egl-9 and rhy-1 inhibit cdo-1 transcription

A) Simplified pathway for sulfur amino acid metabolism beginning with methionine. We highlight the roles of cystathionase (CTH-2), cysteine dioxygenase (CDO-1), and the Moco-requiring sulfite oxidase enzyme (SUOX-1). Human homologs are displayed in purple. Abbreviations are as follows: Met (methionine), Ser (serine), Cth (cystathionine), Cys (cysteine), CSA (cysteinesulfinate), and αKB (α-ketobutyrate). B) Models of Pcdo-1::GFP (upper) and Pcdo-1::CDO-1::GFP (lower) transgenes used in this work are displayed. Boxes indicate exons, connecting lines indicate introns. The cdo-1 promoter is shown. C) egl-9a and rhy-1 gene structures are displayed. Boxes indicate exons and connecting lines are introns. Colored annotations indicate mutations generated or used in our work. Red; chemically-induced mutations that activated Pcdo-1::CDO-1::GFP. Blue; reference null alleles isolated independent of our work. Purple; CRISPR/Cas9-generated mutation that inactivates the prolyl hydroxylase domain of EGL-9. D) Expression of Pcdo-1::GFP transgene is displayed from wild-type, egl-9(sa307), and rhy-1(ok1402) C. elegans animals at the L4 stage of development. Scale bar is 250μm. E) Quantification of GFP expression displayed in Fig. 1D. 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.

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 250μm. B,C) Quantification of the data displayed in Fig. 2A. 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 Fig. 1D,E. They are re-displayed here to allow for clear comparisons to the double mutant strains of interest.

High cysteine activates cdo-1 transcription

A) Expression of the Pcdo-1::GFP transgene is displayed for wild-type, young-adult C. elegans exposed to 100μM supplemental cysteine as well as non-supplemented control animals. Scale bar is 250μm. B) Quantification of the data displayed in Fig. 3A. 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 expression of Pcdo-1::GFP in wild-type C. elegans exposed to 0μM supplemental cysteine is equal to 1 arbitrary unit (a.u.). **, p<0.001, Welch’s t test.

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 where GFP is inserted into endogenous cdo-1, creating a 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 10μm. 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 hours 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 hours 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. Box plots display the median, upper, and lower quartiles and whiskers indicate minimum and maximum data points. Sample size (n) is 10 individuals for each experiment. ****, p<0.0001, multiple unpaired t test with Welch correction. ns indicates no significant difference was identified.

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.

egl-9 inhibits cdo-1 transcription in a 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. 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 transgene is displayed for wild-type, egl-9(sa307, -), vhl-1(ok161), and egl-9(rae276, H487A) C. elegans animals at the L4 stage of development. Scale bar is 250μm. D) Quantification of the data displayed in Fig. 5C. 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.

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 (PHD), 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 promotes the catabolism of cysteine. HIF-1-induced cysteine catabolism requires the activity of rhy-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 for 72 hours on wild-type (black, Moco+) or ΔmoaA mutant (red, Moco-) E. coli. Animal lengths were determined for each condition. Box plots display the median, upper, and lower quartiles and whiskers indicate minimum and maximum data points. Sample size (n) is displayed for each experiment. ****, p<0.0001, multiple unpaired t test with Welch correction. ns indicates no significant difference was identified.

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 250μm. B) Quantification of CDO-1::GFP expression displayed in Fig. S2A. 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<0.0001, ordinary one-way ANOVA with Dunnett’s post hoc analysis.

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 100μM supplemental cysteine. Scale bar is 250μm. B) Quantification of CDO-1::GFP expression displayed in Fig. S3A. 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 0μM supplemental cysteine is 1 arbitrary unit (a.u.). **, p<0.001, Welch’s t test.