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J. Biol. Chem., Vol. 280, Issue 21, 20580-20588, May 27, 2005

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Roles of the HIF-1 Hypoxia-inducible Factor during Hypoxia Response in Caenorhabditis elegans^*

Chuan Shen, Daniel Nettleton, Min Jiang¶, Stuart K. Kim¶, and Jo Anne Powell-Coffman||

From the Department of Genetics, Development, and Cell Biology and the Department of Statistics, Iowa State University, Ames, Iowa 50011 and the ^¶Department of Developmental Biology, Stanford University, Stanford, California 94305

Received for publication, February 18, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 hypoxia-induced 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 oxygen-dependent 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, phosphorylation, and acetylation (7, 9, 10). Oxygen-dependent hydroxylation of the HIF-1 C terminus inhibits binding to the coactivator CBP/p300 (11, 12). Growth factor stimulation may modulate HIF-1 transcriptional activity via small GTPases and mitogen-activated protein kinase cascades (13, 14). The availability of interacting proteins, such as transcriptional coactivators, also influences HIF-1 function. Important questions remain. What other oxygen-sensing molecules control cellular hypoxia response? What are the most important VHL-independent mechanisms for regulating HIF-1 activity? What fraction of the transcriptional response to hypoxia is controlled by HIF-1?

Caenorhabditis elegans has recently proven to be an important model system for studying hypoxia response. The C. elegans homolog of the HIF-1 subunit is hif-1, and the HIF-1/EGL-9/VHL pathway is evolutionarily conserved (6, 15). C. elegans carrying deletions in the hif-1 gene are apparently healthy in standard culture conditions, but they exhibit high levels of lethality in 0.5 or 1% oxygen (15, 16). To identify genes regulated by HIF-1 during response to hypoxia and to determine what fraction of hypoxia-induced gene expression changes is dependent upon hif-1, we conducted genomewide studies of hypoxia-induced changes in mRNA expression in wild-type, hif-1-deficient, and vhl-1-deficient C. elegans. These studies demonstrate that C. elegans hif-1 regulates the majority of early transcriptional responses to hypoxia and provide clear evidence for HIF-1-independent pathways for adaptation to oxygen deprivation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
C. elegans Culture, Microarray Experiments, and Data Analysis—C. elegans were cultured using standard procedures on NGM plates with an Escherichia coli food source (17). For hypoxia treatments, plates containing synchronized third stage larvae were placed either in room air (normoxia control) or a sealed plexiglass chamber with constant gas flow. Compressed air and pure nitrogen were mixed to achieve the appropriate oxygen concentration, as assayed by an oxygen sensor (15). We were most interested in the earliest hif-1-dependent and hif-1-independent responses to hypoxia. Therefore, we assayed mRNA expression at the earliest time point at which the F22B5.4 transcript was consistently induced (4 h at 21 °C in 0.1% oxygen). At the time that this study was conducted, F22B5.4 was the only C. elegans transcript shown to be induced by hypoxia. The F22B5.4 putative 5'-regulatory sequences contain multiple elements similar to the mammalian hypoxia response element (HRE), the DNA binding site for the HIF-1 transcriptional complex, and F22B5.4 is expressed at high levels in egl-9 mutants (6). These data suggested that F22B5.4 might be a direct target for C. elegans HIF-1. For the microarray experiments, three strains were assayed: wild-type N2, hif-1 (ia04), and vhl-1 (ok161). Worms were incubated for 4 h in 21% oxygen or 0.1% oxygen at 21 °C. Animals were quickly harvested in ice-cold M9 buffer, and poly(A) RNA was isolated using established procedures. No more than 3 min elapsed between the removal of plates from the hypoxic chamber and the addition of TRIzol.

