Quiescence enhances survival during viral infection in Caenorhabditis elegans.

Infection causes reduced activity, anorexia, and sleep, which are components of the phylogenetically conserved but poorly-understood sickness behavior. We developed a C. elegans model to study quiescence during chronic infection, using infection with the Orsay virus. The Orsay virus infects intestinal cells yet strongly affects behavior, indicating gut-to-nervous system communication. Infection quiescence has the sleep properties of reduced responsiveness and rapid reversibility. Both the ALA and RIS neurons regulate virus-induced quiescence though ALA plays a more prominent role. Quiescence-defective animals have decreased survival when infected, indicating a benefit of quiescence during chronic infectious disease. The survival benefit of quiescence is not explained by a difference in viral load, indicating that it improves resilience rather than resistance to infection. Orsay infection is associated with a decrease in ATP levels, and this decrease is more severe in quiescence-defective animals. We propose that quiescence preserves energetic resources by reducing energy expenditures and/or by increasing extraction of energy from nutrients. This model presents an opportunity to explore the role of sleep and fatigue in chronic infectious illness.Significance statement Sleepiness and fatigue are cardinal symptoms of infectious disease. We do not understand the mechanisms regulating sleep during sickness, how sickness sleep differs from regular sleep, and whether sickness sleep is beneficial to recovery from illness. We present a model for studying sleep-like quiescence during viral infection in the roundworm C. elegans Sickness quiescence in worms relies on different neural circuitry than other forms of sleep. Quiescence during viral infection increases survival by improving resilience. Loss of quiescence during sickness leads to a drop in ATP levels, suggesting that it improves survival by conserving energy.

Dietary vitamin B12 regulates chemosensory receptor gene expression via the MEF2 transcription factor in Caenorhabditis elegans.

Dynamic changes in chemoreceptor gene expression levels in sensory neurons are one strategy that an animal can use to modify their responses to dietary changes. However, the mechanisms underlying diet-dependent modulation of chemosensory gene expression are unclear. Here, we show that the expression of the srh-234 chemoreceptor gene localized in a single ADL sensory neuron type of Caenorhabditis elegans is downregulated when animals are fed a Comamonas aquatica bacterial diet, but not on an Escherichia coli diet. Remarkably, this diet-modulated effect on srh-234 expression is dependent on the micronutrient vitamin B12 endogenously produced by Comamonas aq. bacteria. Excess propionate and genetic perturbations in the canonical and shunt propionate breakdown pathways are able to override the repressive effects of vitamin B12 on srh-234 expression. The vitamin B12-mediated regulation of srh-234 expression levels in ADL requires the MEF-2 MADS domain transcription factor, providing a potential mechanism by which dietary vitamin B12 may transcriptionally tune individual chemoreceptor genes in a single sensory neuron type, which in turn may change animal responses to biologically relevant chemicals in their diet.

Loss of circRNAs from the crh-1 gene extends the mean lifespan in Caenorhabditis elegans.

Accumulation of circular RNAs (circRNAs) during aging occurs on a genome-wide level for multiple organisms, but its significance is unknown. Generating circRNA loss-of-function mutants is difficult because the vast majority of these RNAs are comprised of exons shared with protein-coding mRNAs. In Caenorhabditis elegans, most circRNAs were previously found to accumulate during aging. Two of the most abundant, age-accumulating circRNAs are generated from exon 4 of the crh-1 gene (circ-crh-1). Here, we found that the biogenesis of circ-crh-1 was regulated by the double-stranded RNA-binding protein ADR-1. We identified Reverse Complementary Match (RCM) sequences in introns flanking circ-crh-1. Using CRISPR-Cas9, we deleted the downstream RCM and found that this completely eliminated expression of the circRNA without affecting linear mRNA expression from the crh-1 gene. Remarkably, worms lacking circ-crh-1 exhibited a significantly longer mean lifespan. Lifespan was partially restored to wild type by expression of circ-crh-1 in neural tissues. Widespread transcriptome alterations in circ-crh-1 mutants were identified using RNA-Seq. Moving forward, intronic RCM deletion using CRISPR should be a widely applicable method to identify lifespan-regulating circRNAs in C. elegans.

