Wang, X. et al. (2017) International Worm Meeting "Can DLC-1 binding teach its partner some new tricks?"

Voronina, E., Wang, X.

[International Worm Meeting, 2017]

Pumilio and FBF (PUF) family RNA binding proteins are highly conserved translational regulators in eukaryotes. FBF-1 and FBF-2, two PUF family proteins in C. elegans, function redundantly in maintaining germline mitotic region. In addition to their common function in mitotic region maintenance and fertility, FBF-1 and FBF-2 have divergent functions. FBF-2 silences its mRNA target through translational repression, while FBF-1 induces clearance of its target mRNAs (Voronina et al., 2012). Additionally, single mutations of fbf-1 and fbf-2 produce distinct effects on the mitotic region size (Lamont et al., 2004). One possibility is that different functions of FBF-1 and FBF-2 are due to association with distinct specific cofactors. Our earlier work identified DLC-1 as a cofactor for FBF-2, not for FBF-1. In vitro, DLC-1 interacts with FBF-2, not FBF-1, due to their sequence divergence. To see if DLC-1 binding is required for FBF-2 function in mitotic region maintenance and fertility, the fbf-2vrm mutant (losing DLC-1 binding sites) transgene was made and crossed into fbf-1 fbf-2 double mutant. We found that comparing to the wild type FBF-2, FBF-2vrm rescued fertility in only 80% of animals, suggesting that its function was compromised but not completely lost. Quantification of the mitotic zone cell number in the rescued gonads indicated that its size does not match either fbf-1 or fbf-2 single mutant. We hypothesize that DLC-1 binding is important for FBF-2-specific function regulating mitotic region size and that without DLC-1 binding, FBF-2vrm functions similarly to FBF-1. In support of this model, we found that FBF-2vrm transgene did rescue fbf-1, but not fbf-2 mutant effect on mitotic region size. We are now testing whether FBF-2vrm promotes clearance of FBF target mRNAs. PUF family phylogeny in five Caenorhabditis species suggests that FBF-1 and FBF-2 evolved from a gene duplication (Liu et al., 2012). Only one FBF homolog exists in both C. japonica and C. brenneri, therefore it would be interesting to explore whether DLC-1 binding to these FBF homologs is conserved, as a way to determine if this was the ancestral function for FBF. Our study develops a paradigm for how duplicated genes can diverge by modifying their interaction partners to fine-tune regulatory networks during animal development.

Olson, J. et al. (2015) International Worm Meeting "The Role of Histone H3 Lysine 9 Trimethylation in Germ Cell Development."

Olson, J., Voronina, E.

[International Worm Meeting, 2015]

Chromatin modifications are covalent modifications of nuclear histone proteins that can result in activation or repression of transcription of DNA. Some chromatin modifications have been linked to fertility and specification of reproductive cells called germ cells. We found that trimethylation of Lysine 9 in Histone H3 becomes prominent in late embryo germ cells of C. elegans, which has not been previously reported. Our research has been focused on identifying the methyltransferase that generates H3K9me3 in germ cells to help determine if H3K9me3 is required for germ cell development and fertility.Previous reports suggested that SET-25 is a major H3K9-trimethylating enzyme in C. elegans (Towbin et al., 2012). We found set-25 mutant embryos lose H3K9me3 in all somatic cells, but maintain H3K9me3 enrichment in the germ cells of later-stage embryos. We employed two strategies to find the H3K9 methyltransferase responsible for the residual H3K9me3 in the germ cells of set-25 mutant. In one approach, we used a double set-25;met-2 mutant, which was reported to completely prevent H3K9 methylation in the embryos (Towbin et al., 2012). We find that the set-25;met-2 double mutant embryos have almost no H3K9me3 in any cells and a fraction of resulting adults manifests temperature dependent sterility. This suggests that trimethylation of H3K9 may be important for germ cell development and fertility.Our second approach was RNAi screen of predicted H3K9 methyltransferases in set-25 mutant background to identify conditions that would disrupt the residual H3K9me3 germ cell enrichment. Through this approach, we identified two other possible regulators of H3K9me3, set-9 and set-26. Both set-9 and set-26 RNAi increased sterility in set-25 worms. Ongoing research includes determining if mutants of set-9 and set-26 affect the levels of H3K9me3 in germ cells to further examine the role of H3K9me3 in fertility. Since H3K9me3 modification has been shown to play a significant role in germ cell development in Drosophila and mouse, we hope our research in C. elegans will identify conserved targets of this regulatory mechanism.

