Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans
- Pediatrics and Rare Diseases Group, Sanford Research, United States
- Department of Pediatrics, Sanford School of Medicine, University of South Dakota, United States
- Department of Molecular Biology, Massachusetts General Hospital, United States
Figures
Figure 1 with 1 supplement
egl-9 and rhy-1 inhibit cdo-1 transcription.
(A) Pathway for sulfur amino acid metabolism beginning with methionine. We highlight the roles of cystathionase (CTH-2/CTH), cysteine dioxygenase (CDO-1/CDO1), and the Moco-requiring sulfite oxidase enzyme (SUOX-1/SUOX). C. elegans enzymes (magenta) and their human homologs (green) are displayed. (B) Pcdo-1::GFP promoter fusion (upper) and Pcdo-1::CDO-1::GFP C-terminal protein fusion (lower) transgenes used in this work are displayed. Boxes indicate exons, connecting lines indicate introns. The cdo-1 promoter is shown as a straight line. (C) egl-9a and rhy-1 gene structures. Boxes indicate exons and connecting lines are introns. Colored annotations indicate mutations generated or used in our work. Magenta; chemically-induced mutations that activated Pcdo-1::CDO-1::GFP fusion protein. Blue; reference null alleles isolated independent of our work. Green; CRISPR/Cas9-generated mutation that inactivates the prolyl hydroxylase domain of EGL-9. (D) Expression of Pcdo-1::GFP transgene is displayed for wild-type, egl-9(sa307), and rhy-1(ok1402) C. elegans animals at the L4 stage. Scale bar is 250 μm. White dotted line outlines animals with basal GFP expression. For GFP imaging, exposure time was 100ms. (E) Quantification of GFP expression displayed in (D). Individual datapoints are shown (circles) as are the mean and standard deviation (red lines). n is 5 individuals per genotype. Data are normalized so that wild-type expression of Pcdo-1::GFP is 1 arbitrary unit (a.u.). ****, p<0.0001, ordinary one-way ANOVA with Dunnett’s post hoc analysis.
Figure 1—figure supplement 1
The Pcdo-1::CDO-1::GFP transgene encodes a functional cysteine dioxygenase enzyme.
moc-1(ok366) cdo-1(mg622) double mutant animals expressing Pcdo-1::CDO-1::GFP or Pcdo-1::CDO-1[C85Y]::GFP transgenes were cultured from synchronized L1 larvae for 72 hr on wild-type (black, Moco+) or ΔmoaA mutant (red, Moco-) E. coli. Animal lengths were determined for each condition. Individual datapoints are shown (circles) as are the mean and standard deviation. Sample size (n) is displayed for each experiment. ****, p<0.0001, multiple unpaired t test with Welch’s correction. ns indicates no significant difference was identified.
Figure 2
cdo-1 transcription is activated by HIF-1 downstream of RHY-1, CYSL-1, and EGL-9.
(A) Expression of the Pcdo-1::GFP transgene is displayed for wild-type, egl-9(sa307), egl-9(sa307) hif-1(ia4) double mutant, egl-9(sa307); cysl-1(ok762) double mutant, rhy-1(ok1402), rhy-1(ok1402); hif-1(ia4) double mutant, and rhy-1(ok1402); cysl-1(ok762) double mutant C. elegans animals at the L4 stage of development. Scale bar is 250 μm. White dotted line outlines animals with basal GFP expression. For GFP imaging, exposure time was 100ms. (B, C) Quantification of the data displayed in (A). Individual datapoints are shown (circles) as are the mean and standard deviation (red lines). n is 5 individuals per genotype. Data are normalized so that wild-type expression of Pcdo-1::GFP is 1 arbitrary unit (a.u.). *, p<0.05, ****, p<0.0001, ordinary one-way ANOVA with Dunnett’s post hoc analysis. Note, wild-type, egl-9(-), and rhy-1(-) images in panel A and quantification of Pcdo-1::GFP in panels B and C are identical to the data presented in (Figure 1D and E). They are re-displayed here to allow for clear comparisons to the double mutant strains of interest.
Figure 3
High levels of cysteine activate cdo-1 transcription and are lethal to hif-1 and cysl-1 mutant animals.
(A) Expression of the Pcdo-1::GFP transgene is displayed for wild-type, egl-9(sa307), and rhy-1(ok1402) young-adult C. elegans exposed to 0 or 100 μM supplemental cysteine. Scale bar is 250 μm. White dotted line outlines animals with basal GFP expression. For GFP imaging, exposure time was 100ms. Supplemental cysteine did not impact the mortality of the animals being imaged. (B) Quantification of the data displayed in (A). Individual datapoints are shown (circles) as are the mean and standard deviation (black lines). n is 6 or 7 individuals per genotype. Data are normalized so that expression of Pcdo-1::GFP in wild-type C. elegans exposed to 0 μM supplemental cysteine is equal to 1 arbitrary unit (a.u.). *, p<0.05, ****, p<0.0001, multiple unpaired t test with Welch’s correction. (C) Quantification of the Pcdo-1::GFP expression is displayed for wild-type young-adult C. elegans exposed to 0, 50, 100, 250, or 500 μM supplemental cysteine. Mean and standard deviation are displayed. n is 6 or 7 individuals per concentration of supplemental cysteine. (D–F) The percentage of animals that survive overnight exposure to (D) 0, (E) 100, or (F) 1000 μM supplemental cysteine. Individual datapoints (circles) represent biological replicates. Three or four biological replicates were performed for each experiment and the total individuals scored amongst all replicates is displayed (n). *, p<0.05, ****, p<0.0001, ordinary one-way ANOVA with Dunnett’s post hoc analysis. ns indicates no significant difference was identified.
