Morton, E. et al. (2019) International Worm Meeting "Exploring the potential role of rDNA copy number in RNAi efficiency using a RIL population of extreme rDNA copy numbers."

Morton, E., Hall, A., Queitsch, C.

[International Worm Meeting, 2019]

Ribosomal RNAs contribute structurally and catalytically to the ribosome and make up the bulk (>80%) of the RNA content of the cell. Equally remarkable are the properties of the genes encoding these rRNAs: loci known as the rDNA. All eukaryotes maintain rDNA genes in large tandemly-repeated arrays. However, the copy number of rDNA units in the arrays varies drastically among species, strains, and individuals. Wild isolates of C. elegans have been characterized with rDNA copy number as low as 70 or as high as 400 copies per haploid genome, qualities that in our hands are stable even under laboratory propagation. We are interested in exploring if this major, largely uncharacterized, source of genomic variation contributes to phenotypic variation. Functionality of the RNA interference pathway is a promising candidate phenotype, due to observations that RNAi pathway components can localize to the nucleolus, an organelle formed around the rDNA, and that RNAi pathways have been implicated in regulation of rRNA expression. To investigate the role that rDNA copy number may have in RNAi efficiency and other phenotypes, as well as explore what genetic background differences may exist to allow such high disparity in copy number, we first characterized two strains: a GFP reporter strain, SEA51, with ~130 copies of the rDNA repeat, and MY1, a wild isolate with ~420 copies of the rDNA. We crossed these two strains to generate a resource of 118 recombinant inbred lines, approximately half of which are homozygous for the 130-copy rDNA locus and half homozygous for the 420-copy locus. We subjected these RILs to a suite of phenotyping assays, including quantification of RNAi sensitivity via growth of worms on embryonic lethal RNAi clones. We observe that the parental strains, MY1 and SEA51, differ in their sensitivity to these RNAi clones, despite the fact that neither parent has the previously-characterized ppw-1 RNAi resistance allele. The RILs exhibited large variation in their RNAi sensitivity, transgressing the parental phenotypes. RNAi sensitivity did not segregate with rDNA copy number, indicating that this locus alone does not account for the parental phenotypes, which likely have multiple contributing loci.

Morton E et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Molecular Characterization of the Heat Shock Transcription Factor HSF-1 and Identification of SUMO as a Regulator of HSF-1 in C. elegans"

Brown K, Chaya T, Lamitina T, Morton E

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The heat shock transcription factor HSF-1 activates transcription of target genes that promote protein homeostasis, implying important roles for HSF-1 in stress tolerance, aging, innate immunity, cancer, and protein diseases. Because active HSF-1 mediates resistance to a wide variety of proteotoxic stressors, drugs that upregulate HSF-1 may be therapeutic for a variety of protein misfolding diseases, such as Huntington's disease. Recent studies suggest, however, that HSF-1 activity may also be involved in undesirable phenotypes, including cellular transformation to a cancerous state. While global regulation of HSF-1 might be undesirable, identification of regulators of HSF-1 may allow us to find more nuanced ways to control its activity. Additionally, while many HSF-1-dependent processes have been extensively studied in the model organism Caenorhabditis elegans, the molecular behavior of HSF-1 has not yet been characterized in this system. We have begun to examine molecular characteristics of HSF-1 in C. elegans using a fluorescently tagged HSF-1. Tagged HSF-1 appears to localize to the nucleus under basal conditions and to condense into nuclear granules upon heat shock. To identify HSF-1 regulators, we conducted a high-throughput RNAi screen on an HSF-1-dependent reporter strain of C. elegans. Our screen covered 17,540 genes and identified 44 regulators of the HSF-1-dependent reporter. RNAi inhibition of these genes did not similarly affect an HSF-1-independent reporter, suggesting that they specifically regulate HSF-1-dependent phenotypes. One gene identified by our screen was smo-1, which encodes the sole C. elegans SUMO homolog. SUMO is a small ubiquitin-like molecule used to post-translationally modify proteins. Repression of smo-1 led to hyper-activation of the HSF1-dependent reporter, but only following activation of the reporter by heat shock. Consistent with this phenotype, smo-1(RNAi) also improved whole-animal thermotolerance. Sequence analysis of HSF-1 revealed a consensus site for sumoylation at the N-terminus, and recent studies from other labs confirm that HSF1 is post-translationally sumoylated. These results suggest that SUMO may directly modify HSF-1 in C. elegans in response to specific activating conditions and that such modifications inhibit HSF-1-regulated gene expression and stress resistance in vivo. Our findings have implications for sumoylation as a future target for therapies intended to regulate HSF-1.