The microarray hybridizations were performed by Min Jiang in the laboratory of Stuart K. Kim at Stanford University (18). cDNA from animals incubated in 21% O₂ (normoxia) was labeled with Cy5, and cDNA from hypoxia-treated worms was labeled with Cy3. There were 18 mRNA samples: (two oxygen concentrations x three genotypes x three independent experiments). Each hybridization compared expression in normoxia versus hypoxia for one genotype, and nine experiments were performed. The raw data and the normalized data are available at the Stanford Microarray Data base. The following criteria were used to filter the data: FLAG = 0; failed = 0; red and green fluorescence intensity at least 2-fold above background. We averaged the log₂ values (hypoxia net intensity/normalized normoxia net intensity) from each experiment. Student's t test p values were determined with the averages for each strain.

To identify putative regulatory elements in hif-1-dependent genes, we used the motif-finding program at bioprospector.stanford.edu (BioProspector). We searched for DNA sequences that were overrepresented in the 18 genes most strongly induced by hypoxia in Table I relative to the 47 hif-1-independent genes. Sequences 200–2,000 bp upstream from each putative translational start were used for this analysis. Gene cluster analyses were performed using tools available at PlantGDB (19).

RNA Blots—Total RNAs were isolated from synchronized L3 stage wild-type (Bristol strain N2) worms, hif-1 (ia04) mutants, vhl-1 (ok161) mutants, and hif-1 (ia04) vhl-1 (ok161) double mutants that had been incubated for 4 h in normoxia or hypoxia (0.5% O₂). The RNA samples were fractionated via agarose-formaldehyde gel electrophoresis and transferred to nylon membranes. DNA probes for RNA blot analyses were amplified using published primers (21). Hybridizations were performed using [-³²P]dCTP-labeled probes, and signals were detected and quantified using a phosphorimager.

GFP Reporter Analysis—To generate the phy-2:GFP construct pSH01, DNA was PCR amplified using the primers Phy2PF2 (PstI) 5'-ggcgctgcagAGACTATAGTCTATAGCTGAAAACG and Phy2PR2 (BamHI) 5'-gcgggatccACTGCTCTCATTCTGAAAGACAAATC. The PCR product includes the phy-2 sequence from –1715 bp to +11 bp relative to the translational start site (23). These regulatory sequences were cloned into GFP vector pPD95.75 (provided by A. Fire, Carnegie Institution of Washington). To create transgenic animals, the DNA was coinjected with the pRF-4 rol-6 marker (24). Five independent transgenic lines were examined.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The primary goals of these studies were to identify hypoxia-induced gene expression changes in C. elegans and to determine which of these responses to hypoxia were regulated by hif-1. Toward these aims, we analyzed mRNA expression patterns in synchronized populations of wild-type worms that were cultured in standard laboratory conditions (normoxia) or in hypoxia. We also assayed mRNA from two mutant strains: (a) animals carrying the strong loss-of-function mutation in hif-1 and (b) C. elegans that carry a deletion in vhl-1 and express the HIF-1 protein at constitutively high levels. RNA was isolated from three independent experiments for each experimental condition (three genotypes; normoxia versus hypoxia). mRNAs were hybridized to whole genome microarrays that contained probes for 17,817 predicted genes (94% of the genome). To identify hypoxia-induced gene expression changes in wild-type worms, we used two criteria. First, we identified genes that exhibited a significant difference in mRNA levels in hypoxia versus normoxia as judged by Student's t test, p value <0.05. Second, we focused on genes that showed an average hypoxia-induced difference in mRNA expression greater than or equal to 2-fold. 110 genes met both of these criteria. To determine which of these gene expression changes were dependent upon hif-1 function, we examined the data from hif-1 (ia04) mutants. 63 of the 110 hypoxia-responsive genes were not significantly regulated by hypoxia in hif-1 (ia04) mutants (p value > 0.05), and they are listed in Table I. In this article, we will refer to this class of genes as "hif-1-dependent." The 47 genes listed in Table II were expressed at significantly higher or lower levels in hypoxia in hif-1-deficient animals (p value < 0.05; Table II), and we will refer to them as "hif-1-independent" genes. In most cases, hypoxia caused increased gene expression, but 6 of the hif-1-dependent genes and 3 of the hif-1-independent genes were down-regulated by hypoxia. A total of 654 genes met less stringent criteria for hypoxia-responsive genes (1.5-fold average difference between hypoxia and normoxia treatments; p < 0.05), and they are listed in the supplemental data.

