Chemical Genetics in C. elegans Identifies Anticancer Mycotoxins Chaetocin and Chetomin as Potent Inducers of a Nuclear Metal Homeostasis Response.

C. elegans numr-1/2 (nuclear-localized metal-responsive) is an identical gene pair encoding a nuclear protein previously shown to be activated by cadmium and disruption of the integrator RNA metabolism complex. We took a chemical genetic approach to further characterize regulation of this novel metal response by screening 41,716 compounds and extracts for numr-1p::GFP activation. The most potent activator was chaetocin, a fungal 3,6-epidithiodiketopiperazine (ETP) with promising anticancer activity. Chaetocin activates numr-1/2 strongly in the alimentary canal but is distinct from metal exposure, because it represses canonical cadmium-responsive metallothionine genes. Chaetocin has diverse targets in cancer cells including thioredoxin reductase, histone lysine methyltransferase, and acetyltransferase p300/CBP; further work is needed to identify the mechanism in C. elegans as genetic disruption and RNAi screening of homologues did not induce numr-1/2 in the alimentary canal and chaetocin did not affect markers of integrator dysfunction. We demonstrate that disulfides in chaetocin and chetomin, a dimeric ETP analog, are required to induce numr-1/2. ETP monomer gliotoxin, despite possessing a disulfide linkage, had almost no effect on numr-1/2, suggesting a dimer requirement. Chetomin inhibits C. elegans growth at low micromolar levels, and loss of numr-1/2 increases sensitivity; C. elegans and Chaetomiaceae fungi inhabit similar environments raising the possibility that numr-1/2 functions as a defense mechanism. There is no direct orthologue of numr-1/2 in humans, but RNaseq suggests that chaetocin affects expression of cellular processes linked to stress response and metal homeostasis in colorectal cancer cells. Our results reveal interactions between metal response gene regulation and ETPs and identify a potential mechanism of resistance to this versatile class of preclinical compounds.

RNAi screening for modulators of an osmo-sensitive gene response to extracellular matrix damage reveals negative feedback and interactions with translation inhibition.

In epidermal tissues, extracellular matrices (ECMs) function as barriers between the organism and environment. Despite being at the interface with the environment, little is known about the role of animal barrier ECMs in sensing stress and communicating with cytoprotective gene pathways in neighboring cells. We and others have identified a putative damage sensor in the C. elegans cuticle that regulates osmotic, detoxification, and innate immune response genes. This pathway is associated with circumferential collagen bands called annular furrows; mutation or loss of furrow collagens causes constitutive activation of osmotic, detoxification, and innate immune response genes. Here, we performed a genome-wide RNAi screen for modulators of osmotic stress response gene gpdh-1 in a furrow collagen mutant strain. RNAi of six genes identified in this screen were tested under other conditions and for effects on other stress responses. The functions of these genes suggest negative feedback within osmolyte accumulation pathways and interactions with ATP homeostasis and protein synthesis. Loss of these gpdh-1 modulators had distinct effects on canonical detoxification and innate immune response genes.

CCR4-NOT subunit CCF-1/CNOT7 promotes transcriptional activation to multiple stress responses in Caenorhabditis elegans.

CCR4-NOT is a versatile eukaryotic protein complex that controls multiple steps in gene expression regulation from synthesis to decay. In yeast, CCR4-NOT has been implicated in stress response regulation, though this function in other organisms remains unclear. In a genome-wide RNAi screen, we identified a subunit of the CCR4-NOT complex, ccf-1, as a requirement for the C. elegans transcriptional response to cadmium and acrylamide stress. Using whole-transcriptome RNA sequencing, we show that the knockdown of ccf-1 attenuates the activation of a broad range of stress-protective genes in response to cadmium and acrylamide, including those encoding heat shock proteins and xenobiotic detoxification. Consistently, survival assays show that the knockdown of ccf-1 decreases C. elegans stress resistance and normal lifespan. A yeast 2-hybrid screen using a CCF-1 bait identified the homeobox transcription factor PAL-1 as a physical interactor. Knockdown of pal-1 inhibits the activation of ccf-1 dependent stress genes and reduces C. elegans stress resistance. Gene expression analysis reveals that knockdown of ccf-1 and pal-1 attenuates the activation of elt-2 and elt-3 under stress that encode master transcriptional co-regulators of stress response in the C. elegans, and that overexpression of ELT-2 can suppress ccf-1's requirement for gene transcription in a stress-dependent manner. Our findings reveal a new role for CCR4-NOT in the environmental stress response and define its role in stress resistance and longevity in C. elegans.