BC Prasad et al. (1999) International C. elegans Meeting "The unc-3 locus encodes an O/E transcription factor, CeO/E, which is required for axonal guidance and chemosensory function."

BC Prasad, RR Reed

[International C. elegans Meeting, 1999]

The O/E family of rHLH transcription factors has been implicated in neurogenesis, axonal pathfinding, muscle formation and B cell maturation. The murine O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis and are expressed in olfactory neurons throughout development where they may regulate components of the olfactory signaling cascade. We have identified a C. elegans O/E homologue (CeO/E) that is encoded by the unc-3 locus. Unc-3 mutants display an uncoordinated phenotype attributed to a severe defasiculation and miswiring of the ventral cord (VNC). Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed transiently in the excitatory VNC motor neurons and throughout development in a pair of chemosensory neurons (ASI amphid neurons). Several observations suggest that the protein may have distinct roles in the two cell types. Although the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI, unc-3 mutants enter the dauer pathway under inappropriate conditions. Although CeO/E possesses highly conserved domains associated with DNA binding in the mammalian homologue, identification of DNA binding sites for the C. elegans protein has not been achieved. We have shown that CeO/E protein can homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. Several unc-3 target genes ( daf-7, unc-17/cha-1, unc-4 ) have been identified by other laboratories and each contains a mammalian binding site in the promoter region. Remarkably, we have been unable to demonstrate CeO/E binding at these sites. We are generating chimeras of O/E-1 and CeO/E to understand the DNA binding specificity of the CeO/E protein and utilizing a yeast-2-hybrid screen with both O/E-1 and CeO/E to identify interacting proteins in C. elegans . These studies should reveal the mechanism of CeO/E transcriptional activation and target gene regulation.

Yuen, Grace J. et al. (2013) International Worm Meeting "Enterococcus infection of C. elegans as a model of innate immunity."

Yuen, Grace J., Ausubel, Frederick M.

[International Worm Meeting, 2013]

Enterococcus is a Gram-positive commensal that is also an important opportunistic pathogen. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both species. We have previously shown that infection with either species leads to gut distention, but only E. faecalis is able to establish a persistent and lethal infection in the nematode. We now provide evidence that at least three canonical C. elegans immune signaling pathways are important for survival during infection with E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by E. faecium infection, mutations in the PMK-1, FSHR-1, and BAR-1 immune signaling pathways lead to an immunocompromised phenotype. This new finding suggests that an active host response is required to keep E. faecium infection "in check" in the worm intestine. To further characterize the C. elegans host response to Enterococcus infections, we used genome-wide transcriptional profiling of nematodes feeding on E. faecalis and E. faecium, as well as two controls, heat-killed E. coli and live Bacillus subtilis, a non-pathogenic Gram-positive. We found that relative to B. subtilis, E. faecalis and E. faecium caused the upregulation of 249 and 166 genes, respectively, of which 105 genes were common to both, comprising the Enterococcus gene signature. Shared by both Enterococcus infection signatures were genes relating to oxidation/reduction, acyl-CoA dehydrogenase/oxidase activity, fatty acid metabolism, and C-type lectins. Additionally, the Enterococcus infection gene signature is fairly distinct from the P. aeruginosa, S. aureus, and C. albicans infection signatures. Furthermore, of the 91 genes that are upregulated in E. faecalis (more virulent) relative to E. faecium (less virulent), 22 are shared with the 77 genes upregulated in worms infected with virulent Microbacterium nematophilum relative to avirulent M. nematophilum (O'Rourke et al., 2006), suggesting that these genes may comprise a "virulence response signature." Studies are underway in understanding the biology of Enterococcus infection in C. elegans and identifying novel Enterococcus-activated pathways.

