Hypoxia-inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) during normal mammalian development or tumor progression. Here, we present evidence that a similar complex mediates response to hypoxia in Caenorhabditis elegans. This complex consists of HIF-1 and AHA-1, which are encoded by C. elegans homologs of the hypoxia-inducible factor (HIF) ␣ and ␤ subunits, respectively. hif-1 mutants exhibit no severe defects under standard laboratory conditions, but they are unable to adapt to hypoxia. Although wild-type animals can survive and reproduce in 1% oxygen, the majority of hif-1-defective animals die in these conditions. We show that the expression of an HIF-1:green fluorescent protein fusion protein is induced by hypoxia and is subsequently reduced upon reoxygenation. Both hif-1 and aha-1 are expressed in most cell types, and the gene products can be coimmunoprecipitated. We conclude that the mechanisms of hypoxia signaling are likely conserved among metazoans. Additionally, we find that nuclear localization of AHA-1 is disrupted in an hif-1 mutant. This finding suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1. M ammals use both systemic and cellular strategies to adapt to decreased oxygen levels during normal development and homeostasis. Hypoxic tissues secrete growth factors to increase vascularization, and individual cells increase anaerobic metabolism to sustain basic cellular functions (1). Hypoxia also plays a central role in tumor biology, as a mass of cancerous cells must adapt to hypoxia and induce angiogenesis to grow and metastasize (2).The majority of the transcriptional responses to hypoxia are mediated by hypoxia-inducible factor (HIF) complexes, which consist of ␣ and ␤ subunits. The HIF␤ subunit is also termed ARNT (aryl hydrocarbon receptor nuclear translocator) (3-7). When cellular oxygen levels are high, the von Hippel-Lindau tumor suppressor protein (VHL) binds directly to the ␣ subunit and targets it for ubiquitination and proteosomal degradation. However, in hypoxic conditions, degradation of HIF␣ is inhibited (8-11). This permits HIF␣ to translocate to the nucleus, dimerize with ARNT, and activate the expression of target genes, which act to increase oxygen delivery or implement metabolic adaptation to hypoxia (2). Despite intensive study, the mechanisms by which cellular oxygen levels are sensed are not well understood. Progress in this field has been limited by the lack of genetic approaches to this important problem.Widely divergent organisms have the ability to adapt to variable oxygen concentrations, which suggests that mechanisms of hypoxic sensing and response may have been established early in evolutionary history. Here, we investigate the molecular mechanisms of hypoxia response in a powerful genetic model organism, the nematode Caenorhabditis elegans. The natural habitat of C. elegans is often hypoxic, and C. elegans can adapt to very low environmental o...

The human hypoxia-inducible transcription factor HIF-1 is a critical regulator of cellular and systemic responses to low oxygen levels. When oxygen levels are high, the HIF-1␣ subunit is hydroxylated and is targeted for degradation by the von Hippel-Lindau tumor suppressor protein (VHL). This regulatory pathway is evolutionarily conserved, and the Caenorhabditis elegans hif-1 and vhl-1 genes encode homologs of the HIF-1␣ subunit and VHL. To understand and describe more fully the molecular basis for hypoxia response in this important genetic model system, we compared hypoxiainduced changes in mRNA expression in wild-type, hif-1-deficient, and vhl-1-deficient C. elegans using whole genome microarrays. These studies identified 110 hypoxia-regulated gene expression changes, 63 of which require hif-1 function. Mutation of vhl-1 abrogates most hif-1-dependent changes in mRNA expression. Genes regulated by C. elegans hif-1 have predicted functions in signal transduction, metabolism, transport, and extracellular matrix remodeling. We examined the in vivo requirement for 16 HIF-1 target genes and discovered that the phy-2 prolyl 4-hydroxylase ␣ subunit is critical for survival in hypoxic conditions. Some HIF-1 target genes negatively regulate formation of stress-resistant dauer larvae. The microarray data presented herein also provide clear evidence for an HIF-1-independent pathway for hypoxia response, and this pathway regulates the expression of multiple heat shock proteins and several transcription factors.During development, homeostasis, or disease states, cellular oxygen levels are often insufficient to meet physiological demands, and this condition is termed hypoxia. Mammalian cells respond to hypoxia by implementing changes in gene expression to increase anaerobic energy production, protect cells from stress, regulate cell survival, and increase local angiogenesis. The requisite changes in gene expression are largely controlled by the hypoxia-inducible factor 1 (HIF-1) 1 transcription factor (1, 2).HIF-1 is a heterodimeric DNA-binding complex, and both subunits are members of the family of transcription factors containing basic-helix-loop-helix and Per-ARNT-Sim domains. The HIF-1␤ subunit is also termed ARNT (aryl hydrocarbon receptor nuclear translocator). Although ARNT can dimerize with other transcription factors, HIF-1␣ is apparently dedicated to a hypoxia response (3-5). When oxygen levels are high, specific proline residues of HIF-1␣ are hydroxylated by oxygendependent enzymes belonging to the EGL-9/PH superfamily of 2-oxoglutarate-dependent dioxygenases (6, 7). Proline hydroxylation in the conserved LXXLAP motif in HIF-1␣ increases its affinity for the von Hippel-Lindau tumor suppressor protein (VHL), which is part of an E3 ubiquitin-ligase complex that targets proteins for proteasomal degradation. Thus, when VHL is disabled by mutation, HIF-1 is expressed at constitutively high levels (8).Cells utilize multiple strategies to regulate HIF-1␣ activity. HIF-1␣ is modified post-translationally by hydroxylation...

