Nuclear hormone receptor NHR-49 acts in parallel with HIF-1 to promote hypoxia adaptation in Caenorhabditis elegans
- Graduate Program in Medical Genetics, University of British Columbia, Canada
- British Columbia Children's Hospital Research Institute, Canada
- Centre for Molecular Medicine and Therapeutics, The University of British Columbia, Canada
- Department of Medical Genetics, University of British Columbia, Canada
- Department of Biochemistry, University of Washington School of Medicine, United States
- Department of Pediatrics, University of Pittsburgh School of Medicine, United States
- Departments of Developmental Biology and Cell Biology and Physiology, University of Pittsburgh School of Medicine, United States
Abstract
The response to insufficient oxygen (hypoxia) is orchestrated by the conserved hypoxia-inducible factor (HIF). However, HIF-independent hypoxia response pathways exist that act in parallel with HIF to mediate the physiological hypoxia response. Here, we describe a hypoxia response pathway controlled by Caenorhabditis elegans nuclear hormone receptor NHR-49, an orthologue of mammalian peroxisome proliferator-activated receptor alpha (PPARα). We show that nhr-49 is required for animal survival in hypoxia and is synthetic lethal with hif-1 in this context, demonstrating that these factors act in parallel. RNA-seq analysis shows that in hypoxia nhr-49 regulates a set of genes that are hif-1-independent, including autophagy genes that promote hypoxia survival. We further show that nuclear hormone receptor nhr-67 is a negative regulator and homeodomain-interacting protein kinase hpk-1 is a positive regulator of the NHR-49 pathway. Together, our experiments define a new, essential hypoxia response pathway that acts in parallel with the well-known HIF-mediated hypoxia response.
Editor's evaluation
The highly conserved protein hypoxia-inducible factor (HIF) is a well-known regulator of animal responses to low-oxygen environments. Using the sophisticated genetic tools of the nematode C. elegans, this paper identifies a parallel mechanism, governed by a different conserved transcription factor, that also provides protection from hypoxia. These findings provide important new insight into the complex genetic architecture of the mechanisms that maintain organismal homeostasis in the face of environmental stress.
https://doi.org/10.7554/eLife.67911.sa0Introduction
Organisms are continuously exposed to endogenous and exogenous stresses, from suboptimal temperatures to foreign substances. Thus, an organism’s ability to mount specific stress responses, including protecting healthy cells from harm or inducing apoptosis when damage to a cell cannot be overcome, is critical for survival. Hypoxia is a stress that occurs when cellular oxygen levels are too low for normal physiological functions. It occurs naturally in cells and tissues during development, as well as in many diseases (Lee et al., 2020; Powell-Coffman, 2010). For example, due to hyperproliferation, inadequate vascularization, and loss of matrix attachment, cancer cells grow in hostile microenvironments featuring hypoxia. Certain cancers thus hijack the hypoxia response to allow growth and metastasis in these harsh conditions (Rankin and Giaccia, 2016; Schito and Semenza, 2016; Zhang et al., 2019), and tumour hypoxia correlates with poor clinical outcome (Keith and Simon, 2007). Most prominently, mutations in the tumour suppressor von Hippel–Lindau (VHL), which inhibits the transcription factor hypoxia-inducible factor (HIF), occur in kidney cancers, and the resulting accumulation of HIF drives tumour growth (Kaelin Jr, 2008; Li and Kim, 2011). In line with a pivotal role of HIF in these cancers are studies showing promising effects of HIF inhibitors in preclinical (Albadari et al., 2019; Chen et al., 2016; Cho et al., 2016) and clinical studies (Fallah and Rini, 2019). However, a better understanding of the transcriptional hypoxia adaptation pathway is needed to pinpoint new drug targets and gain a deeper insight into how cells, tissues, and organisms cope with hypoxia.
The pathways that regulate the response to hypoxia are evolutionarily conserved from the nematode worm Caenorhabditis elegans to humans. As in mammals, a key pathway in C. elegans involves the transcription factor HIF-1, which is critical for the cellular responses to and the defence against hypoxia (Choudhry and Harris, 2018; Jiang et al., 2001). To survive hypoxia, animals activate the EGL-Nine homolog (EGLN)–VHL-HIF pathway (egl-9–vhl-1–hif-1 in C. elegans). In normoxic conditions (21% O2), HIF-1 is degraded and thus inactive. This occurs when EGL-9 adds a hydroxyl group onto a proline residue within HIF-1. The hydroxylated proline promotes binding of the E3 ubiquitin ligase VHL-1, leading to poly-ubiquitination and proteasomal degradation of HIF-1. However, in hypoxic conditions, EGL-9 is rendered inactive; hence, HIF-1 is stabilized and activates a hypoxia adaptation gene expression program (Epstein et al., 2001; Powell-Coffman, 2010).
Although the responses controlled by the HIF-1 master regulator are most studied, evidence for parallel transcriptional programs in hypoxia exists, from C. elegans to mammalian organisms. For example, the transcription factor B lymphocyte-induced maturation protein 1 (BLMP-1) has a hif-1-independent hypoxia regulatory role in C. elegans (Padmanabha et al., 2015), as does the conserved nuclear hormone receptor (NHR) oestrogen-related receptor (dERR) in Drosophila melanogaster (Li et al., 2013), and the cargo receptor sequestosome 1 (SQSTM1/p62) in mammals (Pursiheimo et al., 2009). Thus, despite the evolutionarily conserved and important role of the HIF family, robust and effective hypoxia adaptation requires an intricate network of transcription factors that act in concert. Compared to HIF, there is far less known about the mechanisms by which these pathways contribute to the hypoxia response.
C. elegans NHR-49 is a transcription factor orthologous to mammalian hepatocyte nuclear factor 4 (HNF4) and peroxisome proliferator-activated receptor α (PPARα) (Lee et al., 2016). Similar to these NHRs, it controls lipid metabolism by activating genes involved in fatty acid desaturation and mitochondrial β-oxidation (Pathare et al., 2012; Van Gilst et al., 2005a). By maintaining lipid homeostasis, NHR-49 is able to extend lifespan, a phenotype often associated with stress resistance (Burkewitz et al., 2015; Ratnappan et al., 2014). In addition to regulating lipid metabolism, NHR-49 also regulates putative xenobiotic detoxification genes in a dietary restriction-like state and during starvation (Chamoli et al., 2014; Goh et al., 2018), is required for resistance to oxidative stress (Goh et al., 2018), and activates innate immune response programs upon infection of C. elegans with Staphylococcus aureus (Wani et al., 2021), Pseudomonas aeruginosa (Naim et al., 2021), and Enterococcus faecalis (Dasgupta et al., 2020). Moreover, a recent report showed that nhr-49 is required to increase expression of the catechol-O-methyl-transferase comt-5 in hypoxia, acting downstream of the hypoxia-inhibited receptor tyrosine kinase hir-1 (Vozdek et al., 2018). However, the role of nhr-49 in hypoxia and how it intersects with hif-1 have not been explored.
The detoxification gene flavin mono-oxygenase 2 (fmo-2) is induced in many of the aforementioned stresses in an nhr-49-dependent manner (Dasgupta et al., 2020; Goh et al., 2018; Wani et al., 2021). Interestingly, fmo-2 is also a hif-1-dependent hypoxia response gene (Leiser et al., 2015; Shen et al., 2005), but its dependence on nhr-49 in hypoxia is not known. We hypothesized that nhr-49 may play a role in the worm hypoxia response, in part by regulating fmo-2 expression. Here, we show that nhr-49 is not only required to induce fmo-2, but controls a broad transcriptional response to hypoxia, including the induction of autophagy, a process required within the nhr-49 pathway for survival in hypoxia. Our epistasis experiments indicate that nhr-49 is functionally required independently of hif-1 in hypoxia. Finally, we identify the protein kinase homeodomain-interacting protein kinase 1 (hpk-1) as an upstream activator and the transcription factor nhr-67 as a repressor of the nhr-49 hypoxia response pathway. Together, our data define NHR-49 as a core player in a novel hypoxia response pathway that acts in parallel with HIF-1.
Results
NHR-49 is required to induce the expression of fmo-2 in hypoxia
C. elegans fmo-2 is induced by oxidative stress, starvation, and pathogen infection in an nhr-49-dependent fashion (Dasgupta et al., 2020; Goh et al., 2018; Wani et al., 2021). fmo-2 expression is also induced in a hif-1-dependent manner during hypoxia (0.1% O2; Leiser et al., 2015; Shen et al., 2005). To test whether nhr-49 regulates fmo-2 expression in hypoxia, we quantified fmo-2 mRNA levels in normoxia (21% O2) and hypoxia (0.5% O2) by quantitative reverse transcription PCR (qRT-PCR) in wild-type and mutant animals. The nr2041 allele deletes portions of both the DNA-binding domain and the ligand-binding domain of nhr-49 and is a predicted molecular null allele (Van Gilst et al., 2005b). The ia4 allele deletes exons 2–4 of hif-1 and is also a predicted null allele (Jiang et al., 2001). In wild-type animals, fmo-2 transcript levels increased approximately 40-fold in hypoxia, but this induction was blocked in both nhr-49(nr2041) and hif-1(ia4) mutant animals (Figure 1A). Experiments using a transgenic strain expressing a transcriptional fmo-2p::gfp reporter (Goh et al., 2018) corroborated these observations in vivo. In normoxia, this reporter is weakly expressed in some neurons and in the intestine of transgenic animals, but expression was significantly elevated in the intestine of transgenic animals in hypoxia (Figure 1B and C). High pharyngeal expression made it difficult to quantify neuronal fmo-2p::gfp in hypoxia. Consistent with our qRT-PCR data, loss of nhr-49 abrogated the increase in intestinal upregulation of fmo-2p::gfp animals following hypoxia exposure. We conclude that nhr-49 is required to induce fmo-2 in hypoxia.
Figure 1
nhr-49 regulates fmo-2 induction following exposure to hypoxia.
(A) The graph indicates fold changes of mRNA levels (relative to unexposed wild-type) in L4 wild-type, nhr-49(nr2041), and hif-1(ia4) animals exposed to room air (21% O2) or 0.5% O2 for 3 hr (n = 5). **p<0.01 (two-way ANOVA corrected for multiple comparisons using the Tukey method). (B) Representative micrographs show fmo-2p::gfp and fmo-2p::gfp;nhr-49(nr2041) adult animals in room air or following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2. (C) The graph shows the quantification of intestinal GFP levels in fmo-2p::gfp and fmo-2p::gfp;nhr-49(nr2041) animals following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2 (three repeats totalling >30 individual animals per genotype). **p<0.01, ****p<0.0001 (two-way ANOVA corrected for multiple comparisons using the Tukey method). WT: wild-type. See Source data 1 for (A) and (C).
nhr-49 is required throughout the C. elegans life cycle to promote hypoxia resistance in parallel with hif-1
Wild-type C. elegans embryos can survive a 24 hr exposure to environments with as little as 0.5% O2, dependent on the presence of hif-1 (Jiang et al., 2001; Nystul and Roth, 2004). We wanted to determine if nhr-49, like hif-1, is functionally required for animal survival during hypoxia. We first assessed the ability of embryos to survive for 24 hr in 0.5% O2 and then recover to the L4 or later stage when placed back in normoxia for 65 hr. We found that 86% of wild-type embryos reached at least the L4 stage, while only 25% of nhr-49 and hif-1 null mutant animals reached at least the L4 stage by that time (Figure 2A). The sensitivity of nhr-49 null mutant animals to hypoxia is specific to the loss of nhr-49, as transgenic re-expression of NHR-49 from its endogenous promoter rescues this phenotype (see below). This shows that, like hif-1, nhr-49 is required for embryo survival in hypoxia.
Figure 2 with 1 supplement see all
nhr-49 and hif-1 act in parallel hypoxia response pathways at two stages of the worm life cycle.
(A) The graph shows the average population survival of wild-type, nhr-49(nr2041), hif-1(ia4), and nhr-49(nr2041);hif-1(ia4) worm embryos exposed for 24 hr to 0.5% O2 and then allowed to recover at 21% O2 for 65 hr, counted as the ability to reach at least the L4 stage (five repeats totalling >100 individual animals per genotype). ****p<0.0001 vs. wild-type animals, ⊥p<0.05 vs. nhr-49(nr2041);hif-1(ia4) (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). (B) The graph shows the average developmental success of wild-type, nhr-49(nr2041), hif-1(ia4), and nhr-49(nr2041);hif-1(ia4) larval worms following 48 hr exposure to 0.5% O2 from L1 stage (four repeats totalling >60 individual animals per genotype). ***p<0.001, ****p<0.0001 percent L4 or older vs. wild-type animals (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). (C) The graph shows the average population survival of wild-type, nhr-49(nr2041), and hif-1(ia4) L4 animals following 24 hr exposure to 50 ppm hydrogen sulfide (three repeats totalling 60 individual animals per strain). ****p<0.0001 vs. wild-type animals (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). (D) High-magnification images show nhr-49p::nhr-49::gfp adult worms in wild-type, hif-1(ia4), and hpk-1(pk1393) backgrounds exposed to room air or following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2. Expression is seen in the head, intestine, and hypodermal seam cells (additional repeats in Figure 2—figure supplement 1E and F) (E) The graph shows the average population survival of nhr-49p::nhr49::gfp and nhr-49p::nhr49::gfp;hif-1(ia4) animals and their respective non-GFP sibling embryos exposed for 24 hr to 0.5% O2 and then allowed to recover at 21% O2 for 65 hr, counted as the ability to reach at least the L4 stage (four repeats totalling >100 individual animals per genotype). *p<0.05 (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). YA : young adult; n.s.: not significant; WT: wild-type. See Source data 1 for (A–C, E).
Next, we asked whether nhr-49 acts in the hif-1 hypoxia response pathway or in a separate, parallel response pathway. To address this question, we generated an nhr-49(nr2041);hif-1(ia4) double null mutant. We observed that less than 2% of nhr-49;hif-1 double null mutants reached at least the L4 stage following hypoxia exposure (Figure 2A). This suggests that nhr-49 and hif-1 act in separate, genetically parallel hypoxia response pathways.
To determine if nhr-49 and hif-1 are required for larval development in hypoxia, we exposed newly hatched, first stage (L1) larvae to hypoxia for 48 hr. Following this treatment, 95% of wild-type animals reached at least the L4 stage (Figure 2B). In contrast, only 19% of nhr-49 and only 20% of hif-1 mutant animals, respectively, reached at least the L4 stage, and no nhr-49;hif-1 double null mutant animals survived and developed to L4 (Figure 2B). Together, these results show that nhr-49 is required for worm adaptation to hypoxia in a pathway parallel to that of hif-1 both during embryogenesis and post-embryonically.
In normal conditions, nhr-49 null animals have a shortened lifespan (Van Gilst et al., 2005b). This raised the concern that the defects observed in hypoxia may be an indirect consequence of NHR-49’s normal developmental roles. To test whether the effects observed above were due to a specific requirement for nhr-49 in the hypoxia response, we studied worm development in normoxia. We found that loss of nhr-49 did not affect animal survival from the embryo to at least the L4 stage at 21% O2 (Figure 2—figure supplement 1A, Supplementary file 1). Additionally, although nhr-49 null mutants develop slower than wild-type animals at 21% O2, the majority of animals (88%) develop to at least the L4 stage after 48 hr, which is a significantly higher portion than develop to at least the L4 stage in 0.5% O2 (19%; Figure 2—figure supplement 1B, Supplementary file 2). Together, these data show that although nhr-49 null mutants display mild developmental defects in normoxia, the phenotypes observed are due to the requirement for nhr-49 specifically during hypoxia.
nhr-49 is dispensable for survival in hydrogen sulfide
To assess whether nhr-49 is involved in other responses requiring hif-1, we next asked if it was required for adaptation to hydrogen sulfide (H2S). H2S is produced endogenously and is an important signalling molecule in animals, including in C. elegans (Li et al., 2011). However, exposure to high levels of hydrogen sulfide can be lethal. As in the hypoxia response, hif-1 is a master regulator of the transcriptional response to exogenous hydrogen sulfide, and hif-1 is required for worm survival in 50 ppm H2S (Budde and Roth, 2010; Miller et al., 2011). In contrast, we found that nhr-49 null mutants survive exposure to 50 ppm H2S as well as wild-type control animals (Figure 2C). This suggests that the requirement for nhr-49 is stress specific, and that nhr-49 does not participate in all hif-1-dependent stress responses. This is consistent with previous observations that the hif-1-dependent changes in gene expression in H2S are quite different than those seen in hypoxia (Miller et al., 2011). Additionally, the ability of nhr-49 mutants to readily adapt to H2S provides further evidence that the mild developmental defects of nhr-49 null mutants do not render the animal sensitive to all stresses. Instead, our data indicate that nhr-49’s requirement for hypoxia survival is due to a specific function for this regulator in this particular stress condition.
