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NHR-14 loss of function couples intestinal iron uptake with innate immunity in C. elegans through PQM-1 signaling

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Cite this article as: eLife 2019;8:e44674 doi: 10.7554/eLife.44674

Abstract

Iron is essential for survival of most organisms. All organisms have thus developed mechanisms to sense, acquire and sequester iron. In C. elegans, iron uptake and sequestration are regulated by HIF-1. We previously showed that hif-1 mutants are developmentally delayed when grown under iron limitation. Here we identify nhr-14, encoding a nuclear receptor, in a screen conducted for mutations that rescue the developmental delay of hif-1 mutants under iron limitation. nhr-14 loss upregulates the intestinal metal transporter SMF-3 to increase iron uptake in hif-1 mutants. nhr-14 mutants display increased expression of innate immune genes and DAF-16/FoxO-Class II genes, and enhanced resistance to Pseudomonas aeruginosa. These responses are dependent on the transcription factor PQM-1, which localizes to intestinal cell nuclei in nhr-14 mutants. Our data reveal how C. elegans utilizes nuclear receptors to regulate innate immunity and iron availability, and show iron sequestration as a component of the innate immune response.

https://doi.org/10.7554/eLife.44674.001

Introduction

Iron is an essential nutrient required by nearly all organisms for biological processes including DNA replication, mitochondrial respiration, and oxygen transport (Anderson et al., 2012). As a redox active element, iron also has a role in the production of reactive oxygen species (ROS), which can be deleterious to cellular proteins, DNA and membranes. In humans, tight regulation of cellular iron content is essential, as iron deficiency impairs neurological development in infants and causes anemia in adults, while iron overload leads to cardiomyopathy, diabetes mellitus, and neurodegenerative diseases (Fleming and Ponka, 2012). Excess iron also predisposes humans to infectious diseases, and limiting iron availability to microbes is a critical host innate immune response (Lopez and Skaar, 2018; Weiss and Carver, 2018). Given the dual nature of iron in healthy and diseased states, it is critical for organisms to maintain cellular iron concentration in a narrow physiological range.

C. elegans has emerged as a useful model for studying iron metabolism. In addition to its genetic tractability, ease of maintenance and susceptibility to human pathogens, key proteins involved in mammalian iron uptake, storage and efflux are conserved in C. elegans (Anderson and Leibold, 2014). C. elegans express the divalent metal transporter SMF-3 (Au et al., 2009; Bandyopadhyay et al., 2009; Romney et al., 2011), the iron-storage protein ferritin (FTN-1, FTN-2) (Gourley et al., 2003; Kim et al., 2004; Romney et al., 2008; Valentini et al., 2012) and the iron export protein ferroportin (FPN-1.1–3), which are orthologous to human DMT1 (SLC11A2, NRAMP2), ferritin-H and -L subunits (FTH1, FTL) and ferroportin (SLC40A1), respectively. Iron is transported across the apical enterocyte membrane by SMF-3/DMT1 and is used within the enterocyte for the metalation of iron-containing proteins and by mitochondria for Fe-S cluster biosynthesis (Braymer and Lill, 2017; Rouault, 2015). Iron not utilized is stored in ferritin within the enterocyte or exported across the basolateral enterocyte membrane by ferroportin/FPN-1.1. Ferroportin is the sole iron exporter in mammals (Ward and Kaplan, 2012), unlike C. elegans that express three ferroportin orthologs (FPN-1.1–3) that are not well characterized. Like mammals, intestinal iron absorption in C. elegans is upregulated during iron deficiency by HIF-transcriptional activation of smf-3/DMT1 (C. elegans, HIF-1; human, HIF-2α, EPAS1) (Mastrogiannaki et al., 2009; Romney et al., 2008; Shah et al., 2009). One difference between iron sequestration in C. elegans and vertebrates is that during iron limitation C. elegans ferritin is transcriptionally repressed by HIF-1 (Ackerman and Gems, 2012; Romney et al., 2011), while vertebrate ferritin is translationally repressed by the iron-regulatory proteins (IRPs) (Anderson et al., 2012). In addition to iron, ftn-1 is regulated by the insulin/IGF-like (IIS) pathway (Ackerman and Gems, 2012), the REF-1 transcription factor HLH-29 (Quach et al., 2013), and by the serum/glucocorticoid-regulated kinase (SGK-1) to regulate lipid and iron homeostasis (Wang et al., 2016). The regulation of iron uptake by SMF-3 and iron sequestration by ferritin ensures that cells acquire adequate iron to satisfy their needs while limiting iron toxicity. Precise maintenance of cellular iron homeostasis is underscored by the shortened life span of C. elegans exposed to high iron (Gourley et al., 2003; Valentini et al., 2012).

Iron is required in the establishment of most pathogen infections. Pathogens use a variety of mechanisms to acquire iron from the host, one of which is the synthesis and release of siderophores that bind iron, allowing its uptake by bacteria (Palmer and Skaar, 2016). In response to pathogens, hosts have evolved strategies to limit iron availability that include the production of metal sequestering proteins, the sequestration of iron in ferritin and the regulation of metal transporter function (Lopez and Skaar, 2018; Wessling-Resnick, 2015). C. elegans has provided a valuable genetic model to study host-pathogen interactions because many human pathogens, such as Pseudomonas aeruginosa, Salmonella enterica and Staphylococcus aureus, cause lethal intestinal infections in C. elegans (Cohen and Troemel, 2015). Although C. elegans lack an adaptive immune system, these organisms rely on evolutionarily conserved innate immune systems, such as the p38-mitogen-activated protein kinase (MAPK), insulin and transforming growth factor-β (TGF-β) signaling pathways, for sensing and resolving bacterial infections (Irazoqui et al., 2010; Kim and Ewbank, 2015). Similar to vertebrates, C. elegans use diverse mechanisms to withhold iron and other metals during pathogen infections. FTN-2 provide resistance to S. aureus and E. coli LF82 infections (Simonsen et al., 2011) and SMF-3 provides resistance to S. aureus infections (Bandyopadhyay et al., 2009). In addition, C. elegans respond to pyoverdine, an iron siderophore produced by P. aeruginosa, by upregulation of a mitochondrial surveillance pathway that is triggered by pyoverdine-induced mitochondrial damage (Kang et al., 2018; Kirienko et al., 2015; Tjahjono and Kirienko, 2017). These studies highlight iron limitation as an important component of innate immune response in controlling pathogen infections.

In this report, we identify NHR-14, a nuclear receptor (NR) as a transcriptional repressor of SMF-3-dependent iron uptake and the innate immune response. Loss of nhr-14 promotes the nuclear localization of PQM-1, which binds to GATA-like DAF-16-associated elements (DAEs) in the smf-3 promoter to activate smf-3 transcription. nhr-14 mutants exhibit enhanced resistance to P. aeruginosa infection that in part requires smf-3 as well as other innate immune response genes. Our data implicate NHR-14 as a repressor of iron uptake and the transcriptional innate immune response mediated by PQM-1, showing iron sequestration as an important component of innate immunity in C. elegans.

Results

Loss of NHR-14 rescues low iron growth of hif-1(ia4) mutants

We previously reported that hif-1(ia4) mutant worms are developmentally delayed when grown under iron limiting conditions using the iron chelator, 2,2-bipyridyl (BP), and that this delay was suppressed by elevating intracellular iron by reducing ftn-1 and ftn-2 expression (Romney et al., 2011). To discover novel genes involved in iron homeostasis, a suppressor screen was performed to identify mutations that rescued the low iron phenotype observed in hif-1(ia4) null animals. The ia4 mutation is a 1,231 bp deletion of the second, third, and fourth exons that deletes much of the bHLH and PAS domains in HIF-1, and likely results in complete loss of hif-1 function (Jiang et al., 2001). This screen led to the isolation of several suppressor mutants, of which nhr-14(qa6909) and nhr-14(qa6910) were identified as strong suppressors of the hif-1(ia4) low iron phenotype (Figure 1A). The causative mutation within these lines was determined using whole genome sequencing and Hawaiian strain SNP mapping (see Materials and methods). Both the qa6909 and qa6910 mutations displayed a similar ~1 Mb region of the X chromosome that have a Hawaiian/Bristol reads ratio of close to zero (Figure 1B). Sequence analysis revealed that the qa6909 and qa6910 mutant strains possess distinct mutations within the nuclear-hormone receptor gene nhr-14 (T01B10.4) (Figure 1C). DNA from qa6909 mutants showed a G > A substitution that changes Trp49 to a STOP codon and DNA from qa6910 mutants showed a G > A substitution that changes Gly33 to Arg (Figure 1C).

Mutations in nhr-14 rescue the low iron developmental delay in hif-1(ia4) mutants.

(A) Representative images of wild-type N2 worms, hif-1(ia4) mutants and hif-1(qa6909) and hif-1(qa6910) mutant alleles grown for 5 days on NGM plates containing 25 uM BP (bipyridyl, iron chelator). Worm length is expressed as a percentage of wild-type N2 length (n = 2 biological replicates with 10 worms per strain). Values are expressed as mean ± SEM and were compared by unpaired two-tailed Student’s t-test, *p<0.05. (B) CloudMap SNP Mapping are shown as XY-scatter plots where the ratio of Hawaiian SNPs to Bristol SNPs is represented for the qa6909 and qa6910 mutations. (C) Protein alignment of the DNA-binding domains for human HNF4α and NHR-14 with locations of the qa6909 and qa6910 mutations (adapted from Figure 1 from Antebi, 2006, published under the terms of the Creative Commons Attribution License – CC-By 2.5 license; https://creativecommons.org/licenses/by/2.5/). Amino acid mutations are indicated in red. (D) Representative images of wild-type N2, hif-1(ia4), nhr-14(tm1473) and hif-1(ia4); nhr-14(tm1473) worms grown in NGM or NGM-25 uM BP (low iron) after 5 days. Worm length is expressed as a percentage of wild-type N2-NGM length (one experiment, n = 20 - 30 worms per strain). Values are expressed as mean ± SEM and compared by two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05; ***p < 0.001. Scale bars, 500 um. 

https://doi.org/10.7554/eLife.44674.002

nhr-14 is predicted to encode a protein homologous to the HNF4 family of nuclear receptors, which are transcription factors that regulate gene expression in response to nutritional, metabolic and environmental signals (Taubert et al., 2011). Amino acid sequence alignment between NHR-14 and human HNF4α showed a highly conserved N-terminal zinc finger DNA-binding domain (DBD) and a conserved C-terminal ligand-binding domain (LBD) (Figure 1C). The qa6909 mutation (Trp49STOP) located within the DBD is predicted to result in a truncated protein that lacks a significant portion of the DBD and the entire LBD. The qa6910 missense mutation (Gly33Arg), which is also located within the DBD, changes non-polar glycine to positively-charged arginine (Figure 1C). Gly33 is conserved in HNF4 receptors found in vertebrates and Drosophila, and it is likely that this substitution disrupts DNA binding.

To verify that mutations in nhr-14 are responsible for rescuing the low iron developmental delay of hif-1(ia4) mutants, we crossed nhr-14(tm1473) mutants into the hif-1(ia4) background, and grew hif-1(ia4); nhr-14(tm1473) double mutants on low iron (NGM-BP) plates. The tm1473 mutation is a 409 bp deletion of the third exon and intron that results in a premature stop codon and deletion of the ligand-binding domain that likely results in nhr-14 loss of function. hif-1(ia4); nhr-14(tm1473) double mutants developed normally under iron limitation (Figure 1D), showing that loss of nhr-14 function is responsible for the rescue observed in qa6909 and qa6910 mutant animals.

NHR-14 is highly expressed in intestine and head neurons and is not regulated by iron

Given that the mutations in nhr-14 were identified in a screen for mutants that rescued the low iron developmental delay of hif-1(ia4) mutants, we questioned whether nhr-14 mRNA or protein is regulated by changes in iron status. We generated a transgenic line carrying a nhr-14 fosmid transgene that utilizes the endogenous cis-elements and promoter of nhr-14 to drive the expression of NHR-14 tagged with GFP and 3xFLAG. We validated NHR-14::GFP::FLAG as a functional replacement for endogenous NHR-14 by injecting hif-1(ia4); nhr-14(tm1473) double mutants with the nhr-14 fosmid transgene. These transgenic worms displayed the hif-1(ia4) developmental delay phenotype when grown under iron limitation (Figure 2—figure supplement 1). Fluorescent imaging of NHR-14::GFP::FLAG worms showed that it is highly expressed in intestinal nuclei and in cells in the head (Figure 2A–C). Western blot analysis showed a protein of ~80 kDa in worm lysates consistent with the predicted mass of NHR-14::GFP::FLAG (Figure 2D). No significant change in levels of NHR-14::GFP::FLAG and nhr-14 mRNA were observed in wild-type N2 worms grown in low iron (NGM-BP) or high iron (NGM- ferric ammonium citrate, FAC) compared to NGM (Figure 2D–E). Furthermore, nuclear localization of NHR-14::GFP::FLAG was not affected by iron or by BP (Figure 2—figure supplement 2). The enrichment of nhr-14 in intestinal and head cells is consistent with intestinal and neuronal expression of nhr-14 determined by single-cell RNA sequence and SAGE analyses and (Cao et al., 2017; McGhee, 2007).

Figure 2 with 2 supplements see all
NHR-14 is highly expressed within intestinal and head cell nuclei and is not regulated by iron.