During normal development or during disease, animal cells experience hypoxic (low oxygen) conditions, and the hypoxia-inducible factor (HIF) transcription factors implement most of the critical changes in gene expression that enable animals to adapt to this stress. Here, we examine the roles of HIF-1 in post-mitotic aging. We examined the effects of HIF-1 over-expression and of hif-1 loss-of-function mutations on longevity in C. elegans, a powerful genetic system in which adult somatic cells are post-mitotic. We constructed transgenic lines that expressed varying levels of HIF-1 protein and discovered a positive correlation between HIF-1 expression levels and lifespan. The data further showed that HIF-1 acted in parallel to the SKN-1/NRF and DAF-16/FOXO transcription factors to promote longevity. HIF-1 over-expression also conferred increased resistance to heat and oxidative stress. We isolated and characterized additional hif-1 mutations, and we found that each of 3 loss-of-function mutations conferred increased longevity in normal lab culture conditions, but, unlike HIF-1 over-expression, a hif-1 deletion mutation did not extend the lifespan of daf-16 or skn-1 mutants. We conclude that HIF-1 over-expression and hif-1 loss-of-function mutations promote longevity by different pathways. These data establish HIF-1 as one of the key stress-responsive transcription factors that modulate longevity in C. elegans and advance our understanding of the regulatory networks that link oxygen homeostasis and aging.

Hypoxia-inducible factor (HIF) transcription factors implement essential changes in gene expression that enable animals to adapt to low oxygen (hypoxia). The stability of the C. elegans HIF-1 protein is controlled by the evolutionarily conserved EGL-9/VHL-1 pathway for oxygen-dependent degradation. Here, we describe vhl-1-independent pathways that attenuate HIF-1 transcriptional activity in C. elegans. First, the expression of HIF-1 target genes is markedly higher in egl-9 mutants than in vhl-1 mutants. We show that HIF-1 protein levels are similar in animals carrying strong loss-of-function mutations in either egl-9 or vhl-1. We conclude that EGL-9 inhibits HIF-1 activity, as well as HIF-1 stability. Second, we identify the rhy-1 gene and show that it acts in a novel negative feedback loop to inhibit expression of HIF-1 target genes. rhy-1 encodes a multi-pass transmembrane protein. Although loss-of-function mutations in rhy-1 cause relatively modest increases in hif-1 mRNA and HIF-1 protein expression, some HIF-1 target genes are expressed at higher levels in rhy-1 mutants than in vhl-1 mutants. Animals lacking both vhl-1 and rhy-1 function have a more severe phenotype than either single mutant. Collectively, these data support models in which RHY-1 and EGL-9 function in VHL-1-independent pathway(s) to repress HIF-1 transcriptional activity.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, until now described only in vertebrates, that mediates many of the carcinogenic and teratogenic effects of certain environmental pollutants. Here, we describe orthologs of AHR and its dimerization partner AHR nuclear translocator (ARNT) in the nematode Caenorhabditis elegans, encoded by the genes ahr-1 and aha-1, respectively. The corresponding proteins, AHR-1 and AHA-1, share biochemical properties with their mammalian cognates. Specifically, AHR-1 forms a tight association with HSP90, and AHR-1 and AHA-1 interact to bind DNA fragments containing the mammalian xenobiotic response element with sequence specificity. Yeast expression studies indicate that C. elegans AHR-1, like vertebrate AHR, requires some form of posttranslational activation. Moreover, this requirement depends on the presence of the domains predicted to mediate binding of HSP90 and ligand. Preliminary experiments suggest that if AHR-1 is ligand-activated, its spectrum of ligands is different from that of the mammalian receptor: C. elegans AHR-1 is not photoaffinity labeled by a dioxin analog, and it is not activated by ␤-naphthof lavone in the yeast system. The discovery of these genes in a simple, genetically tractable invertebrate should allow elucidation of AHR-1 function and identification of its endogenous regulators.