Yuen, Grace J. et al. (2011) International Worm Meeting "Enterococcus infection of Caenorhabditis elegans as a model of innate immunity."

Ausubel, Frederick M., Pukkila-Worley, Read, Yuen, Grace J.

[International Worm Meeting, 2011]

Enterococcus is a Gram-positive commensal that is found in the gastrointestinal and biliary tracts of all healthy humans. It is also an important opportunistic pathogen, causing nosocomial urinary tract and wound infections that are often complicated by antibiotic drug resistance. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both strains. We have previously shown that infection with either strain leads to gut distention, but only E. faecalis is able to establish a persistent infection and kill the nematode. We now provide evidence that at least two canonical C. elegans immune signaling pathways are important for survival during infection with both E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by an E. faecium infection, mutations in the PMK-1 and FSHR-1 immune signaling pathways lead to an immunocompromised phenotype, where pmk-1 and fshr-1 mutants die rapidly upon E. faecium feeding. This new finding suggests that an active immune response is required to keep E. faecium infection "in check" in the worm intestine, and that E. faecium is indeed pathogenic to the nematode. We are now using microscopy and genetic studies to understand the biology of the Enterococcus infection in C. elegans and to identify novel Enterococcus-activated immune signaling pathways.

Fuentes, A. et al. (2015) International Worm Meeting "Diet affects neurodegenerative processes in C. elegans."

Fuentes, A., Calixto, A., Garcia, V., Palominos, MF.

[International Worm Meeting, 2015]

C. elegans has been used to understand the dietary impact on development and behavior (Macneil & Walhout., 2013), but very little is known about the role of diet on neurodegenerative processes. To study the effect of diet on neuronal degeneration, we use animals expressing the hyperactivated degenerins mec-4d expressed in the touch receptor neurons (TRNs) and deg-1 expressed in the PVC neurons. We measured the impact of different bacterial diets on neuronal integrity in the respective degeneration models. mec-4d animals with GFP marked TRNs were fed with E. coli OP50, B, HT115 and K12; Commamonas aquatica, Commamonas testosteronii and Bacillus megaterium and their TRN integrity assessed both morphologically (by visual inspection) and functionally (by the touch response) at 72 hours where most axons are degenerated (Calixto et al., 2012). The maximum protection was elicited by E. coli HT115 diet, with up to 50% wild type axons while the control strain E. coli OP50 and its precursor B only protects 3% at 72 hours post hatching. Like in the mec-4d animals, deg-1 animals PVC show a higher percent of functional response when animals are fed E. coli HT115. E. coli HT115 is capable of protecting neurons during large periods of time after adulthood since animals at 168 hours post hatching still showed 16.5 % of wild type axons. E. coli K12, Comammonas aquatica, testosteronii and Bacillus megaterium protected less (between 10 and 17% of wild type axons) than E. coli HT115 and more than E. coli OP50. E. coli HT115 is still capable of generating neuroprotection when diluted 1:100 in E. coli OP50, showing that E. coli OP50 does not produce neurodegeneration. Additionally, this shows that a very small amount of the protective food is necessary to elicit neuronal protection. E. coli HT115 does not produce dietary restriction and supports well animal growth, with 100% animals reaching adulthood at 72 hours vs 100% in E. coli OP50. Dead bacteria produce the same levels of neuronal protection than live bacteria, indicating that the interaction between bacteria and intestine is not required. Our data suggest that E. coli HT115 is capable of triggering pathways involved in neuroprotective processes early in animal development (first 12 hrs after hatching), highlighting the relevance of developmental time when the protective diet is ingested. We are currently performing transcriptomics analysis to identify the bacterial components from E. coli HT115 that mediate neuroprotection.