(A) NHR-14::GFP::FLAG expression in young adult transgenic worms assessed by confocal microscopy (40x). Open arrows, intestinal nuclei; red arrows, head cells. (B) GFP expression of NHR-14::GFP::FLAG in cells in the (B) head and (C) intestine (60x). (D) Western blot analysis of NHR-14::GFP::FLAG non-injected worms (unc-119) versus NHR-14::GFP::FLAG worms grown in NGM, NGM-BP (low iron) and NGM-FAC (high iron) (two biological replicates are shown); right panel, quantification of NHR-14::GFP::FLAG expression (n = 3 biological replicates). The blot was simultaneously probed with FLAG and β-tubulin antibodies. (E) qPCR of nhr-14 expression in wild-type N2 adults grown in NGM, NGM-BP and NGM-FAC normalized to N2 worms grown in NGM (n = 3 biological replicates). Values are expressed as mean ± SEM and are compared by unpaired two-tailed Student’s t test; ns, not significant.

https://doi.org/10.7554/eLife.44674.003

Loss of NHR-14 regulates iron uptake by SMF-3

To determine the mechanism through which nhr-14(tm1473) mutants rescued the low iron developmental delay of hif-1(ia4) mutants, we compared the transcriptional profiles of L4-stage nhr-14(tm1473) mutants with wild-type N2 worms using RNA-seq. We identified 834 differentially expressed genes that changed at least two-fold compared to wild-type N2 worms at a 5% false discovery rate (FDR), 573 of which were upregulated and 261 downregulated in nhr-14(tm1473) mutants (Figure 3—source data 1). Notably, the primary intestinal iron transporter smf-3 is one of the most highly upregulated genes in nhr-14(tm1473) mutants. This observation suggested increased iron uptake as a possible mechanism to explain rescue of hif-1(ia4) mutants under iron limitation.

We confirmed smf-3 upregulation in nhr-14(tm1473) single mutants and in hif-1(ia4); nhr-14(tm1473) double mutants compared to wild-type N2 worms, and smf-3 downregulation in hif-1(ia4) mutants by qPCR (Figure 3A). Consistent with our previous study, hif-1(ia4) and smf-3(ok1035) mutants displayed reduced iron content compared to wild-type N2 worms (Romney et al., 2011) (Figure 3B). Iron content increased in nhr-14(tm1473) mutants consistent with increased smf-3 expression, and decreased in smf-3(ok1035); nhr-14(tm1473) double mutants (Figure 3B). smf-3(ok1035) single mutants, smf-3(ok1035); nhr-14(tm1473) double mutants and smf-3(ok1035); hif-1(ia4); nhr-14(tm1473) triple mutants also displayed a developmental delay under iron limitation showing that smf-3 upregulation in nhr-14(tm1473) mutants is required for the rescue of hif-1(ia4) mutants (Figure 3C). Given that the smf-3(ok1035) single mutant alone displays a developmental delay under iron limitation, this suggests that other genes in nhr-14(tm1473) mutants might also contribute to the rescue of hif-1(ia4) mutants. Further evidence for SMF-3-mediated iron uptake as a mechanism to explain the rescue of hif-1(ia4) mutants under iron limitation is the survival advantage of nhr-14(tm1473) mutants grown in low iron (BP) as compared to wild-type N2 worms (Figure 3D). Collectively, these results are consistent with NHR-14 as a negative regulator of SMF-3-mediated iron uptake.

Loss of NHR-14 regulates iron uptake by SMF-3.

(A) qPCR of smf-3 expression measured in wild-type N2, nhr-14(tm1473) and hif-1(ia4) single mutants, and hif-1(ia4); nhr-14(tm1473) double mutants grown in NGM (n = 3 biological replicates). Values are expressed as fold difference compared to wild-type N2 worms. (B) Total iron content of wild-type N2 worms, hif-1(ia4), nhr-14(tm1473and smf-3(ok1035) single mutants, and smf-3(ok1035); nhr-14(tm1473) double mutants grown in NGM and quantified by ICP-OES (n = 3 biological replicates). For A-B, values are expressed as mean ± SEM, and compared by unpaired two-tailed Student’s t-test for (A) and a two-way ANOVA with Tukey’s multiple comparisons test for (B), *p<0.05, **p<0.01, ***p<0.001. (C) Representative images and quantitation of wild-type N2 worms and indicated mutant strains grown on NGM or NGM-BP (low iron) plates for 5 days. Data are combined from two biological replicates (n = 14–31 worms/strain). Values are expressed as mean worm length ± SEM and compared by two-way ANOVA with Tukey’s multiple comparisons test, ***p<0.001. Scale bar, 500 um. (D) Survival analysis of wild-type N2 and nhr-14(tm1473) worms grown in NGM and NGM-BP (low iron). Median survival time (MS): wild-type N2-NGM (MS = 10 days, n = 52); nhr-14(tm1473)-NGM (MS = 10 days, n = 56); wild-type N2-BP (MS = 9 days, n = 44); nhr-14(tm1473)-BP (MS = 12 days, n = 36), Log-rank Mantel-Cox test, ***p<0.001, wild-type N2-BP versus nhr-14(tm1473)-BP.

https://doi.org/10.7554/eLife.44674.006

PQM-1 acts downstream of NHR-14 to regulate smf-3

We next questioned whether NHR-14 regulates smf-3 expression using transgenic worms expressing a GFP-H2B transcriptional reporter containing 1.5 kb of smf-3 upstream regulatory sequences (Psmf-3::GFP-H2B) (Figure 4A). This region harbors the 118 bp iron-dependent element (IDE) containing two hypoxia-response elements (HREs) and three GATA motifs (WGATAR) that are binding sites for HIF-1 and GATA proteins, respectively. We previously reported that Psmf-3::GFP-H2B upregulation in intestine during iron deficiency is dependent on the smf-3 IDE (Romney et al., 2011). To determine whether NHR-14 regulates smf-3, worms expressing Psmf-3::GFP-H2B were fed empty vector control RNAi (Con) or nhr-14 RNAi, and grown under normal (NGM), low iron (BP) or high iron (FAC) conditions, and GFP expression was assessed in L4 stage worms using the COPAS Biosort (Figure 4B and Figure 4—figure supplement 1). We verified rescue of the low iron developmental delay of hif-1(ia4) mutants by nhr-14 RNAi (Figure 4—figure supplement 2). Consistent with our previous study (Romney et al., 2011), GFP expression increased with BP and decreased with FAC in control-RNAi fed worms, reflecting HIF-1 regulation (Figure 4B). GFP expression is further increased in nhr-14-RNAi fed worms with NGM, NGM-BP and NGM-FAC treatments, indicating that sequences within the smf-3 promoter are important for NHR-14 regulation.

Figure 4 with 3 supplements see all
PQM-1 is downstream of NHR-14 and activates smf-3.

(A) Illustration of the Psmf-3::GFP-H2B transcriptional reporter containing 1.5 kb of 5’-promoter sequences of smf-3. The 118 bp iron-dependent element (IDE) harbors two hypoxia-response elements (HREs) and three GATA-like DAE motifs. Single nucleotide mutations in the DAEs are indicated in red. (B) GFP expression in Psmf-3::GFP-H2B worms fed control (empty vector) RNAi or nhr-14 RNAi grown on NGM, NGM-BP (low iron) or NGM-FAC (high iron) plates and quantified using COPAS Biosort (n = 1000 worms/sample). (C) GFP expression in Psmf-3::GFP-H2B worms harboring a nucleotide mutation (G > C) in each DAE grown on NGM or NGM-BP plates and quantified using COPAS Biosort (n = 1000 worms/sample). (D) Endogenous smf-3 expression in wild-type N2 worms versus pqm-1(ok485) mutants grown on NGM plates. (E) smf-3 expression in wild-type N2 worms and nhr-14(tm1473) mutants fed control RNAi or pqm-1 RNAi. (F) Length of wild-type N2, hif-1(ia4) and pqm-1(ok485) mutants grown on NGM or NGM-BP plates for 4 days. (G) PQM-1::GFP nuclear and cytoplasmic localization in L4 stage and adult worms fed control RNAi or nhr-14 RNAi and quantification of PQM-1::GFP expression. L4 stage, control RNAi (n = 29) and nhr-14 RNAi (n = 44); adults, control RNAi (n = 98) and nhr-14 RNAi (n = 88). For B, E and G, values are expressed as mean ± SEM (n ≥ 3 biological replicates) and compared by two-way ANOVA with Tukey’s multiple comparisons test, **p < 0.01, ***p < 0.001. For CD and F, values are expressed as mean ± SEM (n ≥ 3 biological replicates) and are compared by unpaired two-tailed Student’s t test, *p < 0.05, ***p < 0.001.

https://doi.org/10.7554/eLife.44674.008

To identify transcription factor(s) binding to smf-3 promoter sequences, we examined Wormbase data set containing genome-wide in vivo binding profiles for C. elegans transcription factors (Niu et al., 2011) (Figure 4—figure supplement 3). We found that the transcription factor PQM-1 showed significant association with the smf-3 IDE (IV: 2618542..2619063; qValue 1.7e−32), suggesting that PQM-1 might be involved in smf-3 activation in nhr-14(tm1473) mutants. PQM-1 associates with a GATA-like motif (TGATAAG) termed the DAF-16-associated element (DAE) found in the promoters of DAF-16/FoxO-Class II genes (Tepper et al., 2013). Class II genes are characterized as being downregulated by DAF-16 signaling (Murphy et al., 2003). Given ChIP-seq data showing that PQM-1 associates with the smf-3 promoter, we tested the hypothesis that PQM-1 regulates smf-3. Single mutations in DAE 1, 2 or 3 motifs (TGATAAG to TCATAAG) were associated with reduced GFP expression in untreated and BP-treated worms compared to Psmf-3::GFP-H2B worms carrying wildtype DAEs (Figure 4C—figure supplement 1). Endogenous smf-3 expression was also reduced in pqm-1(ok485) mutants compared to wild-type N2 worms (Figure 4D). Further evidence suggesting that PQM-1 activates smf-3 was shown by reduced smf-3 expression in nhr-14(tm1473) mutants fed pqm-1 RNAi (Figure 4E) and by the low iron developmental delay observed in pqm-1(ok485) mutants (Figure 4F).

Given that PQM-1 transcriptionally activates smf-3, we questioned whether PQM-1 nuclear localization is affected by nhr-14 loss. Previous studies showed that a PQM::GFP::FLAG transgene localized to the nucleus of intestinal cells during larval stages and to the cytoplasm in adults (Dowen et al., 2016; O'Brien et al., 2018; Tepper et al., 2013). In agreement with these studies, PQM-1::GFP::FLAG localized to nuclei in intestinal cells in L4 stage worms fed either control RNAi or nhr-14 RNAi, and to the cytoplasm in adults fed control RNAi. PQM-1::GFP::FLAG, however, remained in intestinal nuclei in adult worms fed nhr-14-RNAi (Figure 4G). Together, these data suggest that PQM-1 activates smf-3 by associating with GATA-like DAE motifs in the smf-3 promoter, and that NHR-14 acts upstream of PQM-1 to inhibit its nuclear translocation and transcriptional activity.

NHR-14 controls expression of innate immune genes

In addition to regulation of intestinal iron uptake, RNA-seq analysis revealed that NHR-14 regulates genes involved in host defense (Figure 5A–B). We identified 573 upregulated genes and 261 downregulated genes in nhr-14(tm1473) mutants versus wild-type N2 worms using the filtering criteria of p-values<0.05 and fold change >1.0 (Figure 3—source data 1). Gene-set enrichment analysis using gene ontology (GO) terms (Huang et al., 2009) showed that upregulated genes were involved in the innate immune response (Figure 5B). Over-represented functional categories within this dataset included C-type lectin (carbohydrate binding proteins), CUB-domain proteins (extracellular and membrane proteins), lysosomes, peptidases, glutathione S-transferases (xenobiotic metabolism) and collagens, all of which are part of a core immune response program in C. elegans (Simonsen et al., 2012) (Figure 5C). Genes that are upregulated in nhr-14(tm1473) mutants include classical immune response genes, such as lysozymes (lys-7lys–1lys-8, lys–2), Class II downstream of daf-16 (dod-21, dod–24, dod-23, dod–19), C-type lectins (clec-41, clec-85, clec-190, clec–150, clec-173, clec–5, clec-63, clec–62), antimicrobial peptides (spp-18, spp-8), and glutathione S-transferases (gst-38, gst-22) (Figure 5D). Using qPCR analysis, we verified nhr-14-dependent upregulation of ten genes within the immune response and iron metabolism groups (Figure 5E). In general, there was positive correlation between RNA-seq and qPCR expression values with the exception of ftn-1, which was significantly upregulated using qPCR, but not in the RNA-seq dataset. No significant change was observed in expression of iron/metal metabolism genes (smf-1, smf-2, ftn-2) and pqm-1 in nhr-14(tm1473) mutants (Figure 5E). Among the 261 downregulated genes, there was enrichment in genes involving cellular organization and body morphogenesis, including sqt-1, noah-1 and sym-1 (Figure 5D and Figure 3—source data 1). Analysis of tissue specific expression of the upregulated and downregulated nhr-14 genes using WormExp v1.0 revealed enrichment in intestine and epidermis, respectively (Figure 5—source data 1).

NHR-14 loss of function regulates an innate immune transcriptional program.

