Hypoxia-inducible factor promotes cysteine homeostasis in Caenorhabditis elegans

Reviewer #1 (Public Review):

Warnhoff et al present a genetic investigation of the response of C. elegans to high dietary cysteine. Using a Pcdo-1::CDO-1::GFP reporter (for a cysteine dioxygenase gene) and unbiased mutagenesis, they identify multiple alleles, including nonsense alleles, in egl-9 and rhy-1, which they validate with reference alleles. Further mutational analysis shows that hif-1 and cysl-1, components of the same established genetic regulatory pathway, also act in cdo-1 regulation. High dietary levels of cysteine activate cdo-1 expression, but loss of cdo-1 does not cause sensitivity to excess dietary cysteine, whereas cysl-1 and hif-1 are completely inviable in these conditions. Using sulfite oxidase suox-1 mutant and double and triple mutant analysis the authors show that the defects caused by suox-1 deletion (which causes sulfite accumulation) are exacerbated by loss of egl-9, which is alleviated by concomitant loss of enzymes cdo-1 / cth-2 or regulators rhy-1 / hif-1, demonstrating that the key issue is cysteine derived sulfites. Further genetic analysis shows that although egl-9 is required for cdo-1 induction, this is only partially dependent on its hydroxylase activity and the egl-9 partner vhl-1 is also only partially involved.

The significance of the findings is that they describe a regulatory pathway by which organisms might respond to high levels of cysteine in vivo.

Strengths
- The genetic analysis is generally well done and convincing, with multiple alleles identified for each gene, several reporters used for cdo-2, etc.
- Genetic analysis using site-directed mutagenesis of egl-9 and cdo-1 with point mtuations is especially nice.
- The data are analyzed and represented properly, and microscopy data have been quantified.
- The paper is also written quite clearly and the figures are easy to understand.

Weaknesses
- The relevance is somewhat unclear. High cysteine levels can be achieved in the laboratory, but, is this relevant in the life of C. elegans? Or is there physiological relevance in humans, e.g. a disease? The authors state "cells and animals fed excess cysteine and methionine", but is this more than a laboratory excess condition? SUOX nonfunctional conditions in humans don't appear to tie into this, since, in that context, the goal is to inactivate CDO or CTH to prevent sulfite production. The authors also mention cancer, but the link to cysteine levels is unclear. In that sense, then, the conditions studied here may not carry much physiological relevance.
- The pathway is described as important for cysteine detoxification, which is described to act via H2S (Figure 6). Much of that pathway has already been previously established by the Roth, Miller, and Horvitz labs as critical for the H2S response. While the present manuscript adds some additional insight such as the additional role of RHY-1 downstream on HIF-1 in promoting toxicity, this study therefore mainly confirms the importance of a previously described signalling pathway, essentially adding a new downstream target rhy-1 -> cysl-1 -> egl-9 -> hif-1 -> sqrd-1/cdo-1. The impact of this finding is reduced by the fact that cdo-1 itself isn't actually required for survival in high cysteine, suggesting it is merely a maker of the activity of this previously described pathway.

Reviewer #2 (Public Review):

The authors investigate the transcriptional regulation of cysteine dioxygenase (CDO-1) in C. elegans and its role in maintaining cysteine homeostasis. They show that high cysteine levels activate cdo-1 transcription through the hypoxia-inducible transcription factor HIF-1. Using transcriptional and translational reporters for CDO-1, the authors propose a negative feedback pathway involving RHY-1, CYSL-1, EGL-9, and HIF-1 in regulating cysteine homeostasis.

Genetics is a notable strength of this study. The forward genetic screen, gene interaction, and epistasis analyses are beautifully designed and rigorously conducted, yielding solid and unambiguous conclusions on the genetic pathway regulating CDO-1. The writing is clear and accessible, contributing to the overall high quality of the manuscript.

Addressing the specifics of cysteine supplementation and interpretation regarding the cysteine homeostasis pathway would further clarify the paper and strengthen the study's conclusions.

First, the authors show that the supplementation of exogenous cysteine activates cdo-1p::GFP. Rather than showing data for one dose, the author may consider presenting dose-dependency results and whether cysteine activation of cdo-1 also requires HIF-1 or CYSL-1, which would be important data given the focus and major novelty of the paper in cysteine homeostasis, not the cdo-1 regulatory gene pathway. While the genetic manipulation of cdo-1 regulators yields much more striking results, the effect size of exogenous cysteine is rather small. Does this reflect a lack of extensive condition optimization or robust buffering of exogenous/dietary cysteine? Would genetic manipulation to alter intracellular cysteine or its precursors yield similar or stronger effect sizes?