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.

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.

Aoki, Scott Takeo et al. (2013) International Worm Meeting "Structural characterization of the P-granule protein scaffold."

Aoki, Scott Takeo, Kimble, Judith

[International Worm Meeting, 2013]

C. elegans P-granules are essential for both the development and maintenance of the germline tissue. Disruption of P-granule formation interferes with proper germ cell proliferation and differentiation, resulting in sterility (1, 2). P-granule assembly requires structural scaffold proteins PGL-1 and PGL-3 (3, 4). These nematode-specific paralogs are sufficient to form granules in cells and multimerize through self-association (5, 6). We aim to understand the structural organization of the P-granule scaffold to better understand how the organelle regulates mRNA trafficking and turnover. We are able to express and purify recombinant PGL-1 and PGL-3. Full-length recombinant protein self-assembles into large soluble aggregates. Protease digestion analyses identify a single domain that dimerizes in solution. We are currently trying to obtain a high-resolution crystal structure of the protease-protected fragment, as well as determine the role of the N- and C- terminal regions in scaffold oligomerization. Several different types of RNA granules are required in eukaryotes for cell homeostasis, differentiation, and response to stress. The fundamental mechanisms involved in P-granule organization will undoubtedly shed light on other granules involved in RNA regulation. References: 1. Updike, D., Strome, S. (2010) J Androl 31: 53-60. 2. Voronina, E., Paix, A., Seydoux, G. (2012) Development 139: 3732-3740. 3. Kawasaki, I., et al. (1998) Cell 94: 635-645. 4. Kawasaki, I., et al. (2004) Genetics 167: 645-661. 5. Updike, D.L., Hachey, S.J., Kreher, J., Strome, S. (2011) J Cell Biol 192: 939-948. 6. Hanazawa, M., Yonetani, M., Sugimoto, A. (2011) J Cell Biol 192: 929-937..

Day, N. et al. (2017) International Worm Meeting "Dynein Light Chain 1 Participates in Post-Transcriptional Regulation of Germline mRNAs."

Day, N., Voronina, E., Ellenbecker, M.

[International Worm Meeting, 2017]

Post-transcriptional regulation in the C. elegans germline is critical for the maintenance of stem cells. RNA binding proteins (RBPs) are involved in maintaining the balance between differentiation and proliferation in these stem cells. Our lab has previously identified dynein light chain 1 (DLC-1) as an important cofactor for the function and localization of the RBP FBF-2, independent of the dynein motor (Wang et al., 2016). DLC-1 is known to have multiple interacting partners, therefore we hypothesize that DLC-1 interacts with and regulates many RNA binding proteins. The interaction network of DLC-1 with germline RBPs is not well understood and necessitates further study. Here we investigate the role of DLC-1 in promoting RBP function and study its role in a post-transcriptional regulatory network. We generated a FLAG-tagged DLC-1 transgene that rescues the loss of function dlc-1 mutant and used this worm to co-immunoprecipitate DLC-1-associated RBPs and their mRNA targets. High throughput sequencing was used to identify what mRNAs associate with DLC-1 in vivo. Using the R package RIPSeeker (Li et al., 2013), we have identified mRNAs that are enriched in the FLAG::DLC-1 pulldown. Our list contains mRNAs that are at least three-fold enriched in the pulldown compared to the input library (Zhao et al., 2010). DLC-1-associated mRNAs are significantly enriched in oogenic transcripts but are depleted in spermatogenic transcripts (Ortiz et al., 2014). Some mRNAs identified in our pulldown were also found in association with specific RBPs reported by other groups, suggesting a potential co-regulatory role of DLC-1. We find that approximately 30% of FBF-2 targets (Prasad et al., 2016) associate with DLC-1, indicating that our RIP-seq approach captures the known interaction and will be useful for studying unknown DLC-1/RBP interactions. We also find that other reported RBP targets vary from 25 to 41% overlap with the DLC-1 associated mRNAs. We expect that overlap of 30% or greater suggests that RBPs may interact with and rely on DLC-1 for function. We are using reporter assays to study how DLC-1 binding influences the expression of a subset of these enriched mRNAs in the germline. Using g:profiler, our gene ontology search of mRNAs enriched in the FLAG::DLC-1 pulldown indicates that DLC-1 may regulate many mRNAs that are responsible for a wide range of functions including: cell fate specification and RNA metabolism. These findings suggest that DLC-1 is an important component of translational regulation of germline mRNAs.

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.

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.

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.

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.