Sexual dimorphism in Caenorhabditis elegans stress resistance.

Physiological responses to the environment, disease, and aging vary by sex in many animals, but mechanisms of dimorphism have only recently begun to receive careful attention. The genetic model nematode Caenorhabditis elegans has well-defined mechanisms of stress response, aging, and sexual differentiation. C. elegans has males, but the vast majority of research only uses hermaphrodites. We found that males of the standard N2 laboratory strain were more resistant to hyperosmolarity, heat, and a natural pro-oxidant than hermaphrodites when in mixed-sex groups. Resistance to heat and pro-oxidant were also male-biased in three genetically and geographically diverse C. elegans strains consistent with a species-wide dimorphism that is not specific to domestication. N2 males were also more resistant to heat and pro-oxidant when keep individually indicating that differences in resistance do not require interactions between worms. We found that males induce canonical stress response genes by similar degrees and in similar tissues as hermaphrodites suggesting the importance of other mechanisms. We find that resistance to heat and pro-oxidant are influenced by the sex differentiation transcription factor TRA-1 suggesting that downstream organ differentiation pathways establish differences in stress resistance. Environmental stress influences survival in natural environments, degenerative disease, and aging. Understanding mechanisms of stress response dimorphism can therefore provide insights into sex-specific population dynamics, disease, and longevity.

Extracellular matrix regulation of stress response genes during larval development in Caenorhabditis elegans.

Mutation or loss of 6 extracellular matrix collagen genes disrupts annular furrows in adult C. elegans cuticles, causes a wide "Dumpy" body morphology, and activates osmotic, detoxification, and antimicrobial defense genes. High environmental osmolarity reduces internal turgor pressure, physically distorts the epidermis, and activates the same stress responses. Collagen gene mutations that cause Dumpy without furrow disruption do not activate stress responses. These results are consistent with an extracellular damage sensor associated with furrows in the adult cuticle that regulates environmental stress responses in adjacent cells. Several cuticle characteristics change between molts, but all stages have annular furrows and express furrow collagen genes. We compared body shape, furrow organization imaged with differential interference contrast microscopy, and stress response gene expression in furrow collagen gene mutants at all postembryonic stages. We find that most body shape and furrow disorganization phenotypes start at the L3 stage and increase in severity with each molt afterwards. Stress response genes were induced the strongest in adults, correlating with the greatest Dumpy and furrow phenotypes. Although weaker than in adults, osmolyte transporter gene hmit-1.1 and antimicrobial gene nlp-29 were also induced in some early larvae that had weak or undetectable cuticle phenotypes. Our data are consistent with progressive cuticle phenotypes in which each new cuticle is at least partially directed by organization of the former cuticle. Gene expression and cuticle data support the role of furrow disruption as a signal in L4 larvae and adults, but also suggest a role for other cuticle organization or epidermal cell effects in early larvae.

An extracellular matrix damage sensor signals through membrane-associated kinase DRL-1 to mediate cytoprotective responses in Caenorhabditis elegans.

We and others previously identified circumferential bands of collagen named annular furrows as key components of a damage sensor in the cuticle of Caenorhabditis elegans that regulates cytoprotective genes. Mutation or loss of noncollagen secreted proteins OSM-7, OSM-8, and OSM-11 activate the same cytoprotective responses without obvious changes to the cuticle indicating that other extracellular proteins are involved. Here, we used RNAi screening to identify protein kinase DRL-1 as a key modulator of cytoprotective gene expression and stress resistance in furrow and extracellular OSM protein mutants. DRL-1 functions downstream from furrow disruption and is expressed in cells that induce cytoprotective genes. DRL-1 is not required for the expression of cytoprotective genes under basal or oxidative stress conditions consistent with specificity to extracellular signals. DRL-1 was previously shown to regulate longevity via a "Dietary Restriction-Like" state, but it functions downstream from furrow disruption by a distinct mechanism. The kinase domain of DRL-1 is related to mammalian MEKK3, and MEKK3 is recruited to a plasma membrane osmosensor complex by a scaffold protein. In C. elegans, DRL-1 contains an atypical hydrophobic C-terminus with predicted transmembrane domains and is constitutively expressed at or near the plasma membrane where it could function to receive extracellular damage signals for cells that mount cytoprotective responses.