A salt-induced kinase is required for the metabolic regulation of sleep.

Many lines of evidence point to links between sleep regulation and energy homeostasis, but mechanisms underlying these connections are unknown. During Caenorhabditis elegans sleep, energetic stores are allocated to nonneural tasks with a resultant drop in the overall fat stores and energy charge. Mutants lacking KIN-29, the C. elegans homolog of a mammalian Salt-Inducible Kinase (SIK) that signals sleep pressure, have low ATP levels despite high-fat stores, indicating a defective response to cellular energy deficits. Liberating energy stores corrects adiposity and sleep defects of kin-29 mutants. kin-29 sleep and energy homeostasis roles map to a set of sensory neurons that act upstream of fat regulation as well as of central sleep-controlling neurons, suggesting hierarchical somatic/neural interactions regulating sleep and energy homeostasis. Genetic interaction between kin-29 and the histone deacetylase hda-4 coupled with subcellular localization studies indicate that KIN-29 acts in the nucleus to regulate sleep. We propose that KIN-29/SIK acts in nuclei of sensory neuroendocrine cells to transduce low cellular energy charge into the mobilization of energy stores, which in turn promotes sleep.

Global accumulation of circRNAs during aging in Caenorhabditis elegans.

BACKGROUND: Circular RNAs (CircRNAs) are a newly appreciated class of RNAs that lack free 5' and 3' ends, are expressed by the thousands in diverse forms of life, and are mostly of enigmatic function. Ostensibly due to their resistance to exonucleases, circRNAs are known to be exceptionally stable. Previous work in Drosophila and mice have shown that circRNAs increase during aging in neural tissues. RESULTS: Here, we examined the global profile of circRNAs in C. elegans during aging by performing ribo-depleted total RNA-seq from the fourth larval stage (L4) through 10-day old adults. Using stringent bioinformatic criteria and experimental validation, we annotated a high-confidence set of 1166 circRNAs, including 575 newly discovered circRNAs. These circRNAs were derived from 797 genes with diverse functions, including genes involved in the determination of lifespan. A massive accumulation of circRNAs during aging was uncovered. Many hundreds of circRNAs were significantly increased among the aging time-points and increases of select circRNAs by over 40-fold during aging were quantified by RT-qPCR. The expression of 459 circRNAs was determined to be distinct from the expression of linear RNAs from the same host genes, demonstrating host gene independence of circRNA age-accumulation. CONCLUSIONS: We attribute the global scale of circRNA age-accumulation to the high composition of post-mitotic cells in adult C. elegans, coupled with the high resistance of circRNAs to decay. These findings suggest that the exceptional stability of circRNAs might explain age-accumulation trends observed from neural tissues of other organisms, which also have a high composition of post-mitotic cells. Given the suitability of C. elegans for aging research, it is now poised as an excellent model system to determine whether there are functional consequences of circRNA accumulation during aging.

Cell-Autonomous and Non-Cell-Autonomous Regulation of a Feeding State-Dependent Chemoreceptor Gene via MEF-2 and bHLH Transcription Factors.

Food and feeding-state dependent changes in chemoreceptor gene expression may allow Caenorhabditis elegans to modify their chemosensory behavior, but the mechanisms essential for these expression changes remain poorly characterized. We had previously shown that expression of a feeding state-dependent chemoreceptor gene, srh-234, in the ADL sensory neuron of C. elegans is regulated via the MEF-2 transcription factor. Here, we show that MEF-2 acts together with basic helix-loop-helix (bHLH) transcription factors to regulate srh-234 expression as a function of feeding state. We identify a cis-regulatory MEF2 binding site that is necessary and sufficient for the starvation-induced down regulation of srh-234 expression, while an E-box site known to bind bHLH factors is required to drive srh-234 expression in ADL. We show that HLH-2 (E/Daughterless), HLH-3 and HLH-4 (Achaete-scute homologs) act in ADL neurons to regulate srh-234 expression. We further demonstrate that the expression levels of srh-234 in ADL neurons are regulated remotely by MXL-3 (Max-like 3 homolog) and HLH-30 (TFEB ortholog) acting in the intestine, which is dependent on insulin signaling functioning specifically in ADL neurons. We also show that this intestine-to-neuron feeding-state regulation of srh-234 involves a subset of insulin-like peptides. These results combined suggest that chemoreceptor gene expression is regulated by both cell-autonomous and non-cell-autonomous transcriptional mechanisms mediated by MEF2 and bHLH factors, which may allow animals to fine-tune their chemosensory responses in response to changes in their feeding state.