Although the hypoxia-responsive genes are located on all six chromosomes, 40% of the hif-1-dependent genes are on chromosome II, and many of these are found in clusters (Fig. 2). Three genes (F45D11.1, F45D11.2, and F45D11.16) are within 50 kb of each other at the left arm of II. Multiple small clusters are located at the center of the chromosome, and a 10-kb domain on the right arm of II contains two hif-1-dependent genes (K10H10.2 and K10H10.4). With one exception (two hif-1-independent genes on chromosome III), hypoxia-responsive genes on the other four autosomes or on the X are not clustered.

We anticipated that some of the hif-1-dependent genes would be direct targets of the HIF-1 transcriptional complex. The mammalian HIF-1 and subunits form a heterodimer and bind to specific DNA sequences (HREs) that contain the sequence 5'-RCGTG (27). A survey of the sequences 200–2,000 bp 5' to the predicted translational start sites shows that the motif 5'-TACGTG is present in 46% of the hif-1-dependent genes (Table I). In comparison, this putative HRE is present in only 23% of hif-1-independent genes (Table II).

Functions of Hypoxia-responsive Genes—Metabolic enzymes are prevalent in both Table I and Table II. For example, pyc-1 encodes pyruvate carboxylase, a key enzyme in gluconeogenesis, and it was induced by hypoxia in an hif-1-independent manner (2.2-fold; p value 0.038). Some glycolytic enzymes were induced at lower levels. F14B4.2 hexokinase, R05F9.6 phosphoglucomutase, T21B10.2 enolase, and R11F4.1 glycerol kinase are expressed 1.4–1.9-fold higher levels in hypoxia relative to normoxia (p < 0.05). Two enzymes predicted to facilitate the conversion of fatty acids to sugars were also induced by hypoxia. C05E4.9/gei-7 (isocitrate lyase) and F54H12.1/aco-2 (aconitate hydratase) were induced 2.8-fold and 1.8-fold, respectively (Table II and supplemental data).

Predicted signaling molecules are another major functional class in both Tables I and II. For example, F26A3.4, an hif-1-independent gene, encodes a protein-tyrosine phosphatase similar to the mammalian mitogen-activated protein kinase phosphatase 6. Additionally, cam-1, a receptor tyrosine kinase orthologous to human ROR1 and ROR2, and the dpf-6 dipeptidyl peptidase, which is predicted to hydrolyze peptide ligands, are both hif-1-dependent genes.

Hypoxia appeared to induce changes in extracellular matrices. The phy-2 prolyl 4-hydroxylase subunit gene and the bli-3 NADPH oxidase were induced by hypoxia, and both have central roles in collagen synthesis (28, 29) (Tables I and II, respectively). Genes encoding three predicted extracellular molecules with lectin domains (C31G12.2, F35D11.10, and F47C12.2) and one predicted chitin-binding protein (K04H4.2) also exhibited hypoxia-responsive mRNA expression (Tables I and II).

Interestingly, we found that egl-9 mRNA levels were induced by hypoxia in an hif-1-dependent manner (Fig. 1A). The egl-9 gene encodes a 2-oxoglutarate-dependent oxygenase, and it is thought to be the sole HIF prolyl hydroxylase (PHD) in C. elegans. Using oxygen as a substrate, EGL-9 hydroxylates HIF-1 in the conserved LXXLAP motif. This modification increases the affinity of HIF-1 for the VHL-1 E3 ligase, which targets HIF-1 for proteosomal degradation. In egl-9-deficient animals, HIF-1 protein is expressed at constitutively high levels (6).

View larger version (95K): [in this window] [in a new window]   FIG. 1. RNA blot analyses of genes regulated by hif-1 or vhl-1. mRNA was isolated from wild-type (WT), hif-1 (ia04), vhl-1 (ok161), or double mutant animals after incubation in 21% oxygen (N) or 0.5% oxygen (H) for 4 h at 21 °C. inf-1 serves as a loading control.

