Garcia-Sanchez, Juan A. et al. (2021) International Worm Meeting "Ubiquitin-related modifying enzymes in the regulation of HLH-30 signaling during S. aureus infection"

Harding, Benjamin, Ewbank, Jonathan J., Belougne, Jerome, Garcia-Sanchez, Juan A., Visvikis, Orane, Loubatier, Celine, Boyer, Laurent

[International Worm Meeting, 2021]

Ubiquitination and ubiquitination-like modifications are conserved post-translational modifications that influence virtually all physiological processes, including innate immunity. They are controlled by hundreds of ubiquitin (Ub) and Ub-like modifying enzymes that regulate their targets by modulating their stability, localization or activity. In recent years, the importance of these enzymes in C. elegans' innate immunity has been revealed, with the identification of key Ub-related regulators of innate immune pathways, as well as Ub-related effectors in the host response (Garcia-Sanchez et al, 2021). Little is known, however, about the role of these processes in the immune response to the intestinal bacterium Staphylococcus aureus, which is coordinated by the transcription factor TFEB/HLH-30 (Visvikis et al, 2014; Najibi et al, 2016). Our research focuses on deciphering the importance of Ub-modifying enzymes in regulating this immune pathway, as well as their role as effectors in the host response. Firstly, we undertook an RNAi-based screen to identify new immune regulatory enzymes. We generated a fluorescent reporter strain which allowed us to screen the effect of 216 enzymes on the activity of HLH-30 during S. aureus infection. We identified 22 genes whose downregulation reduced reporter fluorescence. To distinguish between specific and more general regulators, we performed a parallel screen using the model of epidermal infection by the fungus Drechmeria coniospora, which triggers an immune response mainly controlled by the p38 MAPK/PMK-1 pathway (Zugasti et al, 2014; 2016). We found 6 enzymes that are common to both screens; the remaining 16 enzymes are potentially specific regulators of the HLH-30-dependent immune response to S. aureus. Secondly, to identify Ub-related immune effectors important for the response to S. aureus infection, we analyzed previously published RNA-seq data (Visvikis et al, 2014) and determined that 16 out 19 Ub-related enzymes induced during infection are controlled by HLH-30. Focusing on 3 conserved E3 Ub-ligases, we confirmed their HLH-30 dependent induction by qPCR. Interestingly, using survival assays, we could demonstrate their importance in C. elegans' host defense against S. aureus infection suggesting they play a specific effector role in infection. Altogether, these results demonstrate the importance of Ub-related enzymes in HLH-30 dependent host response to S. aureus infection and have revealed specific and general Ub-related mechanisms in the regulation of immune pathways.

Benjamin Schlager et al. (2006) European Worm Meeting "An Ancestral Module Patterns a Derived Nematode Vulva Equivalence Group"

Ralf Sommer., Benjamin Schlager

[European Worm Meeting, 2006]

Benjamin Schlager and Ralf Sommer. Nematode vulva formation provides a paradigm to study the evolution of pattern formation and cell fate specification. The Caenorhabditis elegans vulva is generated from three of six equipotent cells that form the so-called vulva equivalence group. During evolution, the size of the vulva equivalence group has changed: Panagrellus redivivus has eight, C. elegans six and Pristionchus pacificus only three cells that are competent to form vulval tissue. In P. pacificus, the reduction of the vulva equivalence group is achieved by programmed cell death of individual precursor cells. We have identified the genes controlling this cell death event and the molecular mechanism of the reduction of the vulva equivalence group. Mutations in Ppa-hairy, a gene which is unknown from C. elegans, result in the survival of two precursor cells, which expands the vulva equivalence group. Mutations in Ppa-groucho cause a similar phenotype. Ppa-HAIRY and Ppa-GROUCHO form a molecular module that represses the Hox gene Ppa-lin-39 and thereby, reduces the size of the vulva equivalence group. The C. elegans genome does not encode a similar hairy-like gene and no typical HAIRY/GROUCHO module exists. Instead, the size of the C. elegans vulva equivalence group is regulated by the EGF and WNT signaling pathways.. Therefore the derived vulva equivalence group of P. pacificus is patterned by an ancestral hairy/groucho module.. I will also present a highly speculative model of what kind of adaptive values might be associated with the Pristionchus specific reduction of the size of the vulva equivalence group.

Guang, Shouhong et al. (2009) International Worm Meeting "siRNAs regulate transcription elongation."

Kennedy, Scott, Burkhart, Kirk, Bochner, Aaron, Pavelec, Derek, Guang, Shouhong

[International Worm Meeting, 2009]

siRNAs silence gene expression in the nucleus by recruiting chromatin-modifying factors and targeting nascent transcripts. In S. pombe and A. thaliana, both transcriptional gene silencing (TGS) and co-transcriptional gene silencing (CTGS) programs have been documented. We have conducted a genetic screen to identify factors specifically required for nuclear RNAi in C. elegans. This screen identified several genes, named nuclear RNAi defective (nrde)(1-3). Previously, we reported that nrde-3 is an Argonaute-like protein, transports secondary siRNAs from the cytoplasm to the nucleus, associates with nascent transcripts harboring sequence homology to the guiding siRNAs, and silences gene expression in the nucleus [1]. nrde-2 is a conserved gene containing a SR domain and a domain of unknown function (DUF) 1740 domain. SR proteins play significant roles in pre-mRNA splicing and transcription elongation, suggesting a connection between nuclear RNAi and transcription elongation mediated by the nrde genes. NRDE-2 predominantly localizes in the nucleus. It is not required for endogenous siRNAs production or NRDE-3 mediated transport of siRNAs from the cytoplasm to the nucleus. NRDE-2 physically associates with NRDE-3, and is recruited to nascent transcripts along with NRDE-3 following RNAi. We further examined the mechanism of siRNA-mediated nuclear gene silencing in the nucleus. By utilizing chromatin-immunoprecipitation (ChIP), we determined the relative occupancy of RNA polymerase II at the RNAi targeted sites after feeding dsRNA. We found a nrde dependent accumulation of polymerase II at the genomic sites targeted by RNAi, but not promoter regions, suggesting nuclear RNAi in C. elegans regulates transcription elongation. Consistent with this, RNAi silences the pre-mRNA exclusively 3'' to the region targeted by the dsRNA, in a nrde dependent manner. Currently, we are investigating how NRDEs regulate RNA polymerase II activity and transcription elongation, and what are the endogenous functions of this NRDE pathway. Progress of these experiments will be reported. 1. Guang, S., Bochner, A.F., Pavelec, D.M., Burkhart, K.B., Harding, S., Lachowiec, J., and Kennedy, S., (2008) An Argonaute transports siRNAs from the cytoplasm to the nucleus. Science 321:537-541.