Plasticity of chemoreceptor gene expression: Sensory and circuit inputs modulate state-dependent chemoreceptors.

Animals dramatically modify their chemosensory behaviors when starved, which could allow them to alter and optimize their food-search strategies. Dynamic changes in the gene expression of chemoreceptors may be a general mechanism underlying food and state-dependent changes in chemosensory behaviors. In our recent study,(1) we identified chemoreceptors in the ADL sensory neuron type of C. elegans that are modulated by feeding state and food availability. Here, we highllight our recent findings by which sensory inputs into ADL, neuronal outputs from ADL, and circuit inputs from the RMG interneuron, which is electrically connected to ADL, are required to regulate an ADL-expressed chemoreceptor. This sensory and circuit-mediated regulation of chemoreceptor gene expression is dependent on cell-autonomous pathways acting in ADL, e.g. KIN-29, DAF-2, OCR-2 and calcium signaling, and circuit inputs from RMG mediated by NPR-1. Based on these findings, we propose an intriguing but speculative feedback modulatory circuit mechanism by which sensory perception of food and internal state signals may be coupled to regulate ADL-expressed chemoreceptors, which may allow animals to precisely regulate and fine-tune their chemosensory neuron responses as a function of feeding state.

Long-term imaging of circadian locomotor rhythms of a freely crawling C. elegans population.

BACKGROUND: Locomotor activity is used extensively as a behavioral output to study the underpinnings of circadian rhythms. Recent studies have required a populational approach for the study of circadian rhythmicity in Caenorhabditis elegans locomotion. NEW METHOD: We describe an imaging system for long-term automated recording and analysis of locomotion data of multiple free-crawling C. elegans animals on the surface of an agar plate. We devised image analysis tools for measuring specific features related to movement and shape to identify circadian patterns. RESULTS: We demonstrate the utility of our system by quantifying circadian locomotor rhythms in wild-type and mutant animals induced by temperature cycles. We show that 13 °C:18 °C (12:12h) cycles are sufficient to entrain locomotor activity of wild-type animals, which persist but are rapidly damped during 13 °C free-running conditions. Animals with mutations in tax-2, a cyclic nucleotide-gated (CNG) ion channel, significantly reduce locomotor activity during entrainment and free-running. COMPARISON WITH EXISTING METHOD(S): Current methods for measuring circadian locomotor activity is generally restricted to recording individual swimming animals of C. elegans, which is a distinct form of locomotion from crawling behavior generally observed in the laboratory. Our system works well with up to 20 crawling adult animals, and allows for a detailed analysis of locomotor activity over long periods of time. CONCLUSIONS: Our population-based approach provides a powerful tool for quantification of circadian rhythmicity of C. elegans locomotion, and could allow for a screening system of candidate circadian genes in this model organism.

Feeding state, insulin and NPR-1 modulate chemoreceptor gene expression via integration of sensory and circuit inputs.