Prasad BC et al. (1998) East Coast Worm Meeting "The O/E transcription factor, UNC-3, is required for axonal guidance and ASI function"

Reed RR, Seydoux GC, Prasad BC

[East Coast Worm Meeting, 1998]

The O/E family of vertebrate rHLH transcription factors has been implicated in neurogenesis and axonal pathfinding. The mammalian O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis. A Xenopus O/E homologue, Xcoe2, functions downstream of neurogenin and upstream of neuroD in neurogenesis. The murine O/E members are also expressed in olfactory neurons throughout development and may regulate components of the odorant signal transduction cascade. A C. elegans O/E (CeO/E) homologue has been identified in our laboratory and shown to be encoded by the unc-3 locus, mutations which result in an uncoordinated phenotype. Previous electron microscopic reconstruction of unc-3 mutants revealed that the axons of the ventral cord motor neurons are severely defasiculated, the neuromuscular junctions occur at ectopic sites and the motor neurons receive input from inappropriate interneurons as a result of the defasiculation. Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed in a large subset of the ventral cord motor neurons and in a pair of chemosensory neurons (ASI amphid neurons). The CeO/E protein is expressed transiently, during times of axonogenesis in the ventral nerve cord and throughout development in the ASI neurons. Several observations suggest that the protein may have distinct roles in the two cell types. Specifically, the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI and the unc-3 mutants enter the dauer pathway under inappropriate conditions. There is also evidence for autoregulation of the gene in ASI neurons - the expression of CeO/E is greatly reduced in unc-3 mutants. CeO/E lacks a conserved second repeat helix found that is highly conserved in the mammalian protein. Experiments suggest that CeO/E protein is able to homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. We are currently identifying the binding site for CeO/E by analysis of sequences essential for regulation of expression in the ASI neurons as well as by specific binding site selection methods (Selex). We are using PCR and differential expression methods to isolate downstream targets of CeO/E that might participate in signaling in the ASI neurons and mediate the axonal pathfinding activities in the ventral nerve cord. The isolation of putative cofactors and downstream targets of CeO/E should provide insight into the function of this transcription factor in neuronal differentiation in C. elegans and vertebrates.

Huayue Ye et al. (2008) "Molecular control of the retrieval and prevention functions in stress-induced learning defects from vitamin E administration in Caenorhabditis elegans"

Dayong Wang, Huayue Ye

[2008]

"Vitamin E (α-tocopherol), a potent radical chain breaking antioxidant, is popularly used as ancillary drug for neurodegeneration disease, oxidative stress-related impairment and cognitive dysfunction. In the current work, we explored the thermosensation model to investigate the effects of vitamin E administration on learning behaviors and the related molecular mechanism in Caenorhabditis elegans. In the assay model, administration of 100 μg/mL and 200 μg/mL vitamin E had no significant effects on learning behaviors, whereas relatively high concentration (400 μg/mL) of vitamin E exposure shortened the acquisition period of the association paradigm. Pre-treatment or post-treatment with 200 μg/mL vitamin E could prevent or retrieve and even enhance the UV irradiation- and heavy metal (Al and Pb) exposure- induced learning defects. Moreover, treatment with 200 μg/mL vitamin E could restore the learning formed in the ncs-1 and hen-1 mutant worms, suggesting that vitamin E can largely mimic the function of NCS-1 and HEN-1 in regulating the learning behaviors. In addition, vitamin E could also mimic the function of DEG-1, which plays a key role in the regulation of neurodegeneration, and MEV-1, whose mutation will result an altered sensitivity to oxidative stress. Therefore, our data suggest that the direct association may exist between trace dietary vitamin E intake and learning impairment induced by oxidative stress, and a specific response mechanism may be activated or even be amplified after the administration of vitamin E. Moreover, trace vitamin E administration may ameliorate the metal exposure- and/or the UV irradiation-induced learning impairment at least partially by regulating the NCS-1 functions in learning control in C. elegans."