NHR-49 overexpression compensates for the loss of hif-1 in hypoxia survival
Next, we asked whether the hif-1 and nhr-49 pathways crosstalk in hypoxia. First, we studied NHR-49 levels in hypoxia using the nhr-49p::nhr-49::gfp translational reporter, which expresses a GFP-tagged, full-length NHR-49 fusion protein from its own promoter from an extra-chromosomal array (henceforth referred to as NHR-49::GFP; Ratnappan et al., 2014). Interestingly, we observed an induction of NHR-49::GFP signal in animals exposed to hypoxia (Figure 2D; see also below). Next, we crossed the NHR-49::GFP transgene into the hif-1 mutant background; in the resulting strain, the NHR-49::GFP induction resembled that seen in the wild-type background (Figure 2D, Figure 2—figure supplement 1C and D). Higher-magnification images showed that the NHR-49 induction in hypoxia was similar in the head, intestine, and hypodermal seam cells in the hif-1 null background (Figure 2D, Figure 2—figure supplement 1E and F). In sum, loss of hif-1 does not appear to induce NHR-49 protein levels.
To further explore nhr-49 and hif-1 crosstalk, we tested if NHR-49 was able to rescue the hypoxia survival defects of the hif-1 null mutant. Although NHR-49 overexpression did not protect wild-type embryos from hypoxia (wild-type vs. non-GFP siblings), NHR-49 overexpression restored hif-1 null embryo survival to the level seen in wild-type animals (Figure 2E). Thus, overexpression of NHR-49 compensates for the loss of hif-1, further suggesting that these two transcription factors act in parallel pathways.
The nhr-49-dependent transcriptional response to hypoxia includes hif-1-independent genes
To delineate the genes and biological processes regulated by NHR-49 in hypoxia, we analysed whole-animal transcriptomes of wild-type, nhr-49, and hif-1 mutant animals before and after a 3 hr exposure to hypoxia (0.5% O2) using RNA-sequencing (RNA-seq; Figure 3A and B, Figure 3—figure supplement 1A). Consistent with published microarray data (Shen et al., 2005), we found that hypoxia in wild-type animals upregulated more genes (718) than it downregulated (339); collectively, we refer to these as hypoxia-responsive genes (1,057) (Figure 3A; false discovery rate [FDR] < 0.05, fold regulation ≥2). Despite different experimental setups (harvest stages, oxygen percentage, gene expression profiling technique), we found a significant overlap in hypoxia-induced genes when comparing our data to the data from Shen et al. (Figure 3—figure supplement 1B). Our data also identified several experimentally confirmed hypoxia-inducible genes, such as egl-9, phy-2, nhr-57, F22B5.4, and fmo-2 (Bishop et al., 2004; Shen et al., 2005), validating our approach (Figure 3—figure supplement 1B and C).
Figure 3 with 2 supplements see all
RNA-seq reveals an nhr-49-dependent transcriptional program in hypoxia.
(A, B) Venn diagrams show the overlap of genes regulated by hypoxia (3 hr 0.5% O2; vs. normoxia 21% O2) in wild-type, nhr-49(nr2041), and hif-1(ia4) animals. Numbers indicate the number of differentially (FDR < 0.05, |logFC| ≥ 1) expressed genes in wild-type (grey), nhr-49 (red), and/or hif-1 (blue) animals in hypoxia, with hypoxia-upregulated genes in (A) and hypoxia-downregulated genes in (B). Genes upregulated by hypoxia in wild-type include 83 + 264 + 139 + 232 = 718 total genes. Of these 718 genes, 315 genes are not induced in nhr-49 animals, i.e., they require nhr-49 for induction; these are composed of 83 genes induced in both wild-type and hif-1 animals, but not in nhr-49 animals (grey-blue overlap; these depend only on nhr-49 but not on hif-1; highlighted by black box), and 232 genes induced only in wild-type animals but not in hif-1 or nhr-49 animals (grey; i.e., these are co-dependent on nhr-49 and hif-1). Genes downregulated by hypoxia include 177 genes that require nhr-49 for repression, composed of 51 genes downregulated in wild-type and hif-1 animals, but not in nhr-49 animals (grey-blue overlap; these depend on nhr-49 only); and 126 genes downregulated only in wild-type animals but not in hif-1 or nhr-49 animals (grey; i.e., these are co-dependent on nhr-49 and hif-1). (C) Heatmap of the expression levels of the 83 genes, which are significantly induced over twofold in 21% O2 vs. 0.5% O2 in wild-type and hif-1(ia4) animals, but not in nhr-49(nr2041), i.e., nhr-49-dependent hypoxia response genes. Genes along the y-axis are coloured in each repeat based on their z-scores of the log2-transformed counts per million (CPM) plus 1. Notable genes are highlighted. (D) Network view of the enriched functional categories among the 83 genes, which are significantly induced over twofold in 21% O2 vs. 0.5% O2 in wild-type and hif-1(ia4) animals, but not in nhr-49(nr2041). Edges represent significant gene overlap as defined by a Jaccard coefficient larger than or equal to 25%. The dot size reflects the number of genes in each functional category; colour intensity reflects statistical significance (−log10 p-value). (E) The graph shows the average population survival of wild-type, nhr-49(nr2041), fmo-2(ok2147), acs-2(ok2457), and fmo-2(ok2147);acs-2(ok2457) embryos following 24 hr exposure to 0.5% O2, then allowed to recover at 21% O2 for 65 hr, and counted as the ability to reach at least L4 stage (five or more repeats totalling >100 individual animals per strain). **p<0.01, ****p<0.0001 vs. wild-type animals. Comparison of single mutants to fmo-2(ok2147);acs-2(ok2457) not significant (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). n.s.: not significant; WT: wild-type; FDR: false discovery rate; |logFC|: log2-transformed fold change. See Source data 1 for (E).
Next, we performed functional enrichment profiling to elucidate the biological pathways and processes governed by hypoxia-responsive genes. In wild-type animals, hypoxia-induced genes function mainly in pathways such as detoxification, response to heavy metal stress, and autophagy, whereas hypoxia-repressed genes play roles in processes such as amino acid transport (Figure 3—figure supplement 1D). Interestingly, a set of genes involved in amino acid metabolism was induced while another set was repressed by hypoxia, whereas genes involved in insulin-related metabolism were exclusively repressed (Figure 3—figure supplement 1E).
Then, we performed intersection analysis to identify genes that require nhr-49 and/or hif-1 to respond to hypoxia (Figure 3A and B). We found that 315 upregulated genes (of 718 upregulated in wild type) failed to be upregulated and 177 downregulated genes (of 339 downregulated in wild type) failed to be downregulated in nhr-49 mutants (Figure 3A and B); collectively we call these nhr-49-dependent genes. Of these nhr-49-dependent genes, 83 of the upregulated and 51 of the downregulated genes were hif-1-independent (Figure 3A and B). In line with our above data, fmo-2 was induced in an nhr-49-dependent manner (Figure 3C). However, although our qRT-PCR data (Figure 1A) show that fmo-2 induction is dependent on hif-1, our RNA-seq analysis excluded fmo-2 from the hif-1-dependent set because it retained more than twofold induction in hypoxia vs. normoxia (Figure 3—figure supplement 1C). This suggests that although fmo-2 induction is somewhat dependent on hif-1, it requires nhr-49. Thus, although many hypoxia-responsive genes are controlled by both transcription factors, a subset is nhr-49-dependent but hif-1-independent.
Next, we functionally profiled the 83 genes that exclusively require nhr-49 but not hif-1 for induction in hypoxia using functional enrichment analysis (Figure 3C, Supplementary file 3a). We found that autophagy and detoxification genes were significantly enriched (Figure 3D), suggesting a requirement for nhr-49 to regulate these processes in hypoxia. Interestingly, a separate set of detoxification genes was dependent only on hif-1 (Figure 3—figure supplement 1F and G, Supplementary file 3b), and a third set of detoxification genes was independent of both nhr-49 and hif-1 (Figure 3—figure supplement 1H and I, Supplementary file 3c). This suggests that there may be an additional transcription factor(s) regulating this process in hypoxia.
Our RNA-seq data revealed that the acyl-CoA synthetase gene acs-2 is induced in response to hypoxia in an nhr-49-dependent manner (Figure 3C, Supplementary file 3a). ACS-2 acts in the first step of mitochondrial fatty acid β-oxidation and is strongly induced by NHR-49 during starvation and following exposure to E. faecalis (Dasgupta et al., 2020; Van Gilst et al., 2005a). To validate our RNA-seq data, we quantified acs-2 expression via qRT-PCR. Following hypoxia exposure, acs-2 transcript levels increased approximately 12-fold, and this induction was blocked in the nhr-49 null mutant, but not the hif-1 null mutant (Figure 3—figure supplement 2A). We used a transgenic strain expressing a transcriptional acs-2p::gfp reporter to study this regulation in vivo (Burkewitz et al., 2015). This reporter showed moderate GFP expression in the body of animals under normoxia, but expression increased substantially in the intestine following exposure to hypoxia (Figure 3—figure supplement 2B and C). Consistent with our RNA-seq and qRT-PCR data, loss of nhr-49 blocked transcriptional activation via the acs-2 promoter as GFP was weaker in the intestines of these animals following hypoxia exposure (Figure 3—figure supplement 2B and C). Collectively, these data provide in vivo evidence that nhr-49 is specifically required, and that hif-1 is dispensable, for induction of acs-2 in hypoxia.
Autophagy genes are critical downstream targets of nhr-49 in hypoxia
Next, we wanted to determine which of nhr-49’s downstream transcriptional targets are functionally important for animal survival in hypoxia. We first assessed the ability of fmo-2(ok2147) and acs-2(ok2457) embryos to survive hypoxia as both genes are strongly induced by hypoxia in an nhr-49-dependent manner. Individually, loss of either fmo-2 (60% of embryos develop to at least the L4 stage) or acs-2 (65%) did not significantly decrease embryo viability compared to wild type (79%) (Figure 3E). However, simultaneous loss of both fmo-2 and acs-2 resulted in a significant decrease in survival after hypoxia (47%). None of the mutant animals showed embryo viability defects in normoxia, indicating that the phenotypes observed were specifically due to the requirement of these genes in hypoxia survival (Figure 4—figure supplement 1A, Supplementary file 1). These data suggest that fmo-2 and acs-2 each contribute only modestly to worm survival in hypoxia and are likely not the main factors contributing to nhr-49’s importance in survival to this stress. This resembles previous observations that mutations that disrupt individual hif-1-responsive genes show only minor defects in hypoxia survival (Shen et al., 2005).
Our RNA-seq analysis revealed autophagy as a major biological process modulated by nhr-49 (Figure 3D). Notably, C. elegans show sensitivity to anoxia when the autophagy pathway is disrupted (Samokhvalov et al., 2008), and autophagy is upregulated in anoxia (Chapin et al., 2015). However, the responses to anoxia and hypoxia are mediated by different regulatory pathways (Nystul and Roth, 2004), and it thus was not a priori clear whether autophagy is also required for hypoxia resistance. First, to validate our RNA-seq results, we examined the expression of three autophagy genes with transcriptional (promoter::gfp) reporters. Hypoxia significantly induced GFP fluorescence in worms bearing lgg-1p::gfp, atg-2p::gfp, or epg-3p::gfp reporters (Figure 4A and B, Figure 4—figure supplement 1B and C). Consistent with our RNA-seq results, nhr-49 was required for these inductions, whereas hif-1 was not.
Figure 4 with 1 supplement see all
nhr-49 is required to induce autophagy in response to hypoxia.
(A, B) The figure shows representative micrographs (A) and whole-worm GFP quantification (B) of lgg-1p::gfp adult animals fed EV, nhr-49, hif-1, hpk-1, or nhr-67 RNAi in room air or following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2 (three repeats totalling >30 individual animals per genotype). *p<0.05, ****p<0.0001 (two-way ANOVA corrected for multiple comparisons using the Tukey method). (C, D) The figure shows representative micrographs (C) and quantification (D) of LGG-1::GFP foci in individual hypodermal seam cells in L3 animals in the wild-type, nhr-49(nr2041), and hif-1(ia4) backgrounds, kept in room air or exposed to 5 hr 0.5% O2 (three repeats totalling >110 individual seam cells in at least 15 individual animals per genotype). Micrograph brightness and contrast are matched within genotype, and unmatched between genotypes. ****p<0.0001 (two-way ANOVA corrected for multiple comparisons using the Tukey method). (E, F) The graphs show average population survival of wild-type, nhr-49(nr2041), (E) lgg-2(tm5755) and nhr-49(nr2041);lgg-2(tm5755), and (F) epg-6(tm8366) and nhr-49(nr2041);epg-6(tm8366) animal embryos exposed for 24 hr to 0.5% O2 and then allowed to recover at 21% O2 for 65 hr, counted as the ability to reach at least the L4 stage (three repeats totalling >100 individual animals per genotype). **p<0.01, ***p<0.001, **** p<0.0001 vs. wild-type animals (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). (G) Quantification of LGG-1::GFP foci in individual hypodermal seam cells in L3 animals in second-generation wild-type animals fed EV, hpk-1, or nhr-67 RNAi, kept in room air or exposed to 5 hr 0.5% O2 (three repeats totalling >110 individual seam cells in at least 15 individual animals per genotype). ****p<0.0001 (two-way ANOVA corrected for multiple comparisons using the Tukey method). n.s.: not significant; WT: wild-type. See Source data 1 for (B, D, E– G).
To test whether nhr-49 is required for autophagosome formation in hypoxia, we studied the widely used LGG-1::GFP reporter (Das et al., 2017; Palmisano and Meléndez, 2016; Samokhvalov et al., 2008; Zhang et al., 2015), wherein GFP is tagged to the C-terminus of the autophagosome assembly factor LGG-1 (Atg8 in mammals, LC3 in yeast). In wild-type animals, a 5 hr exposure to hypoxia significantly increased the number of LGG-1::GFP foci in seam cells compared to normoxia exposure (Figure 4C and D). Critically, loss of nhr-49 abrogated the increase in LGG-1::GFP foci following hypoxia exposure, whereas loss of hif-1 did not. This shows that autophagosome formation in hypoxia is dependent on nhr-49, but independent of hif-1.
To determine if upregulation of autophagy by nhr-49 is required for worm survival in hypoxia, we assessed the ability of lgg-2(tm5755) and epg-6(tm8366) mutant embryos to survive hypoxia. Similar to nhr-49 mutant animals, only 41% of lgg-2(tm5755) and 44% of epg-6(tm8366) mutant embryos developed to L4 following exposure to hypoxia (Figure 4E and F). Next, we used epistasis analysis to test whether genes involved in autophagy act in the nhr-49 pathway to promote worm survival in hypoxia. We observed that nhr-49;lgg-1 (38%) and nhr-49;epg-6 (44%) double mutants showed similar survival as does each single null mutant, suggesting that these autophagy genes act in the same pathway as nhr-49 (Figure 4E and F). Each mutant showed normal development from embryo to L4 in normoxia (Figure 4—figure supplement 1D and E, Supplementary file 1), indicating that the phenotypes observed were specifically due to the requirement of these genes in hypoxia survival. To corroborate these results, we also depleted several autophagy genes using feeding RNA interference (RNAi) in the wild-type and nhr-49 null mutant backgrounds and assessed the ability of these embryos to survive hypoxia. RNAi-mediated knockdown of the autophagy genes atg-10 (28%), atg-7 (41%), bec-1 (27%), and epg-3 (38%) caused significant sensitivity to hypoxia in the wild-type background compared to the empty vector (EV) control RNAi treatment (79%; Figure 4—figure supplement 1F). Importantly, the sensitivity of animals did not significantly change when these genes were knocked down in the nhr-49 null mutant background (32, 25, 13, and 13%, respectively, vs. nhr-49(null);EV(RNAi) 21%), suggesting that these genes act in the same pathway as nhr-49. Depletion of these genes by RNAi alone did not cause impaired development from embryo to L4 in normoxia, indicating that the phenotypes observed were specifically due to the requirement of these genes in hypoxia survival (Figure 4—figure supplement 1G, Supplementary file 1). Together, these data show that autophagy is a functionally important nhr-49-regulated process required for worm survival in hypoxia.
NHR-49 expression in multiple tissues is sufficient to promote hypoxia survival
To test if nhr-49 activation is sufficient to promote survival of C. elegans in hypoxia, we studied the nhr-49(et13) gain-of-function strain, which is sufficient to induce fmo-2 (Goh et al., 2018; Lee et al., 2016). After 24 hr of exposure to hypoxia, 86% of wild-type eggs develop to at least L4 stage (Figure 2A), but after 48 hr of hypoxia exposure, only 44% develop to at least L4 stage (Figure 5A). In contrast, 75% of nhr-49(et13) gain-of-function eggs develop to at least L4 stage after 48 hr of hypoxia exposure, indicating that NHR-49 activation is sufficient to improve the population survival of worms in hypoxia.