Feeding state and food availability can dramatically alter an animals' sensory response to chemicals in its environment. Dynamic changes in the expression of chemoreceptor genes may underlie some of these food and state-dependent changes in chemosensory behavior, but the mechanisms underlying these expression changes are unknown. Here, we identified a KIN-29 (SIK)-dependent chemoreceptor, srh-234, in C. elegans whose expression in the ADL sensory neuron type is regulated by integration of sensory and internal feeding state signals. We show that in addition to KIN-29, signaling is mediated by the DAF-2 insulin-like receptor, OCR-2 TRPV channel, and NPR-1 neuropeptide receptor. Cell-specific rescue experiments suggest that DAF-2 and OCR-2 act in ADL, while NPR-1 acts in the RMG interneurons. NPR-1-mediated regulation of srh-234 is dependent on gap-junctions, implying that circuit inputs regulate the expression of chemoreceptor genes in sensory neurons. Using physical and genetic manipulation of ADL neurons, we show that sensory inputs from food presence and ADL neural output regulate srh-234 expression. While KIN-29 and DAF-2 act primarily via the MEF-2 (MEF2) and DAF-16 (FOXO) transcription factors to regulate srh-234 expression in ADL neurons, OCR-2 and NPR-1 likely act via a calcium-dependent but MEF-2- and DAF-16-independent pathway. Together, our results suggest that sensory- and circuit-mediated regulation of chemoreceptor genes via multiple pathways may allow animals to precisely regulate and fine-tune their chemosensory responses as a function of internal and external conditions.

Genome-wide analysis of light- and temperature-entrained circadian transcripts in Caenorhabditis elegans.

Most organisms have an endogenous circadian clock that is synchronized to environmental signals such as light and temperature. Although circadian rhythms have been described in the nematode Caenorhabditis elegans at the behavioral level, these rhythms appear to be relatively non-robust. Moreover, in contrast to other animal models, no circadian transcriptional rhythms have been identified. Thus, whether this organism contains a bona fide circadian clock remains an open question. Here we use genome-wide expression profiling experiments to identify light- and temperature-entrained oscillating transcripts in C. elegans. These transcripts exhibit rhythmic expression with temperature-compensated 24-h periods. In addition, their expression is sustained under constant conditions, suggesting that they are under circadian regulation. Light and temperature cycles strongly drive gene expression and appear to entrain largely nonoverlapping gene sets. We show that mutations in a cyclic nucleotide-gated channel required for sensory transduction abolish both light- and temperature-entrained gene expression, implying that environmental cues act cell nonautonomously to entrain circadian rhythms. Together, these findings demonstrate circadian-regulated transcriptional rhythms in C. elegans and suggest that further analyses in this organism will provide new information about the evolution and function of this biological clock.

Cis-regulatory mechanisms of gene expression in an olfactory neuron type in Caenorhabditis elegans.

The generation of cellular diversity is dependent on the precise spatiotemporal regulation of gene expression by both cis- and trans-acting mechanisms. The developmental principles regulating expression of specific gene subsets in individual cell types are not fully understood. Here we define the cis-regulatory mechanisms driving expression of cell-selective and broadly expressed genes in vivo in the AWB olfactory neuron subtype in C. elegans. We identify an element that is necessary to drive expression of neuron-selective chemoreceptor genes in the AWB neurons, and show that this element functions in a context-dependent manner. We find that the expression of broadly expressed sensory neuronal genes in the AWB neurons is regulated by diverse cis- and trans-regulatory mechanisms that act partly in parallel to the pathways governing expression of AWB-selective genes. We further demonstrate that cis-acting mechanisms driving gene expression in the AWB neurons appear to have diverged in related nematode species. Our results provide insights into the cis-regulatory logic driving cell-specific gene expression, and suggest that variations in this logic contribute to the generation of functional diversity.

The EGL-4 PKG acts with KIN-29 salt-inducible kinase and protein kinase A to regulate chemoreceptor gene expression and sensory behaviors in Caenorhabditis elegans.

The regulation of chemoreceptor (CR) gene expression by environmental signals and internal cues may contribute to the modulation of multiple physiological processes and behavior in Caenorhabditis elegans. We previously showed that KIN-29, a homolog of salt-inducible kinase, acts in sensory neurons to regulate the expression of a subset of CR genes, as well as sensory behaviors. Here we show that the cGMP-dependent protein kinase EGL-4 acts partly in parallel with KIN-29 to regulate CR gene expression. Sensory inputs inhibit both EGL-4 and KIN-29 functions, and KIN-29 function is inhibited in turn by cAMP-dependent protein kinase (PKA) activation. EGL-4 and KIN-29 regulate CR gene expression by antagonizing the gene repression functions of the class II HDAC HDA-4 and the MEF-2 transcription factor, and KIN-29, EGL-4, and PKA target distinct residues in HDA-4 to regulate its function and subcellular localization. While KIN-29 acts primarily via MEF-2/HDA-4 to regulate additional sensory signal-regulated physiological processes and behaviors, EGL-4 acts via both MEF-2-dependent and -independent pathways. Our results suggest that integration of complex sensory inputs via multiple signaling pathways allows animals to precisely regulate sensory gene expression, thereby appropriately modulating physiology and behavior.