Prasad BC et al. (1997) International C. elegans Meeting "THE C. ELEGANS unc-3 GENE ENCODES A HOMOLOGUE OF THE O/E FAMILY OF MAMMALIAN TRANSCRIPTION FACTORS"

Prasad BC, Reed RR, Seydoux GC

[International C. elegans Meeting, 1997]

The expression of specialized signal transduction components in mammalian olfactory neurons is thought to be regulated by the O/E (Olf-1/EBF) family of transcription factors. The O/E proteins are expressed in cells of the olfactory neuronal lineage throughout development, and are also expressed transiently in neurons in the developing nervous system during embryogenesis. We have identified a C. elegans homologue of the mammalian O/E proteins, which displays greater than 80% similarity over 350 amino acids. The CeO/E cDNA maps to cosmid F42D1, previously shown to encode unc-3(1). We demonstrate that CeO/E is the product of the unc-3 gene, mutations in which cause defects in the axonal outgrowth of motor neurons as well as defects in dauer formation, a process requiring chemosensory input. Like its mammalian homologues, CeO/E is expressed in certain chemosensory neurons (ASI amphid neurons) throughout development, and is also expressed transiently in developing motor neurons when these cells undergo axonal outgrowth. Currently, we are defining the DNA sequence binding specificity of the CeO/E protein. These observations suggest that the O/E family of transcription factors play a central and evolutionarily conserved role in the expression of proteins essential for axonal pathfinding and neuronal differentiation. (1) Sean Eddy, WBG 12(5):86 (February 1, 1993)

Ryoichi Saiki et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Caenorhabditis elegans lifespan is profoundly modulated by its diet of E. coli: What are the roles of E. coli metabolism and coenzyme Q?"

Peter Dang, Tarra Bixler, Catherine Clarke, Pamela Larsen, Ryoichi Saiki, Adam Lunceford, Wendy Lee

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. Q is an essential component of respiratory electron transport and a potent lipid soluble antioxidant. Extended lifespans are observed in C. elegans clk-1 mutants with defects in Q biosynthesis. Intriguingly, mice heterozygous for a clk-1 gene disruption also show increased lifespan. C. elegans fed E. coli devoid of Q8 have a significant life span extension when compared to C. elegans fed a standard Q-replete E. coli diet. These results initially suggested that the lack of Q is the important parameter affecting lifespan. However, recent results indicate that feeding respiratory incompetent E. coli, whether Q-replete or Q-less, produces a robust lifespan extension in C. elegans. C. elegans skn-1 mutants fed the respiratory incompetent E. coli diets also show lifespan extension, suggesting that the response is independent of a dietary restriction effect. The respiratory incompetent E. coli diet may be imposed well after the worms reach adulthood, including post-reproductive adults, indicating that lifespan extension operates independently of worm development. Curiously, C. elegans fed the Q-less E. coli diet seem to exhibit more pronounced lifespan extension than when fed respiratory incompetent E. coli that are Q-replete. C. elegans ttx-3 mutants, harboring defects in the gene encoding a LIM homeodomain transcription factor, fail to distinguish the Q-less from the Q-replete respiratory defective E. coli. The difference in life span extension mediated by the two respiratory deficient E. coli diets depends on normal food seeking behavior exhibited by N2 C. elegans. The data suggest that the life span extension of N2 worms fed Q-less E. coli diet is caused by a combination of respiratory deficiency of the E. coli and the food seeking behavior of C. elegans. This work was supported by NIA Grant AG19777, and by the Ellison Medical Foundation.