Figure 5
nhr-49 is sufficient to promote survival in hypoxia and induce some hypoxia response genes.
(A) The graph shows the average population survival of wild-type, nhr-49(nr2041), and nhr-49(et13) worm embryos following 48 hr exposure to 0.5% O2, then allowed to recover at 21% O2 for 42 hr, and counted as the ability to reach at least L4 stage (five repeats totalling >100 individual animals per strain). *p<0.05 vs. wild-type animals, ⊥⊥⊥p<0.001 vs. nhr-49(et13) animals (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). (B) The graph shows the average population survival of nhr-49 tissue-specific rescue worm embryos following 24 hr exposure to 0.5% O2, then allowed to recover at 21% O2 for 65 hr, and counted as the ability to reach at least L4 stage. glp-19p::nhr-49::gfp for intestine, col-12p::nhr-49::gfp for hypodermis, rgef-1p::nhr-49::gfp for neurons, myo-3p::nhr-49::gfp for body wall muscle, and nhr-49p::nhr-49::gfp for endogenous (four or more repeats totalling >50 individual animals per strain). *p<0.05 vs. matching non-GFP siblings. (C) The graph shows fold changes of mRNA levels (relative to wild type) in L4 wild-type and nhr-49(et13) animals (n = 3). *p<0.05, ***p<0.001 vs. wild-type animals (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). WT: wild-type. See Source data 1 for (A–C).
NHR-49 is expressed in multiple tissues, including the intestine, neurons, muscle, and hypodermis (Ratnappan et al., 2014). Neuronal NHR-49 is sufficient to extend lifespan in some contexts and regulates genes in distal tissues (Burkewitz et al., 2015), but where the protein acts to regulate the response to hypoxia is unknown. As described above, NHR-49::GFP imaging indicated that NHR-49 protein levels are induced in the intestine, neurons, and hypodermis during hypoxia (Figure 2D, Figure 2—figure supplement 1E and F). Hence, we asked if the expression of NHR-49 in any one of these tissues could rescue the hypoxia survival defects of the nhr-49 mutant (Naim et al., 2021). Comparing the survival of each tissue-specific NHR-49::GFP rescue strain to their respective non-GFP siblings, we found that expressing nhr-49 in the intestine, neurons, hypodermis, body wall muscle, or from its endogenous promoter was sufficient to restore population survival to wild-type levels (Figure 5B). Taken together, these data suggest that NHR-49 can act in multiple somatic tissues, perhaps cell non-autonomously, to regulate the organismal hypoxia response.
To determine if NHR-49 activity alone is sufficient to induce expression of hypoxia response genes, we tested if the nhr-49(et13) gain-of-function mutant strain showed upregulation of nhr-49-dependent hypoxia response genes identified in our RNA-seq analysis in the absence of stress (Figure 5C). In line with previous findings (Goh et al., 2018; Lee et al., 2016), nhr-49 was sufficient to induce fmo-2 and acs-2 expression on its own. However, other hypoxia-inducible nhr-49 regulated genes involved in autophagy and detoxification (Supplementary file 3a) were not induced in the nhr-49(et13) gain-of-function mutant. It is possible that nhr-49 regulates autophagy indirectly, or that the et13 mutation, which has combined gain- and loss-of-function properties (Lee et al., 2016), cannot induce these tested autophagy genes. It is also possible that, to induce these genes, NHR-49 acts in concert with another hypoxia-responsive transcription factor or requires binding of a hypoxia-associated signalling molecule or post-translational modification by a hypoxia-regulated factor, which is not activated in the nhr-49(et13) mutant. Together, this shows that NHR-49 is sufficient to extend the survival of worms in hypoxia in various tissues, but the gain-of-function strain is only able to induce certain response genes without the presence of stress.
The nuclear hormone receptor NHR-67 negatively regulates the nhr-49 hypoxia response
Cellular stress response pathways are intricate networks involving a multitude of proteins. Activation or repression of downstream response genes thus often requires signalling via additional factors such as kinases and transcription factors. To identify factors acting in the nhr-49-regulated hypoxia response pathway, we focused on proteins that have previously been reported to physically interact with NHR-49 (Reece-Hoyes et al., 2013); such proteins might be regulators of NHR-49. One potential NHR-49-binding protein is NHR-67, the sole C. elegans orthologue of the D. melanogaster tailless and vertebrate NR2E1 proteins (Gissendanner et al., 2004). NHR-67 is important in neural and uterine development (Fernandes and Sternberg, 2007; Verghese et al., 2011), but a role for this NHR in stress responses has not yet been described. Our RNA-seq data showed that nhr-67 mRNA expression is modestly increased during hypoxia in wild-type animals and much more substantially induced in the nhr-49 null background (Figure 6A), suggesting a possible regulatory interaction between these two NHRs in hypoxia. To explore this interaction further, we used feeding RNAi to knock down nhr-67 in normoxia and hypoxia, and observed how this affected the expression of the fmo-2p::gfp and acs-2p::gfp transcriptional reporters. Compared to EV(RNAi), knockdown of nhr-67 significantly induced both reporters even in the absence of stress, suggesting a repressive role for nhr-67 on these genes (Figure 6B–E). In hypoxia, nhr-67(RNAi) resulted in even higher expression of these reporters. In both normoxia and hypoxia, increased expression of the reporters was dependent on nhr-49 as loss of nhr-49 abrogated the GFP induction (Figure 6B–E). The nhr-49(et13) gain-of-function mutation is sufficient to induce expression of the fmo-2p::gfp reporter in non-stressed conditions (Goh et al., 2018), although it does not alter nhr-67 expression under normoxic conditions (Figure 6—figure supplement 1A). Knockdown of nhr-67 further increased the expression of the fmo-2p::gfp reporter in the nhr-49(et13) background in both normoxia and hypoxia (Figure 6—figure supplement 1B and C). Together, these data suggest that nhr-67 negatively regulates the expression of the hypoxia response genes fmo-2 and acs-2 in both normoxic and hypoxic conditions, and that this regulation is dependent on nhr-49.
Figure 6 with 1 supplement see all
nhr-67 is a negative regulator of the nhr-49-dependent hypoxia response pathway.
(A) The graph shows the average transcript levels in counts per million (CPM) of nhr-67 mRNA in L4 wild-type, nhr-49(nr2041), and hif-1(ia4) animals exposed to 0.5% O2 for 3 hr or kept at 21% O2 (n = 3). **p <0.01 (two-way ANOVA corrected for multiple comparisons using the Tukey method). (B–E) Representative micrographs and quantification of intestinal GFP levels in fmo-2p::gfp and fmo-2p::gfp;nhr-49(nr2041) (B, C) and acs-2p::gfp and acs-2p::gfp;nhr-49(nr2041) (D, E) adult animals fed EV RNAi or nhr-67 RNAi following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2 (three repeats totalling >30 individual animals per strain). *p<0.05, ***p<0.001, ****p<0.0001 (two-way ANOVA corrected for multiple comparisons using the Tukey method). (F) Representative micrographs show nhr-49p::nhr-49::gfp adult animals fed EV, nhr-49, or nhr-67 RNAi following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2. (G) The graph shows quantification of whole-worm GFP levels in nhr-49p::nhr-49::gfp animals fed EV, nhr-49, or nhr-67 RNAi following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2 (three or more repeats totalling >30 individual animals per strain). ****p<0.0001 (two-way ANOVA corrected for multiple comparisons using the Tukey method). n.s.: not significant; WT: wild type. See Source data 1 for (A, C, E, G).
Above, we showed that autophagy induction in hypoxia is nhr-49 dependent. To test whether nhr-67 regulates autophagy genes in hypoxia, we examined the activity of the lgg-1p::gfp, atg-2p::gfp, and epg-3p::gfp reporters after nhr-67 knockdown. Compared to the EV(RNAi) control, nhr-67 knockdown unexpectedly blocked the induction of epg-3p::gfp by hypoxia (Figure 4—figure supplement 1C), but did not alter lgg-1p::gfp or atg-2p::gfp induction by hypoxia (Figure 4A and B, Figure 4—figure supplement 1B). Next, we assessed whether nhr-67 regulates autophagosome formation in hypoxia. Following a 5 hr exposure to hypoxia, the number of LGG-1::GFP foci increased significantly and similarly in both EV(RNAi) control and in nhr-67(RNAi) animals (Figure 4G). This suggests that, although nhr-67 is required for the induction of epg-3, its role in autophagy regulation in hypoxia is minor.
As a negative regulator of some nhr-49-dependent hypoxia response genes, it is possible that nhr-67 acts upstream of nhr-49 or directly on the promoter of acs-2 and fmo-2. To determine how nhr-67 regulates this response, we used feeding RNAi to knock down nhr-67 and observed expression of the NHR-49::GFP translational fusion protein. Whole-animal NHR-49::GFP expression was increased in both normoxia and hypoxia following knockdown of nhr-67, with the highest increase observed in the intestine (Figure 6F and G). This suggests that nhr-67 negatively regulates NHR-49, but in hypoxia, an increase in NHR-49 protein levels may in turn repress nhr-67, suggesting a negative feedback loop. The effects seen on fmo-2 and acs-2 expression are likely a consequence of NHR-67’s effect on NHR-49.
Loss-of-function mutations in nhr-67 cause early L1 lethality or arrest (Fernandes and Sternberg, 2007), so we used feeding RNAi to study nhr-67’s functional requirements in hypoxia. We assessed the ability of nhr-67(RNAi) embryos to survive hypoxia and recover, as described above. Only 58% of nhr-67 knockdown embryos survived to at least L4 stage compared to the EV(RNAi) animals (82%; Figure 6—figure supplement 1D). Next, we used epistasis analysis to test whether nhr-67 acts in the nhr-49 pathway. We observed that nhr-49(null);nhr-67(RNAi) animals showed similar survival (17%) as do nhr-49;EV(RNAi) animals (29%), suggesting that these two genes act in the same pathway. In contrast, hif-1(null);nhr-67(RNAi) animals showed significantly reduced survival (16%) compared to hif-1;EV(RNAi) animals (46%), consistent with the view that hif-1 and nhr-49 act in separate pathways (Figure 6—figure supplement 1D). The majority of nhr-67(RNAi) animals were able to reach at least L4 stage in normoxia (98%), resembling EV(RNAi) animals (94%; Figure 6—figure supplement 1E, Supplementary file 1). Thus, although nhr-67 appears to perform a negative regulatory role on the NHR-49-dependent hypoxia pathway, it, too, is functionally required for survival in hypoxia. Taken together, these data show that nhr-67 is a functionally important negative regulator of the nhr-49-dependent hypoxia response, although it does not equally control all NHR-49-regulated hypoxia response genes.
The kinase hpk-1 positively regulates nhr-49-dependent hypoxia response genes and is required for survival in hypoxia
Many stress response pathways involve upstream kinases that phosphorylate transcription factors, including PMK-1 and GSK-3, which phosphorylate the oxidative stress response regulator SKN-1 (Blackwell et al., 2015), and AKT-1/2, which phosphorylate DAF-16 in the insulin/IGF signalling pathway (Ogg and Ruvkun, 1998). To identify factors acting in the nhr-49-dependent hypoxia response pathway, we studied kinases that we found to potentially act in the nhr-49-dependent oxidative stress response (Doering & Taubert, manuscript in preparation). We depleted each kinase using feeding RNAi to determine if any treatment prevented fmo-2p::gfp induction in hypoxia in the worm intestine. As expected, nhr-49 RNAi diminished this intestinal fluorescence compared to the EV(RNAi) (Figure 7A and B). Of the kinases tested, RNAi knockdown of the nuclear serine/threonine kinase hpk-1 significantly decreased intestinal fmo-2p::gfp expression following hypoxia exposure (Figure 7A and B), phenocopying nhr-49 knockdown. Knockdown of hpk-1 also significantly reduced intestinal expression of the acs-2p::gfp reporter in hypoxia (Figure 7C and D) and reduced expression of fmo-2p::gfp in the nhr-49(et13) background in normoxia (Figure 7—figure supplement 1A and B). In comparison, hif-1 RNAi significantly decreased the expression of the fmo-2p::gfp reporter in hypoxia (Figure 7A–B) but did not alter it in the nhr-49(et13) background (Figure 7—figure supplement 1A, B), and actually increased expression of the acs-2p::gfp reporter in hypoxia (Figure 7C and D). We corroborated the hpk-1 data using qRT-PCR in wild-type animals and in a hpk-1(pk1393) mutant. The pk1393 allele deletes the majority of the kinase domain of hpk-1 and is a predicted molecular null allele (Raich et al., 2003). In hypoxia, the expression of both acs-2 and fmo-2 was significantly reduced by loss of hpk-1, phenocopying loss of nhr-49 (Figure 7E). Together, these data suggest that, like nhr-49, hpk-1 is required for upregulation of fmo-2 and acs-2 in response to hypoxia.
Figure 7 with 1 supplement see all
hpk-1 is a positive regulator within the nhr-49-dependent hypoxia response pathway.
(A–D) Representative micrographs and quantification of intestinal GFP levels in fmo-2p::gfp (A, B) and acs-2p::gfp (C, D) adult animals fed EV, nhr-49, hif-1, or hpk-1 RNAi following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2 (three or more repeats totalling >30 individual animals per strain). *p<0.05, **p<0.01, ****p<0.0001 vs. EV(RNAi) (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). (E) The graph shows fold changes of mRNA levels in L4 wild-type, nhr-49(nr2041), and hpk-1(pk1393) animals exposed to 0.5% O2 for 3 hr (n = 4). **p<0.01, ***p<0.001 (two-way ANOVA corrected for multiple comparisons using the Tukey method). (F) The graph shows the average population survival of wild-type, nhr-49(nr2041), hpk-1(pk1393), and nhr-49(nr2041);hpk-1(pk1393) embryos following 24 hr exposure to 0.5% O2, then allowed to recover at 21% O2 for 65 hr, and counted as the ability to reach at least L4 stage (four repeats totalling >100 individual animals per strain). ***p<0.001, ****p<0.0001 vs. wild-type animals. Comparison of single mutants to nhr-49(nr2041);hpk-1(pk1393) not significant (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). (G) The graph shows the average population survival of wild-type, hif-1(ia4), hpk-1(pk1393), and hif-1(ia4);hpk-1(pk1393) embryos following 24 hr exposure to 0.5% O2, then allowed to recover at 21% O2 for 65 hr, and counted as the ability to reach at least L4 stage (four repeats totalling >100 individual animals per strain). ****p<0.0001 vs. wild-type animals, ⊥⊥⊥p<0.001 vs. hif-1(ia4);hpk-1(pk1393) (ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). n.s.: not significant; WT: wild-type. See Source data 1 for (B, D, E–G).
hpk-1 regulates autophagy in response to dietary restriction in C. elegans (Das et al., 2017). To determine if hpk-1 is involved in the regulation of autophagy in response to hypoxia, like nhr-49, we examined the expression of the lgg-1p::gfp, atg-2p::gfp, and epg-3p::gfp transcriptional reporters. Similar to nhr-49, induction of all three autophagy genes in hypoxia required hpk-1 (Figure 4A and B, Figure 4—figure supplement 1B and C). Next, we assessed whether hpk-1 is necessary for autophagosome formation in hypoxia. Following a 5 hr exposure to hypoxia, the number of LGG-1::GFP foci was not changed compared to the hpk-1(RNAi) normoxia control (Figure 4G). This shows that, like nhr-49, hpk-1 is required for the induction of autophagy genes and autophagosome formation in hypoxia.
To determine if hpk-1 is functionally required for animal survival in hypoxia, we assessed the ability of hpk-1 mutant embryos to survive hypoxia. Similar to nhr-49 mutant animals, only 45% of hpk-1 mutant embryos developed to L4 (wild-type animals 92%; Figure 7F). We used epistasis analysis to test the hypothesis that hpk-1 acts in the nhr-49 pathway to coordinate a transcriptional response to hypoxia. We observed that the nhr-49;hpk-1 double null mutant showed similar survival (26%) to each of the single null mutants, suggesting that these two genes act in the same hypoxia response pathway (Figure 7F). In contrast, the hif-1;hpk-1 double null mutant was significantly impaired (<2%) compared to each of the single null mutants alone, consistent with the view that these two genes act in separate response pathways (Figure 7G). Each mutant showed normal development from embryo to L4 in normoxia, indicating that the phenotypes observed were specifically due to the requirement of these genes in hypoxia survival (Figure 7—figure supplement 1C and D, Supplementary file 1). Taken together, these experiments show that hpk-1 is required for embryo survival in hypoxia, consistent with it playing a role as an activator of the nhr-49-dependent response pathway.