Synchronous and collaborative online concept mapping of membrane transport.

The rush to remote learning during the COVD-19 pandemic has caused instructors to rapidly adapt mechanisms of learning. Here, I describe an online concept mapping activity for membrane transport mechanisms that can be accomplished by students working together remotely and either synchronously or asynchronously.

RNA processing errors triggered by cadmium and integrator complex disruption are signals for environmental stress.

BACKGROUND: Adaptive responses to stress are essential for cell and organismal survival. In metazoans, little is known about the impact of environmental stress on RNA homeostasis. RESULTS: By studying the regulation of a cadmium-induced gene named numr-1 in Caenorhabditis elegans, we discovered that disruption of RNA processing acts as a signal for environmental stress. We find that NUMR-1 contains motifs common to RNA splicing factors and influences RNA splicing in vivo. A genome-wide screen reveals that numr-1 is strongly and specifically induced by silencing of genes that function in basal RNA metabolism including subunits of the metazoan integrator complex. Human integrator processes snRNAs for functioning with splicing factors, and we find that silencing of C. elegans integrator subunits disrupts snRNA processing, causes aberrant pre-mRNA splicing, and induces the heat shock response. Cadmium, which also strongly induces numr-1, has similar effects on RNA and the heat shock response. Lastly, we find that heat shock factor-1 is required for full numr-1 induction by cadmium. CONCLUSION: Our results are consistent with a model in which disruption of integrator processing of RNA acts as a molecular damage signal initiating an adaptive stress response mediated by heat shock factor-1. When numr-1 is induced via this pathway in C. elegans, its function in RNA metabolism may allow it to mitigate further damage and thereby promote tolerance to cadmium.

A Damage Sensor Associated with the Cuticle Coordinates Three Core Environmental Stress Responses in Caenorhabditis elegans.

Extracellular matrix barriers and inducible cytoprotective genes form successive lines of defense against chemical and microbial environmental stressors. The barrier in nematodes is a collagenous extracellular matrix called the cuticle. In Caenorhabditis elegans, disruption of some cuticle collagen genes activates osmolyte and antimicrobial response genes. Physical damage to the epidermis also activates antimicrobial responses. Here, we assayed the effect of knocking down genes required for cuticle and epidermal integrity on diverse cellular stress responses. We found that disruption of specific bands of collagen, called annular furrows, coactivates detoxification, hyperosmotic, and antimicrobial response genes, but not other stress responses. Disruption of other cuticle structures and epidermal integrity does not have the same effect. Several transcription factors act downstream of furrow loss. SKN-1/Nrf and ELT-3/GATA are required for detoxification, SKN-1/Nrf is partially required for the osmolyte response, and STA-2/Stat and ELT-3/GATA for antimicrobial gene expression. Our results are consistent with a cuticle-associated damage sensor that coordinates detoxification, hyperosmotic, and antimicrobial responses through overlapping, but distinct, downstream signaling.

F-Box Protein XREP-4 Is a New Regulator of the Oxidative Stress Response in Caenorhabditis elegans.