(A) Volcano plot of differentially transcribed genes in nhr-14(tm1473) mutants normalized to wild-type N2. The x-axis denotes the fold-changes and the y-axis denotes -log10 of wald test adjusted p-values. The horizontal line represents the p-value threshold of <0.05. (B) Gene ontology (GO) analysis of genes that are upregulated in nhr-14(tm1473) mutants normalized to wild-type N2 worms. (C) DAVID analysis for over-represented functional categories of upregulated genes in nhr-14(tm1473) mutants normalized to wild-type N2 worms. (D) Heat map showing the top 20 upregulated and 20 downregulated genes that are differentially expressed between nhr-14(tm1473) mutants and wild-type N2 worms. The columns represent three biological replicates of wild-type N2 and nhr-14(tm1473) mutants, and the rows represent individual genes. (E) qPCR validation of selected innate immune and iron-related genes that are upregulated in nhr-14(tm1473) mutants (n ≥ 3 biological replicates). Values are expressed as fold difference compared to wild-type N2 worms ± SEM and compared by unpaired two-tailed Student’s t test *p<0.05, **p<0.01, ***p<0.001. (F) Motif enrichment analysis for upregulated and downregulated genes in nhr-14(tm1473) mutants.

https://doi.org/10.7554/eLife.44674.012

An unbiased search for cis-regulatory elements in the 1 kb promotor regions of upregulated and downregulated nhr-14 genes revealed the presence of a GATA motif in 341 out of 573 upregulated genes and a tandem-direct repeat motif in 14 out of 261 downregulated genes (Figure 5F and Figure 3—source data 1). The tandem-repeat is of interest as vertebrate HNF4α activates transcription by binding to DNA consisting of two distinct half-site motifs in either direct, inverted or everted configurations (Fang et al., 2012).

Given that the transcriptional profile of nhr-14(tm1473) mutants is enriched for immune response genes, we examined the overlap between upregulated nhr-14 genes and previously published genes that are induced by infection to various pathogenic bacteria. We found overlap between genes upregulated in nhr-14(tm1473) mutants and genes upregulated in response to P. aeruginosa (Shapira et al., 2006; Troemel et al., 2006), S. aureus (Visvikis et al., 2014), Pseudomonas luminescens (Wong et al., 2007), Enterococcus faecalis (Wong et al., 2007) and Serratia marcescens (Engelmann et al., 2011) (Figure 6A and Figure 6—source data 1). These genes include lysozymes, C-type lectins, and antimicrobial proteins.

Overlap between pathogen and innate immune pathway datasets and nhr-14(tm1473) mutants.

(A) Number of overlapping genes between nhr-14(tm1473) mutants and published pathogen infection models. (B) Number of overlapping genes between nhr-14(tm1473) mutants and published innate immune pathway datasets.

https://doi.org/10.7554/eLife.44674.014

The pathogen response in C. elegans is controlled through three distinct signaling pathways that converge on inducing specific innate immune transcriptional program (Cohen and Troemel, 2015; Irazoqui et al., 2010). We compared our nhr-14 RNA-seq dataset with p38-MAPK (Troemel et al., 2006), TGFβ (Roberts et al., 2010), ELT-2 (Block et al., 2015) and DAF-2/DAF-16 (Murphy et al., 2003) innate immune pathway datasets, and found expression overlap with these pathways (Figure 6B and Figure 6—source datas 2 and 3). Notably, overlap occurred with the DAF-16/FoxO-Class II subset of genes that include the downstream-of-daf-16 (dod) group of which many are innate immune genes (Murphy et al., 2003) and classic antimicrobial genes (Figure 6B and Figure 6—source data 3. As Class II genes harbor GATA-like DAE motifs that are PQM-1 binding sites (Tepper et al., 2013), and GATA motifs are enriched in the promoters of upregulated nhr-14 genes, this suggested that some of these genes may be regulated by PQM-1. Analysis of ChIP-seq data revealed significant PQM-1 association to 15 out of the top 25 upregulated genes in nhr-14(tm1473) mutants, while only 3 out of 25 for the downregulated subset showed significant PQM-1 association (Niu et al., 2011) (Figure 6—source data 3).

We next questioned whether other upregulated nhr-14 genes in addition to smf-3 require PQM-1 for expression. qPCR analysis showed reduced expression of Class II genes dod-19, dod-24, clec-41, gst-38 and oac-14 in nhr-14(tm1473); pqm-1 RNAi mutants compared with nhr-14(tm1473); control RNAi worms. The Class II genes ins-7 and lys-2 and Class I genes lys-7 and ftn-1 were not changed in nhr-14(tm1473); pqm-1 RNAi mutants (Figure 7). Together, these data implicate NHR-14 as a repressor of the transcriptional innate immune response and specific DAF-16/FoxO-Class II genes mediated in part by PQM-1.

PQM-1 regulates DAF-16/FoxO-Class II genes downstream of nhr-14.

qPCR analysis of upregulated nhr-14 genes in nhr-14(tm1473); pqm-1 RNAi mutants and nhr-14(tm1473); Con RNAi worms (n = 3 biological replicates). Values are expressed as fold difference compared to nhr-14(tm1473); Con RNAi mutants ± SEM and are compared by unpaired two-tailed Student’s t test, *p<0.05.

https://doi.org/10.7554/eLife.44674.019

Comparison of upregulated nhr-14 genes with C. elegans mutant datasets using WormExp v1.0 showed significant overlap with genes upregulated in nhr-8(hd117) mutants and hyl-2 mutants (Figure 6—source data 4). nhr-8 encodes a nuclear receptor that regulates cholesterol and bile acid homeostasis (Magner et al., 2013) and hyl-2 encodes ceramide synthase that is involved in oxygen deprivation and regulation of innate immune genes (Ladage et al., 2016). Downregulated nhr-14 genes showed significant overlap with genes downregulated in elo-5(gk208) mutants (Kniazeva et al., 2015). elo-5 encodes a fatty acid elongase that produces monomethyl branched-chain fatty acids that function in intestine to promote sensory neuron maturation (Kniazeva et al., 2015). These analyses suggest a role for NHR-14 in lipid metabolism.

Loss of NHR-14 is required for host defense

Given that RNA-seq data revealed enrichment of innate immune response genes, DAF-16/FoxO-Class II suppressed genes and iron metabolism genes in nhr-14(tm1473) mutants, we tested the sensitivity of nhr-14(tm1473) and smf-3(ok1035) single mutants, and smf-3(ok1035); nhr-14(tm1473) double mutants to pathogenic P. aeruginosa strain PA14. PA14 causes a lethal intestinal infection in C. elegans similar to PA14 infection in mammals (Kirienko et al., 2014), and thus has become a useful model to study immune host defenses critical for resistance to pathogen infection (Cornelis and Dingemans, 2013). For our studies, the ‘slow’ killing assay was used that involves the colonization of P. aeruginosa in intestine (Tan et al., 1999). Compared to wild-type N2 worms, nhr-14(tm1473) mutants displayed enhanced resistance to PA14 infection, while smf-3(ok1035) mutants were hypersensitive (Figure 8A and Figure 8—source data 1). smf-3(ok485); nhr-14(tm1473) double mutants displayed increased resistance to PA14 compared to smf-3(ok1035) single mutants. These data show that nhr-14 partially suppresses the sensitivity of smf-3(ok1035) to PA14, suggesting that other genes in addition to smf-3 contribute to the resistance of nhr-14(tm1473) mutants to PA14.

Loss of NHR-14 confers resistance to P. aeruginosa infection.

(A) Survival analysis for the indicated mutants and wild-type N2 worms infected with P. aeruginosa PA14. Data are combined from three biological replicates. Median survival (MS): wild-type N2 (MS = 3 days, n = 68); nhr-14(tm1473) (MS = 4 days, n = 78); pqm-1(ok485) (MS = 3 days, n = 68); smf-3(ok1035) (MS = 2 days, n = 66); smf-3(ok1035); nhr-14(tm1473) (MS = 3 days, n = 59); nhr-14(tm1473); Con (RNAi) (MS = 4 days, n = 47); nhr-14(tm1473); pqm-1 (RNAi) (MS = 3 days, n = 52). Median survival of wild-type N2 worms grown in OP50 is 10 days (see Figure 3D). All survival curves are significantly different from one another, p<0.05, except for wild-type N2 vs pqm-1(ok485) p=0.835; wild-type N2 vs smf-3(ok1035); nhr-14(tm1473) p=0.621; nhr-14(tm1473) vs nhr-14 Con RNAi; Log-rank Mantel-Cox test. (B) qPCR analysis of indicated genes in wild-type N2 worms after feeding on non-pathogenic E. coli OP50 or PA14 for 24 hr. Values are expressed as mean fold change relative to OP50 ± SEM (n = 3 biological replicates) and compared by unpaired two-tailed Student’s t test, **p<0.01, ***p<0.001. (C) Western blot analysis of lysates prepared from NHR-14::GFP::FLAG worms after feeding on OP50 or PA14 for 24 hr (n = 3 biological replicates). (D) PQM-1::GFP::FLAG expression in adult worms exposed to OP50 and PA14 for 24 hr. Quantification of nuclear and diffuse cytoplasmic localization of PQM-1::GFP:FLAG exposed to OP50 (n = 134 worms) and PA14 (n = 169 worms). Values are expressed as percentage PQM-1::GFP::FLAG localization. (E) qPCR analysis of indicated genes in nhr-14(tm1473) and pqm-1(ok485) mutants after feeding on OP50 or PA14 for 24 hr. Values are expressed as mean PA14/OP50 fold change relative to wild-type N2 worms ± SEM (n = 3 biological replicates) and compared by unpaired two-tailed Student’s t test **p<0.01, ***p<0.001. (F) Iron content in wild-type N2 worms and indicated mutants after feeding on OP50 or PA14 for 24 hr as measured using ICP-MS. Values are expressed as mean ± SEM (n = 3–5 biological replicates) and compared by two-way ANOVA with Tukey’s multiple comparisons test, *p<0.05, **p<0.01, ***p<0.001.

https://doi.org/10.7554/eLife.44674.020

We also analyzed the resistance of the pqm-1(ok485) single mutant and nhr-14(tm1473); pqm-1 RNAi mutants to PA14. pqm-1(ok485) mutants displayed a wild-type sensitivity to PA14. nhr-14(tm1473); pqm-1 RNAi mutants were more sensitive to PA14 than nhr-14(tm1473) single mutants, indicating that PQM-1 is required for nhr-14(tm1473) enhanced PA14 resistance (Figure 8A and Figure 8—source data 1).

We tested whether PA14 affects the expression of nhr-14, smf-3, ftn-1 and ftn-2 in wild-type N2 worms. The DAF-16/FoxO-Class II dod-24 gene was also examined as it is known to be induced by PA14 (Shapira et al., 2006; Troemel et al., 2006). PA14 exposure (24 hr) repressed nhr-14 and ftn-1 and induced smf-3 and dod-24, but did not affect ftn-2 or pqm-1 compared to non-pathogenic OP50 E. coli (Figure 8B). PA14-mediated suppression of nhr-14 mRNA correlated with reduced NHR-14::GFP::FLAG expression in transgenic worms exposed to PA14 for 24 hr (Figure 8C). We also found that PQM-1::GFP::FLAG accumulated in the nucleus upon PA14 exposure in adult worms (Figure 8D), consistent with PQM-1 nuclear localization in adult worms fed nhr-14 RNAi (Figure 4G).

We next questioned whether nhr-14 or pqm-1 is required for expression of smf-3, ftn-1, ftn-2 and dod-24 during a PA14 infection. For these experiments, we assessed the expression of the above genes in nhr-14(tm1473) and in pqm-1(ok485) mutants after exposure to OP50 and PA14 for 24 hr. PA14 induced expression of smf-3, dod-24 and ftn-1 in nhr-14(tm1473) mutants relative to wild-type N2-PA14/OP50 levels, and notably, these genes were repressed by PA14 in pqm-(ok485) mutants (Figure 8E). ftn-2 was unchanged in nhr-14(tm1473) and pqm-1(ok485) mutants, and pqm-1 was unchanged in nhr-14(tm1473) mutants exposed to either OP50 or PA14 (Figure 8F). These data show that NHR-14 repression and PQM-1 are required for PA14 induction of smf-3, ftn-1 and dod-24.

Iron is required for pathogen survival and infectivity, and both pathogens and hosts have evolved specialized mechanisms to control iron availability (Hood and Skaar, 2012). We postulated that P. aeruginosa-mediated NHR-14 repression may provide a strategy to limit iron to the pathogen in the intestinal lumen by increasing iron uptake by SMF-3. If so, iron content would increase in wild-type N2 worms exposed to PA14. As predicted, iron content increased in wild-type N2 worms exposed to PA14 compared to OP50, and was further increased in nhr-14(tm1473) mutants exposed to either OP50 or PA14 (Figure 8F). The low iron content observed in smf-3(ok1035) single mutants and smf-3(ok1035); nhr-14(tm1473) double mutants was unaffected by PA14 exposure (Figure 8E and Figure 3B). Iron content was lower in pqm-1(ok485) mutants than nhr-14(tm473) mutants, and was unaffected by PA14. Together, these data show that NHR-14 repression and PQM-1 activation are required for PA14 induction of SMF-3-dependent iron uptake and FTN-1 iron storage, demonstrating iron sequestration as a component of the innate immune response in C. elegans (Figure 9).

Proposed model for NHR-14-PQM-1 signaling pathway in intestinal regulation of innate immunity and iron metabolism in C. elegans.