NHR-49 is regulated post-transcriptionally in hypoxia in an hpk-1-dependent fashion
To test our hypothesis that HPK-1 activates NHR-49 in hypoxia, we examined whether NHR-49 is induced by hypoxia and whether hpk-1 is involved in this regulation. NHR-49 and HPK-1 protein levels are increased in response to tert-butyl hydroperoxide and/or heat shock, respectively, but mRNA levels remain unchanged (Das et al., 2017; Goh et al., 2018). Similarly, we observed that nhr-49 and hpk-1 mRNA levels were not increased upon exposure to hypoxia (Figure 8A). Consistent with this, a transcriptional reporter of the hpk-1 promoter fused to GFP (Das et al., 2017) was also not induced following hypoxia exposure (Figure 8—figure supplement 1A and B). These data show that the transcription of neither nhr-49 nor hpk-1 is induced in hypoxia.
Figure 8 with 1 supplement see all
NHR-49 is induced in hypoxia in an hpk-1-dependent fashion.
(A) The graph shows the average fold changes of mRNA levels (relative to unexposed wild type) in L4 wild-type animals exposed to 0.5% O2 for 3 hr (n = 3 or 4; ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method). (B) Representative micrographs show nhr-49p::nhr-49::gfp and nhr-49p::nhr-49::gfp;hpk-1(pk1393) adult animals following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2. (C) The graph shows the quantification of whole-worm GFP levels in nhr-49p::nhr-49::gfp and nhr-49p::nhr-49::gfp;hpk-1(pk1393) animals following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2 (three repeats totalling >30 individual animals per strain). ****p<0.0001 (two-way ANOVA corrected for multiple comparisons using the Tukey method). n.s.: not significant; WT: wild-type. See Source data 1 for (A, C).
We considered the possibility that NHR-49 may be regulated post-transcriptionally. To assess NHR-49 protein levels, we again used the translational NHR-49::GFP reporter to measure the expression of the fusion protein in response to hypoxia. As described above, the whole-worm NHR-49::GFP signal was modestly, but significantly, elevated upon exposure to hypoxia (Figure 8B and C). Interestingly, although hpk-1 null mutation had no effect on NHR-49::GFP levels in normoxia, it abrogated the upregulation of the NHR-49::GFP signal by hypoxia (Figure 8B and C). Higher magnification images showed that NHR-49 is upregulated in the head, intestine, and hypodermal seam cells during hypoxia, and loss of hpk-1 abrogated NHR-49 induction in all three tissues (Figure 2D, Figure 2—figure supplement 1E and F). This suggests that NHR-49 is regulated post-translationally in response to hypoxia, and that hpk-1 may be involved in this regulation. Taken together, these data show that hpk-1 is a functionally important upstream positive regulator of the nhr-49-dependent hypoxia response.
Discussion
Animals, tissues, and cells must be able to rapidly, flexibly, and reversibly adapt to a plethora of stresses. Past studies have identified many stress response factors, often termed master regulators. However, recent studies indicate that stress response regulation requires the intricate interactions of multiple factors as part of networks that provide regulatory redundancy and flexibility. NHR-49 is a transcription factor that promotes longevity and development by regulating lipid metabolism and various stress responses (Chamoli et al., 2014; Dasgupta et al., 2020; Goh et al., 2018; Naim et al., 2021; Wani et al., 2021). Our data show that NHR-49 coordinates a part of the transcriptional response to hypoxia. The NHR-49 pathway operates in parallel to the canonical HIF-1 hypoxia response pathway. Besides nhr-49, this pathway includes nhr-67 and hpk-1. The former interacts with NHR-49 (Reece-Hoyes et al., 2013), potentially forming a regulatory NHR-NHR heterodimer that modulates NHR-49 activity. During normoxia, nhr-67 acts to repress NHR-49; however, during hypoxia, an increase in NHR-49 protein levels in turn represses nhr-67 levels, forming a feedback loop that may serve to reinforce NHR-49 activity. In contrast to nhr-67, the upstream kinase HPK-1 positively regulates at least part of the NHR-49-dependent hypoxia response, either directly or indirectly, as it is required to survive hypoxia and to activate the NHR-49-regulated hypoxia response genes, including fmo-2, acs-2, and autophagy genes. Downstream, NHR-49 and HPK-1 induce autophagy, which is essential to promote hypoxia survival. Collectively, our experiments delineate a hypoxia response pathway that contains distinct upstream and downstream components and is just as essential for hypoxia survival as the parallel hif-1 pathway (Figure 9).
Figure 9
Model of the new NHR-49 hypoxia response pathway and its interaction with HIF-1 signalling.
The proposed model of how NHR-49 regulates a new hypoxia response parallel to HIF-1. During normoxia, the transcription factor NHR-67 negatively regulates NHR-49. However, during hypoxia, NHR-49 represses nhr-67, and the kinase HPK-1 positively regulates NHR-49, possibly directly or indirectly. This allows NHR-49 to activate its downstream hypoxia response target genes, including fmo-2, acs-2, and autophagy genes, whose induction is required for worm survival to hypoxia. (Figure created with https://biorender.com/, Toronto, ON, Canada).
NHR-49 controls a novel hypoxia response pathway that is parallel to canonical HIF signalling
nhr-49 is required to induce fmo-2 in various stresses and infection models (Chamoli et al., 2014; Dasgupta et al., 2020; Goh et al., 2018; Naim et al., 2021; Wani et al., 2021). Similarly, HIF-1 regulates fmo-2 in several C. elegans longevity paradigms (Leiser et al., 2015), and fmo-2 is induced in hypoxia, specifically 0.1% O2 exposure, in a hif-1-dependent manner (Leiser et al., 2015; Shen et al., 2005). This raised the possibility that hif-1 also promoted fmo-2 expression in hypoxia (0.5% O2) in L4 or older worms, and, more generally, that nhr-49 might act through hif-1 in the hypoxia response. However, several lines of evidence support a model whereby HIF-1 and NHR-49 are core components of parallel signalling networks (Figure 9). First, hif-1 and nhr-49 interact genetically in hypoxia survival experiments, suggesting that they work in parallel genetic pathways (Figure 2A, B and E). Second, our transcriptome analysis identified sets of genes that are regulated exclusively by HIF-1 or NHR-49 (Figure 3A and B). Third, the kinase hpk-1 and the transcription factor nhr-67 show synthetic genetic interaction with hif-1, but not with nhr-49 (Figure 7F and G, Figure 6—figure supplement 1D). In support of our study, a recent publication (Vozdek et al., 2018) showed that nhr-49 is required to induce the hif-1-independent hypoxia response gene comt-5 both in 0.5% O2 and in a strain mutant for the kinase hir-1. In hypoxia, HIR-1 coordinates remodelling of the extracellular matrix independently of HIF-1 (Vozdek et al., 2018). Thus, although our RNA-seq results did not identify comt-5 as a target of NHR-49 in hypoxia, this study supports the idea of a nhr-49 hypoxia response pathway that acts in parallel with hif-1.
Homeodomain-interacting protein kinases in hypoxia
Our efforts to map additional components of the NHR-49 hypoxia response pathway, especially factors acting in concert with NHR-49, revealed HPK-1 (Figure 9). Homeodomain-interacting protein kinases (HIPKs) are a family of nuclear serine/threonine kinase that can phosphorylate transcription factors (Rinaldo et al., 2007; Rinaldo et al., 2008). The worm’s only HIPK orthologue, hpk-1, regulates development and the response to DNA damage, heat shock, and dietary restriction (Berber et al., 2013; Berber et al., 2016; Das et al., 2017; Rinaldo et al., 2007). Notably, hpk-1 regulates autophagy in response to dietary restriction as it is necessary to induce autophagosome formation and autophagy gene expression (Das et al., 2017). Here, we show that hpk-1 is an upstream regulator of the nhr-49-dependent hypoxia response pathway. Our data suggest that HPK-1 promotes the accumulation of NHR-49 protein in hypoxia, leading to induction of NHR-49-dependent hypoxia response genes. This includes the induction of autophagy genes and autophagosome formation in hypoxia (Figure 4A, B and G, Figure 4—figure supplement 1B and C). In line with our model, mammalian HIPK2 is induced in and required to protect cardiomyocytes from hypoxia/reoxygenation induced injury (Dang et al., 2020). In contrast, in breast cancer cells, HIPK2 is degraded during periods of low oxygen via association with the E3 ubiquitin ligase SIAH2 (Calzado et al., 2009); this degradation of HIPK2 is necessary as the protein normally represses the expression of HIF-1α by binding at its promoter (Nardinocchi et al., 2009). Thus, protecting cells from hypoxic injury may be a conserved, albeit cell-type-specific, role of HIPKs. Future experiments may reveal how HPK-1 regulates NHR-49, perhaps by examining direct phosphorylation and activation of the NHR-49 protein by HPK-1.
Paradoxical regulation of the β-oxidation gene acs-2 by hypoxia
Mitochondria consume cellular oxygen to produce energy and thus must adapt to limited oxygen availability. In particular, mitochondrial β-oxidation, the consumption of oxygen to catabolize fatty acids for energy production, is repressed in hypoxia in favour of anaerobic respiration. For example, the heart and skeletal muscle of mice and rats show decreased expression of key β-oxidation enzymes in acute hypoxia (Kennedy et al., 2001; Morash et al., 2013). In C. elegans, the acyl-CoA synthetase acs-2 is part of the mitochondrial β-oxidation pathway, where it functions in the first step to activate fatty acids. NHR-49 activates acs-2 expression during starvation, when β-oxidation is induced (Van Gilst et al., 2005b). Considering this, acs-2 expression would be expected to be downregulated in hypoxia due to reduced β-oxidation. Paradoxically, however, we found that acs-2 is strongly induced in hypoxia and that this regulation depends on nhr-49 (Figure 3C, Figure 3—figure supplement 2A–C). Examination of other fatty acid β-oxidation enzymes in our RNA-seq data showed that acs-2 is the only enzyme induced. This suggests that, during hypoxia, ACS-2 is not feeding its product fatty acyl-CoA into the β-oxidation cycle, but perhaps produces fatty acyl-CoA for anabolic functions needed for survival in or recovery from low oxygen, such as phospholipid or triglyceride synthesis (reviewed in Tang et al., 2018). Similar functions have been observed in human macrophages, which, during hypoxia, decrease β-oxidation but increase triglyceride synthesis (Boström et al., 2006).
In line with the repression of β-oxidation in hypoxia (Boström et al., 2006; Kennedy et al., 2001; Morash et al., 2013), there is evidence supporting a HIF-dependent downregulation of the mammalian NHR-49 homolog PPARα, which promotes β-oxidation (Atherton et al., 2008). For example, in human hepatocytes and mouse liver sections, HIF-2α accumulation in hypoxia directly suppresses PPARα expression (Chen et al., 2019). Additionally, HIF-1α suppresses PPARα protein and mRNA levels during hypoxia in intestinal epithelial cells, and the PPARA promoter contains a HIF-1α DNA-binding consensus motif, suggesting direct control of PPARA by HIF transcription factors (Narravula and Colgan, 2001).
Some evidence suggests alternative actions of PPARα. Knockdown of PPARα attenuates the ability of Phd1 (a homolog of C. elegans egl-9) knockout myofibers to successfully tolerate hypoxia (Aragonés et al., 2008), suggesting that PPARα is an important regulator of the hypoxia response downstream of Phd1. Along these lines, PPARα protein levels increase in the muscle of Phd1 knockout mice (Aragonés et al., 2008) and following hypoxic exposure in mouse hearts (Morash et al., 2013). Similarly, we show that NHR-49 protein levels increase in response to hypoxia (Figures 2D, 8B and C, Figure 2—figure supplement 1C–F), and that NHR-49 is a vital regulator of a hypoxia response that works in parallel with HIF-1. Together, these data suggest that, similar to evidence from studies in mammalian systems, NHR-49 levels are increased and required in hypoxia, and may be regulating acs-2 for functions other than fatty acid β-oxidation.
NHR-49 promotes autophagy activation to achieve hypoxia survival
During stress, damaged cellular components can be cleared or recycled via autophagy, a key process regulated by nhr-49 in hypoxia (Figures 3D and 4). Autophagy is part of an adaptive response to hypoxia. During periods of low oxygen, cells switch from aerobic mitochondrial respiration to anaerobic glycolysis. To meet this increased glycolytic demand, the autophagy machinery promotes the activity and cell surface expression of the glucose transporter GLUT1 to increase cellular glucose uptake (Roy et al., 2017). In addition, hypoxia causes improper protein folding in the endoplasmic reticulum (ER), activating the unfolded protein response (UPR). Although the exact mechanism is unknown, it is thought that autophagy and the UPR are activated simultaneously during stress to restore homeostasis, and that autophagy can assist in alleviating ER stress when the UPR is disrupted or overwhelmed (reviewed in Yan et al., 2015).
In mammals, PPARα activates autophagy in response to various stresses, including in neurons to clear Aβ in Alzheimer’s disease (Luo et al., 2020), and in the liver during inflammation (Jiao et al., 2014) and starvation (Lee et al., 2014). Proper regulation of autophagy is also a requirement in hypoxic conditions. Knockdown or genetic mutation of various C. elegans autophagy genes showed that they are required for worm survival when worms experience anoxia and elevated temperatures combined (Samokhvalov et al., 2008). Similarly, Zhang et al. found that mitochondrial autophagy (mitophagy) is induced by hypoxia in mouse embryo fibroblasts. This process requires the expression of BNIP3 (Bcl-2/E1B 19 kDa-interacting protein 3), an autophagy regulator, which is induced in a HIF-1-dependent manner (Zhang et al., 2008). In agreement with this, our RNA-seq data showed a 3.8-fold induction of the C. elegans BNIP3 homolog dct-1 in hypoxia; however, this induction was dependent on neither nhr-49 nor hif-1. The above study also found that the autophagy genes Beclin-1 and Atg5 are induced and required for cell survival in hypoxia (Zhang et al., 2008). Here, we show for the first time that autophagy is both induced and required for C. elegans adaptation and survival to 0.5% O2. The C. elegans orthologue of Beclin-1, bec-1, and the worm lgg-2, epg-6, epg-3, atg-7, and atg-10 genes, which are involved in the completion of the autophagosome along with atg-5/Atg5, are required for worm embryo survival to hypoxia in an nhr-49-dependent manner (Figure 4E and F, Figure 4—figure supplement 1F and G). In addition, we show that both nhr-49 and hpk-1 are required to induce the expression of autophagy genes and autophagosome formation during hypoxia, processes that are independent of hif-1. In agreement with our findings, Valko et al. recently reported that the formation of autophagosomes by hypoxia is independent of hif-1/sima in Drosophila melanogaster (Valko et al., 2021).
Cell non-autonomous functions of NHR-49 in hypoxia
Cell non-autonomous regulation occurs in many pathways in C. elegans. For example, HIF-1 acts in neurons to induce fmo-2 expression in the intestine to promote longevity (Leiser et al., 2015). NHR-49 is expressed in the intestine, neurons, muscle, and hypodermis (Ratnappan et al., 2014). Re-expression of nhr-49 in any one of these tissues is sufficient to enhance worm survival upon infection with the pathogens S. aureus (Wani et al., 2021) and to promote longevity in germline-less animals (Naim et al., 2021), but NHR-49 acts only in neurons to promote survival upon infection by P. aeruginosa (Naim et al., 2021). We thus aimed to identify the key tissue wherein NHR-49 promotes hypoxia survival. Surprisingly, we found that nhr-49 expression in any of the intestine, neurons, hypodermis, or body wall muscle is sufficient for whole-animal survival to hypoxia (Figure 5B), suggesting that NHR-49 can act in a cell non-autonomous fashion to execute its effects. Possibly, a signalling molecule whose synthesis is promoted by NHR-49 activity in any tissue promotes organismal hypoxia adaptation. It is also possible that ectopic overexpression of NHR-49 shifts metabolism in the tissue wherein it is expressed, releasing metabolites that promote organismal hypoxia adaptation.
In sum, we show here that NHR-49 regulates a novel hypoxia response pathway parallel to HIF-1 and controls an important transcriptional response for worm survival in hypoxia. If the mammalian NHR-49 homologs PPARα and HNF4 play similar roles in the cellular response to hypoxia, our discovery could lead to the identification and development of new targets for drugs and therapies for diseases exhibiting hypoxic conditions.