The transcription factor SKN-1 (Skinhead family member-1) in Caenorhabditis elegans is a homolog of the mammalian Nrf-2 protein and functions to promote oxidative stress resistance and longevity. SKN-1 mediates protection from reactive oxygen species (ROS) via the transcriptional activation of genes involved in antioxidant defense and phase II detoxification. Although many core regulators of SKN-1 have been identified, much remains unknown about this complex signaling pathway. We carried out an ethyl methanesulfonate (EMS) mutagenesis screen and isolated six independent mutants with attenuated SKN-1-dependent gene activation in response to acrylamide. All six were found to contain mutations in F46F11.6/xrep-4 (xenobiotics response pathways-4), which encodes an uncharacterized F-box protein. Loss of xrep-4 inhibits the skn-1-dependent expression of detoxification genes in response to prooxidants and decreases survival of oxidative stress, but does not shorten life span under standard culture conditions. XREP-4 interacts with the ubiquitin ligase component SKR-1 and the SKN-1 principal repressor WDR-23, and knockdown of xrep-4 increases nuclear localization of a WDR-23::GFP fusion protein. Furthermore, a missense mutation in the conserved XREP-4 F-box domain that reduces interaction with SKR-1 but not WDR-23 strongly attenuates SKN-1-dependent gene activation. These results are consistent with XREP-4 influencing the SKN-1 stress response by functioning as a bridge between WDR-23 and the ubiquitin ligase component SKR-1.

The Skp1 Homologs SKR-1/2 Are Required for the Caenorhabditis elegans SKN-1 Antioxidant/Detoxification Response Independently of p38 MAPK.

SKN-1/Nrf are the primary antioxidant/detoxification response transcription factors in animals and they promote health and longevity in many contexts. SKN-1/Nrf are activated by a remarkably broad-range of natural and synthetic compounds and physiological conditions. Defining the signaling mechanisms that regulate SKN-1/Nrf activation provides insights into how cells coordinate responses to stress. Nrf2 in mammals is regulated in part by the redox sensor repressor protein named Keap1. In C. elegans, the p38 MAPK cascade in the intestine activates SKN-1 during oxidative stress by promoting its nuclear accumulation. Interestingly, we find variation in the kinetics of p38 MAPK activation and tissues with SKN-1 nuclear accumulation among different pro-oxidants that all trigger strong induction of SKN-1 target genes. Using genome-wide RNAi screening, we identify new genes that are required for activation of the core SKN-1 target gene gst-4 during exposure to the natural pro-oxidant juglone. Among 10 putative activators identified in this screen was skr-1/2, highly conserved homologs of yeast and mammalian Skp1, which function to assemble protein complexes. Silencing of skr-1/2 inhibits induction of SKN-1 dependent detoxification genes and reduces resistance to pro-oxidants without decreasing p38 MAPK activation. Global transcriptomics revealed strong correlation between genes that are regulated by SKR-1/2 and SKN-1 indicating a high degree of specificity. We also show that SKR-1/2 functions upstream of the WD40 repeat protein WDR-23, which binds to and inhibits SKN-1. Together, these results identify a novel p38 MAPK independent signaling mechanism that activates SKN-1 via SKR-1/2 and involves WDR-23.

Inhibition of the oxidative stress response by heat stress in Caenorhabditis elegans.

It has long been recognized that simultaneous exposure to heat stress and oxidative stress shows a synergistic interaction that reduces organismal fitness, but relatively little is known about the mechanisms underlying this interaction. We investigated the role of molecular stress responses in driving this synergistic interaction using the nematode Caenorhabditis elegans To induce oxidative stress, we used the pro-oxidant compounds acrylamide, paraquat and juglone. As expected, we found that heat stress and oxidative stress interact synergistically to reduce survival. Compared with exposure to each stressor alone, during simultaneous sublethal exposure to heat stress and oxidative stress the normal induction of key oxidative-stress response (OxSR) genes was generally inhibited, whereas the induction of key heat-shock response (HSR) genes was not. Genetically activating the SKN-1-dependent OxSR increased a marker for protein aggregation and decreased whole-worm survival during heat stress alone, with the latter being independent of HSF-1. In contrast, compared with wild-type worms, inactivating the HSR by HSF-1 knockdown, which would be expected to decrease basal heat shock protein expression, increased survival during oxidative stress alone. Taken together, these data suggest that, in C. elegans, the HSR and OxSR cannot be simultaneously activated to the same extent that each can be activated during a single stressor exposure. We conclude that the observed synergistic reduction in survival during combined exposure to heat stress and oxidative stress is due, at least in part, to inhibition of the OxSR during activation of the HSR.

Characterization of skn-1/wdr-23 phenotypes in Caenorhabditis elegans; pleiotrophy, aging, glutathione, and interactions with other longevity pathways.

