Berkyurek, A.C. et al. (2017) International Worm Meeting "piChIP: A novel tool to dissect the role of chromatin factors during transgenerational epigenetic inheritance (TEI) in the C. elegans germline."

Akay, A., Berkyurek, A.C., Dimaggio, P., Miska, E.

[International Worm Meeting, 2017]

Non DNA-sequence based inheritance has been observed in many organisms from microbes to men and likely impacts public health. The mechanisms of non DNA-sequence based inheritance remain largely unknown. However, some examples of non DNA-sequence based inheritance in mammals, plants and invertebrates are linked to the control of selfish, transposable elements in the genome. However, current knowledge in this field is mostly restricted to molecular genetics experiments. Here we propose to discover the mechanistic basis of multi-generational non DNA-sequence based inheritance, also referred to as transgenerational epigenetic inheritance (TEI), using the laboratory animal model Caenorhabditis elegans. 21U Piwi-interacting RNAs (piRNAs) target transposable element mRNAs and induce a secondary siRNA response in the germline by recruiting histone methyltransferases SET-25 and SET-32. In this pathway, our goal is to identify chromatin and non-coding RNA "epigenetic" marks that transfer information from generation to generation in the germline. Towards this goal we have established a germline-specific single locus system that we named piRNA-related insertional chromatin immunoprecipitation (piChIP). To create a locus-specific ChIP system, we used the LacI-lacO system by targeting the piRNA sensor which encodes for a GFP-H2A construct with a piRNA target site in the 3' end. In order to immunoprecipitate (IP) the piRNA sensor, we introduced DNA binding elements upstream of the sensor. By expressing a protein that specifically recognizes the DNA binding elements on the piRNA sensor, novel factors such as non-coding RNAs, histone post-translational modifications (PTMs) and proteins could be isolated and characterized with wild type and mutant strains that are defective to express a fully functional piwi protein PRG-1 or argonaute HRDE-1 during different stages of the TEI. Using piChIP, our specific aims are: Aim-1: Identification of novel non-coding RNAs in the germline nuclear RNAi pathway. Method: RNA-seq Aim-2: Identification of novel proteins and histone PTMs in the germline nuclear RNAi pathway. Method: SILAC proteomics and Mass Spectrometry

Berkyurek AC et al. (2021) EMBO J "The RNA polymerase II subunit RPB-9 recruits the integrator complex to terminate Caenorhabditis elegans piRNA transcription."

Butter F, Miska EA, Berkyurek AC, Cunha Navarro I, Weick EM, Lampersberger L, Nischwitz E, Braukmann F, Akay A, Sarkies P, Beltran T, Furlan G, Price J

[EMBO J, 2021]

PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. We show that rpb-9 initiates heritable piRNA-mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co-opted an ancient machinery for high-fidelity transcription.

Furlan, G. et al. (2019) International Worm Meeting "Investigating the molecular mechanisms of SET-24 in the C. elegans germline."

Jordan, D., Miska, E., Berkyurek, A.C., Mars, J., Furlan, G.

[International Worm Meeting, 2019]

Germline proliferation and maintenance are essential processes in development and are required to ensure the proper transmission of genetic and epigenetic information to the offspring. In the C. elegans germline, Argonaute-mediated small RNA pathways direct genome surveillance to ensure proper inheritance and maintain fertility. Two of these are the piRNA pathway, which guides the repression of parasitic genomic elements, and the germline nuclear RNAi pathway, which mediates environmentally-induced transgenerational gene silencing. Defects in these pathways lead to sterility, with mutant individuals exhibiting a mortal germline (Mrt) phenotype, in which a progressive decline in fertility accumulates across generations, ultimately resulting in complete sterility. This phenotype is enhanced by environmental stress conditions such as high temperatures. Interestingly, the heat-dependent Mrt phenotype typical of small RNA pathway mutants can be found in some C. elegans wild isolates. Recent work (Frezal, et al., 2018) has identified the set-24 gene as the major causal locus of the strong Mrt phenotype observed in specific wild strains. Set-24 encodes a germline-specific protein with an N-terminal SET domain. Set-24 mutant animals reach sterility in less than 20 generations when chronically exposed to restrictive (25 deg C) temperatures. Preliminary results indicate that SET-24 is dispensable for piRNA activity and global H3K9me3 deposition, but it is required for the transgenerational maintenance of silencing via the nuclear RNAi pathway, as inferred from the progressive re-expression of a germline-specific GFP transgene after GFP RNAi. Ongoing work is aimed at elucidating the contribution of SET-24 to the nuclear RNAi pathway by identifying SET-24 interactors, antagonists and targets. This will broaden our understanding of how epigenetic memory is established and transmitted at the molecular level.