Figure 4 with 2 supplements
Hypodermal CDO-1 accumulates in the cytoplasm when egl-9 or rhy-1 are inactive and is sufficient to promote sulfur amino acid metabolism.
(A) Diagram of cdo-1(rae273), a CRISPR/Cas9-generated allele with GFP inserted into the cdo-1 gene, creating a functional C-terminal CDO-1::GFP fusion protein expressed from the native cdo-1 locus. (B) Differential interference contrast (DIC) and fluorescence imaging are shown for wild-type, egl-9(sa307), and rhy-1(ok1402) C. elegans expressing CDO-1::GFP encoded by cdo-1(rae273). Scale bar is 10 μm. For GFP imaging, exposure time was 200 ms. An anterior segment of the Hyp7 hypodermal cell is displayed. CDO-1::GFP accumulates in the cytoplasm and is excluded from the nuclei. (C) moc-1(ok366), moc-1(ok366) cdo-1(mg622), and moc-1(ok366) cdo-1(rae273) animals were cultured from synchronized L1 larvae for 72 hr on wild-type (black, Moco+) or ΔmoaA mutant (red, Moco-) E. coli. (D) moc-1(ok366) cdo-1(mg622) double mutant animals expressing Pcol-10::CDO-1::GFP or Pcol-10::CDO-1[C85Y]::GFP transgenes were cultured for 48 hr on wild-type (black, Moco+) or ΔmoaA mutant (red, Moco-) E. coli. Two independently derived strains were tested for each transgene. For panels C and D, animal lengths were determined for each condition. Individual datapoints are shown (circles) as are the mean and standard deviation. Sample size (n) is 10 individuals for each experiment. ****, p<0.0001, multiple unpaired t test with Welch’s correction. ns indicates no significant difference was identified.
Figure 4—figure supplement 1
A functional CDO-1::GFP fusion protein is induced by loss of egl-9 or rhy-1.
(A) Expression of CDO-1::GFP from the cdo-1(rae273) allele is displayed for wild-type, egl-9(sa307), and rhy-1(ok1402) animals at the L4 stage of development. Scale bar is 250 μm. For GFP imaging, exposure time was 100ms. (B) Quantification of CDO-1::GFP expression displayed in (A). Individual datapoints are shown (circles) as are the mean and standard deviation (red lines). n is 7 individuals per genotype. Data are normalized so that wild-type expression of CDO-1::GFP is 1 arbitrary unit (a.u.). **, p<0.01, ordinary one-way ANOVA with Dunnett’s post hoc analysis.
Figure 4—figure supplement 2
CDO-1::GFP encoded by cdo-1(rae273) is induced by supplemental cysteine.
(A) Expression of CDO-1::GFP from the cdo-1(rae273) allele is displayed for wild-type young adult animals exposed to 0 or 100 μM supplemental cysteine. Scale bar is 250 μm. For GFP imaging, exposure time was 100ms. Supplemental cysteine did not impact the mortality of the animals being imaged. (B) Quantification of CDO-1::GFP expression displayed in (A). Individual datapoints are shown (circles) as are the mean and standard deviation (red lines). n is 6 individuals per genotype. Data are normalized so that wild-type expression of CDO-1::GFP exposed to 0 μM supplemental cysteine is 1 arbitrary unit (a.u.). ****, p<0.0001, unpaired t test with Welch’s correction.
Figure 5
egl-9 inhibits cdo-1 transcription in a largely prolyl-hydroxylase and VHL-1-independent manner.
(A) The pathway of HIF-1 processing during normoxia is displayed. EGL-9 uses O2 as a substrate to hydroxylate (-OH) HIF-1 on specific proline residues. Prolyl hydroxylated HIF-1 is bound by VHL-1 which facilitates HIF-1 polyubiquitination and targets HIF-1 for degradation by the proteasome. (B) Amino acid alignment of the EGL-9 prolyl hydroxylase domain from C. elegans, D. melanogaster, M. musculus, and H. sapiens. ‘*’ indicate perfect amino acid conservation while ‘.’ indicates weak similarity amongst species compared. Highlighted (red) is the catalytically essential histidine 487 residue in C. elegans. Alignment was performed using Clustal Omega (EMBL-EBI). (C) Expression of Pcdo-1::GFP promoter fusion transgene is displayed for wild-type, egl-9(sa307, -), egl-9(rae276, H487A), and vhl-1(ok161) C. elegans animals at the L4 stage of development. Scale bar is 250 μm. White dotted line outlines animals with basal GFP expression. For GFP imaging, exposure time was 500ms. (D) Quantification of the data displayed in (C). Individual datapoints are shown (circles) as are the mean and standard deviation (red lines). n is 6 individuals per genotype. Data are normalized so that wild-type expression of Pcdo-1::GFP is 1 arbitrary unit (a.u.). ***, p<0.001, ****, p<0.0001, ordinary one-way ANOVA with Tukey’s multiple comparisons test. ns indicates no significant difference was identified.