Materials and methods
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | OP50 | Caenorhabditis Genetics Center (CGC) | ||
| Strain, strain background (E. coli) | HT115 | Caenorhabditis Genetics Center (CGC) | ||
| Genetic reagent (Caenorhabditis elegans) | N2 | Caenorhabditis Genetics Center (CGC) (Brenner, 1974) | ||
| Genetic reagent (C. elegans) | nhr-49(nr2041) I | PMID:15719061 (Van Gilst et al., 2005a) | STE68; RRID:WB-STRAIN:WBStrain00034504 | |
| Genetic reagent (C. elegans) | eavEx20[fmo-2p::gfp+rol-6(su1006)] | PMID:29508513 (Goh et al., 2018) | VE40 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041) I; eavEx20[fmo-2p::gfp+rol-6(su1006)] | This study | STE129 | |
| Genetic reagent (C. elegans) | hif-1(ia4) V | PMID:11427734 (Jiang et al., 2001) | ZG31; RRID:WB-STRAIN:WBStrain00040824 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041) I; hif-1(ia4) V | This study | STE130 | |
| Genetic reagent (C. elegans) | fmo-2(ok2147) IV | PMID:26586189 (Leiser et al., 2015) | VC1668; RRID:WB-STRAIN:WBStrain00036780 | |
| Genetic reagent (C. elegans) | acs-2(ok2457) V | PMID:21704635 (Zhang et al., 2011) | RB1899 | |
| Genetic reagent (C. elegans) | fmo-2(ok2147) IV; acs-2(ok2457) V | This study | STE131 | |
| Genetic reagent (C. elegans) | nhr-49(et13) I | PMID:27618178 (Lee et al., 2016) | STE110 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041) I;glmEx5 [nhr-49p::nhr-49::gfp+myo-2p::mCherry] | PMID:34156142 (Naim et al., 2021) | AGP33a | |
| Genetic reagent (C. elegans) | nhr-49(nr2041) I; glmEx9 [gly-19p::nhr-49::gfp+myo-2p::mCherry] | PMID:34156142 (Naim et al., 2021) | AGP65 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041)I; glmEx11 [col-12p::nhr-49::gfp+myo-2p::mCherry] | PMID:34156142 (Naim et al., 2021) | AGP53 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041)I; glmEx13 [rgef-1p::nhr-49::gfp+myo-2p::mCherry] | PMID:34156142 (Naim et al., 2021) | AGP51 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041)I; glmEx8 [myo-3p::nhr-49::gfp+myo-2p::mCherry] | PMID:34156142 (Naim et al., 2021) | AGP63 | |
| Genetic reagent (C. elegans) | wbmEx57 [acs-2p::gfp+rol-6(su1006)] | PMID:25723162 (Burkewitz et al., 2015) | WBM170 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041) I; wbmEx57 [acs-2p::gfp+rol-6(su1006)] | PMID:25723162 (Burkewitz et al., 2015) | WBM169 | |
| Genetic reagent (C. elegans) | glmEx5 (nhr-49p::nhr-49::gfp+myo-2p::mCherry) | PMID:25474470 (Ratnappan et al., 2014) | AGP25f | |
| Genetic reagent (C. elegans) | hif-1(ia4) V; glmEx5 (nhr-49p::nhr-49::gfp+myo-2p::mCherry) | This study | STE140 | |
| Genetic reagent (C. elegans) | hpk-1(pk1393) X; glmEx5 (nhr-49p::nhr-49::gfp+myo-2p::mCherry) | This study | STE142 | |
| Genetic reagent (C. elegans) | hpk-1(pk1393) X | PMID:12618396 (Raich et al., 2003) | EK273; RRID:WB-STRAIN:WBStrain00007138 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041) I; hpk-1(pk1393) X | This study | STE132 | |
| Genetic reagent (C. elegans) | hif-1(ia4) V; hpk-1(pk1393) X | This study | STE133 | |
| Genetic reagent (C. elegans) | nhr-49(et13) I; eavEx20[fmo-2p::gfp+rol-6(su1006)] | PMID:29508513 (Goh et al., 2018) | STE117 | |
| Genetic reagent (C. elegans) | artEx12 [hpk-1p::gfp+rol-6(su1006)] | PMID:29036198 (Das et al., 2017) | AVS394 | |
| Genetic reagent (C. elegans) | dpy-5(e907) I; sEx14068 [rCes atg-2::GFP+pCeh361] | PMID:15338614 (McKay et al., 2003) | BC14068 | |
| Genetic reagent (C. elegans) | dpy-5(e907) I; sEx13567 [rCes lgg-1::GFP+pCeh361] | PMID:15338614 (McKay et al., 2003) | BC13567 | |
| Genetic reagent (C. elegans) | dpy-5(e907) I; sEx10273 [rCes epg-3::GFP+pCeh361] | PMID:15338614 (McKay et al., 2003) | BC10273 | |
| Genetic reagent (C. elegans) | adIs2122 [lgg-1p::GFP::lgg-1 + rol-6(su1006)] | PMID:17785524 (Kang et al., 2007) | DA2123 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041) I; adIs2122 [lgg-1p::GFP::lgg-1 + rol-6(su1006)] | This study | STE143 | |
| Genetic reagent (C. elegans) | hif-1(ia4) V; adIs2122 [lgg-1p::GFP::lgg-1 + rol-6(su1006)] | This study | STE144 | |
| Genetic reagent (C. elegans) | lgg-2(tm5755) IV | PMID:24374177 (Manil-Ségalen et al., 2014) | RD220 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041) I; lgg-2(tm5755) IV | This study | STE145 | |
| Genetic reagent (C. elegans) | epg-6(tm8366) III | This study, non-outcrossed mutant obtained from NBRP; PMID:19934255 | STE147 | |
| Genetic reagent (C. elegans) | nhr-49(nr2041) I; epg-6(tm8366) III | This study | STE146 | |
| Sequence-based reagent | Source BioScience | PMID:11099033 | RNAi clones | |
| Sequence-based reagent | fmo-2_F | This paper | qPCR primer | GGAACAAGCGTGTTGCTGT |
| Sequence-based reagent | fmo-2_R | This paper | qPCR primer | GCCATAGAGAAGACCATGTCG |
| Sequence-based reagent | acs-2_F | This paper | qPCR primer | AGTGAGACTTGACAGTTCCG |
| Sequence-based reagent | acs-2_R | This paper | qPCR primer | CTTGTAAGAGAGGAATGGCTC |
| Sequence-based reagent | nhr-49_F | This paper | qPCR primer | TCCGAGTTCATTCTCGACG |
| Sequence-based reagent | nhr-49_R | This paper | qPCR primer | GGATGAATTGCCAATGGAGC |
| Sequence-based reagent | hpk-1_F | This paper | qPCR primer | TGTCAAAGTGAAGCCGCTGG |
| Sequence-based reagent | hpk-1_R | This paper | qPCR primer | CGGCGCCAGTTCGTGTAGTA |
| Sequence-based reagent | nhr-67_F | This paper | qPCR primer | GAGGATGATGCGACGAGTAG |
| Sequence-based reagent | nhr-67_R | This paper | qPCR primer | TGGTCTTGAAGAGGAAGGGGA |
| Sequence-based reagent | act-1_F | This paper | qPCR primer | GCTGGACGTGATCTTACTGATTACC |
| Sequence-based reagent | act-1_R | This paper | qPCR primer | GTAGCAGAGCTTCTCCTTGATGTC |
| Sequence-based reagent | tba-1_F | This paper | qPCR primer | GTACACTCCACTGATCTCTGCTGACAAG |
| Sequence-based reagent | tba-1_R | This paper | qPCR primer | CTCTGTACAAGAGGCAAACAGCCATG |
| Sequence-based reagent | ubc-2_F | This paper | qPCR primer | AGGGAGGTGTCTTCTTCCTCAC |
| Sequence-based reagent | ubc-2_R | This paper | qPCR primer | CGGATTTGGATCACAGAGCAGC |
| Sequence-based reagent | oac-14_F | This paper | qPCR primer | TTCCAGCGACTTTTCTTTCG |
| Sequence-based reagent | oac-14_R | This paper | qPCR primer | CCCAGGATTGCTTCAATCAG |
| Sequence-based reagent | cyp-13A11_F | This paper | qPCR primer | ACACGTGGACACTTCACTATG |
| Sequence-based reagent | cyp-13A11_R | This paper | qPCR primer | TTCCGATACACTGTCGAGGTC |
| Sequence-based reagent | cyp-25A3_F | This paper | qPCR primer | agaatcgttgctccaaaacac |
| Sequence-based reagent | cyp-25A3_R | This paper | qPCR primer | ttcaaaatctccaggaacagg |
| Sequence-based reagent | ugt-20_F | This paper | qPCR primer | CCGACAAATCCCAGAGAGACA |
| Sequence-based reagent | ugt-20_R | This paper | qPCR primer | TGTCCAAAAAGAAGTACTCAACG |
| Sequence-based reagent | atg-2_F | This paper | qPCR primer | AGATGTCCGCCATAGTCTGC |
| Sequence-based reagent | atg-2_R | This paper | qPCR primer | TCTTCCTGAGCAGCGAGTTC |
| Sequence-based reagent | epg-9_F | This paper | qPCR primer | CGACGAAAACCGAGATTCCC |
| Sequence-based reagent | epg-9_R | This paper | qPCR primer | TGAGCCAGCGATTGTTTGTG |
| Sequence-based reagent | lgg-2_F | This paper | qPCR primer | GCAGTTTACCACTTATGGATCGC |
| Sequence-based reagent | lgg-2_R | This paper | qPCR primer | CGTTCATTGACGAGCAGGAAG |
| Sequence-based reagent | atg-13_F | This paper | qPCR primer | AAGCAGCTGAAAACTGCTCC |
| Sequence-based reagent | atg-13_R | This paper | qPCR primer | CGGAGAACGAATTGACGTGTT |
| Sequence-based reagent | Random primers | Invitrogen | 48190-011 | |
| Sequence-based reagent | dNTPs | Fermentas | R0186 | |
| Chemical compound, drug | Carbenicillin | BioBasic | CDJ469 | |
| Chemical compound, drug | IPTG | Santa Cruz | sc-202185B | CAS 367-93-1 |
| Chemical compound, drug | Tetracycline | BioBasic | TB0504 | |
| Chemical compound, drug | RNAseOUT | Invitrogen | 10777-019 | |
| Chemical compound, drug | Fast SYBR Master Mix | Life Technologies | 4385612 | |
| Chemical compound, drug | Levamisole | Sigma | L9756 | |
| Chemical compound, drug | H2S | AirGas, Seattle, WA | X02NI99CP581327 | |
| Chemical compound, drug | 5000 ppm O2 balanced with N2 | Praxair Canada | NI OX5000C−T | |
| Software, algorithm | ImageJ | PMID:22930834 | https://imagej.nih.gov/ij/index.html | |
| Software, algorithm | Trimmomatic version 0.36 | PMID:24695404 | RRID:SCR_011848 | |
| Software, algorithm | Salmon version 0.9.1 | PMID:28263959 | RRID:SCR_017036 | https://combine-lab.github.io/salmon/ |
| Software, algorithm | tximport | PMID:26925227 | RRID:SCR_016752 | https://github.com/mikelove/tximport |
| Software, algorithm | edgeR | PMID:19910308 | RRID:SCR_012802 | http://bioconductor.org/packages/edgeR/ |
| Software, algorithm | eVITTA | PMID:34019643 | https://tau.cmmt.ubc.ca/eVITTA/ |
Nematode strains and growth conditions
Request a detailed protocolWe cultured C. elegans strains using standard techniques on nematode growth media (NGM) plates. To avoid background effects, each mutant was crossed into our lab N2 strain; original mutants were backcrossed to N2 at least six times, except lgg-2 and epg-6 mutants, which were backcrossed four times. Escherichia coli OP50 was the food source in all experiments except for RNAi experiments, where we used E. coli HT115. All experiments were carried out at 20°C. Worm strains used in this study are listed in the Key resources table. For synchronized worm growths, we isolated embryos by standard sodium hypochlorite treatment. Isolated embryos were allowed to hatch overnight on unseeded NGM plates until the population reached a synchronized halted development at L1 stage via short-term fasting (12–24 hr). Synchronized L1 stage larvae were then transferred to OP50 seeded plates and grown to the desired stage.
Feeding RNAi
Request a detailed protocolRNAi was performed on NGM plates supplemented with 25 μg/ml carbenicillin (BioBasic CDJ469), 1 mM IPTG (Santa Cruz CAS 367-93-1), and 12.5 μg/ml tetracycline (BioBasic TB0504; NGM-RNAi plates), and seeded with appropriate HT115 RNAi bacteria. The RNAi clones were from the Ahringer library (Source BioScience) and were sequenced prior to use.
RNA isolation and qRT-PCR analysis
Request a detailed protocolSynchronized L1 worms were allowed to grow on OP50 plates for 48 hr to L4 stage, then either kept in 21% O2 or transferred to 0.5% O2 for 3 hr and rapidly harvested. RNA isolation was performed as previously described (Goh et al., 2014). 2 μg total RNA was used to generate cDNA with Superscript II reverse transcriptase (Invitrogen 18064-014), random primers (Invitrogen 48190-011), dNTPs (Fermentas R0186), and RNAseOUT (Invitrogen 10777-019). Quantitative PCR was performed in 10 μl reactions using Fast SYBR Master Mix (Life Technologies 4385612), 1:10 diluted cDNA, and 5 μM primer, and analysed with an Applied Biosystems StepOnePlus machine. We analysed the data with the ΔΔCt method. For each sample, we calculated normalization factors by averaging the (sample expression)/(average reference expression) ratios of three normalization genes, act-1, tba-1, and ubc-2. The reference sample was EV(RNAi), wild-type, or 21% O2, as appropriate. We used one-way or two-way ANOVA to calculate the statistical significance of gene expression changes and corrected for multiple comparisons using the Tukey method. Primers were tested on serial cDNA dilutions and analysed for PCR efficiency prior to use. All data originate from three or more independent biological repeats, and each PCR reaction was conducted in technical triplicate. Sequences of qRT-PCR primers are listed in the Key resources table.
Analysis of fluorescent reporter lines via DIC and fluorescence microscopy
Request a detailed protocolTo analyse fluorescence in reporter lines, egg lays were performed on NGM plates seeded with OP50 or RNAi plates seeded with the appropriate HT115 RNAi culture. Worms were allowed to grow to adulthood. Plates were then kept in 21% O2 or transferred to 0.5% O2 for 4 hr and allowed to recover for 1 hr in normoxia before imaging to allow for GFP maturation; hence, these assays are technically post-hypoxia experiments. Worms were collected into M9 buffer containing 0.06% levamisole (Sigma L9756) for immobilization on 2% (w/v) agarose pads for microscopy. We captured images at ×10 magnification on a CoolSnap HQ camera (Photometrics) attached to a Zeiss Axioplan 2 compound microscope, followed by MetaMorph Imaging Software with Autoquant 3D digital deconvolution. For higher resolution images, we used the Hamamatsu ORCA-Flash4.0 LT+ Digital CMOS camera attached to a Leica SP8X confocal microscope at ×40 magnification. All images for the same experiment were captured at the same exposure time. Images were analysed using ImageJ software (https://imagej.nih.gov/ij/download.html), with fluorescence calculated by taking the difference of the background fluorescence from the mean intestinal or whole-worm fluorescence. For experiments imaging the fmo-2p::gfp and acs-2p::gfp reporters, intestinal fluorescence was measured. For experiments imaging hpk-1p::gfp, nhr-49p::nhr-49::gfp, lgg-1p::gfp, atg-2p::gfp, or epg-6p::gfp, whole-worm fluorescence was measured. For each experiment, at least three independent trials were performed with a minimum of 30 worms per condition.
Autophagosome formation measurement
Request a detailed protocolAutophagosome formation was measured by counting fluorescent foci in the hypodermal seam cells of animals expressing the translational LGG-1::GFP reporter (Das et al., 2017; Zhang et al., 2015). L3 worms were either kept in 21% O2 or transferred to 0.5% O2 for 5 hr. Worms were collected into 1 M NaN3 for immobilization on 2% (w/v) agarose pads for microscopy, and the Leica SP8X microscope was used as above at ×63 magnification. For each experiment, at least three independent trials were performed with a minimum of 15 worms scored for GFP foci, totalling at least 110 individual seam cells per condition. For the micrographs shown in Figure 4C, image brightness and contrast were adjusted in ImageJ to best visualize the number of foci present in each seam cell; importantly, the same adjustment was consistently applied throughout the whole image. The same brightness and contrast settings were applied to images within each genotype (i.e., comparing hypoxia-exposed and normoxia-exposed animals of the same genotype), but different settings were used for different genotypes.
NHR-49 transgenic strains
Request a detailed protocolTo construct the nhr-49p::nhr-49::gfp containing plasmid, a 6.6 kb genomic fragment of the nhr-49 gene (including a 4.4 kb coding region covering all nhr-49 transcripts and a 2.2 kb promoter region) was cloned into the GFP expression vector pPD95.77 (Addgene #1495), as reported previously (Ratnappan et al., 2014). For generating tissue-specific constructs, the nhr-49 promoter was replaced with tissue-specific promoters using SbfI and SalI restriction enzymes to create plasmids for expressing NHR-49 in the muscle (myo-3p::nhr-49::gfp), intestine (gly-19p::nhr-49::gfp), hypodermis (col-12p::nhr-49::gfp), and neurons (rgef-1p::nhr-49::gfp). 100 ng/μl of each plasmid was injected, along with pharyngeal muscle-specific myo-2p::mCherry as a co-injection marker (25 ng/μl), into the nhr-49(nr2041) mutant strain using standard methods (Mello and Fire, 1995). Strains were maintained by picking animals that were positive for both GFP and mCherry.