Under basal conditions, NHR-14 represses smf-3, specific DAF-16/FoxO-Class II genes and innate immune response genes to promote growth and development. Upon exposure to P. aeruginosa, NHR-14 function/activity is reduced, leading to PQM-1 nuclear localization and transcriptional activation of smf-3 and DAF-16/FoxO-Class II through the GATA-like DAE. SMF-3 mediates Fe(II) uptake after the reduction of Fe(III) by an unknown membrane associated ferrireductase (yellow oval). Increased cellular iron activates ftn-1 transcription through a HIF-1-dependent mechanism (Ackerman and Gems, 2012; Romney et al., 2011), leading to the sequestration of iron within FTN-1. We suggest that increased iron uptake by SMF-3 and sequestration by FTN-1 may provide a strategy to limit P. aeruginosa acquisition of intestinal luminal iron and serves as a component of the host innate immune response. How NHR-14 senses pathogen stress and the identity of NHR-14 downstream effectors remain to be determined.

https://doi.org/10.7554/eLife.44674.022

Discussion

Using a genetic suppressor screen to rescue the low iron developmental delay phenotype of hif-1(ia4) mutants, we identified NHR-14 as a transcriptional repressor of SMF-3-dependent iron uptake and innate immunity in C. elegans. Loss of nhr-14 increases SMF-3-mediated iron uptake and induces innate immune response genes that contribute to the resistance of nhr-14(tm1473) mutants to P. aeruginosa. These transcriptional responses are regulated in part by the transcription factor PQM-1. Our data support a model whereby C. elegans respond to pathogen infection by reducing NHR-14 abundance, thus allowing PQM-1 to translocate to the nucleus of intestinal cells to activate genes critical for innate immunity and iron uptake. Several C. elegans nuclear receptors, such as DAF-12 (Liu et al., 2013), NHR-49 (Sim and Hibberd, 2016), NHR-25 (Ward et al., 2014), and NHR-114 and NHR-144 (Yuen and Ausubel, 2018), regulate innate immune response genes; however, there is no evidence to date indicating a role for these nuclear receptors in iron metabolism. NHR-14 may thus be unique among nuclear receptors in coupling innate immunity with iron sequestration as a strategy to limit iron to invading intestinal pathogens.

Originally discovered to be induced in response to paraquat-mediated stress (Tawe et al., 1998), PQM-1 has been shown to be responsible for the activation of the DAF-16/FoxO-Class II subset of IIS genes (Tepper et al., 2013). Under normal conditions, the IIS pathway is upregulated by insulin-like peptides via activation of the DAF-2 tyrosine kinase receptor that culminates in the transcription factor DAF-16/FoxO phosphorylation and its cytoplasmic retention. Under stress, IIS signaling is reduced, DAF-16/FoxO is dephosphorylated and driven into the nucleus to activate Class I genes (stress response) and repress Class II genes (metabolism/growth) (Murphy et al., 2003; Tepper et al., 2013). PQM-1 antagonizes DAF-16/FoxO nuclear localization upon increased insulin signaling, resulting in the activation of Class II genes through binding to the DAE (Tepper et al., 2013). In larval stage worms, PQM-1 localizes to the nucleus of intestinal cells, but in adults, PQM-1 becomes mostly cytoplasmic (Dowen et al., 2016; O'Brien et al., 2018; Tepper et al., 2013). Consistent with these studies, we found PQM-1::GFP localized to the nucleus in L4 stage worms fed either control RNAi or nhr-14 RNAi; however, PQM-1::GFP remained nuclear in adults fed nhr-14 RNAi. These data suggest that PQM-1 nuclear localization caused by loss of nhr-14 in adults may be a mechanism to maintain growth via Class II gene activation as well as defend against stress in aged worms. Other signaling pathways are known to regulate PQM-1 subcellular localization, including mTORC/SGK-1, which promotes PQM-1 cytoplasmic localization in intestinal cells (Dowen et al., 2016), and HSP-90, which promotes PQM-1 nuclear localization in neuronal and intestinal cells (O'Brien et al., 2018). PQM-1 has also been shown to have a role in reproductive aging (Templeman et al., 2018).

The enrichment of Class II and innate immune response genes in nhr-14(tm1473) mutants suggest that these mutants may have a survival advantage when challenged with pathogens. As predicted, nhr-14(tm1473) mutants displayed enhanced resistance to P. aeruginosa that require PQM-1. P. aeruginosa infection reduced NHR-14 abundance in wild-type N2 adults, which led to PQM-1 nuclear localization, activation of smf-3 and specific DAF-16/FoxO-Class II genes and increased iron content. The observation that not all DAF-16/FoxO-Class II genes are regulated by PQM-1 in nhr-14(tm1473) mutants suggests other pathways are involved in their regulation. Our data are consistent with a model whereby increased iron uptake by SMF-3 may be a strategy to limit iron to pathogens in the intestinal lumen. It is also possible that iron helps to maintain iron homeostasis during a pathogen infection as it is required by enzymes involved in critical cellular processes.

We found that pqm-1(ok485) mutants displayed similar sensitivity to P. aeruginosa PA14 as wild-type N2 worms consistent with a recent report (O'Brien et al., 2018), but conflicts with two other studies showing pqm-1 is required for resistance to PA14 (Joshi et al., 2016; Shapira et al., 2006). The reason for the conflicting data is unclear, but may be caused by different background strains or different conditions used for pathogen survival assays.

In vertebrates, natural resistance-associated macrophage protein 1 (NRAMP1), a paralog of DMT1, is found in the phagosome membrane of macrophages, where it transports Fe(II) and Mn(II) out of phagosomes, thus restricting growth of pathogens residing within these compartments by limiting iron availability (Lopez and Skaar, 2018; Wessling-Resnick, 2015). DMT1 is highly expressed in duodenal enterocytes where it is a key regulator of intestinal iron absorption (Fleming et al., 1998; Gunshin et al., 1997). The intestinal mucosa also serves as the first line of defense against pathogens. Upon a pathogen infection in intestine, the innate immune response is triggered by the detection of microorganism-associated molecular patterns (MAMPs) via pattern-recognition receptors (PRR) (e.g. Tol/Toll-like and NOD-like receptors) that activate pathways regulating the production of anti-microbial proteins and cytokines, as well as the recruitment of acute inflammatory cells (Lopez and Skaar, 2018; Santaolalla et al., 2011). Several studies have shown that DMT1 is induced in response to TNFα (tumor necrosis factor α) and IFNγ (interferon γ) in duodenal enterocytes and bronchial epithelial cells (Laftah et al., 2006; Wang et al., 2005). It was speculated in these studies that the increase in DMT1 is a means to sequester iron to prevent microbial access to host iron and limit pathogenesis. Further investigation is needed to determine a role for DMT1-mediated iron uptake in intestinal innate immunity in vertebrates.

How NHR-14 senses pathogens remain to be determined. Unlike vertebrates, C. elegans lack clear pattern-recognition receptors orthologs, and its single Toll-like receptor is not necessary for immune defense (Cohen and Troemel, 2015; Stuart et al., 2013). A surveillance pathway has been identified in C. elegans that detects microbial-derived toxin damage to core cellular processes, such as translation and mitochondrial respiration, and induces signaling pathways controlling innate immune genes (Pukkila-Worley, 2016). The ligand-binding domain of NHR-14 may sense and bind pathogen-derived molecules or endogenous-produced ligands that facilitate NHR-14 stability or coregulator binding to regulate target genes. NHR-14 is also highly expressed in head neurons, suggesting that neuronal NHR-14 may detect small molecules that could modulate the innate immune response in intestine. NHR-14 was reported to function as an estrogen receptor (Mimoto et al., 2007), but conflicts with other studies showing that estrogen and bisphenol A did not affect NHR-14 function (Allard and Colaiácovo, 2010; Fischer et al., 2012).

Of the 284 NRs in C. elegans, 269 are derived from a HNF4-related ancestor (Robinson-Rechavi et al., 2005). This is unlike mammals that express two HNF4 receptors (HNF4α and HNF4γ) and Drosophila melanogaster that express a single HNF4 (Taubert et al., 2011). Mammalian HNF4α is expressed in liver, kidney, intestine, and the colon where it activates genes involved in glucose, fatty acid, and xenobiotic metabolism (Gonzalez, 2008). HNF4α has been shown to control iron homeostasis in liver and hepatoma cells through the regulation of transferrin, transferrin receptor-1 and hepcidin genes (Matsuo et al., 2015; Truksa et al., 2009). The expansion of the HNF4-related NRs in C. elegans suggests that they may have evolved dedicated functions that are performed by HNF4 in higher organisms (Taubert et al., 2011). Studies have shown that in the plant parasitic nematode Meloidogyne incognita (Abad et al., 2008) and in the human parasitic nematode Brugia malayi (Ghedin et al., 2007) the number of NRs are reduced to 92 and 27, respectively. Of note, NHR-14 is one of four HNF4-related nuclear receptors that is conserved in both parasites, suggesting that NHR-14 may be important for parasite infectivity.

In summary, the identification of NHR-14 adds a new layer of regulation that links iron metabolism with the innate immune response and provides new insights by which C. elegans responds to pathogen infections.

Materials and methods

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional information
Strain (Caenorhabditis elegans)C. elegans strains used in this study are listed in Supplementary file 1 -Table 1This paper
Strain (Escherichia coli)OP50C. elegans Genetics CenterRRID: WB-STRAIN:OP50
Strain (Escherichia coli)HT115(DE3)C. elegans Genetics CenterRRID: WB-STRAIN:HT115(DE3)
Strain (Pseudomonas aeruginosa)PA14OtherRRID: WB-STRAIN:PA14Ausubel Laboratory,
The Massachusetts General Hospital
Genetic reagentnhr-14 RNAi (T01B10)Kamath et al., 2003; PMID: 12828945Ahringer RNAi feeding library
Genetic reagentpqm-1 RNAi (F40F8.7)Kamath et al., 2003; PMID: 12828945Ahringer RNAi feeding library
AntibodyMonoclonal anti-FLAG M2 antibodySigma AldrichCat# F3165; RRID: AB_259529
AntibodyMonoclonal anti β-tubulin antibodyMP Biomedicals LLCCat# 08691261; RRID: AB_2335131
Sequence-based reagent - TaqMan Assaysmf-3Thermofisher ScientificCe02461546_g1
Sequence-based reagent - TaqMan Assayspp-18Thermofisher ScientificCe02457997_g1
Sequence-based reagent - TaqMan Assaynhr-14Thermofisher ScientificCe02420450_g1
Sequence-based reagent - TaqMan Assayact-2Thermofisher ScientificCe02507510_s1
Sequence-based reagent - TaqMan Assaydod-19Thermofisher ScientificCe02470201_m1
Sequence-based reagent - TaqMan Assaydod-24Thermofisher ScientificCe02466435_g1
Sequence-based reagent - TaqMan Assaylys-2Thermofisher ScientificCe02480494_g1
Sequence-based reagent - TaqMan Assaylys-7Thermofisher ScientificCe02473444_g1
Sequence-based reagent - TaqMan Assayclec-41Thermofisher ScientificCe02483609_g1
Sequence-based reagent - TaqMan Assaygst-38Thermofisher ScientificCe02486571_g1
Sequence-based reagent - TaqMan Assayftn-1Thermofisher ScientificCe02477612_g1
Sequence-based reagent - TaqMan Assayftn-2Thermofisher ScientificCe02415799_g1
Sequence-based reagent - TaqMan Assaysmf-1Thermofisher ScientificCe02496641_g1
Sequence-based reagent - TaqMan Assaysmf-2Thermofisher ScientificCe02496629_g1
Sequence-based reagent - TaqMan Assaynhr-23Thermofisher ScientificCe02405513_g1
Sequence-based reagent - TaqMan Assaypqm-1Thermofisher ScientificCe02438957_g1
Sequence-based reagent - TaqMan Assaytba-1Thermofisher ScientificCe02412618_gH
Sequence-based reagent - TaqMan Assayins-7Thermofisher ScientificCe02458078_g1
Sequence-based reagent - TaqMan Assaydod-23Thermofisher ScientificCe02435949_g1
Sequence-based reagent - TaqMan Assayoac-14Thermofisher ScientificCe02499634_g1
Recombinant DNAL440 plasmidAddgenePlasmid # 1654; https://www.addgene.org/1654/ RRID: Addgene_1654
Recombinant DNANHR-14::3xFLAG::GFP transgene fosmidSarov et al., 2006;PMID: 22901814, TransgeneOMEWBGene0000361
Chemical compound, drug2,2′-BipyridylSigma AldrichCat# D216305
Chemical compound, drugFerric Ammonium CitrateSigma AldrichCat# RES20400-A7
Chemical compound, drug5-fluoro-2-deoxyuridine (FUdR)Sigma AldrichCat# F0503
Chemical compound, drugCoomassie Plus Protein Assay ReagentThermofisher ScientificCat# 23236
Chemical compound, drugIsopropyl β-D-1-thiogalactopyranoside (IPTG)Thermofisher ScientificCat# 15529019
Chemical compound, drugTRIzol ReagentInvitrogenCat# 15596018
Software, algorithmPrism 6 and 7.04Graphpadhttps://www.graphpad.com/ RRID: SCR_002798
Software, algorithmImage JNational Institute of Healthhttps://imagej.nih.gov/ij/
Software, algorithmExcel 2013Microsoft Professional 2013 Spreadsheet Softwarehttps://products.office.com/en-us/excel
Software, algorithmBioProspectorLiu et al., 2001http://ai.stanford.edu/~xsliu/BioProspector/
Software, algorithmDAVID Bioinformatics Resources 6.8Huang et al., 2009; PMID: 19131956https://david.ncifcrf.gov/home.jsp
Commercial assay or kitWestern Lighting Plus-ECL ChemiluminescencePerkin ElmerCat# WP20005
Commercial assay or kitqScript XLT cDNA SuperMixQuantaBioCat# 95161–025
Commercial assay or kitSuperScript III First-Strand Synthesis SuperMixInvitrogenCat# 18080400
Commercial assay or kitPierce Coomassie Plus (Bradford) Assay ReagentThermofisher ScientificCat# 23238
Commerical assay or kitPierce BCA Protein AssayThermofisher ScientificCat# 23227

C. elegans strains and culture

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Strains obtained from the CGC and those generated for this study are listed in Supplementary file 1 and Key Resources Table. nhr-14(tm1473) mutants were backcrossed 6x to wild-type N2 worms. These worms displayed normal development and brood size. hif-1(ia4); nhr-14(tm1473) and smf-3(ok1035); nhr-14(tm1473)double mutants, and smf-3(ok1035); hif-1(ia4); nhr-14(tm1473) triple mutants were constructed and verified using standard genetic and molecular methods (Brenner, 1974). Worms were grown on nematode growth medium (NGM) agar plates seeded with Escherichia coli OP50 bacteria at 22°C unless otherwise noted. For iron experiments, worms synchronized by hypochlorite treatment at the L4 stage to young adult stage were grown overnight on NGM plates and then transferred to NGM plates supplemented with 6.6 mg/ml ferric ammonium citrate (FAC) or 25 uM 2,2’-bipyridyl (BP, iron chelator) for rescue experiments and 100 uM BP for RNAi experiments for times indicated in each experiment. The pH of FAC-NGM agar was adjusted to pH 7.0. For synchronizing worms, eggs were obtained by treating gravid adults with alkaline hypochlorite. Eggs were allowed to hatch overnight in Egg Buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 25 mM HEPES) and arrest in the L1 stage. Synchronized L1 stage worms were grown on NGM plates seeded with OP50 until reaching the L4 stage.