KIN-29 SIK regulates chemoreceptor gene expression via an MEF2 transcription factor and a class II HDAC.

The expression of individual chemoreceptor (CR) genes in Caenorhabditis elegans is regulated by multiple environmental and developmental cues, possibly enabling C. elegans to modulate its sensory responses. We had previously shown that KIN-29, a member of the salt-inducible kinase family, acts in a subset of chemosensory neurons to regulate the expression of CR genes, body size and entry into the alternate dauer developmental stage. Here, we show that KIN-29 regulates these processes by phosphorylating the HDA-4 class II histone deacetylase (HDAC) and inhibiting the gene repression functions of HDA-4 and an MEF-2 MADS domain transcription factor. MEF-2 binds directly to the CR gene regulatory sequences, and is required only to repress but not activate CR gene expression. A calcineurin phosphatase antagonizes the KIN-29/MEF-2-regulated pathway to modulate levels of CR gene expression. Our results identify KIN-29 as a new regulator of MEF2/HDAC functions in the nervous system, reveal cell-specific mechanisms of action of this pathway in vivo and demonstrate remarkable complexity in the regulation of CR gene expression in C. elegans.

Shotgun cloning of transposon insertions in the genome of Caenorhabditis elegans.

We present a strategy to identify and map large numbers of transposon insertions in the genome of Caenorhabditis elegans. Our approach makes use of the mutator strain mut-7, which has germline-transposition activity of the Tc1/mariner family of transposons, a display protocol to detect new transposon insertions, and the availability of the genomic sequence of C. elegans. From a pilot insertional mutagenesis screen, we have obtained 351 new Tc1 transposons inserted in or near 219 predicted C. elegans genes. The strategy presented provides an approach to isolate insertions of natural transposable elements in many C. elegans genes and to create a large-scale collection of C. elegans mutants.

Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions.

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms.

Hyperactivation of the G12-mediated signaling pathway in Caenorhabditis elegans induces a developmental growth arrest via protein kinase C.

The G(12) type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells. However, little is known about the molecular nature of the components that act in the G(12)-signaling pathway in an organism. We characterized a C. elegans Galpha subunit gene, gpa-12, which is a homolog of mammalian G(12)/G(13)alpha, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G(12)QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G(12) participates, we screened for suppressors of the G(12)QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCtheta/delta. TPA-1 mediates the action of the tumor promoter PMA. Expression of G(12)QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G(12) signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G(12) proteins.

Proteins interacting with Caenorhabditis elegans Galpha subunits.

To identify novel components in heterotrimeric G-protein signalling, we performed an extensive screen for proteins interacting with Caenorhabditis elegans Galpha subunits. The genome of C. elegans contains homologues of each of the four mammalian classes of Galpha subunits (Gs, Gi/o, Gq and G12), and 17 other Galpha subunits. We tested 19 of the GGalpha subunits and four constitutively activated Galpha subunits in a largescale yeast two-hybrid experiment. This resulted in the identification of 24 clones, representing 11 different proteins that interact with four different Galpha subunits. This set includes C. elegans orthologues of known interactors of Galpha subunits, such as AGS3 (LGN/PINS), CalNuc and Rap1Gap, but also novel proteins, including two members of the nuclear receptor super family and a homologue of human haspin (germ cell-specific kinase). All interactions were found to be unique for a specific Galpha subunit but variable for the activation status of the Galpha subunit. We used expression pattern and RNA interference analysis of the G-protein interactors in an attempt to substantiate the biological relevance of the observed interactions. Furthermore, by means of a membrane recruitment assay, we found evidence that GPA-7 and the nuclear receptor NHR-22 can interact in the animal.