FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity.

Hypoxia-inducible factor 1 (HIF-1) plays an essential role in cellular and systemic oxygen homeostasis (Iyer et al. 1998). HIF-1 activates transcription of genes whose protein products either function to increase O2 availability by promoting erythropoiesis (e.g., erythropoietin) and angiogenesis [e.g., vascular endothelial growth factor (VEGF)] or mediate adaptive intracellular responses to O2 deprivation such as increased glycolytic metabolism (e.g., glucose transporters and glycolytic enzymes) (for review, see Semenza 1999). However, experimental evidence suggests that HIF-1-regulated gene products (e.g., NIP3) may also mediate hypoxia-induced apoptosis under certain conditions (Carmeliet et al. 1998; Bruick 2000). It is not known what determines the battery of genes that are activated by HIF-1 in response to hypoxia in a given cell type and whether this response will lead to adaptation or apoptosis. Because HIF-1 activity is induced by hypoxia in all cell types, the interaction of HIF-1 with other proteins is likely to play a major role in determining its biological activity. Because of the important role played by HIF-1 in cancer and ischemic cardiovascular disease (for review, see Semenza 2000), identification of interacting proteins may also provide novel therapeutic targets. HIF-1 is a heterodimer consisting of HIF-1α and HIF-1β subunits (Wang et al. 1995; Wang and Semenza 1995). Whereas HIF-1β is constitutively expressed, HIF-1α mRNA expression, protein half-life, and transactivation domain function are all regulated by the cellular O2 concentration in vivo (for review, see Semenza 1999). The C-terminal half of HIF-1α contains several important regulatory domains. First, amino acids 429–608 (Fig. ​(Fig.1A)1A) encompass sequences that mediate O2-dependent ubiquitination of HIF-1α (Huang et al 1998; Sutter et al. 2000) via binding of the von Hippel-Lindau (VHL) tumor suppressor protein, which is the recognition component of an E3 ubiquitin-protein ligase (Cockman et al. 2000; Kamura et al. 2000; Ohh et al. 2000) that targets HIF-1α for proteasomal degradation (Salceda and Caro 1997). Binding of VHL is dependent on hydroxylation of Pro 564 in HIF-1α via an enzymatic process that requires O2 as well as iron and is inhibited by cobalt chloride (Ivan et al. 2001; Jaakkola et al. 2001). VHL also binds to other residues in HIF-1α (Yu et al. 2001), but the functional significance of these interactions remains to be determined. Finally, HIF-1α interacts with p53 (An et al. 1998), which recruits MDM2, another E3 ubiquitin-protein ligase, and this interaction modulates HIF-1α stability under hypoxic conditions (Ravi et al. 2000). Figure 1 Identification of FIH-1 by yeast two-hybrid screen. (A) Structure of HIF-1α and the bait and prey proteins. HIF-1α contains basic helix–loop–helix (bHLH) and PAS domains that are required for dimerization and DNA binding, . In addition to the domain regulating protein stability, HIF-1α contains two transactivation domains (Jiang et al. 1997; Pugh et al. 1997), TAD-N (residues 531–575) and TAD-C (residues 786–826), which bind coactivators including CBP, p300, SRC-1, and TIF-2 (Arany et al. 1996; Ema et al. 1999; Carrero et al. 2000). Residues 576–785 comprise an inhibitory domain the deletion of which increases transactivation domain function especially under nonhypoxic conditions (Jiang et al. 1997). Transactivation domain function is negatively regulated by O2 independently of protein stability. Exposure of cells to hypoxia, cobalt chloride, or the iron chelator desferrioxamine induces both HIF-1α protein stabilization and transactivation (Jiang et al. 1997; Pugh et al. 1997), suggesting the existence of a common molecular mechanism. We have utilized the yeast two-hybrid assay to screen for proteins that interact with HIF-1α and modulate its biological activity. Here, we report the identification and characterization of FIH-1 (factor inhibiting HIF-1), a negative regulator of HIF-1α transactivation domain function. We demonstrate that FIH interacts with HIF-1α as well as with VHL and that both FIH-1 and VHL inhibit HIF-1α transactivation domain function. The involvement of VHL provides a unifying mechanism for the O2-dependent regulation of HIF-1α protein stability and transcriptional activity.