Reporter constructs

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Psmf-3::GFP-H2B was generated by PCR amplification of sequences 1,500 bp upstream from the initiation ATG of smf-3 (Y69A2AR.4) using primers containing SalI and NheI restrictions sites, cloned into TOPO Zeroblunt (Invitrogen) followed by digestion and insertion into Sal1 and NheI sites of pAP.10 as previously described (Romney et al., 2011). The GATA-like DAE 1–3 mutants were generated by mutation of GATA >CATA in Psmf-3::GFP-H2B using the QuikChange Site-Directed Mutagenesis kit (Stratagene). Transgenic strains were generated using a standard microinjection protocol. Each DAE construct (20 ng/µl), a selection plasmid pBX-1 (100 ng/µl) and salmon sperm DNA (25 ng/ul) were coinjected into pha-1 animals. Transgenic animals were obtained after growth at 25°C. Five independent lines were established for Psmf-3(DAE1)::GFP-H2B, Psmf-3(DAE2)::GFP-H2B and Psmf-3(DAE3)::GFP-H2B), and all showed similar GFP patterns.

Generation of NHR-14::FLAG::GFP transgenic worms

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The expression pattern of NHR-14 was determined using transgenic animals expressing a fosmid (40 kb) encoding a NHR-14::3xFLAG::GFP transgene (TransgeneOme) (Sarov et al., 2006). This fosmid contains the entire nhr-14 locus as well as large amounts of flanking DNA critical for endogenous nhr-14 expression. The fosmid was injected into ttTi5606; unc-119 worms at 250 ng/ul and three independent lines were established. Line three was used for experiments in Figure 2A-D.

EMS screen and SNP mapping

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Ethyl methanesulfonate (EMS) mutagenesis was performed as previously described (Brenner, 1974). Briefly, L4 stage hif-1(ia4) worms (Jiang et al., 2001) were treated with 50 mM EMS in M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl) for 4 hr. Mutagenized worms were washed five times to remove excess EMS, plated on NGM plates and grown until gravid. Gravid F1s were then bleached, and F2 eggs plated on NGM plates. Gravid F2 worms were transferred to 25 uM BP plates and removed after laying F3 eggs. F3 progeny that rescued the low iron phenotype of hif-1(ia4) mutants (determined by body length measurements using ImageJ) were scored as suppressors. We identified 16 suppressors from 15,000 haploid genomes using EMS, six that fully and 10 that partially rescued the hif-1(ia4) low iron phenotype. We focused on two mutations, qa6909 and qa6910, that strongly suppressed the hif-1(ia4) low iron phenotype. The other four strong suppressors were sequenced and were not mutations in nhr-14.

Whole genome sequencing-single nucleotide polymorphism (WGS-SNP) mapping was used to identify the causative mutations in qa6909 and qa6910 (Doitsidou et al., 2010). hif-1(ia4) mutants were crossed into the polymorphic Hawaiian strain CB4856 six times. Hawaiian hif-1(ia4) males were crossed with qa6909 and qa6910 mutants and F1 progeny were moved to fresh NGM plates. Gravid F1 adults were allowed to lay eggs overnight on NGM plates containing 25 uM BP. Adults were removed the next day and ~50 F2 recombinants that reached adulthood within five days were singled to NGM plates and DNA from their F3 and F4 progeny was purified using the PureLink Genomic DNA Mini kit (Invitrogen) and pooled. WGS was performed using an Illumina HiSeq sequencer. Hawaiian SNP positions were mapped on the genomes of suppressor mutants qa6909 and qa6910 using the single-step SNP-mapping pipeline CloudMap (Minevich et al., 2012).

Rescue experiments

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Five gravid adults from each strain were picked to either NGM or NGM-25 uM BP plates and allowed to lay eggs overnight. Adults were removed the following day and progeny were incubated for five days at 22°C after which images were collected using a Leica M205 FA microscope and Leica DFC 310 FX camera (Leica Microsystems). Progeny were scored based on length using Image J.

RNAi

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RNAi clones nhr-14 (T01B10) and pqm-1 (F40F8.7) were obtained from the Ahringer RNAi feeding library (Kamath et al., 2003). Empty vector (L4440) was used as a control. All RNAi clones were verified by sequencing. Bacterial strains (HT115 DE3) were grown overnight in Luria-Bertani medium containing 50 µg/ml ampicillin and seeded onto NGM or NGM-BP plates containing 100 µg/ml ampicillin and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce the gene of interest. Bacteria were then incubated at room temperature overnight to induce dsRNA. Five hif-1(ia4) gravid adults were allowed to lay eggs overnight on NGM or NGM-25 uM BP RNAi plates at 20°C. Adults were removed the following day and progeny were scored for rescue against BP as described above. For qPCR experiments, 20–30 gravid adults were placed on RNAi plates for 48–72 hr. The progeny that reached L3 and L4 stages were harvested for RNA isolation.

For nhr-14(tm1473); pqm-1 RNAi experiments, L1 stage synchronized worms were seeded onto RNAi plates and incubated at 22°C to obtain adult hermaphrodites (P0). Gravid hermaphrodites from P0 were used to obtain a L1 stage synchronized population (F1), which was seeded onto pqm-1 RNAi plates for a second round. Gravid F1 hermaphrodites were synchronized to obtain a L1 stage population (F2) and were seeded on pqm-1 RNAi plates for a third round. Worms were allowed to reach L4 stage and then harvested for qPCR experiments.

qPCR

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For indicated conditions and time-points, worms were washed off the plate with M9 buffer followed by three further washes to remove external bacteria, and rocked for 0.5 hr in M9 buffer to clear bacteria from the gut. RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. cDNA synthesis was performed using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) or qScript XLT cDNA SuperMix (QuantaBio). After reverse transcription, qPCR was performed using TaqMan Gene Expression Assays (Applied Biosystems) and analyzed using an Applied Biosystems 7900 HT qPCR instrument. The cycle threshold (Ct) value for each transcript was normalized to either act-1, act-2b or nhr-23. For pathogen experiments, all values were normalized using control genes nhr-23 or tba-1. The comparative Ct method was used to quantify transcript abundance. At least three biological replicates with duplicate technical replicates were used for qPCR experiments. TaqMan Assays are listed in Key Resources Table.

RNA-seq analysis

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Wild-type N2 and nhr-14(tm1473) L1 stage worms were grown on NGM plates seeded with OP50 and harvested at L4 stage (48 hr at 22°C). Three biological replicates for each strain were used for RNA-seq analysis. Worms were collected by centrifugation and washed with M9 buffer to remove, and RNA was extracted using a modified TRIzol procedure. Briefly, the aqueous phase was mixed with an equal volume of 70% EtOH and added to RNeasy spin columns (Qiagen). RNA concentration and quality were determined using a NanoDrop spectrophotometer followed by further quality control with the Bioanalyzer RNA 9000 Nano Chip. Library construction was performed using the Illumina TruSeq RNA Sample Preparation Kit v2 (RS-122–2001 and RS-122–2002) and was sequenced with the Illumina HiSeq (50 cycle, single-end). Sequenced reads were aligned to the Ce10 (WormBase Build 220) transcriptome index using Novoalign, and differential expression was determined using Useq 8.7.4 (Nix et al., 2008). Transcripts with a log2 ratio of 1 or higher at a false discovery rate (FDR) ≤ 0.05 were considered differentially expressed. Gene enrichment analysis was performed using GOrilla (Eden et al., 2009), which determines enriched GO terms at the top of a ranked list of genes. BioProspector (Liu et al., 2001) was used for de novo motif finding with the width parameter set to 16. Consistent motifs were identified using 1000 bp upstream of upregulated and downregulated genes.

Pathogen infection and lifespan analysis

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P. aeruginosa strain PA14 slow-kill (SK) assays were performed as previously described (Tan et al., 1999). PA14 was cultured in Luria broth and seeded on slow-kill plates. Eggs from gravid adults were obtained by alkaline hypochlorite treatment, allowed to hatch overnight in S-basal medium, and arrested in the L1 stage. Synchronized L1 stage worms were grown on NGM plates seeded with OP50 until reaching the L4 stage. Each strain of L4 stage worms were transferred to NGM or SK plates containing 75 uM 5-fluoro-2-deoxyuridine (FUdR) to prevent growth of progeny and incubated at 22°C. C. elegans viability was scored every day using a dissecting microscope to detect dead worms (Figure 8—source data 1). For iron content quantification, worms were harvested after 24 h PA14 exposure and processed for ICP-MS analysis.

Lifespan analysis of nhr-14(tm1473) and wild-type N2 worms grown under low iron conditions was performed by transferring L4 stage worms to NGM and NGM-25 uM BP plates containing 75 uM FUdR to prevent growth of progeny. Assays were carried out at 22°C and worms were scored every day. For pathogen and lifespan analysis, worms were scored as dead if they did not move, pump, or respond to gentle prodding. Survival analyses were analyzed by Kaplan-Meir method, and statistical significance was assessed using the Mantel-Cox log-rank test (GraphPad Prism).

Western blot analysis

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At indicated time points, worms were washed off the plate with ddH2O and washed three times to remove bacteria. Worms were resuspended in lysis buffer (20 mM HEPES pH 7.5, 25 mM KCl, 0.5% NP-40) and disrupted by two 5 s pulses using an Ultrasonic Processor (Sonics) at 50% amplitude. Protein concentrations were determined using Coomassie Plus Protein Assay Reagent (Thermo Scientific). Protein samples were incubated for 10 min at 95°C in NuPage LDS Sample Buffer (Invitrogen) containing 10 mM DTT then subjected to electrophoresis using NuPage 4–12% Bis-Tris gels (Invitrogen) and transferred to Amersham Hybond ECL nitrocellulose (GE Healthcare). Blots were incubated with mouse anti-FLAG antibody (Sigma cat# F3165) along with monoclonal β-tubulin antibody (MP Biomedicals LLC cat# 08691261) in 1x Tris-buffered saline (0.01% Tween-20) containing 5% non-fat dry milk. Horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratories) were visualized using Western Lighting Plus-ECL Chemiluminescence Substrate (Perkin Elmer). Three biological replicates were performed and NHR-14::GFP::FLAG was quantified by densitometry analysis using Image J (NIH).

Iron content quantification

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Synchronized L1 stage worms were grown on NGM plates seeded with OP50 until reaching the L4 stage. Worms were washed extensively with M9 buffer and incubated in M9 buffer at room temperature for 2 hr to allow for purging of the gut followed by three rinses with ddH2O. Worms were pelleted and dried, and metal analysis determined by inductively-coupled plasma-optical emission spectroscopy (ICP-OES) (Children’s Hospital Oakland Research Institute (CHORI) Elemental Analysis Facility). Empty tubes were run in parallel to serve as controls. Iron content was normalized to sulfur content. For pathogen experiments, worms were harvested in water, washed and homogenized for metal analysis. Protein concentration in lysates was measured using the BCA Protein Assay (Thermo Fisher Scientific) and 100 µg of lysate was digested overnight in 5:1 HNO3:H2O2, dried, and the pellet resuspended in 2% HNO3 for metal analysis by inductively-coupled plasma mass spectrometry (ICP-MS) (University of Utah, Center for Iron and Hematology Disorders, Iron and Heme Core). At least 3–5 biological experiments were performed for each strain with triplicate technical replicates. Calibration standard solutions for determination of Fe, Zn, Cu and Mn were prepared from Agilent multi-element calibration standard-2A. Iron content was normalized to protein.

COPAS biosort

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Animals expressing Psmf-3::GFP-H2B containing 1,500 bp of smf-3 promoter sequences (Romney et al., 2011), Psmf-3(mDAE1)::GFP-H2B, Psmf-3(mDAE2)::GFP-H2B and Psmf-3(mDAE3)::GFP-H2B reporters were synchronized by hypochlorite treatment, and arrested L1 stage larvae were grown on 10 cm NGM plates containing nhr-14 RNAi or control (empty vector) RNAi for 48 hr described above. Worms were washed in M9 buffer and transferred to nhr-14 RNAi or control RNAi plates supplemented with either 100 μM BP or 6.6 mg/mL FAC and grown overnight. Worms were collected by centrifugation and washed several times in M9 buffer to remove bacteria and debris. Worms were analyzed using COPAS Biosort where L4 stage to young adult worms were gated based on extinction and time of flight parameters (TOF). Extinction and TOF parameters were held constant for subsequent GFP fluorescence acquisition throughout all conditions. GFP fluorescence for 1000 worms was analyzed using FlowJo.

