independently confer degradation of HIF-1␣, deletion of this entire region is required to give rise to a stable HIF-1␣, capable of heterodimerization, DNA-binding, and transactivation in the absence of hypoxic signaling. Conversely, the ODD domain alone confers oxygendependent instability when fused to a stable protein, Gal4. Hence, the ODD domain plays a pivotal role for regulating HIF-1 activity and thereby may provide a means of controlling gene expression by changes in oxygen tension.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is characterized by the development of multiple vascular tumors and is caused by inactivation of the von Hippel-Lindau protein (pVHL). Here we show that pVHL, through its beta-domain, binds directly to hypoxia-inducible factor (HIF), thereby targeting HIF for ubiquitination in an alpha-domain-dependent manner. This is the first function to be ascribed to the pVHL beta-domain. Furthermore, we provide the first direct evidence that pVHL has a function analogous to that of an F-box protein, namely, to recruit substrates to a ubiquitination machine. These results strengthen the link between overaccumulation of HIF and development of VHL disease.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is critical for hypoxic induction of a number of physiologically important genes. We present evidence that regulation of HIF-1 activity is primarily determined by the stability of the HIF-1␣ protein.Both HIF-1␣ and HIF-1␤ mRNAs were constitutively expressed in HeLa and Hep3B cells with no significant induction by hypoxia. However, the HIF-1␣ protein was barely detectable in normoxic cells, even when HIF-1␣ was overexpressed, but was highly induced in hypoxic cells, whereas HIF-1␤ protein levels remained constant, regardless of pO 2 . Hypoxia-induced HIF-1 binding as well as the HIF-1␣ protein were rapidly and drastically decreased in vivo following an abrupt increase to normal oxygen tension. Moreover, short pre-exposure of cells to hydrogen peroxide selectively prevented hypoxia-induced HIF-1 binding via blocking accumulation of HIF-1␣ protein, whereas treatment of hypoxic cell extracts with H 2 O 2 had no effect on HIF-1 binding. These observations suggest that an intact redox-dependent signaling pathway is required for destablization of the HIF-1␣ protein. Hypoxia-inducible factor 1 (HIF-1) 1 was identified by Semenza, Wang, and co-workers (1-5) as a transcription factor in hypoxic cells that binds specifically to a 3Ј enhancer of the gene encoding erythropoietin and to promoters/enhancers in other genes important in adaptation to hypoxia, such as those encoding tyrosine hydroxylase (6), vascular endothelial growth factor (7), glycolytic enzymes (8 -10), and glucose transporters (11). HIF-1 activity can be induced by hypoxia in a wide variety of cells, as demonstrated by specific binding to oligonucleotides containing a HIF-1 response element (3) and by transactivation of reporter genes (12).HIF-1 is composed of a 120-kDa ␣ subunit and a 91-94-kDa ␤ subunit (4). When activated, the HIF-1 heterodimer recognizes an 8-base pair DNA sequence 5Ј-TACGTGCT-3Ј in the erythropoietin enhancer, interacting with both DNA strands in the major groove (13). Recent cloning of HIF-1␣ and HIF-1␤ genes (5) showed that they are members of the basic helix-loophelix-PAS family of transcription factors. HIF-1␣ is a novel protein, whereas HIF-1␤ is the previously cloned and characterized aryl hydrocarbon receptor nuclear translocator (ARNT) (14).HIF-1 DNA binding, along with the expression of HIF-1-responsive genes, can be induced by cobaltous ion and desferrioxamine with kinetics similar to that of hypoxia induction (3, 13). HIF-1 activation, irrespective of stimulus, is blocked by pretreatment of cells with cycloheximide (2, 3), an inhibitor of protein synthesis as well as by 2-aminopurine (13), a protein kinase inhibitor. Treatment of hypoxic cell extracts with alkaline phosphatase abolishes DNA binding (13). Thus phosphorylation may be required for binding. More recently, Wang et al. (5) reported that both HIF-1␣ mRNA and HIF-1␤ mRNA were barely detectable in normoxic cells but were highly induced following exposure to hypoxia, with rapid decay upo...