Two distinct roles for EGL-9 in the regulation of HIF-1-mediated gene expression in Caenorhabditis elegans

Abstract

Oxygen is critically important to metazoan life, and the EGL-9/PHD enzymes are key regulators of hypoxia (low oxygen) response. When oxygen levels are high, the EGL-9/PHD proteins hydroxylate hypoxia-inducible factor (HIF) transcription factors. Once hydroxylated, HIFalpha subunits bind to von Hippel-Lindau (VHL) E3 ligases and are degraded. Prior genetic analyses in Caenorhabditis elegans had shown that EGL-9 also acted through a vhl-1-independent pathway to inhibit HIF-1 transcriptional activity. Here, we characterize this novel EGL-9 function. We employ an array of complementary methods to inhibit EGL-9 hydroxylase activity in vivo. These include hypoxia, hydroxylase inhibitors, mutation of the proline in HIF-1 that is normally modified by EGL-9, and mutation of the EGL-9 catalytic core. Remarkably, we find that each of these treatments or mutations eliminates oxygen-dependent degradation of HIF-1 protein, but none of them abolishes EGL-9-mediated repression of HIF-1 transcriptional activity. Further, analyses of new egl-9 alleles reveal that the evolutionarily conserved EGL-9 MYND zinc finger domain does not have a major role in HIF-1 regulation. We conclude that C. elegans EGL-9 is a bifunctional protein. In addition to its well-established role as the oxygen sensor that regulates HIF-1 protein levels, EGL-9 inhibits HIF-1 transcriptional activity via a pathway that has little or no requirement for hydroxylase activity or for the EGL-9 MYND domain.

Figure 1.—

EGL-9 functions and models tested in this study. (A) EGL-9 regulates HIF-1 by two pathways, and they are illustrated here. First, EGL-9 controls oxygen-dependent degradation of HIF-1 (labeled pathway 1). EGL-9 hydroxlates HIF-1 on a conserved proline residue (P621), and this enables binding of HIF-1 to the VHL-1 E3 ligase. HIF-1 is then degraded. Molecular oxygen, Fe(II), and 2-oxoglutarate are required for the hydroxylation reaction. EGL-9 also suppresses expression of HIF-1 targets by a second pathway that does not require VHL-1 (labeled pathway 2 here). (B) Initial alternative models for the VHL-1-independent functions of EGL-9 (pathway 2). Each model predicts a different combination of experimental outcomes. Model a postulates that pathway 2 (like pathway 1) requires hydroxylation of HIF-1 proline 621. Model b is that EGL-9 hydroxylates a different target to inhibit HIF-1 transcriptional activity. This model predicts that all EGL-9 functions would be abrogated by mutations or treatments that eliminated EGL-9 hydroxylase activity. Model c is that EGL-9 represses HIF-1-mediated transcription by a mechanism that does not require EGL-9 hydroxylase activity.

Figure 2.—

The HIF-1(P621G) mutation does not prevent egl-9-mediated repression of HIF-1 activity. (A) In control experiments, the transgene encoding wild-type hif-1, rescued expression of Pnhr-57∷GFP in hif-1(ia04) mutant animals, as measured by protein blots. The egl-9(sa307) loss-of-function mutation dramatically increased expression of the Pnhr-57∷GFP reporter. +, the wild-type allele. (B and C) To determine whether the HIF-1(P621G) mutation abrogated all regulation by egl-9, the expression of two HIF-1 targets, Pnhr-57∷GFP and K10H10.2, were compared in egl-9(+) and egl-9(sa307) animals. These experiments were conducted in hif-1(ia04) mutants rescued by the hif-1(P621G)∷tag transgene. (B) The HIF-1(P621G) mutation did not prevent repression of Pnhr-57∷GFP by egl-9. Pnhr-57∷GFP expression was assayed by protein blots. This result was consistent across three independently isolated hif-1(P621G)∷tag transgenic lines (iaIs32, iaIs33, and iaIs34) (P < 0.01 in each case). (C) The hif-1(P621G) iaIs32 mutation does not abolish regulation of K10H10.2 expression by egl-9. Quantitative RT–PCR experiments established a significant difference in K10H10.2 mRNA levels between egl-9(+) and egl-9(sa307) (P < 0.05).