Microscopy

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Transgenic worms carrying NHR-14::GFP::FLAG fosmid were imaged at 40x and 60x using an Olympus FV1000 confocal microscope and camera using Olympus FV10-ASW 3.1 confocal imaging software. PQM-1::GFP::FLAG worms were imaged at either 20x or 40x using a Leica DM6000B microscope. Following acquisition, images were rotated, cropped and sized using Adobe Photoshop.

Statistical analysis

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Statistical analyses were performed using GraphPad software Prism 6 or 7.04 and Excel. Results are expressed as the mean ± SEM. Data were analyzed by an unpaired two-tailed Student’s t-test or two-way ANOVA with Tukey’s multiple comparison test. Survival comparisons were performed using the Mantel-Cox log-rank test. All data were evaluated at the significance level p≤0.05. Biological replicates reflect different sources of material and/or experiments performed on different days. Statistical details for experiments are indicated in the figure legends.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
    Nuclear hormone receptors in C. elegans
    1. A Antebi
    (2006)
    WormBook, 10.1895/wormbook.1.64.1, 18050471.
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
    The genetics of Caenorhabditis elegans
    1. S Brenner
    (1974)
    Genetics 77:71–94.
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
    Signaling in the innate immune response
    1. DH Kim
    2. JJ Ewbank
    (2015)
    WormBook : The Online Review of C. Elegans Biology pp. 1–35.
    https://doi.org/10.1895/wormbook.1.83.2
  36. 36
  37. 37
  38. 38
  39. 39
  40. 40
  41. 41
    BioProspector: discovering conserved DNA motifs in upstream regulatory regions of co-expressed genes
    1. X Liu
    2. DL Brutlag
    3. JS Liu
    (2001)
    Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing pp. 127–138.
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
    The C. elegans intestine
    1. JD McGhee
    (2007)
    WormBook, 10.1895/wormbook.1.133.1, 18050495.
  48. 48
  49. 49
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58
  59. 59
  60. 60
  61. 61
  62. 62
  63. 63
  64. 64
  65. 65
  66. 66
  67. 67
  68. 68
  69. 69
  70. 70
  71. 71
  72. 72
  73. 73
  74. 74
  75. 75
  76. 76
  77. 77
  78. 78
  79. 79
  80. 80
  81. 81
  82. 82
  83. 83
    Ferroportin-mediated iron transport: expression and regulation
    1. DM Ward
    2. J Kaplan
    (2012)
    Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1823:1426–1433.
    https://doi.org/10.1016/j.bbamcr.2012.03.004
  84. 84
  85. 85
  86. 86
  87. 87

Decision letter

  1. Tadatsugu Taniguchi
    Senior Editor; Institute of Industrial Science, the University of Tokyo, Japan
  2. Bruno Lemaître
    Reviewing Editor; École Polytechnique Fédérale de Lausanne, Switzerland

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "NHR-14 integrates iron uptake and innate immunity in Caenorhabditis elegans through PQM-1 signaling" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

All the reviewers found the manuscript to be very interesting, but the lack of rigor or in-depth analysis in many areas makes it unsuitable for publication in its current form. The manuscript could be improved by going more in depth on the metal biology or by strengthening the innate immunity aspects. The diversity of the reviewer's suggestions indicate that the revision will likely take more than two months, the time limit in eLife for revision. This is why the paper was rejected. Nevertheless, the authors could consider re-submitting their article to eLife as a new submission once the reviewer's comments have been addressed in full.

Reviewer #1:

The authors had previously discovered a role for the C. elegans HIF1a homolog in regulating intestinal iron homeostasis during iron deficiency by activating and inhibiting genes involved in iron uptake and storage. Taking advantage of a developmental delay phenotype of hif-1(ia4) mutants on low iron media, the authors performed a suppressor screen and uncovered two mutations in a nuclear hormone receptor gene, nhr-14, that rescued the delay. NHR-14 transcript and protein levels were unaffected by iron levels. RNA-seq anaylsis comparing gene expression profiles between wildtype animals and nhr-14 mutants revealed that nhr-14 regulates the iron transport gene smf-3, and innate immunity genes. Analysis of the smf-3 promoter and mining modENCODE ChIP-seq data suggested that PQM-1 acts downstream of NHR-14 to regulate smf-3. The authors explored the role of NHR-14 in innate immunity, demonstrating that in addition to regulating immune genes, nhr-14 mutants were resistant to P. aeruginosa and S. enterica infection, and that bacterial infection downregulated NHR-14 protein levels. Finally, the authors showed that knockdown of the nhr-14 ortholog, HNF4a, in human cells resulted in a similar upregulation of the smf-3 homolog (DMT1). This is an interesting study looking at how nuclear hormone receptors coordinate iron availability and innate immunity, but the manuscript suffers from a lack of details in the genetic analyses, and a lack of exploration of the roles of NHR-14 in innate immunity required to show that the gene expression signature is not simply a consequence of disrupting a core cellular process.

Substantive concerns

1) Elements of the manuscript were difficult to evaluate, as many details about the screen and the genetics were lacking. For the screen, how many genomes were screened? How many mutants were recovered? Was the screen saturating? As this was the first report of a screen, these details should be provided. Additionally, were the suppressors, and the nhr-14(tm1473) pqm-1(ok485) mutants backcrossed? If so, how many times?

2) There is a complete lack of characterization of the nhr-14 mutant strains that hampers evaluation of many elements of the manuscript. Do these strains have normal broodsizes? Do they develop at a normal rate? Do they have a normal lifespan? These questions are particularly important for interpreting the pathogen data. What is the nhr-14(tm1473) allele? Is it a null? Are the three nhr-14 mutants phenotypically equal? If so, that would be particularly interesting as the insertion in the DNA binding domain would be predicted to disrupt DNA binding, but not the activation domains or ligand binding domain, and would support that direct gene regulation by NHR-14 contributes to the suppression of the hif-1a arrest on low iron media.

3) The innate immunity sections are problematic. Melo and Ruvkun (2012) demonstrated that RNAi disruption of core cellular processes induces expression of detoxification and innate immune effectors, even in the absence of toxins or pathogens. Similar to point 1, much more information about the nhr-14 strains needs to be provided to convince the reader that this is specifically an immune phenotype and not due to affecting a core process. The authors claim that NHR-14 is the first C. elegans NHR to be involved in innate immunity (and iron metabolism), but Ward et al., 2014, demonstrated that inactivation of nhr-25 causes sensitivity to a range of stresses and pathogens. Several experiments would help strengthen the immunity sections: i) lifespan on OP50 as a baseline for comparing the pathogen sensitivity data; ii) NHR-14 overexpression, which would be predicted to cause increased sensitivity to pathogens. Kirienko et al., 2013, reported a liquid killing assay for PA14 that involves different pathogenesis mechanisms than plate-based assays and is particularly dependent on iron. This assay would be very useful to evaluate the importance of NHR-14-mediated iron regulation in the context of immunity.

4) A major weakness of the paper is lack of any mechanism of how NHR-14 is regulating PQM-1 and in turn SMF-3, or how pathogenic stress regulates NHR-14 levels. An INS-7 overexpression experiment would be very straightforward.

Reviewer #2:

Elucidating mechanisms that organisms use to mediate iron homeostasis through the regulation of transcription factors and transport proteins is an important objective in the field of metal biology. This manuscript details the interaction of two transcription factors that link iron homeostasis with the response of C. elegans to infection by pathogenic bacteria. The authors performed an innovative forward genetic screen leading to the identification of two mutations in the nhr-14 gene. They used rigorous methods to demonstrate that mutations in nhr-14 cause the phenotype. They characterized the phenotype of nhr-14 mutant animals, including the rescue of the hypersensitivity to low iron phenotype of the hif-1 mutant, an increase in metal content for iron and manganese, an increase in the expression of many genes including smf-3 and multiple genes linked to innate immunity, and an alteration in the localization of the transcription factor PQM-1, and resistance to bacterial infections. They also demonstrate that the loss of the human homolog of NHR-14 causes an increase in the expression of another iron transport protein in vertebrate cells, suggesting a conservation of function of the identified nuclear receptor. The impact and quality of the work found within this manuscript would make it a strong candidate for eLife if the authors address the following concerns:

Major Issues:

1) The authors identified mutations in nhr-14 as suppressors of the low iron hypersensitivity of hif-1, which is an innovative discovery technique. They used powerful positional cloning approaches to clearly demonstrate that the mutations in nhr-14 are the cause of the enhanced growth in low iron. The major question that arises is how does mutation of nhr-14 rescue the hif-1 growth defect, and a key approach is to analyze the phenotype of nhr-14 mutations in an otherwise wild type background. This manuscript would benefit from experiments that compare the growth of nhr-14 mutants to wild type animals in two or three concentrations of BP and two or three concentrations of FAC. If the nhr-14 mutations do indeed act by enhancing iron uptake, then it is predicted that they will enhance growth in BP compared to wild-type animals, and they may be hypersensitive to FAC compared to wild type animals. If these effects are not observed, it would call into question the model that the nhr-14 mutation acts by enhancing iron uptake.

2) The authors argue that the cellular site of action of nhr-14 is intestinal cells, as shown in Figure 9. This conclusion appears to be based on the localization of a NHR-14:GFP fusion protein, as shown in Figure 2D. However, the fusion protein is also expressed in other cell types – at a minimum, the red arrows highlight expression in head neurons. It is not possible to judge from the single image how many other cell types may also express NHR-14. Thus, if the conclusion about cellular site of action is based solely on expression data, then an alternative possibility is that the key site of action is the head neurons, or other cell types that may not be visible in this single image. If the authors want to draw a conclusion about the cellular site of action, then they should do experiments to express NHR-14 in a specific cell type using a heterologous promoter, such as an intestinal specific promoter. I do not think determining the cellular site of action is essential for the main conclusions of the paper, so another approach is for the authors to deemphasize the conclusion that the site of action is intestinal cells.

3) The authors investigated the exciting hypothesis that nhr-14 is regulated by dietary iron. However, the results shown in Figure 2 are negative and support the conclusion that both the transcriptional and translational levels of NHR-14 are unaffected by dietary iron. However, a possible unexplored explanation for the function of NHR-14 in iron homeostasis is that the localization of NHR-14 is altered in response to changes in supplemental iron, since some nuclear receptors are regulated by nuclear localization. The functional characterization of NHR-14 would be more complete with an examination of the localization change when animals expressing the tagged version of NHR-14 are exposed to excess and limited iron conditions.

4) The conclusion that nhr-14 acts through regulation of smf-3 to increase the growth of hif-1 mutants should be considered more rigorously. The authors clearly demonstrate that nhr-14 mutants have higher levels of smf-3 transcript, and SMF-3 is a known iron import protein, leading to the hypothesis that this is the critical target gene for the hif-1 rescue. I acknowledge this is a reasonable and even plausible hypothesis. However, the transcriptional analysis of nhr-14 mutant animals demonstrates that hundreds of transcripts are altered, so in principle any one of these changes, or many of these changes in combination might mediate the hif-1 rescue. The critical issue is what additional evidence can test the smf-3 hypothesis. The authors create a smf-3; nhr-14; hif-1 triple mutant and the rescue is lost, in that these triple mutants do not grow in low iron. My concern is that the smf-3 single mutants may not be able to grow in low iron, in which case this experiment is not really informative. For example, if the authors create a triple mutant with nhr-14, hif-1 and any mutation that causes L1 arrest, they will get this result. To interpret this experiment, the authors should analyze the smf-3 single mutant in BP. If the smf-3 single mutant displays arrested growth, then I think there is no clear interpretation of the double mutant of smf-3 with hif-1 and the triple mutant of smf-1, hif-1 and nhr-14. These data would therefore not support the conclusion that NHR-14 functions in iron homeostasis through smf-3.

5) In Figure 3B, there is a significant increase in the amount of manganese in animals with a mutation in nhr-14. This raises the possibility that the change in manganese levels causes any or all of the nhr-14 mutant phenotypes. It would be helpful if the authors addressed this possibility, certainly in the text and possibly experimentally. Have the authors examined the phenotypes of nhr-14 mutants with respect to excess and limited manganese?

6) The model that NHR-14 works in combination with PQM-1 to reduce the iron in the intestinal lumen through SMF-3 is speculative. For example, there are no measurements of the level of iron in the intestinal lumen. Alternative models are that it is changes in iron in the body of the worm that influence pathogen resistance, or that there is a non-iron based mechanism of pathogen resistance. One possible test of the model could be examining the effect of adding supplemental iron to the pathogenicity of the tested bacteria. This would help to test the prediction that decreasing the iron in the lumen is protective to these animals. In a similar vein, the model suggests that nhr-14 mutant animals are resistant because there is an increase in the expression of smf-3 in these animals. What are the levels of smf-3 expression in mutant animals exposed to pathogenic bacteria? This could be included as part of Figure 7D and 7F. These data would also provide support for the current model.

7) Looking at the pathogen resistance data, it was surprising that the authors decided to pursue further phenotype analysis of the nhr-14 mutant using PA14 rather than S. enterica as the pathogenic bacteria tested. The lifespan phenotype associated with S. enterica was much stronger. The data would be more compelling if Figure 7I was performed with S. enterica. Also in Figure 7I, the intermediate phenotype of the double mutant is hard to interpret and does not support the idea that NHR-14 acts upstream of smf-3. If the loss of NHR-14 makes animals resistant through SMF-3, then the double mutant of nhr-14 and smf-3 would be expected to be as hypersensitive as the single mutant of smf-3.