The SKN-1/Nrf transcription factors are master regulators of oxidative stress responses and are emerging as important determinants of longevity. We previously identified a protein named WDR-23 as a direct repressor of SKN-1 in C. elegans. Loss of wdr-23 influences stress resistance, longevity, development, and reproduction, but it is unknown if WDR-23 influences development and reproduction solely through SKN-1 and the mechanisms by which SKN-1 promotes stress resistance and longevity are poorly defined. Here, we characterize phenotypes of wdr-23 and skn-1 manipulation and explore the role of glutathione. We provide evidence that diverse wdr-23 phenotypes are dependent on SKN-1, that beneficial and detrimental phenotypes of wdr-23 and skn-1 can be partially decoupled, and that SKN-1 activation delays degenerative tissue changes during aging. We also show that total glutathione levels are substantially elevated when the wdr-23/skn-1 pathway is activated and that skn-1 is required for preserving this cellular antioxidant during stress and aging. Alternatively, total glutathione was not elevated in worms with reduced insulin/IGF-1-like signaling or dietary restriction suggesting that SKN-1 ensures longevity via different mechanisms under these conditions. Lastly, genetic interaction data revise our understanding of which skn-1 variants are required for longevity during dietary restriction.

Direct interaction between the WD40 repeat protein WDR-23 and SKN-1/Nrf inhibits binding to target DNA.

SKN-1/Nrf transcription factors activate cytoprotective genes in response to reactive small molecules and strongly influence stress resistance, longevity, and development. The molecular mechanisms of SKN-1/Nrf regulation are poorly defined. We previously identified the WD40 repeat protein WDR-23 as a repressor of Caenorhabditis elegans SKN-1 that functions with a ubiquitin ligase to presumably target the factor for degradation. However, SKN-1 activity and nuclear accumulation are not always correlated, suggesting that there could be additional regulatory mechanisms. Here, we integrate forward genetics and biochemistry to gain insights into how WDR-23 interacts with and regulates SKN-1. We provide evidence that WDR-23 preferentially regulates one of three SKN-1 variants through a direct interaction that is required for normal stress resistance and development. Homology modeling predicts that WDR-23 folds into a ß-propeller, and we identify the top of this structure and four motifs at the termini of SKN-1c as essential for the interaction. Two of these SKN-1 motifs are highly conserved in human Nrf1 and Nrf2 and two directly interact with target DNA. Lastly, we demonstrate that WDR-23 can block the ability of SKN-1c to interact with DNA sequences of target promoters identifying a new mechanism of regulation that is independent of the ubiquitin proteasome system, which can become occupied with damaged proteins during stress.

Phylogenetic, expression, and functional analyses of anoctamin homologs in Caenorhabditis elegans.

Ca(2+)-activated Cl(-) channels (CaCCs) are critical to processes such as epithelial transport, membrane excitability, and signal transduction. Anoctamin, or TMEM16, is a family of 10 mammalian transmembrane proteins, 2 of which were recently shown to function as CaCCs. The functions of other family members have not been firmly established, and almost nothing is known about anoctamins in invertebrates. Therefore, we performed a phylogenetic analysis of anoctamins across the animal kingdom and examined the expression and function of anoctamins in the genetically tractable nematode Caenorhabditis elegans. Phylogenetic analyses support five anoctamin clades that are at least as old as the deuterostome/protosome ancestor. This includes a branch containing two Drosophila paralogs that group with mammalian ANO1 and ANO2, the two best characterized CaCCs. We identify two anoctamins in C. elegans (ANOH-1 and ANOH-2) that are also present in basal metazoans. The anoh-1 promoter is active in amphid sensory neurons that detect external chemical and nociceptive cues. Within amphid neurons, ANOH-1::GFP fusion protein is enriched within sensory cilia. RNA interference silencing of anoh-1 reduced avoidance of steep osmotic gradients without disrupting amphid cilia development, chemotaxis, or withdrawal from noxious stimuli, suggesting that ANOH-1 functions in a sensory mode-specific manner. The anoh-2 promoter is active in mechanoreceptive neurons and the spermatheca, but loss of anoh-2 had no effect on motility or brood size. Our study indicates that at least five anoctamin duplicates are evolutionarily ancient and suggests that sensory signaling may be a basal function of the anoctamin protein family.