Interestingly, many of the hif-1-independent genes listed in Table II are transcription factors and heat shock proteins. 17% (8/47) of hif-1-independent genes are predicted transcriptional regulators. In comparison, only 5% (3/63) of the hif-1-dependent genes are likely transcription factors, and this is similar to the prevalence of predicted transcription factors in the complete genome (31). The C. elegans heat shock protein genes C12C8.1, F08H9.4, F44E5.5, and F44E5.4 were all induced 4.5–5.6-fold by hypoxia, and induction of these heat shock proteins does not require hif-1 function (Table II).

Many other C. elegans researchers have performed microarray experiments to identify genes that were expressed differentially during specific developmental stages or in response to diverse environmental stresses. Computational analyses of these data have resulted in the identification of "mountains" of coexpressed genes (32). We note that 43% of the hif-1-independent (20/47) and 13% of the hif-1-dependent genes (8/63) are in "mountain 1," an 1,818-gene cluster enriched in genes with neuronal or muscle function.

Essential Functions for Individual hif-1-dependent Genes— Mutants lacking hif-1 function exhibit a much higher level of embryonic and larval lethality in hypoxic conditions compared with wild-type C. elegans. Thus, we anticipated that certain individual HIF-1 target genes might also be critical for survival in hypoxia. To test this hypothesis, we used existing mutant strains or RNAi to examine the requirement for 16 individual hif-1-dependent genes in hypoxia. We tested 7 strains carrying loss-of-function mutations in hif-1-dependent genes, and 5 of the strains exhibited a statistically significant decrease in embryonic or larval viability in 0.5% oxygen relative to wild-type animals. As shown in Fig. 3, animals carrying loss-of-function mutations in fmo-12, fmo-14, vab-1, or inx-2 were slightly more vulnerable to hypoxic stress than were wild-type worms.

The most dramatic hypoxia-dependent phenotypes were evident in animals carrying a strong loss-of-function mutation in the phy-2 prolyl 4-hydroxylase subunit. Prior studies have shown that the phy-2 prolyl 4-hydroxylase subunit has a central role in collagen synthesis, and genetic analyses have shown that in normal culture conditions the developmental functions of phy-2 could also be provided by dpy-18 (23, 33). As shown in Fig. 3, only 22.3% of phy-2 (ok177) mutant animals successfully completed embryogenesis in 0.5% oxygen. 97% of wild-type animals and 58.2% of hif-1 (ia04) embryos hatched in the same conditions. Animals lacking dpy-18 function were relatively healthy in hypoxia (94% hatch; n = 91). These data show that dpy-18 cannot completely compensate for loss of phy-2 function when oxygen is limiting.

When phy-2 (ok177) mutants were allowed to complete embryogenesis in normal culture conditions and then transferred to a hypoxic chamber (0.5% oxygen) to complete development, 79% were sterile as adults (n = 38). This was unexpected because expression of a phy-2:lacZ transgene has been reported to be predominantly hypodermal (23). To examine the phy-2 expression pattern in live animals, we constructed a phy-2:GFP reporter. phy-2:GFP was strongly expressed in the hypodermis, excretory cell, and spermatheca (Fig. 4). These data are consistent with the hypothesis that collagen synthesis in the spermatheca is important to fertility and embryonic viability.

We used RNAi to examine the requirement for an additional 9 hif-1-dependent genes. These assays required culturing the animals on plates containing isopropyl 1-thio--D-galactopyranoside and tetracycline, and worms grown on control bacteria in these conditions showed increased sensitivity to hypoxia (Fig. 5). Worms treated with RNAi for the genes F22B5.4 (unknown function), nhr-57 (nuclear hormone receptor), K10H10.2 (cysteine synthase), or M05D6.5 (protein containing a hypoxia-inducible domain) exhibited higher levels of embryonic lethality in 0.5% oxygen (Fig. 5).