Reviewer #3:

The paper is well written, the methods are very diverse and appropriately used, and the approach is sound and thorough. The topic is trendy, the interpretations and conclusions are fair and the scientific contribution is significant.

The authors describe a new role for the transcription factor NHR-14, attracting attention to the somewhat understudied extended family of 269 C. elegans HNF4-derived transcription factors. They find a key role for NHR-14 in gut infection handling through iron status modulation (intake by the DMT1/NRAMP2 orthologue SMF-3 and sequestration by FTN-1) and transcriptional regulation of a set of innate immunity-associated genes. Notably, they also identify PQM-1 has a downstream effector that shuttles between the cytosol and the nucleus of enterocytes to directly regulates smf-3 expression.

This study hence connects PQM-1, which is gaining recognition as a major transcription factor in the modulation of C. elegans fat handling, iron metabolism and ageing, to innate immunity. Although expected and supported by two other recent studies, it is an important step toward an understanding of C. elegans innate immunity regulation. Moreover, the upstream role of NHR-14 could not have been inferred from the current literature and it contributes significantly to piecing together the complex network that regulates trade-offs between energy handling and immune responses.

There is a great potential for further studies trying to delineate the exact role of NHR-14 in resistance to infection (it seems specific to certain infections), its upstream regulators (there are hints from mammalian studies, notably specific fatty acids) and how it modulates PQM-1 shuttling, and indirectly DAF-16 shuttling. Connections between immunity, fat metabolism, lipid signalling and aging are being noticed more and more and this study contributes to solving the puzzle.

On a more technical note, I liked the fact that the authors made the effort to accumulate corroborative evidence without relying abusively on a single technical approach. The genetic work is also appreciated (double and triple mutants will make excellent tools for the community). I also liked a couple of points of detail that helped convince me of the interpretations. For instance, the qRT-PCR results give fold increases that match very well the fold increases seen by RNAseq. Also, in Figure 7I, nhr-14 seems to be able to increase smf-3 resistance to P. aeruginosa infection, which is consistent with the idea that NHR-14 increases infection resistance through other genes than smf-3 and iron sequestration alone.

General concerns.

A couple of side issues may have been neglected to ensure that the picture presented is not blown out of proportions (see specific concerns).

The raw and analysed data from the RNAseq and lists of overlapping genes between different datasets should be available. If not, the claims of the authors cannot be verified. It is also common practice to make these datasets available upon publication. It would help the peer-review process if at least the analysed data used in the manuscript were made available to the reviewers.

Although the methods are generally sound, some should be expanded upon to allow for replication of the results. Numbers of animals and/or replicates are low for some experiments and would require additional repeats to meet desirable standards. When it comes to biological replicates, their definition is unclear. One would expect that each biological replicate would be a different population on a different day as interday variability is far greater for C. elegans populations compared to intraday variability, but the low numbers might indicate that biological replicates came from the same day. Clarification is required on this issue.

Specific concerns.

First, the title is slightly misleading. Although the authors provided evidence that a NHR-14 – PQM-1 axis does integrate elements of iron metabolism through SMF-3, the case is less strong when it comes to innate immunity. Indeed, no clear immunity pathway has been defined as it is mostly based on observed enrichment in innate immunity-associated genes in RNAseq analysis without hierarchy. Moreover, direct regulation of innate immunity genes by PQM-1 has not been verified (using biochemical assays, qRT-PCR under infection conditions, or transcriptional reporters in pqm-1 mutant, and/or WT animals under infection conditions). "Integrate" is a too strong word as it suggests pivotal role that is not demonstrated.

Second, SMF-1 and SMF-2 are two other DMT1 homologues in C. elegans and SMF-1 is expressed in the gut just as SMF-3 is. Yet there is no comment on SMF-1 potential role here and its possible regulation by nhr-14, which should have been checked and reported.

The specificity of NHR-14 action on PQM-1 is not clearly established especially in the absence of pathway between the two. Several other high-ranking intestinal transcription factors (ELT-2, DAF-16, SKN-1, ATF-7, NHR-49) have been involved in gut immunity in worms and could have been looked at more carefully, especially when transgenic reporters are available to look at nuclear shuttling (SKN-1::GFP and DAF-16::GFP for instance).

The role of pqm-1 in infection resistance is not clearly established, while nhr-14 and smf-3 are.

HIF-1 is mentioned initially because nhr-14 was found in a suppressor screen for the hif-1 growth defect under iron deprivation, but it is not discussed later, nor is it connected to the findings.

Suggested additional experiments.

– Infections assays with P. aeruginosa and S. enterica on pqm-1 mutants are necessary to demonstrate a functional role of PQM-1 in innate immunity (high priority).

– To corroborate this the PQM-1::GFP strain should be exposed to P. aeruginosa and nuclear shuttling should be observed (high priority).

– Make sure there are at least 3 independent experiments (different days) with a minimum of 30 worms per strain for the infection assays. A good idea would be to simply do the additional experiments required with nhr-14, WT and pqm-1 mutants in parallel (high priority).

C. elegans gut bacterial infections are performed at adult stage because larvae usually do not take up bacteria. It is essential that the PQM-1::GFP nuclear localisation shift seen in L3 larvae on nhr-14 RNAi is replicated in adult worms to convince that PQM-1 can act upon gut bacterial infection to regulate innate immunity genes (high priority).

– qRT-PCR of smf-1, -2 and -3 in nhr-14 and in pqm-1 at least (double mutants nhr-14;pqm-1 welcome) to demonstrate the specificity of SMF-3 regulation (if time and money are an issue, it is not the highest priority experiment)

– qRT-PCR of pqm-1 in nhr-14 mutant vs WT to confirm the RNAseq data (low priority, up to the good will of the authors)

– Look at DAF-16::GFP vs PQM-1 shuttling in control, nhr-14, and pqm-1 RNAi to confirm the antagonistic shuttling between DAF-16 and PQM-1 and the specificity of the effect of nhr-14 on PQM-1 vs DAF-16 or ELT-2 (medium priority, depending on which other experiments are done)

– P. luminescens, E. faecalis, S. marscens, S. aureus infections would have made sense in Figure 7 given that the authors compare their RNAseq data to datasets on worms infected by these pathogens. This seems particularly valid as Table 2 seems to point toward a more specific role of nhr-14 downregulation in resistance against certain pathogens and not others (low priority, up to the good will of the authors).

– Inclusion of the elt-2 microarray dataset published by Block DH et al., 2015 would make sense in Figure 6 and Table 2, considering that the present article did briefly investigate elt-2. (medium priority).

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "NHR-14 loss of function couples intestinal iron uptake with innate immunity in C. elegans through PQM-1 signaling" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Tadatsugu Taniguchi as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Elucidating mechanisms that organisms use to mediate iron homeostasis through the regulation of transcription factors and transport proteins is an important objective in the field of metal biology. This manuscript details the interaction of two transcription factors that link iron homeostasis with the response of C. elegans to infection by pathogenic bacteria. The authors performed an innovative forward genetic screen leading to the identification of two mutations in the nhr-14 gene. They used rigorous methods to demonstrate that mutations in nhr-14 cause the phenotype. They showed NHR-14 is expressed in intestinal cells and other cells in the head and does not appear to be regulated by iron. They characterized the phenotype of nhr-14 mutant animals, including the rescue of the hypersensitivity to low iron phenotype of the hif-1 mutant, an increase in metal content for iron, an increase in the expression of many genes including smf-3 and multiple genes linked to innate immunity, nuclear localization of PQM-1, and resistance to bacterial infections. PQM-1 appears to interact directly with the smf-3 promoter. The findings lead to the proposal that pathogenic infections down regulate nhr-14, leading to increased expression of SMF-3, which imports iron into intestinal cells, thereby depriving the pathogen of iron in the lumen of the intestine and limiting its capacity for proliferation. While largely conclusions are supported by the results, some additional experiments are needed to reinforce the major findings.

Essential revisions:

1) Transcriptome analysis. A main concern is with the analysis presented in Figure 5. Apart from the fact that DAVID has not been updated (https://david.ncifcrf.gov/helps/update.html), it contains far less information than C. elegans-specific tools such as WormExp. Running their list through WormExp reveals several very significant overlaps with other datasets, including nhr-8 mutants that merit at least discussion if not investigation.

Further, the authors write, "Among the 261 downregulated genes, there was enrichment in genes involving cellular organization and body morphogenesis, including sqt-1, noah-1 and sym-1 (Figure 5D and Figure 3—source data 1)".

But what is really striking is the number of genes that normally undergo a sharp drop of expression at the L4 to adult transition, many of them expressed in the epidermis. As can be visualized at https://elegans.mdc-berlin.de/cel_ex.html, this applies, for example, to sqt-1, col-17, rol-8 (the top 3 in Figure 5D), as well as to the heterochronic gene lin-42. There is no comment on this very remarkable pattern, nor the simple experiments that might address the question of how it might be related to the continued nuclear localization of PQM-1 into adulthood.

On the same line, the authors write, "Interestingly, we observed less overlap between nhr-14 upregulated genes with Serratia marscens [marcescens] (Wong et al., 2007). The basis for this difference is unknown, but is likely a result of a highly specialized response to S. marscens [marcescens]". If they had used WormExp, they would have seen a very significant overlap with Serratia-induced genes in independent study (Engelmann et al., 2011). And reading that study, there is even the following, "In the current analysis, the results of our previous study of the response to S. marcescens using oligo-arrays [22] stood out. As this was not the case for the results for the response to 2 other bacterial pathogens, and given the underrepresentation in the S. marcescens data set of 'common response genes' [22], this presumably reflects an experimental difference in the strength of the infection for the samples prepared for analysis using oligo-arrays". In other words, the authors' conclusion is unlikely to be correct. And that makes more sense since the screens that used C. elegans to identify S. marcescens virulence factors highlighted a role for iron (see for example the Abstract of PMID: 12660152, and 25070509).

2) The authors clearly demonstrate that nhr-14 mutants have higher levels of smf-3 transcript, and SMF-3 is a known iron import protein, leading to the hypothesis that this is the critical target gene for the hif-1 rescue. I acknowledge this is a reasonable and even plausible hypothesis. However, the transcriptional analysis of nhr-14 mutant animals demonstrates that hundreds of transcripts are altered, so in principle any one of these changes, or many of these changes in combination might mediate the hif-1 rescue. The critical issue is what additional evidence can test the smf-3 hypothesis. The authors create a smf-3; nhr-14; hif-1 triple mutant and the rescue is lost, in that these triple mutants do not grow in low iron. My concern is that the smf-3 single mutants also do not grow in low iron. So, yes nhr-14 requires smf-3 to rescue hif-1, but this is somewhat different from proving that nhr-14 rescues by increasing smf-3 activity. The authors should acknowledge that because the smf-3 single mutant cannot grow in low iron, there are multiple interpretations for the triple mutant.

3) Additional controls:

3A) Figure 3. Results shown in panels B, C, and D lack essential genotype that has to be tested, namely nhr-14,smf-3 double mutant. This genotype is essential to prove that the phenotypes of nhr-14 mutants are mostly mediated by the elevated level of smf-3.

3B) Figure 5E and D. It is nice to see enrichment in PQM-1 binding motif in the promoters of the upregulated genes, but experimentally do these genes really require PQM-1 for their expression? What will be their level of induction in nhr-14, pqm-1 double mutant? I think this experiment is important to prove that pqm-1 regulates these genes downstream of nhr-14.

3C) Figure 6A. This experiment lacks nhr-14, pqm-1 double mutant. Again, this is essential to prove epistatic relations between the two genes.

https://doi.org/10.7554/eLife.44674.028

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

All the reviewers found the manuscript to be very interesting, but the lack of rigor or in-depth analysis in many areas makes it unsuitable for publication in its current form. The manuscript could be improved by going more in depth on the metal biology or by strengthening the innate immunity aspects. The diversity of the reviewer's suggestions indicate that the revision will likely take more than two months, the time limit in eLife for revision. This is why the paper was rejected. Nevertheless, the authors could consider re-submitting their article to eLife as a new submission once the reviewer's comments have been addressed in full.

Based on the reviewers’ suggestions and comments, we made substantial changes to the manuscript, including addition of new iron pathogen experiments, reanalysis of RNA-seq data and addition of genetic details, which has resulted in a stronger manuscript.

In this manuscript, we report on the identification of a relatively uncharacterized nuclear receptor-1 (NHR-14) that couples innate immunity with iron sequestration. NHR-14 shares homology with vertebrate HNF4. We identified nhr-14 in a genetic suppressor screen conducted to identify genes that rescues the developmental delay of hif-1 mutants grown under iron limitation. We found that nhr-14 loss of function rescues the hif-1 developmental phenotype through increased expression of the intestinal iron importer SMF-3. Transcriptome studies using nhr-14 mutants revealed enrichment of innate immune response genes, many of which are Class 2 genes reported by Murphy et al. (Nature 2003) to be repressed by DAF-16/FOXO during reduced insulin signaling. More recently, Tepper et al. (Cell 2011) discovered that Class 2 genes were activated by the PQM-1 transcription factor during reduced insulin signaling. Using genetic and biochemical approaches, we showed that smf-3 is a PQM-1 target gene. Given the innate immune response and iron signatures in nhr-14 mutants, we showed that nhr-14 loss of function is required for pathogen resistance and that iron sequestration is a component of the pathogen innate immune response. Several nuclear receptors are known regulators of innate immunity in C. elegans; however, NHR-14 is unique among these receptors in coupling innate immunity with iron sequestration. Our data provide new knowledge into how C. elegans use nuclear receptors to regulate innate immunity and iron availability, and show iron sequestration as an important component of the innate immune response.