Second, there remain several major questions regarding the interpretation of the cysteine homeostasis pathway. How much specificity is involved for the RHY-1/CYSL-1/EGL-9/HIF-1 pathway to control cysteine homeostasis? Is the pathway able to sense cysteine directly or indirectly through its metabolites or redox status in general? Given the very low and high physiological concentrations of intracellular cysteine and glutathione (GSH, a major reserve for cysteine), respectively, there is a surprising lack of mention and testing of GSH metabolism. In addition, what are the major similarities and differences of cysteine homeostasis pathways between C. elegans and other systems (HIF dependency, transcription vs post-transcriptional control)? These questions could be better discussed and noted with novel findings of the current study that are likely C. elegans specific or broadly conserved.

Reviewer #3 (Public Review):

There has been a long-standing link between the biology of sulfur-containing molecules (e.g., hydrogen sulfide gas, the amino acid cysteine, and its close relative cystine, et cetera) and the biology of hypoxia, yet we have a poor understanding of how and why these two biological processes and are co-regulated. Here, the authors use C. elegans to explore the relationship between sulfur metabolism and hypoxia, examining the regulation of cysteine dioxygenase (CDO1 in humans, CDO-1 in C. elegans), which is critical to cysteine catabolism, by the hypoxia inducible factor (HIF1 alpha in humans, HIF-1 in C. elegans), which is the key terminal effector of the hypoxia response pathway that maintains oxygen homeostasis. The authors are trying to demonstrate that (1) the hypoxia response pathway is a key regulator of cysteine homeostasis, specifically through the regulation of cysteine dioxygenase, and (2) that the pathway responds to changes in cysteine homeostasis in a mechanistically distinct way from how it responds to hypoxic stress.

Briefly summarized here, the authors initiated this study by generating transgenic animals expressing a CDO-1::GFP protein chimera from the cdo-1 promoter so that they could identify regulators of CDO-1 expression through a forward genetic screen. This screen identified mutants with elevated CDO-1::GFP expression in two genes, egl-9 and rhy-1, whose wild-type products are negative regulators of HIF-1, raising the possibility that cdo-1 is a HIF-1 transcriptional target. Indeed, the authors provide data showing that cdo-1 regulation by EGL-9 and RHY-1 is dependent on HIF-1 and that regulation by RHY-1 is dependent on CYSL-1, as expected from other published findings of this pathway. The authors show that exogenous cysteine activates cdo-1 expression, reflective of what is known to occur in other systems. Moreover, they find that exogenous cysteine is toxic to worms lacking CYSL-1 or HIF-1 activity, but not CDO-1 activity, suggesting that HIF-1 mediates a survival response to toxic levels of cysteine and that this response requires more than just the regulation of CDO-1. The authors validate their expression studies using a GFP knockin at the cdo-1 locus, and they demonstrate that a key site of action for CDO-1 is the hypodermis. They present genetic epistasis analysis supporting a role for RHY-1, both as a regulator of HIF-1 and as a transcriptional target of HIF-1, in offsetting toxicity from aberrant sulfur metabolism. The authors use CRISPR/Cas9 editing to mutate a key amino acid in the prolyl hydroxylase domain of EGL-9, arguing that EGL-9 inhibits CDO-1 expression through a mechanism that is largely independent of the prolyl hydroxylase activity.

Overall, the data seem rigorous, and the conclusions drawn from the data seem appropriate. The experiments test the hypothesis using logical and clever molecular genetic tools and design. The sample size is a bit lower than is typical for C. elegans papers; however, the experiments are clearly not underpowered, so this is not an issue. The paper is likely to drive many in the field (including the authors themselves) into deeper experiments on (1) how the pathway senses hypoxia and sulfur/cysteine/H2S using these distinct mechanisms/modalities, (2) how oxygen and sulfur/cysteine/H2S homeostasis influence one another, and (3) how this single pathway evolved to sense and respond to both of these stress modalities.

Major strengths of the paper include (1) the use of the powerful whole animal C. elegans model to reveal results that have meaning in vivo, (2) the careful demonstration through mutant rescue experiments that key transgenes have functional activity, (3) the use of CRISPR/Cas9 editing to mutate a critical residue in the catalytic domain of the EGL-9 prolyl hydroxylase, (4) transgenic rescue experiments that show that CDO-1 operates in the hypodermis with regard to the larval arrest phenotype, and (5) the thorough epistatic analysis of different pathway mutants.