Molecular Links between Hypoxia Response and Dauer Formation—Some of the genes induced by hypoxia also regulate entry into the dauer phase, an alternative larval stage characterized by low metabolic rate and resistance to environmental stress. Mutations that favor dauer formation generally also confer longevity and resistance to certain environmental stresses. At sufficiently high temperatures, oxygen deprivation is toxic to wild-type C. elegans, and this experimental regimen has been termed hypoxic death. Interestingly, specific mutations in the daf-2 insulin-like growth factor receptor gene confer resistance to hypoxic death (34). Loss-of-function mutations in daf-2 also cause constitutive dauer formation and increased longevity. These phenotypes are suppressed by mutations that decrease the function of DAF-16, a forkhead transcription factor (35, 36). In our microarray experiments, daf-16 mRNA was induced 1.7–1.9-fold by hypoxia in wild-type, hif-1 (ia04), or vhl-1 (ok161) worms (p < 0.02 to p < 0.05) (supplemental data). We performed RNA blot analyses to validate these findings, and these experiments showed that expression of daf-16 mRNA was 2-fold higher in worms treated with 0.5% oxygen for 4 h compared with normoxia-treated worms (Fig. 1C). Thus, increased daf-16 expression is a common feature of both hypoxia response and of perturbations in the DAF-2 signaling pathway which lead to constitutive dauer formation, increased longevity, and enhanced stress resistance (Fig. 6).

View larger version (54K): [in this window] [in a new window]   FIG. 2. Genomic distribution of hypoxia-responsive genes. Genes that are induced by hypoxia in wild-type, but not hif-1 (ia04) animals, are listed in red. Genes induced by hypoxia independently of hif-1 function are listed in blue.

VHL-1 Regulates HIF-1 Function and Other Unknown Pathway(s)—Prior studies had demonstrated that C. elegans lacking vhl-1 function express HIF-1 protein at constitutively high levels (6). Thus, we predicted that hypoxia-dependent changes in gene expression induced by HIF-1 would be abrogated in vhl-1-defective mutants but that inactivation of vhl-1 would have little effect on hif-1-independent genes. Our data generally validate these models. For 60 of the 63 hif-1-dependent genes, regulation by hypoxia was diminished in vhl-1 (ok161) mutants, and 50 of the genes did not show a significant difference (p > 0.05) between mRNA levels in hypoxia and normoxia. In contrast, 39 of the 47 genes in Table II exhibited significant regulation by hypoxia in vhl-1 mutants.

Close examination of the raw data also provided evidence that VHL has hif-1-independent functions. The gene R03A10.4 (predicted glutamine-phenylpyruvate transaminase) was not regulated by hypoxia, and mutation of hif-1 had little effect on its expression. However, animals lacking vhl-1 function expressed R03A10.4 at nearly undetectable levels (Fig. 1D). Thus, VHL-1 appears to act through an HIF-1-independent mechanism to positively regulate the expression of R03A10.4.

View larger version (38K): [in this window] [in a new window]   FIG. 3. Viability assays in normoxia (21% oxygen) and hypoxia (0.5% oxygen). Animals carrying strong loss-of-function mutations in downstream targets of HIF-1 were incubated in 0.5% oxygen (hypoxia) or room air (normoxia) for 24 h and then assayed for successful completion of embryogenesis (A) and survival to adulthood as described previously (16) (B). Numerical values are listed in Table S2 (supplemental data). Asterisks indicate that the viability of hypoxia-treated mutants is significantly lower than that of wild-type (WT).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In humans response to hypoxia plays a central role in angiogenesis, tumor development, and cardiovascular disease. The hypoxia-inducible factor is thought to be the most important transcriptional regulator of mammalian hypoxic response during normal development and disease states (1, 14). Studies from other research groups have identified many genes regulated by HIF-1. Some of the genes that are transcriptionally activated by mammalian HIF-1 induce systemic responses to oxygen deprivation, such as increased red blood cell production and angiogenesis. Other HIF-1 targets modify the extracellular matrix, regulate glucose metabolism, or contribute to cellular survival in stressful conditions (41).

View larger version (29K): [in this window] [in a new window]   FIG. 4. phy-2:GFP expression in an adult hermaphrodite. The positions of the vulva and the spermathecas are indicated with a star and an s, respectively.