Figure 3.—

Differential effects of hypoxia or iron chelators on the two EGL9 pathways. (A) In control experiments, hypoxia (0.5% oxygen, 1 hr) or the iron chelator 2, 2′-dipyridyl (DIP, 200 μm, 4 hr) increased HIF-1 protein to levels similar to those caused by loss-of-function mutations in vhl-1 or egl-9. The bar graph shows HIF-1 protein levels relative to that in untreated vhl-1(+) and egl-9(+) animals. In these strains, the only functional copy of hif-1 is the epitope-tagged transgene. The error bars indicate the standard errors from three independent biological replicates. (B) To determine whether hydroxylase inhibitors had similar effects on HIF-1 target genes as a mutation in egl-9, expression of the reporter was assayed by protein blots. Hypoxia or DIP treatment increased expression of the reporter to levels found in vhl-1 loss-of-function mutants, but the treatments did not completely phenocopy the effects of a loss-of-function mutation in egl-9. In the bar graph, Pnhr-57∷GFP levels are shown relative to that in untreated vhl-1(+) and egl-9(+) animals. The vhl-1(ok161) and egl-9(sa307) mutations are strong loss-of-function alleles. +, the wild-type allele. The bars represent average values from three independent replicates, and the error bars reflect standard error. The statistics were comparing the hypoxia or DIP treated data to untreated data with the same genotype. Asterisks represent statistically significant differences between treatment and room air for animals of the same genotype. *P < 0.05; **P < 0.01.

Figure 4.—

The egl-9(H487A) mutation does not abolish egl-9-mediated repression of HIF-1 activity. (A) Alignment of human PHD2 and C. elegans EGL-9 at the region around EGL-9 His487. (B) Diagrams of egl-9 minigenes. The minigenes are fusions of genomic and cDNA sequences. Boxes represent coding regions. GFP is fused in frame to egl-9. The H487A mutation disrupts the Fe(II) binding pocket in EGL-9 and impairs EGL-9 catalytic activity. (C) In egl-9(sa307) mutants, HIF-1 destabilization was rescued by the transgene coding for wild-type egl-9(iaIs38), but not by the egl-9(H487A) transgenes (iaEx101, iaEx104, or iaEx110). AHA-1 protein levels have been shown to be unaffected by severe loss-of-function mutations in egl-9 or vhl-1 (Shen et al. 2006), and AHA-1 serves as a loading control. (D and E) Transgenes expressing either wild-type egl-9 or egl-9(H487A) can suppress expression of HIF-1 targets in an egl-9(sa307) background. (D) In strains carrying the wild-type egl-9 transgene, repression of Pnhr-57∷GFP is more effective and is inhibited by DIP. In strains expressing egl-9(H487A), DIP does not have a significant effect. The bar graph shows averages of Pnhr-57∷GFP protein levels from three biological replicates, normalized to expression levels in an egl-9(sa307) mutant. The asterisk indicates DIP causes a statistically significant difference in the expression of the reporter (*P < 0.05) and NS is no significant difference. (E) The wild-type egl-9 transgene and the egl-9(H487) mutant transgene were able to repress K10H10.2 mRNA levels in an egl-9(sa307) mutant. K10H10.2 mRNA levels were also assayed in wild-type N2 and in vhl-1-deficient animals for comparison. The bar graph shows the relative K10H10.2 mRNA levels in each strain, compared to egl-9(sa307) and the error bars reflect standard error. NS, no significant difference. *P < 0.05; **P < 0.01.

Figure 5.—

Characterization of egl-9 loss-of-function alleles. (A) Diagram of egl-9 exons and introns and description of new mutations. Boxes represent exons for the predominant egl-9 mRNA isoform, and the exons encoding the MYND or hydroxylase domains are filled. Lines represent deleted sequences in the gk277, sa307, or ok478 alleles; arrows indicate the positions of the ia58 and ia61 mutations; and the position of the ia60 transposon insertion is shown. The alleles isolated in this study are described in the table. (B) Relative effects of egl-9 mutations on HIF-1 protein levels. These animals carry the hif-1(ia04) deletion mutation, and hif-1 function is restored by the hif-1∷tag transgene. The numbers above the lanes reflect HIF-1 levels relative to those detected in egl-9(sa307) mutants, as determined from three replicate experiments. (C) Expression of the Pnhr-57∷GFP reporter in egl-9 mutants, relative to animals homozygous for the egl-9(sa307), a strong loss-of-function egl-9 allele. The bars represent average values relative to those in egl-9(sa307), from three independent replicates, and the error bars reflect standard error.

Figure 6.—

The egl-9(gk277) mutation removes the MYND zinc finger domain, but has little effect on HIF-1 protein levels or on expression of HIF-1 targets. (A) The egl-9(gk277) mutation does not significantly increase expression of HIF-1 protein in vhl-1(+) or vhl-1(ok161). (B and C) The egl-9(gk277) mutation has a significant effect on the expression of HIF-1 target genes, but these effects are much smaller than the severe egl-9(sa307) mutation. The bar graphs show the expression of the Pnhr-57∷GFP reporter (B) and the endogenous HIF-1 target gene K10H10.2 (C) in each strain, relative to the values for animals carrying wild-type alleles of vhl-1 and egl-9. The bars represent average values from three independent replicates, and the error bars reflect standard error. NS, no significant difference. *P < 0.05.