Osterli, E. et al. (2017) International Worm Meeting "Investigating the interaction of DLC-1 and GLD-1 in regulation of gene expression."

Osterli, E., Voronina, E., Ellenbecker, M.

[International Worm Meeting, 2017]

The regulation of RNA-binding protein (RBP) activity in cells is a central question in gene expression studies. One important RBP in C. elegans germline is GLD-1; this particular protein prompts the germ cell switch from mitotic proliferation to differentiation thus acting as a tumor suppressor. Previous research in our lab identified a small protein, DLC-1, as a cofactor of an RBP FBF-2. We hypothesized that DLC-1 may also promote the RNA regulatory function of GLD-1 because DLC-1 interacts with many cellular proteins. We tested our hypothesis on several strains of C. elegans that express fluorescent reporter proteins under control of GLD-1. These reporters are normally repressed by GLD-1 during differentiation. When we knock down DLC-1, we can see how it affects the reporter's repression. Fluorescence microscopy revealed that dlc-1 knockdown resulted in derepression of a subset of the reporters during differentiation. Our hypothesis that DLC-1 promotes GLD-1 functions is supported by the derepression of the reporters when DLC-1 is knocked down because loss of DLC-1 results in loss of GLD-1 function. Identifying this relationship between DLC-1 and GLD-1 is important for understanding stem cell balance. When the stem cell balance between mitotic proliferation and differentiation is altered, it can result in serious consequences such as unchecked cell proliferation, tumor formations, infertility, and cancer. Understanding the mechanism(s) by which DLC-1 promotes GLD-1 would be extremely relevant to advancing our understanding of certain human diseases like cancer.

Osterli, E. et al. (2019) International Worm Meeting "Investigating the role of the dynein motor in the activation of the MAP kinase pathway."

Osterli, E., Day, N., Voronina, E.

[International Worm Meeting, 2019]

The ubiquitous mitogen-activated protein kinase (MAPK) pathway regulates many different biological processes and plays critical roles in multiple diseases including cancer (Kim and Choi, 2015). In Caenorhabditis elegans this pathway also is required for the transition of meiotic cells to progress to oocytes (Lee et al., 2007). Previous work from our lab identified a pachytene exit defect in dlc-1 mutant worms (Ellenbecker et al., 2019). This observation of a disrupted transition from meiotic cells to differentiate into oocytes prompted the investigation of the potential role of the dlc-1 within the MAPK signaling pathway. DLC-1 (dynein light chain) is a small protein that can function either dependently or independently of the dynein motor and neither functions are suggested to be associated with the MAPK pathway. We hypothesize that the dynein motor is involved with the MAPK pathway and through its involvement contributes to the normal progression of meiosis to oogenesis. Western blot analysis of dlc-1 mutant worms revealed that activated MAPK in somatic cells was unaffected but decreased in the germline. In wild type C. elegans germlines, active MAPK is found near the end of the meiotic pachytene and in the oocytes. Since we are interested in the meiotic pachytene, further immunostaining analysis was necessary. Here we report that active MAPK levels in the meiotic pachytene decreased in all treatments that disrupted dynein motor function. dlc-1 and dhc-1 (dynein heavy chain) mutants express the decreased active MAPK phenotype 35% and 80% of the time respectively. Additionally, we performed RNA interference (RNAi) on N2 worms and noted an even more drastic decrease in active MAPK expression with different components of the dynein motor. dnc-1 (dynactin subunit) and dyci-1 (dynein intermediate chain) RNAi experiments showed a decreased expression down to 15% and 3% respectively. These results suggest that by disrupting the dynein motor the MAPK pathway is affected, and the active form of MAPK is decreased. We then wanted to know if the decreased levels of active MAPK were a result of a deficiency in total MAPK. To answer this question, we probed the germlines for total MAPK and found that the total MAPK levels did not decrease in any of the treatments that disrupted dynein motor function. This allows us to make the conclusion that the depletion of active MAPK in seen in the germline is not due to a loss of total MAPK. Further analysis must be done to fully understand whether this disruption of activated MAPK is dependent on the dynein motor or microtubule transport. Nevertheless, these findings show a potential new role of the dynein motor in the activation of the MAPK pathway, which was previously unheard of. Since the MAPK pathway is known to play roles in diseases like cancer, identifying new contributors to its activation is relevant to advancing our understanding of the disease.

Ellenbecker, M. et al. (2017) International Worm Meeting "Analysis of DLC-1 mediated regulation of the tumor suppressor protein GLD-1."

Osterli, E., Voronina, E., Ellenbecker, M.

[International Worm Meeting, 2017]

Dynein light chain (DLC-1) is a component of the dynein motor complex involved in transport of molecules and organelles along microtubules. Additionally, DLC-1 interacts with a diversity of cellular proteins and we propose that it functions as an allosteric regulator in ribonucleoprotein complexes. We previously found that DLC-1 promotes the activity of the RNA-binding protein FBF-2 in the stem cell region of the C. elegans germline. Since DLC-1 interacts with many cellular proteins, we hypothesized that DLC-1 may facilitate GLD-1, a component of the regulatory network that controls decision between stem cell proliferation and differentiation. GLD-1 is an RNA-binding protein that promotes germ cell differentiation during animal development by binding to and repressing translation of target mRNAs. This RNA regulatory function is essential for preventing ectopic proliferation in germ cells. Our genetic studies show that disruption of the DLC-1/GLD-1 interaction using dlc-1 RNA interference predisposes the nematode to tumor formation and this phenotype is also observed when the DLC-1 binding site on GLD-1 is mutated. We find that knockdown of DLC-1 does not affect GLD-1 levels. DLC-1 helps regulate translation of a subset of GLD-1 target mRNAs and this regulation is not dependent upon the dynein motor related function of DLC-1. Biochemical assays show that DLC-1 binds a conserved peptide motif located on the unstructured N-terminal domain of GLD-1 (YSQTP). By contrast, mutation of a conserved phosphorylated residue on the N-terminal domain (S22A) does not inhibit DLC-1 binding GLD-1. Transgenic worm experiments suggest that the DLC-1/GLD-1 binding interaction enables GLD-1 function in vivo. GLD-1 mutant worms unable to bind DLC-1 exhibit disorganized germline phenotype and the SQT to AAA mutation is associated with an increase in sterility. Together, these data support the hypothesis that DLC-1 facilitates the RNA regulatory and tumor suppressor functions of GLD-1 through a direct protein interaction. Mammalian Quaking proteins are GLD-1 orthologs and function as potent glioblastoma multiforme tumor suppressors by regulating the TGF beta signaling network that controls cell growth, proliferation and differentiation. We are currently using biochemical and molecular biology techniques to determine if this regulatory system is conserved in mammals.

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)