View larger version (36K): [in this window] [in a new window]   FIG. 5. Bacteria-mediated RNAi of nine downstream targets of HIF-1. The percentage of C. elegans that completed embryogenesis and hatched is shown. Numerical values are listed in Table S3 (supplemental data).

View larger version (30K): [in this window] [in a new window]   FIG. 6. Models for hypoxia signaling and response. A, proteasomal degradation of HIF-1 is mediated by EGL-9 and VHL-1 (6). B, response to hypoxia is mediated by HIF-1 and by HIF-1-independent pathway(s). The four largest functional categories of genes induced by each pathway are shown. HIF-1 positively regulates the expression of npc-1 and cam-1, which are negative regulators of dauer formation. This is consistent with constitutive dauer formation of hif-1 (ia04) mutants at 27 °C. daf-16 mRNA expression is induced by hypoxia independently of hif-1 function.

The Oxygen-requiring PHDs phy-2 and egl-9 are hif-1-dependent Genes—The prolyl 4-hydroxylase enzymes that modify collagen in the endoplasmic reticulum require oxygen as a substrate. C. elegans has been shown to encode three prolyl 4-hydroxylase subunits. The expression pattern of phy-2 overlaps with that of two other subunits, phy-1 and phy-3, in the hypodermis and in the spermatheca, respectively. The majority of the proteins in the worm cuticle are collagens, and double mutants lacking phy-1 and phy-2 function are inviable (23, 33, 43). As shown in Fig. 1, C. elegans phy-2 mRNA was induced by hypoxia in an hif-1-dependent manner. Transcriptional regulation of the prolyl 4-hydroxylase subunits by HIF appears to be evolutionarily conserved (44). We have shown that phy-2-defective animals exhibit lethality and sterility in hypoxic conditions. These data fit a model in which phy-2 function, and perhaps increased transcription of phy-2, is required to offset decreased availability of oxygen substrate in hypoxic conditions.

The EGL-9 PHD modifies HIF-1, thereby increasing its affinity for the VHL-1 E3 ligase that targets HIF-1 for proteosomal degradation (6). Data in Table I and Fig. 1 show that egl-9 mRNA levels are regulated by C. elegans hif-1. This is in agreement with a recently published independent study (42). Positive regulation of egl-9 transcription by HIF-1 should help to maintain EGL-9 activity when oxygen substrate is limiting, and it may create a negative feedback loop to attenuate HIF-1 activation (Fig. 6). In humans, three PHD genes (EGL-9 homologs) have been identified, and PHD2 and PHD3 are induced by hypoxia (6, 45, 46). Thus, this regulatory mechanism is evolutionarily conserved.

In mammals, HIF-1 also induces the expression of CITED2, which competes with CBP/p300 for binding to HIF-1, thereby inhibiting transcriptional activation by HIF-1. HIF-1 transcriptional repressors have not yet been identified in C. elegans. The C. elegans homologs of HIF-1 and CBP have been shown to bind each other in yeast two-hybrid assays (47), but it is not yet known whether interaction of the native proteins is regulated by environmental oxygen concentrations. hif-1-independent Pathway(s) for Hypoxia Response—Relatively little is known about the oxygen sensors and regulatory pathways that mediate transcriptional responses independent of HIF. The data presented in Table II and Fig. 1B clearly demonstrate that there are hif-1-independent mechanisms for hypoxia adaptation in C. elegans. Consistent with these findings, others have shown recently that the transcription of certain hsp16 small heat shock proteins is induced by hypoxia, and this induction does not require hif-1 function (48).

For all 47 of the hif-1-independent genes, hypoxia causes significant differences in expression in the absence of hif-1 function, but in some cases, such as the F26A3.4 phosphatase, the gene expression change is 2-fold less than that seen in wild-type animals (Fig. 1B). This supports a model in which some genes are regulated by hypoxia via both hif-1-dependent and hif-1-independent pathways.