Hypoxia sensitivity assays
Request a detailed protocolHypoxic conditions were maintained using continuous flow chambers, as previously described (Fawcett et al., 2012). Compressed gas tanks (5000 ppm O2 balanced with N2) were certified as standard to within 2% of indicated concentration from Praxair Canada (Delta, BC). Oxygen flow was regulated using Aalborg rotameters (Aalborg Instruments and Controls, Inc, Orangeburg, NY). Hypoxic chambers (and room air controls) were maintained in a 20°C incubator for the duration of the experiments.
For embryo survival assays, gravid first-day adult worms (picked as L4 the previous day) were allowed to lay eggs for 1–4 hr on plates seeded with 15 μl OP50 or appropriate HT115 RNAi bacteria the previous day. Adults were removed, and eggs were exposed to 0.5% O2 for 24 hr or 48 hr. Animals were scored for developmental success (reached at least L4 stage) after being placed back into room air for 65 hr (following 24 hr exposure) or 42 hr (following 48 hr exposure). For RNAi survival assays, worms were grown for one generation from egg to adult on the appropriate HT115 RNAi bacteria before their progeny was used for the egg lay.
For larval development assays, gravid adult worms (picked as L4 the previous day) were allowed to lay eggs for 2 hr and kept at 20°C for 13–17 hr to allow hatching (egg lays for nhr-49(nr2041) strains with embryonic developmental delays were performed 2 hr earlier to ensure synchronization with wild-type worms). Freshly hatched L1 worms were transferred to plates seeded with 15 μl OP50 the previous day and exposed to 0.5% O2 for 48 hr. Animals were placed back into room air and immediately scored for stage.
For all normoxia (21% O2) comparison experiments, methods were as described above except plates were kept in room air for the duration (instead of being exposed to 0.5% O2).
Hydrogen sulfide sensitivity assay
Request a detailed protocolConstruction of H2S chambers was as previously described (Fawcett et al., 2012; Miller and Roth, 2007). In short, 5000 ppm H2S (balanced with N2) was diluted with room air to a final concentration of 50 ppm and monitored with a custom H2S detector, as described (Miller and Roth, 2007). Compressed gas mixtures were obtained from Airgas (Seattle, WA) and certified as standard to within 2% of the indicated concentration. Survival assays were performed in three independent trials with 20 L4 animals picked onto OP50 seeded plates. Plates were exposed to 50 ppm H2S for 24 hr in a 20°C incubator, then returned to room air to score viability. Animals were scored 30 min after removal from H2S, and plates with dead animals were re-examined after several hours to ensure animals had not reanimated.
RNA sequencing
Request a detailed protocolSynchronized L1 wild-type, nhr-49(nr2041), and hif-1(ia4) worms were allowed to grow on OP50 plates to L4 stage, then either kept in 21% O2 or transferred to 0.5% O2 for 3 hr. RNA was isolated from whole worms as described above (immediately following hypoxia exposure). RNA integrity and quality were ascertained on a BioAnalzyer. Construction of strand-specific mRNA sequencing libraries and sequencing (75 bp PET) on an Illumina HiSeq 2500 machine was done at the Sequencing Services facility of the Genome Sciences Centre, BC Cancer Agency, Vancouver BC, Canada (https://www.bcgsc.ca/services/sequencing-services). We sequenced >20 million reads per sample. The raw FASTQ reads obtained from the facility were trimmed using Trimmomatic version 0.36 (Bolger et al., 2014) with parameters LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. Next, the trimmed reads were aligned to the NCBI reference genome WBcel235 WS277 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000002985.6/) using Salmon version 0.9.1 (Patro et al., 2017) with parameters -l A -p 8 --gcBias. Then, transcript-level read counts were imported into R and summed into gene-level read counts using tximport (Soneson et al., 2015). Genes not expressed at a level greater than one count per million (CPM) reads in at least three of the samples were excluded from further analysis. The gene-level read counts were normalized using the trimmed mean of M-values (TMM) in edgeR (Robinson et al., 2010) to adjust samples for differences in library size. Differential expression analysis was performed using the quasi-likelihood F-test with the generalized linear model (GLM) approach in edgeR (Robinson et al., 2010). Differentially expressed genes (DEGs) were defined as those with at least a two-fold difference between two individual groups at an FDR < 0.05. RNA-seq data have been deposited at NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under the record GSE166788.
Functional enrichment analysis and visualization were performed using the Overrepresentation Analysis (ORA) module with the default parameters in easyGSEA in the eVITTA toolbox (https://tau.cmmt.ubc.ca/eVITTA/; input December 14, 2020; Cheng et al., 2021). easyVizR in the eVITTA toolbox was used to visualize the overlaps and disjoints in the DEGs (input December 14, 2020).
Data availability
RNA-seq data have been deposited at NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under the record GSE166788. All data generated or analysed during this study are included in the manuscript and Supplementary files. Raw data points from each N are shown in figures wherever possible. See transparent reporting form for details.
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NCBI Gene Expression OmnibusID GSE166788. NHR-49 controls a HIF-1 independent hypoxia adaptation pathway in Caenorhabditis elegans.
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NCBI Gene Expression OmnibusID GSE2836. Hypoxia response.
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Decision letter
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Douglas PortmanReviewing Editor; University of Rochester, United States
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Piali SenguptaSenior Editor; Brandeis University, United States
Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.
Decision letter after peer review:
Thank you for submitting your article "Nuclear Hormone Receptor NHR-49 controls a HIF-1-independent hypoxia adaptation pathway in Caenorhabditis elegans" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Piali Sengupta as the Senior Editor. The reviewers have opted to remain anonymous.
The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.
All three reviewers agree that your findings are interesting and have the potential to provide important new insight into hypoxia responses in C. elegans. However, there are a number of areas where the reviewers feel that the conclusions your paper draws are not fully supported by the data. Additionally, for the paper to provide the level of biological insight that would be appropriate for eLife, the reviewers feel some additional issues need to be addressed.
Based on the individual reviewer comments below, the following points must be addressed in a revised version.
(1) Autophagy. The reviewers feel that the link between hypoxia, nhr-49, and autophagy is one of the most important insights provided by your work. However, the role of autophagy in hypoxia, and its reliance on nhr-49, needs to be better substantiated.
a. As pointed out by Reviewers 2 and 3, the sample size for the experiments in Figure 3F and Supp Figure 4B are quite small. Together with the high variance in your measurements, it is unlikely that these experiments have the power to detect differences that could be present when comparing WT vs nhr-49 backgrounds. Please increase sample sizes here, ideally guided by power size calculations. As Reviewer 3 suggests, it may also be useful to consider altering the experimental conditions.
b. Please confirm the changes in expression of autophagy genes, and their dependence on nhr-49, by RT-PCR.
c. As suggested by reviewer 1, please examine autophagosome formation directly (e.g., with LGG-1::GFP) under hypoxic conditions, in WT, nhr-49, and hif-1 animals.
(2) Site of action of NHR-49. Reviewers 1 and 2 raise concerns about the interpretation and significance of these findings.
a. Please provide additional information on the site of NHR-49 induction by hypoxia, e.g., with high-magnification images.
b. Please confirm the specificity of the protective effects of NHR-49 overexpression by (i) examining body-wall-specific and pharyngeal-muscle-specific overexpression as suggested by Reviewer 2 OR (ii) using tissue-specific RNAi as suggested by Reviewer 1.
(3) RNAseq data. As suggested by Reviewer 1, please clarify the analysis presented in Figure 3A,B and, if possible, compare your results with other hypoxia transcriptome data that may have been previously reported.
(4) Interactions between NHR-67 and NHR-49.
a. As suggested by Reviewers 1 and 2, please examine transcriptional reporters for nhr-49 and nhr-67 in hypoxia and normoxia, in both wild-type and nhr-49/67 mutants.
b. Please investigate genetic interactions: is nhr-67 synthetic lethal with hif-1 and/or nhr-49 in the context of hypoxia survival?
c. As Reviewer 2 suggests, it would also be useful to determine whether specific subsets of hypoxia response genes are dependent on nhr-67, though this is not considered essential.
(5) Regulation of NHR-49 by HPK-1.
a. The reviewers consider the proposed mechanism to be quite speculative. If possible, it would be ideal for you to determine whether NHR-49 becomes phosphorylated under hypoxia, and whether this depends on hpk-1, as suggested by Reviewer 2, or whether the localization of NHR-49 changes under these conditions, as suggested by Reviewer 3. Alternatively, please modify the text and figures to make it clear that this part of your model has not been tested and that the regulation of nhr-49 by hpk-1 could be indirect.
b. To substantiate the connections between hpk-1, nhr-49, and autophagy, please determine whether the upregulation of autophagy genes requires hpk-1.
(6) In a number of places, the interpretation of your results should be softened. One example of this is lines 150-151 (Figure S1B). ~95% of WT animals are adults at 48 hr but 85% of nhr-49 mutants are still L3 and L4. However, you claim that "mutants did not significantly develop slower than wildtype". If so, a different test needs to be carried out, or sample sizes need to be increased, because it seems clear that there is a difference in developmental rate between these groups. Other examples where conclusions need to be tempered are provided in the reviewer comments below.
In addition to these points, please also consider the other issues raised by the reviewers (see below) as you prepare your revision.
Reviewer #1 (Recommendations for the authors):
1. To address Major Concern 2, the authors should confirm their RNA-seq results on new samples via qRT-PCR and also test whether autophagosomes are formed under hypoxic conditions (LGG-1::GFP), which should be dependent on nhr-49 but not hif-1 (if the authors model is true). The authors should expand their discussion to explain how increased autophagy would protect against hypoxia (metabolic shift for anaerobic respiration, decreased proteome reduces energetic demands, etc.).
2. To address Major Concerns 3 and 4, the authors should use tissue specific RNAi or hairpins to inactivate nhr-49 and assess whether loss in any one tissue compromises hypoxic response/survival. Additionally, if ectopic overexpression of NHR-49 within one tissue does indirectly protect other tissues through an adaptive response, one would predict that overexpressing NHR-49 in one tissue would mute the induction of NHR-49 a neighboring tissue after hypoxic treatment. This could be tested easily via combining their nhr-49p::NHR-49::GFP reporter and tissue specific overexpression of NHR-49. The authors should consider testing this possibility.
3. I strongly suggest avoiding the use of "worms" in formal manuscripts and use "animals" instead. It's jargon and diminishes the impact of C. elegans research when read by scientists using other systems.
Reviewer #2 (Recommendations for the authors):
– Figure 3F – These results are very crucial for the main conclusion of this study. Since RNAi produces more variable results across biological replicates, this experiment should be repeated with some of the autophagy mutants such as unc-51 etc.
– The sample sizes for the RNAi experiments in Figure 3F do not seem to be enough to capture the differences in hypoxia survival between WT and nhr-49 mutant conditions: 41% vs 25% (atg-7), 27% vs 13% (bec-1) and 38% vs 13% (epg-3). None of these differences were detected as statistically significant. This is likely due to n = 4 for RNAi treatments of the nhr-49 mutant and very high variability across biological replicates. The authors should at least double their sample sizes for these conditions and test whether there are any significant differences in hypoxia survival between WT and nhr-49 mutant when autophagy is inhibited. Similarly, in Supplementary Figure 4B, some of the autophagy gene RNAi conditions seem to reduce survival by 25% (with some data points showing greater than 50% reduction) even in normoxia. None of these differences were detected as statistically significant, which again is likely due to low sample sizes and high variability across replicates. These experiments need to be carefully designed so that there is enough statistical power to discern whether nhr-49 and autophagy are regulating hypoxia survival via the same or independent pathways and whether autophagy inhibition reduces survival also in normoxia.
– For tissue-specific rescue of nhr-49 (data shown in Figure 4B), the authors should also perform a body wall muscle-specific rescue experiment. Since nhr-49 is expressed in the muscles, it will be informative to know whether body wall muscle-specific nhr-49 function is sufficient to improve hypoxia survival at the organism level. In addition, a pharyngeal muscle-specific nhr-49 rescue might serve as a good negative control, where tissue-specific rescue might not be able to affect whole organism hypoxia survival.
– To confirm the bidirectional regulation between nhr-49 and nhr-67, the authors should create a gfp transcriptional reporter for nhr-67 and test whether nhr-49 inhibition affects nhr-67 expression in hypoxia and normoxia. The RNA-seq data shown in Figure 5A suggests that NHR-49 possibly regulates nhr-67 expression only in hypoxic conditions, but it will be informative to know whether this regulation is restricted to specific tissues. In comparison, NHR-67 seems to be regulating nhr-49 expression predominantly in the intestine in both normoxia and hypoxia (line 309).
– The physiological relevance of the bidirectional negative regulation between nhr-49 and nhr-67 is not clear. Inhibition of both nhr genes reduces survival in hypoxic conditions (Supplementary Figure 5D). While this study has found downstream targets of nhr-49 that promote hypoxia survival, it is not clear how nhr-67 fits into this regulation. In this regard, the authors should investigate two directions: (1) does nhr-67 also show synthetic lethal interactions with hif-1 and nhr-49 in the context of hypoxia survival (similar to Figure 2A)?, and (2) does nhr-67 inhibition in hypoxic conditions prevent the activation of a specific subset of hypoxia response genes, such as genes involved in detoxification and autophagy?
– The authors propose that survival during hypoxia is predominantly due to the upregulation of autophagy genes, but not due to upregulation of fmo-2 and acs-2 (lines 222-224). However, they have only tested whether hpk-1 removal alters the expression of fmo-2 and acs-2 genes (Figures 6A-E). To mechanistically elucidate whether HPK-1 and NHR-49 are acting via the same pathway to promote hypoxia survival (as suggested in Figure 6F), it should be tested whether hpk-1 mutation impairs the upregulation of autophagy genes during hypoxia.
– The role of HPK-1 in stabilizing NHR-49 during hypoxia is a novel finding that is of broad interest. In its current form, the proposed mechanism is quite speculative. Since this regulation is central to the proposed HIF-1-independent hypoxia response pathway, the authors should investigate whether NHR-49 is phosphorylated in low oxygen conditions in an HPK-1-dependent manner. A simple way to test this would be to use the nhr-49::gfp translational reporter strain and prepare whole worm extracts in normoxia and hypoxia conditions. An immunoblot using GFP antibody would show whether there is an upward shift in the NHR-49::GFP band size in hypoxic conditions, which should be reversible after CIP treatment of the whole worm extracts. This experiment can also be performed in the hpk-1 mutant background to validate where the NHR-49 phosphorylation is dependent on HPK-1. An alternative way to test this would be immunoprecipitating NHR-49::GFP protein in normoxia and hypoxic conditions and immunoblotting with phospho-Ser and phospho-Thr antibodies.
Reviewer #3 (Recommendations for the authors):
Question on results presented by author:
– Expression of fmo-2 increases in hypoxia (0.5%) in a nhr-49 and hif-1 dependent manner; approach was qRT-PCR and Pfmo-2::gfp reporter in wildtype and mutants.
– Figure 1A- large deviation in fmo-2 mRNA fold change in.5% hypoxia. Any experimental reasons why such is observed?
– The nhr-49 mutant does not survive 24 hrs 0.5% hypoxia (exposed as embryos) well or if exposed as L1 larvae, the post hypoxia larvae do not progress to L4 stage (24-hour exposure, allowed to develop to L4 stage). The nhr-1 mutant a similar survival rate as hif-1 mutants; whereas the double mutant was significantly lower. These data suggest separate pathways and that nhr-49 is required for adaptation to hypoxia.
– Were the nhr-49 L1 larvae exposed to hypoxia arrested or growing slow after hypoxia exposure (eventually reach L4/adult stage)?
– The hydrogen sulfide experiment separated out the role nhr-49 has in stress responses that hif-1 has.
– Authors took a transcriptomic approach (N2, nhr-49 and hif-1 mutants, normoxia/hypoxia). Results show 315 upregulated and 177 downregulated were dependent on nhr-49; 83 upregulated and 51 downregulated were hif-1 independent. Subset of gene expression responses to hypoxia are nhr-49 dependent. Based on transcriptomic data – of the 83 genes that require nhr-49 but not hif-1 for upregulation in hypoxia genes classified as autophagy and detoxification were enriched (Figure 3D).
– Supplementary files- present these as excel files or tables that communicate more data (wormbase ID, gene identification, gene name, gene description, classification based on GO terms or perhaps use wormCat and if category is significantly represented). This will be helpful to the community.
– Hypoxia induced acs-2 expression in a nhr-49 dependent manner is interesting, acs-2 expression was variable in the hif-1 mutant- ideas why? Due to variability in methodology (was RNA collected immediately after hypoxia exposure?)
– Important question- which nhr-49 regulated genes are critical for hypoxia survival. The fmo-2;acs-2 double mutant sensitive to hypoxia (embryo); individually hypoxia survival was overall not reduced. Reduction in autophagy genes via RNAi reduced hypoxia survival (atg-10, atg-7, atg-7, bec-1 and epg-3) in N2 and nhr-49 background.