We believe that our work is of current interest as several of the genes in nhr-14 mutants have been identified in other stress response pathways in C. elegans. For example, O’Brien et al., 2018, identified PQM-1 as a regulator of transcellular chaperone signaling in neurons and in intestine. Several of the key components of neuronal and intestinal signaling pathways and are upregulated in nhr-14 mutants. Of interest, nhr-14 expression is also enriched in neuronal and intestinal cells, providing further evidence suggesting a role for NHR-14 in these pathways. In another paper, Jiang, et al. (eLife2018) identified a genetic pathway that regulates hypothermia stress, and several of the key genes in this pathway, including a transcription factor and proteases, are upregulated in nhr-14 mutants. It is thus likely that NHR-14 has a function in these pathways.

As NHR-14-PQM-1 represents a new innate immunity pathway, future studies are needed to identify other key components in this pathway and endogenous or exogenous ligands that regulate NHR-14 transcriptional function, and to determine the role of NHR-14 in the stress response pathways described above.

[Editors' note: the author responses to the re-review follow.]

Essential revisions:

1) Transcriptome analysis. A main concern is with the analysis presented in Figure 5. Apart from the fact that DAVID has not been updated (https://david.ncifcrf.gov/helps/update.html), it contains far less information than C. elegans-specific tools such as WormExp. Running their list through WormExp reveals several very significant overlaps with other datasets, including nhr-8 mutants that merit at least discussion if not investigation.

We thank the reviewer for this suggestion.

We compared upregulated and downregulated nhr-14 genes with WormExpv1.0 “mutant” category. We found that 148 upregulated nhr-14 genes out of 568 genes overlapped with genes upregulated in nhr-8 mutants. Also, of interest, 156 upregulated nhr-14 genes overlapped with hyl-2 (encoding ceramide synthase) mutants. Together, these observations suggest a role for nhr-14 in fat metabolism. Iron and lipid metabolism are related and many enzymes involved in lipid metabolism require iron for activity. We added the above gene comparisons to a supplemental table (Figure 6—source data 4).

Using WormExp “tissue” category and Cao et al., 2017 single-cell PCR dataset, we found that downregulated nhr-14 genes are enriched in hypodermis and neurons and nhr-14 upregulated genes are enriched in intestine and neurons. These data are consistent with enriched expression of the NHR-14::GFP::FLAG transgene in intestine and head cells (Figure 3B-D). WormExp “tissue” dataset overlap with upregulated and downregulated nhr-14 genes in reported in Figure 5—source data 1.

Further, the authors write, "Among the 261 downregulated genes, there was enrichment in genes involving cellular organization and body morphogenesis, including sqt-1, noah-1 and sym-1 (Figure 5D and Figure 3—source data 1)".

But what is really striking is the number of genes that normally undergo a sharp drop of expression at the L4 to adult transition, many of them expressed in the epidermis. As can be visualized at https://elegans.mdc-berlin.de/cel_ex.html, this applies, for example, to sqt-1, col-17, rol-8 (the top 3 in Figure 5D), as well as to the heterochronic gene lin-42. There is no comment on this very remarkable pattern, nor the simple experiments that might address the question of how it might be related to the continued nuclear localization of PQM-1 into adulthood.

We examined the developmental expression of the top 50 downregulated nhr-14 genes using MDC-BIMSB. In agreement with this reviewer observations, we found that expression of 42 out of the top 50 downregulated nhr-14 genes were sharply downregulated at the late L4 stage, while only 3 were upregulated. The 3 upregulated genes were enriched in intestine, while downregulated nhr-14 genes were enriched in seam and non-seam hypodermal cells (determined from Cao, et al., 2017 single-cell RNA-seq).

We also examined the developmental expression of the top 50 upregulated nhr-14 genes and found most genes are also downregulated at late L4 (20 genes were unchanged or not in the dataset and 6 were upregulated), but not as sharply as the downregulated nhr-14 genes. Of interest, the 6 genes upregulated at late L4 were DAF-16/FoxO Class II genes (dod-19, dod-21, dod-23, dod-24, oac-14, C32H11.9). qPCR analysis revealed that expression of dod-19, dod-24, oac-14 and clec-41 (dod -21 and C32H11.9 were not assessed) were reduced in nhr-14; pqm-1 (RNAi) mutants, indicating that PQM-1 regulate these genes downstream of nhr-14. These new data are reported in Figure 7. See comment 3B below.

We appreciate the reviewer’s effort in uncovering these interesting observations regarding the developmental expression of nhr-14 genes. We are pursuing how loss of nhr-14 causes the nuclear accumulation of PQM-1 in adults, and how it affects gene expression and aging. However, we prefer not to discuss the developmental expression of upregulated and downregulated nhr-14 genes in this manuscript as it would require inclusion of more datasets as well as experiments. These experiments would require a considerable amount of work to do rigorously. We feel that these experiments are beyond the scope of our manuscript, which is to understand the link between nhr-14 regulation of innate immunity and iron metabolism.

On the same line, the authors write, "Interestingly, we observed less overlap between nhr-14 upregulated genes with Serratia marscens [marcescens] (Wong et al., 2007). The basis for this difference is unknown, but is likely a result of a highly specialized response to S. marscens [marcescens]". If they had used WormExp, they would have seen a very significant overlap with Serratia-induced genes in independent study (Engelmann et al., 2011). And reading that study, there is even the following, "In the current analysis, the results of our previous study of the response to S. marcescens using oligo-arrays [22] stood out. As this was not the case for the results for the response to 2 other bacterial pathogens, and given the underrepresentation in the S. marcescens data set of 'common response genes' [22], this presumably reflects an experimental difference in the strength of the infection for the samples prepared for analysis using oligo-arrays". In other words, the authors' conclusion is unlikely to be correct. And that makes more sense since the screens that used C. elegans to identify S. marcescens virulence factors highlighted a role for iron (see for example the Abstract of PMID: 12660152, and 25070509).

We agree and revised this section. We compared the Engelmann et al., 2011 Serratia dataset with upregulated nhr-14 genes and found that out of 2124 genes in the Engelmann dataset, 237 overlapped with upregulated nhr-14 genes. These data are shown in Figure 6A and listed in Figure 6—source data 1. The Wong et al., 2007 Serratia dataset was deleted.

2) The authors clearly demonstrate that nhr-14 mutants have higher levels of smf-3 transcript, and SMF-3 is a known iron import protein, leading to the hypothesis that this is the critical target gene for the hif-1 rescue. I acknowledge this is a reasonable and even plausible hypothesis. However, the transcriptional analysis of nhr-14 mutant animals demonstrates that hundreds of transcripts are altered, so in principle any one of these changes, or many of these changes in combination might mediate the hif-1 rescue. The critical issue is what additional evidence can test the smf-3 hypothesis. The authors create a smf-3; nhr-14; hif-1 triple mutant and the rescue is lost, in that these triple mutants do not grow in low iron. My concern is that the smf-3 single mutants also do not grow in low iron. So, yes nhr-14 requires smf-3 to rescue hif-1, but this is somewhat different from proving that nhr-14 rescues by increasing smf-3 activity. The authors should acknowledge that because the smf-3 single mutant cannot grow in low iron, there are multiple interpretations for the triple mutant.

We agree and added the sentence:”Given that the smf-3(ok1035) single mutant alone displays a developmental delay under iron limitation, this suggests that other genes in nhr-14(tm1473) mutants might also contribute to the rescue of hif-1(ia4) mutants”.

3) Additional controls:

3A) Figure 3. Results shown in panels B, C, and D lack essential genotype that has to be tested, namely nhr-14,smf-3 double mutant. This genotype is essential to prove that the phenotypes of nhr-14 mutants are mostly mediated by the elevated level of smf-3.

We agree and included iron content of the smf-3(ok1035); nhr-14(tm1473) double mutants (Figure 3B) as well as an image and growth analysis of this mutant on NGM and NGM-BP (low iron) (Figure 3C). We did not include the smf-3; nhr-14 double mutant to the life span analysis in panel D because of its poor growth under iron limitation.

3B) Figure 5E and D. It is nice to see enrichment in PQM-1 binding motif in the promoters of the upregulated genes, but experimentally do these genes really require PQM-1 for their expression? What will be their level of induction in nhr-14, pqm-1 double mutant? I think this experiment is important to prove that pqm-1 regulates these genes downstream of nhr-14.

We agree and used qPCR to measure expression of several upregulated nhr-14 genes in nhr-14(tm1473); pqm-1 (RNAi) and nhr-14(tm1473); Control RNAi worms. qPCR analysis showed reduced expression of Class II genes dod-19, dod-24, clec-41, gst-38 and oac-14 in nhr-14; pqm-1 (RNAi) mutants vs nhr-14; Con (RNAi) worms. The expression of Class II genes ins-7 and lys-2 and Class I genes lys-7 and ftn-1 were not changed in nhr-14; pqm-1 (RNAi) mutants. These data are shown in Figure 7. See response to comment #1.

3C) Figure 6A. This experiment lacks nhr-14, pqm-1 double mutant. Again, this is essential to prove epistatic relations between the two genes.

We agree and measured the survival nhr-14(tm1473); pqm-1 (RNAi) worms after infection with PA14. We found that the nhr-14(tm1473); pqm-1 (RNAi) mutants are more sensitive to PA14 that the nhr-14 single mutant, suggesting that PQM-1 is required for nhr-14(tm1473) resistance. These data are shown in Figure 8A (graph) and Figure 8—source data 1 (survival analysis).

https://doi.org/10.7554/eLife.44674.029

Article and author information

Author details

  1. Malini Rajan

    1. Department of Medicine, Division of Hematology, University of Utah, Salt Lake City, United States
    2. Molecular Medicine Program, University of Utah, Salt Lake City, United States
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing
    Contributed equally with
    Cole P Anderson, Paul M Rindler and Steven Joshua Romney
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7653-4223
  2. Cole P Anderson

    1. Molecular Medicine Program, University of Utah, Salt Lake City, United States
    2. Department of Oncological Sciences, University of Utah, Salt Lake City, United States
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft
    Contributed equally with
    Malini Rajan, Paul M Rindler and Steven Joshua Romney
    Competing interests
    No competing interests declared
  3. Paul M Rindler

    1. Department of Medicine, Division of Hematology, University of Utah, Salt Lake City, United States
    2. Molecular Medicine Program, University of Utah, Salt Lake City, United States
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing
    Contributed equally with
    Malini Rajan, Cole P Anderson and Steven Joshua Romney
    Competing interests
    No competing interests declared
  4. Steven Joshua Romney

    1. Department of Medicine, Division of Hematology, University of Utah, Salt Lake City, United States
    2. Molecular Medicine Program, University of Utah, Salt Lake City, United States
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Methodology, Writing—review and editing
    Contributed equally with
    Malini Rajan, Cole P Anderson and Paul M Rindler
    Competing interests
    No competing interests declared
  5. Maria C Ferreira dos Santos

    1. Department of Medicine, Division of Hematology, University of Utah, Salt Lake City, United States
    2. Molecular Medicine Program, University of Utah, Salt Lake City, United States
    Contribution
    Formal analysis, Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  6. Jason Gertz

    1. Department of Oncological Sciences, University of Utah, Salt Lake City, United States
    2. Huntsman Cancer Institute, University of Utah, Salt Lake City, United States
    Contribution
    Software, Formal analysis, Methodology
    Competing interests
    No competing interests declared
  7. Elizabeth A Leibold

    1. Department of Medicine, Division of Hematology, University of Utah, Salt Lake City, United States
    2. Molecular Medicine Program, University of Utah, Salt Lake City, United States
    3. Department of Oncological Sciences, University of Utah, Salt Lake City, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    betty.leibold@genetics.utah.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1000-9503

Funding

NIH Office of the Director (R01DK068602)

  • Elizabeth A Leibold

NIH Office of the Director (T32DK007115)

  • Cole P Anderson
  • Paul M Rindler

NIH Office of the Director (R00HG006922)

  • Jason Gertz

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by the NIH awards R01DK068602 (EAL), T32DK007115 (CPA and PMR) and R00HG006922 (JG) and University of Utah SEED grant (EAL). This work was supported by the NIDDK Cooperative Hematology Specialized Core Center award U54DK110858, specifically the Iron and Heme Core for assistance with metal analysis, and by the High-Throughput Genomics and Bioinformatic Analysis Shared Resource at Huntsman Cancer Institute at the University of Utah NIH award P30CA042014. Strains were provided by the Caenorhabditis Genetics Center (CGC) and nhr-14(tm1473) was obtained from the National Bioresource Project, Tokyo Womens Medical University. NHR-14::GFP::FLAG fosmid was provided by TransgeneOme. We thank Ms. Diana Lim for figure preparation and the reviewers for comments and suggestions. The authors have declared that no competing interests exist.

Senior Editor

  1. Tadatsugu Taniguchi, Institute of Industrial Science, the University of Tokyo, Japan

Reviewing Editor

  1. Bruno Lemaître, École Polytechnique Fédérale de Lausanne, Switzerland

Publication history

  1. Received: April 8, 2019
  2. Accepted: September 17, 2019
  3. Accepted Manuscript published: September 18, 2019 (version 1)
  4. Version of Record published: October 4, 2019 (version 2)
  5. Version of Record updated: October 15, 2019 (version 3)

Copyright

© 2019, Rajan 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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