A negative-feedback loop between the detoxification/antioxidant response factor SKN-1 and its repressor WDR-23 matches organism needs with environmental conditions.

Negative-feedback loops between transcription factors and repressors in responses to xenobiotics, oxidants, heat, hypoxia, DNA damage, and infection have been described. Although common, the function of feedback is largely unstudied. Here, we define a negative-feedback loop between the Caenorhabditis elegans detoxification/antioxidant response factor SKN-1/Nrf and its repressor wdr-23 and investigate its function in vivo. Although SKN-1 promotes stress resistance and longevity, we find that tight regulation by WDR-23 is essential for growth and reproduction. By disabling SKN-1 transactivation of wdr-23, we reveal that feedback is required to set the balance between growth/reproduction and stress resistance/longevity. We also find that feedback is required to set the sensitivity of a core SKN-1 target gene to an electrophile. Interestingly, the effect of feedback on target gene induction is greatly reduced when the stress response is strongly activated, presumably to ensure maximum activation of cytoprotective genes during potentially fatal conditions. Our work provides a framework for understanding the function of negative feedback in inducible stress responses and demonstrates that manipulation of feedback alone can shift the balance of competing animal processes toward cell protection, health, and longevity.

In vitro and in vivo characterization of a tunable dual-reactivity probe of the Nrf2-ARE pathway.

The cell utilizes the Keap1/Nrf2-ARE signaling pathway to detoxify harmful chemicals in order to protect itself from oxidative stress and to maintain its reducing environment. When exposed to oxidative stress and xenobiotic inducers, the redox sensitive Keap1 is covalently modified at specific cysteine residues. Consequently, the latent transcription factor Nrf2 is stabilized and translocates into the nucleus, where it transactivates the expression of detoxification genes through binding to the antioxidant response element (ARE). In the pursuit of potent and bioavailable activators of the ARE, we validated hits from a pathway-directed high-throughput screening campaign by testing them in cell culture and a reporter strain of a whole animal model, Caenorhabditis elegans. These studies allowed us to identify AI-3 as an ARE activator that induces cytoprotective genes in human cells and in worms, which also translated into in vivo activity in mice. AI-3 is an electrophilic ARE activator with two thiol sensitive sites toward a nucleophilic aromatic substitution, and SAR studies indicated the tunability of the system. Tandem LC-MS analysis revealed that AI-3 alkylates Keap1 primarily at Cys151, while AI-3 is reactive toward additional cysteine residues at higher doses in vitro and in vivo. The immediate effects of such alkylation included the disruption of Keap1-Cul3 (low [AI-3]) and/or Keap1-Nrf2 (high [AI-3]) interactions that both led to the stabilization of Nrf2. This further translated into the downstream Nrf2-ARE regulated cytoprotective gene activation. Collectively, AI-3 may become a valuable biological tool and may even provide therapeutic benefits in oxidative stress related diseases.

Physiological and molecular mechanisms of salt and water homeostasis in the nematode Caenorhabditis elegans.

Intracellular salt and water homeostasis is essential for all cellular life. Extracellular salt and water homeostasis is also important for multicellular organisms. Many fundamental mechanisms of compensation for osmotic perturbations are well defined and conserved. Alternatively, molecular mechanisms of detecting salt and water imbalances and regulating compensatory responses are generally poorly defined for animals. Throughout the last century, researchers studying vertebrates and vertebrate cells made critical contributions to our understanding of osmoregulation, especially mechanisms of salt and water transport and organic osmolyte accumulation. Researchers have more recently started using invertebrate model organisms with defined genomes and well-established methods of genetic manipulation to begin defining the genes and integrated regulatory networks that respond to osmotic stress. The nematode Caenorhabditis elegans is well suited to these studies. Here, I introduce osmoregulatory mechanisms in this model, discuss experimental advantages and limitations, and review important findings. Key discoveries include defining genetic mechanisms of osmolarity sensing in neurons, identifying protein damage as a sensor and principle determinant of hypertonic stress resistance, and identification of a putative sensor for hypertonic stress associated with the extracellular matrix. Many of these processes and pathways are conserved and, therefore, provide new insights into salt and water homeostasis in other animals, including mammals.