– Line 235, line 238, Figure 3F- standard deviation not noted in text but the figure indicates it is large – authors have an indication as to why individual experiments vary, what is this due to? Have authors tried longer hypoxia exposures? This may intensify the hypoxia sensitivity and reduce variability in capacity to survive/develop after hypoxia exposure. I am not suggesting to redo all the hypoxia exposure experiments but something to keep in mind and consider to determine essential nature of the autophagy process in hypoxia.
– What is role of autopagy in adaptation to hypoxia? This section a bit disconnected from the fmo-2, acs-2 expression work.
– Line 227- did authors confirm expression changes, in nhr-49 dependent manner, of the autophagy genes using RT-PCR?
– nhr-49(et23) gain of function experiment is of interest.
– Elaborate and provide more details about what is known about this gf mutation. Unclear what a combined gain and loss of function properties indicate (environment dependent?).
– Line 280-281- unclear how other proteins that interact with NHR-49 were "studied"? What was done to identify NHR-67? May need to reword if no experiments conducted to identify NHR-67. Line 285, were other transcription factors that are upregulated in response to hypoxia screened through (RNAi) or did authors focus on nhr-67?
– nhr-67(RNAi) induced expression of fmo-2 and acs-2 (gfp reporters), indicating a negative regulation.
– Did nhr-67(RNAi) have an impact on the autophagy gene expression?
– Include text to provide better logic authors used to assess kinases as the next step in the study. The transition to these studies lacking. Why kinases? Provide text to transition and provide the rationale/relationship between oxidative stress and hypoxia and these particular kinases. Line 328, list the genes in a table with functional description or list in text.
– What is role of these kinases relative to NHR-49; epistasis suggests acting in same pathway. Figure 7- did authors observe any difference in the NHR-49 localization in the hpk-1 mutant? Impact on NHR-49 seemed minimal in hpk-1 mutant. Perhaps images to assess tissue expression better more convincing.
– Figure 8- data didn't demonstrate that HPK-1 dysfunction impacts autophagy genes; are reporters available or assess with RT-PCR.
– Line 545,546- may want to note in result that these assays are actually post-hypoxia experiments. The worm is recovering, unclear if the reporters are upregulated during hypoxia or if this is a post hypoxia expression.
– Wondering why the animal time in hypoxia differed between the RT-PCR/RNA-seq experiments and fluorescent reporter assays (3hrs, 4hrs, respectively; line 527 and 545).
– What was the genome coverage for RNA sequencing experiments? Can authors communicate if RNA isolation was immediately after hypoxia exposure or if animals were exposed to a normoxia recovery time.
https://doi.org/10.7554/eLife.67911.sa1Author response
Based on the individual reviewer comments below, the following points must be addressed in a revised version.
(1) Autophagy. The reviewers feel that the link between hypoxia, nhr-49, and autophagy is one of the most important insights provided by your work. However, the role of autophagy in hypoxia, and its reliance on nhr-49, needs to be better substantiated.
a. As pointed out by Reviewers 2 and 3, the sample size for the experiments in Figure 3F and Supp Figure 4B are quite small. Together with the high variance in your measurements, it is unlikely that these experiments have the power to detect differences that could be present when comparing WT vs nhr-49 backgrounds. Please increase sample sizes here, ideally guided by power size calculations. As Reviewer 3 suggests, it may also be useful to consider altering the experimental conditions.
We agree that the evidence in our initial manuscript did not convincingly link autophagy gene function to hypoxia survival and the nhr-49 pathway. To overcome variability issues with RNAi, we instead studied functional requirements with mutants in two autophagy genes: lgg-2 and epg-6. Analysis of lgg-2 and epg-6 single mutants and double mutants of these genes with the nhr-49 mutant showed that mutation of each of these genes reduced hypoxia resistance, which was not exacerbated by concomitant nhr-49 mutation. This provides strong support for our conclusion that autophagy contributes to hypoxia adaptation via the nhr-49 pathway. These new results are presented in Figure 4E, F. The RNAi data have been moved to Figure 4—figure supplement 1F, G.
b. Please confirm the changes in expression of autophagy genes, and their dependence on nhr-49, by RT-PCR.
We appreciate the need to validate RNA-seq data and attempted to quantify gene expression changes with qRT-PCR. In this experiment, we observed a general increase in autophagy gene expression in wild-type worms exposed to hypoxia; however, these changes were not significant, likely because the increase in expression of each individual gene is relatively small (Author response image 1) .
Author response image 1
qRT-PCR reveals that increase of autophagy gene expression is not significant.
The graph indicates fold changes of mRNA levels in L4 stage wild-type animals exposed to room air (21% O2) or 0.5% O2 for 3 hr (n = 3). Statistics: two-way ANOVA corrected for multiple comparisons using the Tukey method.
As an alternative approach, we studied previously characterized transcriptional reporters (promoter-GFP fusions) of three autophagy genes and quantified their induction in normoxia vs. hypoxia following knockdown of nhr-49, hif-1, hpk-1, or nhr-67. The data show that all three autophagy genes are significantly induced approximately 1.5–2 fold in hypoxia, and that nhr-49 and hpk-1, but not nhr-67 and hif-1, are required for these inductions. This validates our RNA-seq data, and clearly links nhr-49 and hpk-1 to the regulation of autophagy genes in hypoxia. We have added these new data in Figure 4A, B and Figure 4—figure supplement 1B, C.
c. As suggested by reviewer 1, please examine autophagosome formation directly (e.g., with LGG-1::GFP) under hypoxic conditions, in WT, nhr-49, and hif-1 animals.
We thank the reviewer for this excellent suggestion. As requested, we used the LGG-1::GFP reporter to assess autophagosome formation in hypoxia in the WT, nhr-49, and hif-1 mutant backgrounds. Consistent with our gene regulation and functional data, we observed a clear requirement for nhr-49, but not hif-1, in the upregulation of autophagosome formation in hypoxia (new Figure 4C, D).
In addition, we performed a similar experiment using RNAi for hpk-1 and nhr-67. This analysis revealed that hpk-1, but not nhr-67, is required for LGG-1::GFP induction in hypoxia, (Figure 4G).
Collectively we believe that these experiments provide strong support of one of the most important conclusions of our study: that nhr-49 and hpk-1, but not hif-1, are required for autophagy induction and hence survival in hypoxia.
(2) Site of action of NHR-49. Reviewers 1 and 2 raise concerns about the interpretation and significance of these findings.
a. Please provide additional information on the site of NHR-49 induction by hypoxia, e.g., with high-magnification images.
As requested, we have included additional high-magnification images assessing NHR-49 induction in hypoxia. This shows that NHR-49::GFP is induced in the head region, in the hypodermis, and in the intestine (new Figure 2D and Figure 2—figure supplement 1E, F).
b. Please confirm the specificity of the protective effects of NHR-49 overexpression by (i) examining body-wall-specific and pharyngeal-muscle-specific overexpression as suggested by Reviewer 2 OR (ii) using tissue-specific RNAi as suggested by Reviewer 1.
i. We were able to confirm a similar protective effect of NHR-49 rescue in body-wall muscle as in other tissues; these data have been added to Figure 5B. However, we were not able to obtain a pharynx-specific rescue strain as attempts to generate this strain failed for unknown reasons.
ii. We appreciate the suggestion of the reviewer for this interesting experiment and tried it as requested. However, in our hypoxia/normoxia survival assays, the tissue-specific RNAi strains featured baseline sickness and large variability, which prevented us from reliably detecting effects of nhr-49 RNAi on hypoxia survival (see Author response image 2). For this reason, we did not include these results in the manuscript.
Author response image 2
Tissue specific RNAi experiments resulted in highly variable hypoxia resistance.
The graph shows average population survival of rde-1(ne219) (control, RNAi deficient), OLB11 (rde-1(ne219);(pOLB11(elt-2p::rde-1) + pRF4(rol-6(su1006))); intestine-specific RNAi; McGhee et al., 2009), NR350 (rde-1(ne219) V; kzIs20 [hlh-1p::rde-1 + sur-5p::NLS::GFP]; muscle-specific RNAi; Qadota et al., 2007), and NR222 (rde-1(ne219) V; kzIs9 [(pKK1260) lin-26p::NLS::GFP + (pKK1253) lin-26p::rde-1 + rol-6(su1006)]; hypodermis-specific RNAi; Qadota et al., 2007) strains grown on control empty vector RNAi (EV(RNAi)) or nhr-49(RNAi). Worm embryos were exposed for 24 hr to 0.5% O2 and then allowed to recover at 21% O2 for 65 hr, and then scored for ability to reach at least the L3 stage (four repeats totalling >100 individual animals per genotype) (left). As control, we determined developmental success of the same strains on the same RNAi conditions grown in normoxia (right). Statistics: ordinary one-way ANOVA corrected for multiple comparisons using the Tukey method.
(3) RNAseq data. As suggested by Reviewer 1, please clarify the analysis presented in Figure 3A,B and, if possible, compare your results with other hypoxia transcriptome data that may have been previously reported.
We apologize for the confusion in our analysis and description. We have revised the figure legends of Figure 3A, B and the corresponding text on lines 290-295 to better explain which genes belong in which groups.
We also appreciate the idea of comparing our RNA-seq data to previously generated C. elegans hypoxia transcriptome data. Surprisingly, we were not able to find many such datasets. RNA-seq data of wild-type worms exposed to hypoxia have been published (Vozdek et al., 2018). However, the data were not deposited in GEO or similar databases and, upon request, the corresponding author was unable to provide complete gene lists or raw unprocessed data for reanalysis. The only other paper that we found with hypoxia-normoxia transcriptome data is Shen et al., 2005. Comparing our gene list to the list from this paper, we found that 24 of 110 genes identified by Shen are also hypoxia-inducible in our study (new Figure 3—figure supplement 1B). This may appear low, but likely reflects the fact that Shen et al., studied different conditions and used different analysis and processing approaches (L3 larval stage by Shen, L4 by us; 0.1% O2 by Shen, 0.5% O2 by us; microarrays by Shen, RNA-seq by us). We note that our RNA-seq successfully identified several genes that have been shown by other studies to be hypoxia inducible in C. elegans, including nhr-57, egl-9, fmo-2, and F22B5.4 (Bishop et al., 2004; Shen et al., 2005). In sum, we are confident that our hypoxia RNA-seq analysis is of high quality and hope that it will serve as a reference study for future investigations in this area.
(4) Interactions between NHR-67 and NHR-49.
a. As suggested by Reviewers 1 and 2, please examine transcriptional reporters for nhr-49 and nhr-67 in hypoxia and normoxia, in both wild-type and nhr-49/67 mutants.
We thank the reviewers for this suggestion. To examine nhr-67 expression following knockdown of nhr-49, we used the MU1268 strain, which contains an extrachromosomal array wherein the promoter and the first three exons of nhr-67 are linked to GFP (Gissendanner et al., 2004). However, although we did detect significant increase of nhr-67p::gfp in hypoxia in both EV(RNAi) and nhr-49(RNAi) conditions, GFP expression in these worms was extremely low, even after nhr-49 knockdown in hypoxia, and thus we would prefer not to include these data in the manuscript (Author response image 3) .
Author response image 3
Analysis of nhr-67 and nhr-49 expression.
(Left) The graph shows the quantification of GFP levels in nhr67p::gfp animals in normoxia or following 4 hr exposure to 0.5% O2 and 1 hr recovery in 21% O2, on control empty vector RNAi (EV(RNAi)), nhr-49(RNAi), or hif-1(RNAi) (three repeats totalling >30 individual animals per genotype). ** p <0.01 (two-way ANOVA corrected for multiple comparisons using the Tukey method). (Right) The graph indicates relative levels of nhr-49 mRNA in L4 control empty vector RNAi (EV(RNAi)) and nhr-49(RNAi) animals exposed to room air (21% O2) or 0.5% O2 for 3 hr (n = 3). Statistics: two-way ANOVA corrected for multiple comparisons using the Tukey method.
To assess nhr-49 expression following knockdown of nhr-67, we attempted to generate an nhr-49p::gfp transcriptional reporter. However, numerous attempts by two labs (Taubert, Ghazi) failed to generate the appropriate expression vector. As an alternative approach, we assessed nhr-49 mRNA levels in hypoxia and normoxia in the control (empty vector, EV) and nhr-67(RNAi) backgrounds. Consistent with our conclusion that NHR-49 is regulated post-transcriptionally in response to stress, mRNA expression level was similar in all these conditions (Author response image 3).
b. Please investigate genetic interactions: is nhr-67 synthetic lethal with hif-1 and/or nhr-49 in the context of hypoxia survival?
As requested, we tested genetic interactions between these three transcriptional regulators. Specifically, we quantified embryo survival in hypoxia in nhr-49(-);nhr67(RNAi) and hif-1(-);nhr67(RNAi) worm embryos. In line with our previous data, these new experiments showed that nhr-67 acts within the nhr-49 response pathway but shows synthetic lethality with hif-1 (see updated Figure 6—figure supplement 1D).
c. As Reviewer 2 suggests, it would also be useful to determine whether specific subsets of hypoxia response genes are dependent on nhr-67, though this is not considered essential.
We appreciate the interest in the role nhr-67 plays in the hypoxia response. In our revised manuscript, we include new data showing that nhr-67 does not appear to play a regulatory role in autophagy during hypoxia, via both transcriptional reporter assays and autophagosome formation (LGG-1::GFP foci) (new Figures 4A, B, G, Figure 4—figure supplement 1B, C). We have also shown that nhr-67 is important in the negative regulation of fmo-2 and acs-2 (Figures 6B-E). Further exploration of genes and processes regulated by nhr-67 via RNA-seq analysis will be an interesting future direction.
In sum, although some of these experiments were not successful, the available data agree with our conclusion – that NHR-67 acts in the new NHR-49 pathway and in parallel to HIF-1.
(5) Regulation of NHR-49 by HPK-1.
a. The reviewers consider the proposed mechanism to be quite speculative. If possible, it would be ideal for you to determine whether NHR-49 becomes phosphorylated under hypoxia, and whether this depends on hpk-1, as suggested by Reviewer 2, or whether the localization of NHR-49 changes under these conditions, as suggested by Reviewer 3. Alternatively, please modify the text and figures to make it clear that this part of your model has not been tested and that the regulation of nhr-49 by hpk-1 could be indirect.
We attempted to investigate how HPK-1 may regulate NHR-49, but pilot experiments to study effects on NHR-49 did not reliably identify bands that might reflect phosphorylated NHR-49. We have instead substantially toned down our conclusions to indicate the speculative nature of this part of our model (see text line 747-750 and Figure 9).
b. To substantiate the connections between hpk-1, nhr-49, and autophagy, please determine whether the upregulation of autophagy genes requires hpk-1.
We agree that the role of hpk-1 in autophagy regulation needed further testing, and have done as requested. As noted in the response to comment 1b, hypoxia induction of autophagy gene expression (promoter-GFP reporters) and of autophagosome formation (LGG-1::GFP foci) showed strong reliance on hpk-1, supporting our core findings (Figure 4A, B, G, Figure 4—figure supplement 1B, C).
(6) In a number of places, the interpretation of your results should be softened. One example of this is lines 150-151 (Figure S1B). ~95% of WT animals are adults at 48 hr but 85% of nhr-49 mutants are still L3 and L4. However, you claim that "mutants did not significantly develop slower than wildtype". If so, a different test needs to be carried out, or sample sizes need to be increased, because it seems clear that there is a difference in developmental rate between these groups. Other examples where conclusions need to be tempered are provided in the reviewer comments below.
We apologize for overinterpretation and have changed the wording in this (line 187-209) and other instances.
In addition to these points, please also consider the other issues raised by the reviewers (see below) as you prepare your revision.
Reviewer #1 (Recommendations for the authors):
1. To address Major Concern 2, the authors should confirm their RNA-seq results on new samples via qRT-PCR and also test whether autophagosomes are formed under hypoxic conditions (LGG-1::GFP), which should be dependent on nhr-49 but not hif-1 (if the authors model is true). The authors should expand their discussion to explain how increased autophagy would protect against hypoxia (metabolic shift for anaerobic respiration, decreased proteome reduces energetic demands, etc.).
As requested, we have performed validation experiments for our RNA-seq and studied autophagosomes, as detailed above (Editor Comment #1). To address putative protective effects of autophagy in hypoxia, we have edited the text in the discussion on lines 892-900.
2. To address Major Concerns 3 and 4, the authors should use tissue specific RNAi or hairpins to inactivate nhr-49 and assess whether loss in any one tissue compromises hypoxic response/survival. Additionally, if ectopic overexpression of NHR-49 within one tissue does indirectly protect other tissues through an adaptive response, one would predict that overexpressing NHR-49 in one tissue would mute the induction of NHR-49 a neighboring tissue after hypoxic treatment. This could be tested easily via combining their nhr-49p::NHR-49::GFP reporter and tissue specific overexpression of NHR-49. The authors should consider testing this possibility.