Major weaknesses of the paper include (1) the over-reliance on genetic approaches, (2) the lack of novelty regarding prolyl hydroxylase-independent activities of EGL-9, and (3) the lack of biochemical approaches to probe the underlying mechanism of the prolyl hydroxylase-independent activity of EGL-9.

Major Issues We Feel the Authors Should Address:

1. One particularly glaring concern is that the authors really do not know the extent to which the prolyl hydroxylase activity is (or is not) impacted by the H487A mutation in egl-9(rae276). If there is a fair amount of enzymatic activity left in this mutant, then it complicates interpretation. The paper would be strengthened if the authors could show that the egl-9(rae276) eliminates most if not all prolyl hydroxylase activity. In addition, the authors may want to consider doing RNAi for egl-9 in the egl-9(rae276) mutant as a control, as this would support the claim that whatever non-hydroxylase activity EGL-9 may have is indeed the causative agent for the elevation of CDO-1::GFP. Without such experiments, readers are left with the nagging concern that this allele is simply a hypomorph for the single biochemical activity of EGL-9 (i.e., the prolyl hydroxylase activity) rather than the more interesting, hypothesized scenario that EGL-9 has multiple biochemical activities, only one of which is the prolyl hydroxylase activity.

2. The authors observed that EGL-9 can inhibit HIF-1 and the expression of the HIF-1 target cdo-1 through a combination of activities that are (1) dependent on its prolyl hydroxylase activity (and subsequent VHL-1 activity that acts on the resulting hydroxylated prolines on HIF-1), and (2) independent of that activity. This is not a novel finding, as the authors themselves carefully note in their Discussion section, as this odd phenomenon has been observed for many HIF-1 target genes in multiple publications. While this manuscript adds to the description of this phenomenon, it does not really probe the underlying mechanism or shed light on how EGL-9 has these dual activities. This limits the overall impact and novelty of the paper.

3. Cysteine dioxygenases like CDO-1 operate in an oxygen-dependent manner to generate sulfites from cysteine. CDO-1 activity is dependent upon availability of molecular oxygen; this is an unexpected characteristic of a HIF-1 target, as its very activation is dependent on low molecular oxygen. Authors neither address this in the text nor experimentally, and it seems a glaring omission.

4. The authors determined that the hypodermis is the site of the most prominent CDO-1::GFP expression, relevant to Figure 4. This claim would be strengthened if a negative control tissue, in the animal with the knockin allele, were shown. The hypodermal specific expression is a highlight of this paper, so it would make this article even stronger if they could further substantiate this claim.

Minor issues to note:

Mutants for hif-1 and cysl-1 are sensitive to exogenous cysteine levels, yet loss of CDO-1 expression is not sufficient to explain this phenomenon, suggesting other targets of HIF-1 are involved. Given the findings the authors (and others) have had showing a role for RHY-1 in sulfur amino acid metabolism, shouldn't the authors consider testing rhy-1 mutants for sensitivity to exogenous cysteine?

The cysteine exposure assay was performed by incubating nematodes overnight in liquid M9 media containing OP50 culture. The liquid culture approach adds two complications: (1) the worms are arguably starving or at least undernourished compared to animals grown on NGM plates, and (2) the worms are probably mildly hypoxic in the liquid cultures, which complicates the interpretation.

An easily addressable concern is the wording of one of the main conclusions: that cdo-1 transcription is independent of the canonical prolyl hydroxylase function of EGL-9 and is instead dependent on one of EGL-9's non-canonical, non-characterized functions. There are several points in which the wording suggests that CDO-1 toxicity is independent of EGL-9. In their defense, the authors try to avoid this by saying, "EGL-9 PHD," to indicate that it is the prolyl hydroxylase function of EGL-9 that is not required for CDO-1 toxicity. However, this becomes confusing because much of the field uses PHD and EGL-9/EGLN as interchangeable protein names. The authors need to be clear about when they are describing the prolyl hydroxylase activity of EGL-9 rather than other (hypothesized) activities of EGL-9 that are independent of the prolyl hydroxylase activity.

The authors state in the text, "the egl-9; suox-1 double mutants are extremely sick and slow growing." We appreciate that their "health" assay, based on the exhaustion of food from the plate, is qualitative. We also appreciate that it is a functional measure of many factors that contribute to how fast a population of worms can grow, reproduce, and consume that lawn of food. However, unless they do a lifespan assay and/or measure developmental timing and specifically determine that the double mutant animals themselves are developing and/or growing more slowly, we do not think it is appropriate to use the words "slow growing" to describe the population. As they point out, the rate of consumption of food on the plate in their health assay is determined by a multitude and indeed a confluence of factors; the growth rate is one specific one that is commonly measured and has an established meaning.