Figure 6
Model for the regulation of cysteine metabolism by HIF-1.
(A) Proposed genetic pathway for the regulation of cdo-1. rhy-1, cysl-1, and egl-9 act in a negative-regulatory cascade to control activity of the HIF-1 transcription factor, which activates transcription of cdo-1. (B) Under basal conditions (inactivated HIF-1), EGL-9 negatively regulates HIF-1 through 2 distinct pathways; one pathway is dependent upon O2, prolyl hydroxylation (Pro-OH), and VHL-1, while the second acts independently of these canonical factors. Under these conditions cdo-1 transcription is kept at basal levels and cysteine catabolism is not induced. (C) During conditions where HIF-1 is activated (high H2S), CYSL-1 directly binds and inhibits EGL-9, preventing HIF-1 inactivation. Active HIF-1 binds the cdo-1 promoter, driving transcription and promoting CDO-1 protein accumulation. High CDO-1 levels promote the catabolism of cysteine leading to production of sulfites (SO32-) that are toxic during Moco or SUOX-1 deficiency. HIF-1-induced cysteine catabolism requires the activity of rhy-1.
Tables
Table 1
Growth of C. elegans strains on standard laboratory conditions.
C. elegans strains and their corresponding mutations are displayed. For each strain, 5 L4-stage animals were seeded onto standard NGM petri dishes seeded with a monoculture of E. coli OP50. Petri dishes were monitored until the animals (and their progeny) depleted the lawn of E. coli. This was recorded as ‘days for population to starve petri dish’. The average of these experiments is displayed for each C. elegans strain as is the standard deviation (SD) and the number of biological replicates (n). Significant differences in population growth were determined by appropriate comparisons to either suox-1(gk738847) (GR2269) or egl-9(sa307); suox-1(gk738847) (USD421) using an ordinary one-way ANOVA with Dunnett’s post hoc analysis. No test indicates a statistical comparison was not made. Note, the data for USD421 are displayed twice in the table to allow for ease of comparison.
| C. elegans strain | Genetic locus 1 | Genetic locus 2 | Genetic locus 3 | Days for population to starve petri dish ±SD (n) | Significant difference compared to GR2269 (Adjusted p Value) |
|---|---|---|---|---|---|
| N2 | Wild type | Wild type | Wild type | 5±1 (8) | No test |
| GR2269 | Wild type | suox-1(gk738847) | Wild type | 8±1 (4) | No test |
| JT307 | egl-9(sa307) | Wild type | Wild type | 7±1 (5) | No test |
| USD421 | egl-9(sa307) | suox-1(gk738847) | Wild type | 19±5 (11) | 0.0003 (***) |
| USD926 | egl-9(rae276) | Wild type | Wild type | 6±1 (3) | No test |
| USD937 | egl-9(rae276) | suox-1(gk738847) | Wild type | 9±1 (3) | 0.93 (ns) |
| USD512 | rhy-1(ok1402) | Wild type | Wild type | 6±0 (4) | No test |
| USD414 | rhy-1(ok1402) | suox-1(gk738847) | Wild type | 7±0 (4) | 0.99 (ns) |
| CB5602 | vhl-1(ok161) | Wild type | Wild type | 6±0 (4) | No test |
| USD422 | vhl-1(ok161) | suox-1(gk738847) | Wild type | 8±1 (4) | 0.99 (ns) |
| C. elegans strain | Genetic locus 1 | Genetic locus 2 | Genetic locus 3 | Days for population to starve petri dish ±SD (n) | Significant difference compared to USD421 (Adjusted p value) |
| USD421 | egl-9(sa307) | suox-1(gk738847) | Wild type | 19±5 (11) | No test |
| USD430 | egl-9(sa307) | suox-1(gk738847) | cdo-1(mg622) | 7±0 (6) | <0.0001 (****) |
| USD433 | egl-9(sa307) | suox-1(gk738847) | cth-2(mg599) | 9±1 (4) | <0.0001 (****) |
| USD432 | egl-9(sa307) | suox-1(gk738847) | rhy-1(ok1402) | 7±1 (4) | <0.0001 (****) |
| USD434 | egl-9(sa307) | suox-1(gk738847) | cysl-1(ok762) | 16±1 (9) | 0.1 (ns) |
| USD431 | egl-9(sa307) | suox-1(gk738847) | hif-1(ia4) | 9±1 (6) | <0.0001 (****) |
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Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans
eLife 12:RP89173.
https://doi.org/10.7554/eLife.89173.3