With regards to the use of tissue specific RNAi, please see above (Editor Comment # 2b).
With regards to the second proposed experiment, this is not feasible because the tissue-specific NHR-49 overexpression strains also all bear the NHR-49 GFP fusion as well as the same transgenic marker; these existing strains can therefore not be used to generate the proposed double transgenic strains by crossing. In addition, the nhr-49p::NHR-49::GFP strain already substantially overexpresses NHR-49, and we hesitate introducing another gain-of-function transgene into this genetic context; interpretation of resulting data might be tricky, as pointed out by the reviewer themselves. We have therefore opted not to pursue this line of investigation.
3. I strongly suggest avoiding the use of "worms" in formal manuscripts and use "animals" instead. It's jargon and diminishes the impact of C. elegans research when read by scientists using other systems.
We appreciate the comment and have corrected the terminology throughout the manuscript.
Reviewer #2 (Recommendations for the authors):
– Figure 3F – These results are very crucial for the main conclusion of this study. Since RNAi produces more variable results across biological replicates, this experiment should be repeated with some of the autophagy mutants such as unc-51 etc.
We thank this reviewer for this suggestion. As requested, we have done these experiments using mutants in autophagy genes. Please see above (Editor Comment #1a) for details.
– The sample sizes for the RNAi experiments in Figure 3F do not seem to be enough to capture the differences in hypoxia survival between WT and nhr-49 mutant conditions: 41% vs 25% (atg-7), 27% vs 13% (bec-1) and 38% vs 13% (epg-3). None of these differences were detected as statistically significant. This is likely due to n = 4 for RNAi treatments of the nhr-49 mutant and very high variability across biological replicates. The authors should at least double their sample sizes for these conditions and test whether there are any significant differences in hypoxia survival between WT and nhr-49 mutant when autophagy is inhibited. Similarly, in Supplementary Figure 4B, some of the autophagy gene RNAi conditions seem to reduce survival by 25% (with some data points showing greater than 50% reduction) even in normoxia. None of these differences were detected as statistically significant, which again is likely due to low sample sizes and high variability across replicates. These experiments need to be carefully designed so that there is enough statistical power to discern whether nhr-49 and autophagy are regulating hypoxia survival via the same or independent pathways and whether autophagy inhibition reduces survival also in normoxia.
We agree and in our revised manuscript have redone this analysis with autophagy mutant strains, which strongly supports our conclusions. Please see above for details (Editor Comment #1a).
– For tissue-specific rescue of nhr-49 (data shown in Figure 4B), the authors should also perform a body wall muscle-specific rescue experiment. Since nhr-49 is expressed in the muscles, it will be informative to know whether body wall muscle-specific nhr-49 function is sufficient to improve hypoxia survival at the organism level. In addition, a pharyngeal muscle-specific nhr-49 rescue might serve as a good negative control, where tissue-specific rescue might not be able to affect whole organism hypoxia survival.
As requested we have added experiments with a body wall muscle-specific rescue strain, which also provides functional rescue. Please see above for details (Editor Comment # 2b).
– To confirm the bidirectional regulation between nhr-49 and nhr-67, the authors should create a gfp transcriptional reporter for nhr-67 and test whether nhr-49 inhibition affects nhr-67 expression in hypoxia and normoxia. The RNA-seq data shown in Figure 5A suggests that NHR-49 possibly regulates nhr-67 expression only in hypoxic conditions, but it will be informative to know whether this regulation is restricted to specific tissues. In comparison, NHR-67 seems to be regulating nhr-49 expression predominantly in the intestine in both normoxia and hypoxia (line 309).
As requested we have further investigated the relationship between NHR-49 and NHR-67. Please see above (Editor Comment #4a).
– The physiological relevance of the bidirectional negative regulation between nhr-49 and nhr-67 is not clear. Inhibition of both nhr genes reduces survival in hypoxic conditions (Supplementary Figure 5D). While this study has found downstream targets of nhr-49 that promote hypoxia survival, it is not clear how nhr-67 fits into this regulation. In this regard, the authors should investigate two directions: (1) does nhr-67 also show synthetic lethal interactions with hif-1 and nhr-49 in the context of hypoxia survival (similar to Figure 2A)?, and (2) does nhr-67 inhibition in hypoxic conditions prevent the activation of a specific subset of hypoxia response genes, such as genes involved in detoxification and autophagy?
We further examined crosstalk between nhr-67 and the other two regulators; in response to (1), our data suggest that nhr-67 loss interacts genetically with hif-1 loss, showing that they act in separate pathways; in response to (2), we observed that depletion of nhr-67 did not affect the induction of autophagy gene reporters or autophagosomes by hypoxia. For details, please see above (Editor Comment #4b, 4c).
– The authors propose that survival during hypoxia is predominantly due to the upregulation of autophagy genes, but not due to upregulation of fmo-2 and acs-2 (lines 222-224). However, they have only tested whether hpk-1 removal alters the expression of fmo-2 and acs-2 genes (Figures 6A-E). To mechanistically elucidate whether HPK-1 and NHR-49 are acting via the same pathway to promote hypoxia survival (as suggested in Figure 6F), it should be tested whether hpk-1 mutation impairs the upregulation of autophagy genes during hypoxia.
As requested, we not only tested whether hpk-1 regulates the expression of autophagy related genes in hypoxia, but also assessed whether it is required for autophagosome formation, both of which excitingly is the case, in agreement with our model. For details, please see above (Editor Comment #1b, 3b).
– The role of HPK-1 in stabilizing NHR-49 during hypoxia is a novel finding that is of broad interest. In its current form, the proposed mechanism is quite speculative. Since this regulation is central to the proposed HIF-1-independent hypoxia response pathway, the authors should investigate whether NHR-49 is phosphorylated in low oxygen conditions in an HPK-1-dependent manner. A simple way to test this would be to use the nhr-49::gfp translational reporter strain and prepare whole worm extracts in normoxia and hypoxia conditions. An immunoblot using GFP antibody would show whether there is an upward shift in the NHR-49::GFP band size in hypoxic conditions, which should be reversible after CIP treatment of the whole worm extracts. This experiment can also be performed in the hpk-1 mutant background to validate where the NHR-49 phosphorylation is dependent on HPK-1. An alternative way to test this would be immunoprecipitating NHR-49::GFP protein in normoxia and hypoxic conditions and immunoblotting with phospho-Ser and phospho-Thr antibodies.
We attempted to investigate NHR-49 phosphorylation but were unable to conclusively identify phosphorylated NHR-49. In line with the suggestion of the editor, we have therefore tempered our interpretation of the role of HPK-1 in NHR-49 regulation. For details, please see above (Editor Comment #5a).
Reviewer #3 (Recommendations for the authors):
Question on results presented by author:
– Expression of fmo-2 increases in hypoxia (0.5%) in a nhr-49 and hif-1 dependent manner; approach was qRT-PCR and Pfmo-2::gfp reporter in wildtype and mutants.
– Figure 1A- large deviation in fmo-2 mRNA fold change in.5% hypoxia. Any experimental reasons why such is observed?
We agree with the reviewer that these deviations are rather large; however, importantly, they are statistically significant. We do not have a clear explanation for the large variability – worms are all at the same stage, and are exposed for same time. Potentially, post-hypoxia harvesting procedures vary by minutes such that changes manifest due to normoxia exposure during the washing and harvesting procedure.
– The nhr-49 mutant does not survive 24 hrs 0.5% hypoxia (exposed as embryos) well or if exposed as L1 larvae, the post hypoxia larvae do not progress to L4 stage (24-hour exposure, allowed to develop to L4 stage). The nhr-1 mutant a similar survival rate as hif-1 mutants; whereas the double mutant was significantly lower. These data suggest separate pathways and that nhr-49 is required for adaptation to hypoxia.
– Were the nhr-49 L1 larvae exposed to hypoxia arrested or growing slow after hypoxia exposure (eventually reach L4/adult stage)?
We did not test whether arrested worms eventually reach adulthood. Anecdotally, we have observed that worms able reach the L3 or L4 stage eventually develop into adults, whereas animals arrested at the L1 or L2 stages remain permanently arrested.
– The hydrogen sulfide experiment separated out the role nhr-49 has in stress responses that hif-1 has.
– Authors took a transcriptomic approach (N2, nhr-49 and hif-1 mutants, normoxia/hypoxia). Results show 315 upregulated and 177 downregulated were dependent on nhr-49; 83 upregulated and 51 downregulated were hif-1 independent. Subset of gene expression responses to hypoxia are nhr-49 dependent. Based on transcriptomic data – of the 83 genes that require nhr-49 but not hif-1 for upregulation in hypoxia genes classified as autophagy and detoxification were enriched (Figure 3D).
– Supplementary files- present these as excel files or tables that communicate more data (wormbase ID, gene identification, gene name, gene description, classification based on GO terms or perhaps use wormCat and if category is significantly represented). This will be helpful to the community.
We agree with the reviewer that these files will be important for the community and provide them in our revised manuscript as Supplementary File 3.
– Hypoxia induced acs-2 expression in a nhr-49 dependent manner is interesting,
acs-2 expression was variable in the hif-1 mutant- ideas why? Due to variability in methodology (was RNA collected immediately after hypoxia exposure?)
We do not know why acs-2 expression is somewhat variable. Methodology was kept as consistent between individual experimental repeats as possible.
– Important question- which nhr-49 regulated genes are critical for hypoxia survival. The fmo-2;acs-2 double mutant sensitive to hypoxia (embryo); individually hypoxia survival was overall not reduced. Reduction in autophagy genes via RNAi reduced hypoxia survival (atg-10, atg-7, atg-7, bec-1 and epg-3) in N2 and nhr-49 background.
– Line 235, line 238, Figure 3F- standard deviation not noted in text but the figure indicates it is large – authors have an indication as to why individual experiments vary, what is this due to? Have authors tried longer hypoxia exposures? This may intensify the hypoxia sensitivity and reduce variability in capacity to survive/develop after hypoxia exposure. I am not suggesting to redo all the hypoxia exposure experiments but something to keep in mind and consider to determine essential nature of the autophagy process in hypoxia.
We agree that it was important to provide further evidence showing that autophagy genes are important for hypoxia adaptation and have done as requested. For details, please see above (Editor Comment #1a).
We appreciate the idea to performing longer hypoxia survival assays to reduce variability, and we have done so in Figure 5A. However, this also reduces WT survival, and therefore is not suitable for all assays.
– What is role of autopagy in adaptation to hypoxia? This section a bit disconnected from the fmo-2, acs-2 expression work.
We agree that this needed to be evaluated further and have extensively done so. Please see above (Reviewer #1, Major Suggestion #1).
– Line 227- did authors confirm expression changes, in nhr-49 dependent manner, of the autophagy genes using RT-PCR?
As requested, in our revision, we confirm gene expression changes by means of promoter-GFP reporter analysis. For details, please see above (Editor Comment #1b).
– nhr-49(et23) gain of function experiment is of interest.
– Elaborate and provide more details about what is known about this gf mutation. Unclear what a combined gain and loss of function properties indicate (environment dependent?).
We agree that this was somewhat unclear. This mutant has been characterized in detail in Lee et al., 2016. We have added a reference to that extent.
– Line 280-281- unclear how other proteins that interact with NHR-49 were "studied"? What was done to identify NHR-67?
We apologize for the oversight. Interaction between NHR-49 and NHR-67 was identified in a previous study (Reece-Hoyes et al., 2013). We have added text to that extent on lines 523-526.
May need to reword if no experiments conducted to identify NHR-67. Line 285, were other transcription factors that are upregulated in response to hypoxia screened through (RNAi) or did authors focus on nhr-67?
We apologize that this was not clear. We examined in our RNA-seq data the expression of TFs known to interact with NHR-49 (from Reece-Hoyes et al., 2013) and noticed that nhr-67 showed an interesting pattern of regulation. We have clarified this further in the manuscript on lines 523-526.
– nhr-67(RNAi) induced expression of fmo-2 and acs-2 (gfp reporters), indicating a negative regulation.
– Did nhr-67(RNAi) have an impact on the autophagy gene expression?
As requested we addressed whether nhr-67 loss affects autophagy gene regulation (new Figure 4A, B, Figure 4—figure supplement 1B, C). For details, please see above (Editor Comment #1b).
– Include text to provide better logic authors used to assess kinases as the next step in the study. The transition to these studies lacking. Why kinases? Provide text to transition and provide the rationale/relationship between oxidative stress and hypoxia and these particular kinases. Line 328, list the genes in a table with functional description or list in text.
We apologize for the unclear rationale. We investigated kinases because they are known to act upstream of many stress responsive transcription factors. We have added text to this extent on lines 633-636.
With regards to the genes tested, we did not screen them for hypoxia related action. Instead, we performed a separate screen for a different project on NHR-49 in stress response pathways (manuscript currently in preparation). For this reason, we prefer not to disclose the full list of genes identified in the present manuscript, wherein we focused solely on HPK-1.
– What is role of these kinases relative to NHR-49; epistasis suggests acting in same pathway. Figure 7- did authors observe any difference in the NHR-49 localization in the hpk-1 mutant? Impact on NHR-49 seemed minimal in hpk-1 mutant. Perhaps images to assess tissue expression better more convincing.
We agree and provide higher magnification images. For details, please see above (Editor Comment #2a).
– Figure 8- data didn't demonstrate that HPK-1 dysfunction impacts autophagy genes; are reporters available or assess with RT-PCR.
As requested, we used transcriptional reporters to provide comprehensive evidence that HPK-1 affects the regulation of autophagy genes in hypoxia as well as autophagosome formation. For detail, please see above (Editor Comment #1a, b).
– Line 545,546- may want to note in result that these assays are actually post-hypoxia experiments. The worm is recovering, unclear if the reporters are upregulated during hypoxia or if this is a post hypoxia expression.
We appreciate the astute comment of the reviewer. In pilot experiments, anecdotally, we observe slight upregulation directly after hypoxia; however, most of the GFP is made only once the worms return to normoxia because many of the translation steps involved in GFP production require oxygen. In addition, oxygen is required for maturation of the GFP fluorophore. We have added text to this extent in the methods on lines 1014-1016.
– Wondering why the animal time in hypoxia differed between the RT-PCR/RNA-seq experiments and fluorescent reporter assays (3hrs, 4hrs, respectively; line 527 and 545).
We used a longer timepoint in the experiments with GFP-reporters because not only mRNA but also GFP protein has to be produced. We performed several pilot experiments and settled on the incubation times where we observed the most difference for each type of experiment.
– What was the genome coverage for RNA sequencing experiments? Can authors communicate if RNA isolation was immediately after hypoxia exposure or if animals were exposed to a normoxia recovery time.
Worms were frozen immediately after hypoxia, with no recovery time. We sequenced >20 million reads per sample. Details of this procedure have been added to the methods section on lines 1111-1112.
https://doi.org/10.7554/eLife.67911.sa2Article and author information
Author details
Funding
National Institutes of Health (R56AG066682)
- Arjumand Ghazi
Natural Sciences and Engineering Research Council of Canada (RGPIN-2018-05133)
- Stefan Taubert
National Institutes of Health (R01AG051659)
- Arjumand Ghazi
Cancer Research Society (22727)
- Stefan Taubert
BC Children's Hospital Foundation
- Kelsie RS Doering
- Stefan Taubert
Canada Research Chairs
- Stefan Taubert
National Institutes of Health (R01AG044378)
- Dana L Miller
Natural Sciences and Engineering Research Council of Canada
- Kelsie RS Doering
Canadian Institutes of Health Research (PJT-153199)
- Stefan Taubert
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Acknowledgements
We thank the Taubert, Miller, and Ghazi labs for comments on the manuscript. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). Some strains were provided by The National BioResource Project (NBRP). Grant support was from The Canadian Institutes of Health Research (CIHR; PJT-153199 to ST), the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN-2018-05133 to ST), the Cancer Research Society (CRS; to ST), and the National Institutes of Health (NIH; R01AG051659 and R56AG066682 to AG, R01AG044378 to DM). KRSD was supported by NSERC CGS-M, NSERC CGS-D, and BCCHR scholarships, and ST by a Canada Research Chair and a BCCHR IGAP award.
Senior Editor
- Piali Sengupta, Brandeis University, United States
Reviewing Editor
- Douglas Portman, University of Rochester, United States
Version history
- Preprint posted: February 24, 2021 (view preprint)
- Received: February 26, 2021
- Accepted: March 12, 2022
- Accepted Manuscript published: March 14, 2022 (version 1)
- Version of Record published: March 28, 2022 (version 2)
- Version of Record updated: January 5, 2023 (version 3)
Copyright
© 2022, Doering et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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Nuclear hormone receptor NHR-49 acts in parallel with HIF-1 to promote hypoxia adaptation in Caenorhabditis elegans
eLife 11:e67911.
https://doi.org/10.7554/eLife.67911
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