To compare hypoxia-induced changes in mRNA levels in different genetic backgrounds, we assayed animals from a defined developmental stage (L3) and at a single early time point. We expect that assays performed at other oxygen concentrations or other developmental stages will reveal additional hypoxia-responsive genes. Early HIF-1 targets likely control later responses to oxygen deprivation. 67 of the hypoxia-responsive genes in Tables I and II have known or predicted functions, and 26 of these are expected to mediate transcription or intracellular protein-protein interactions. For example, F26A3.4 (listed at the top of Table II) encodes a protein-tyrosine phosphatase similar to the mammalian mitogen-activated protein kinase phosphatase 6. Predicted transcription factors induced by hypoxia include the nuclear hormone receptors nhr-57 and nhr-88 and the T-box family members tbx-33 and tbx-38.

Oxygen Levels and the Dauer Decision—Dauers are resistant to several types of stress, including oxygen deprivation (34). Recent analyses have also shown that the putative HIF-1 binding site (HRE) is overrepresented in the putative regulatory sequences of genes up-regulated in dauers (49). This had led to the hypothesis that HIF-1 may act in concert with DAF-16 (the forkhead transcription factor that promotes longevity and dauer formation) to regulate some of the genes that confer resistance to hypoxic stress (49). As illustrated in Figs. 1 and 6, hypoxia increases both daf-16 expression and HIF-1 activity. Interestingly, some of the genes induced by HIF-1 inhibit dauer formation, and hif-1-defective animals form dauer-like larvae at high temperatures, even in the presence of abundant food. Because hif-1 mutants are unable to acclimate to higher temperatures (40), dauer formation may allow survival of stressful conditions that might otherwise be lethal to animals that are not expressing HIF-1 target genes at sufficient levels.

Some responses to oxygen stress are post-translational. A recent study demonstrated that C. elegans will aerotax from 21% oxygen to 7% oxygen, and this response is mediated by gcy-35, which encodes an oxygen-sensing guanylate cyclase (50). C. elegans are standardly cultured in the laboratory in room air (21% oxygen). In the wild, C. elegans inhabits the soil, and the oxygen levels in soil containing ample supplies of its bacterial food source could be substantially below 21%. In future studies, it will be interesting to investigate whether the expression of hypoxia-responsive genes varies significantly between 21% oxygen and the lower oxygen levels preferred by C. elegans.

The microarray experiments reported here were not designed to compare gene expression patterns in vhl-1 mutant and wild-type animals directly, but we did find one gene that was regulated by VHL-1 but not HIF-1. An independent study has recently been published which identified additional C. elegans genes regulated by vhl-1 independently of hif-1 (42). This is especially interesting because mutations have been identified in the mammalian VHL tumor suppressor protein which predispose individuals to carcinomas, but do not impair HIF-1 ubiquitination (51, 52). Further studies of HIF-1 and VHL homologs in C. elegans may shed light on the shared and distinct cellular pathways that regulate tumorigenesis.

	FOOTNOTES

 
^* This work was supported by grants from the National Institutes of Health and Howard Hughes Medical Institute (to S. K. K.) and an American Heart Association established investigator grant (to J. A. P.-C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Tables S1–S3.

^|| To whom correspondence should be addressed: 2108 Molecular Biology Bldg., Iowa State University, Ames, IA 50011-3260. Tel.: 515-294-3906; E-mail: japc{at}iastate.edu.

¹ The abbreviations used are: HIF-1, hypoxia-inducible factor 1; CBP, cAMP-response element-binding protein-binding protein; E3, ubiquitin-protein isopeptide ligase; eEF-2, elongation factor 2; GFP, green fluorescent protein; HRE, hypoxia response element; PHD, prolyl hydroxylase; RNAi, RNA interference; VHL, von Hippel-Lindau.

	ACKNOWLEDGMENTS

 
Mutant strains were obtained from the Caenorhabditis Genetics Center, which is supported by the National Institutes of Health National Center for Research Resources. Yuji Kohara and colleagues provided cDNA constructs. We are grateful to Qunfeng Dong, who generated the graphic shown in Fig. 2.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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