Nicole F Liachko et al. (2005) International Worm Meeting "Characterization of putative DAF-16 target genes"

Siu Sylvia Lee, Nicole F Liachko

[International Worm Meeting, 2005]

The forkhead transcription factor DAF-16 is a key mediator of longevity, metabolism, and development. An informatic search using the known DAF-16 DNA recognition sequence to predict putative DAF-16 target genes has been performed (1). To further characterize the predicted DAF-16 target genes, we are optimizing a chromatin immunoprecipitation (ChIP) procedure using larvae and adult worms to demonstrate the presence of DAF-16 at the promoter of the target genes. In addition, we are using a promoter GFP fusion strategy to examine the expression of a subset of the DAF-16 target genes in worms. This approach allows study of when and where the target genes are expressed, as well as study of the dynamic regulation of the expression of the target genes in reponse to environmental stresses. Preliminary results show cell-type and developmental stage specific expression of the genes of interest. (1) S.S. Lee, S. Kennedy, A.C. Tolonen, G. Ruvkin, Science 300, 644 (2003).

Steven J Husson et al. (2005) International Worm Meeting "Caenorhabditis elegans: in search of neuropeptides by mass spectrometry"

Steven J Husson, Geert Baggerman, Liliane Schoofs, Elke Clynen, Arnold De Loof, Tom Janssen

[International Worm Meeting, 2005]

Peptides are the original signalling molecules in metazoan nervous systems. They are also ubiquitous in nervous systems that employ classical neurotransmitters where they serve as modulators, hormones and transmitters. Understanding the role of neuropeptides is however unfortunately hindered by the absence of primary sequence information and knowledge about their post-translational modifications. This sequence information has arrived very slowly, due to the huge efforts required in tissue collection and purification to ultimately isolate and functionally characterize a peptide. In total, only 12 C. elegans peptides have been biochemical isolated and identified to date, all of which are FMRFamide-related peptides (FaRPs). The major advance of using C. elegans as a model organism in neuropeptide research is the availability of its genomic sequence [1] from which 23 FMRFamide like peptide (flp) genes [2,3] and 32 neuropeptide-like protein (nlp) genes [4] were predicted. The genomic database also represents an indispensable foundation to perform a high throughput peptidomic study. By using two-dimensional nanoscale liquid chromatography, tandem mass spectrometry and database mining, we analysed a mixed stage C. elegans extract in which we identified 28 FaRPs and 20 NLP peptides. In addition, we were able to identify 15 entirely novel peptides derived from 10 precursors that were not identified or predicted from the genome in any way previously. Some of the peptides display profound sequence similarities with neuropeptides from other animals, suggesting that they have a long evolutionary history. The present identification of novel endogenous peptides offers opportunities for a greater understanding of neuropeptide biology in C. elegans. Since this nanoscale approach worked very well for an organism as tiny as C. elegans, we are convinced that virtually any organism of which the genome has been sequenced can be analyzed by this peptidomics technology. [1] The C. elegans Sequencing Consortium (1998) Science, 282: 2012-2018. [2] Li, C, Nelson, L.S., Kim, K, Nathoo, A, Hart, A.C. (1999) Ann. N.Y. Acad. Sci., 897: 239-252. [3] Kim, K., Li, C. (2004) J. Comp. Biol., 475: 540-550. [4] Nathoo, A.N., Moeller, R.A., Westlund, B.A., Hart, A.C. (2001) PNAS, 98: 14000-14005.

Peter Juo et al. (2005) International Worm Meeting "CDK5 regulates the synaptic abundance of the glutamate receptor GLR-1 in the ventral nerve cord of C. elegans"

Tom Harbaugh, Josh Kaplan, Peter Juo, Gian Garriga

[International Worm Meeting, 2005]

Glutamate is the major excitatory neurotransmitter in the brain and the abundance of postsynaptic glutamate receptors at synapses regulates the strength of synaptic transmission. We are interested in identifying genes and mechanisms involved in regulating glutamate receptor levels at the postsynaptic membrane. We show here that the cyclin-dependent kinase CDK5 can regulate the synaptic abundance of the glutamate receptor GLR-1. GLR-1 is a C. elegans non-NMDA-type glutamate receptor that is expressed and localized to synapses in ventral cord interneurons (1-4). CDK5 is a serine/threonine kinase whose activity is largely restricted to the nervous system. The activity of CDK5 requires association with a cyclin-like regulatory subunit called p35 (5). CDK5 plays a role in many cellular processes including cell migration, axon outgrowth, and neurodegeneration (5). Synaptic GLR-1 decreases in loss-of-function mutants of cdk-5 or of the regulatory subunit p35 in the ventral nerve cord. Conversely, overexpression of cdk-5 increases GLR-1 in a manner that is dependent on its kinase activity and on p35. We have previously shown that the PDZ protein LIN-10/MINT1 regulates the abundance of GLR-1 at synapses (3). GLR-1 accumulates in lin-10 null mutants (3), whereas overexpression of LIN-10/MINT1 results in a decrease in GLR-1. Our data indicate that CDK5 can regulate LIN-10/MINT1 levels in a bi-directional manner. We are currently testing whether CDK5 regulation of GLR-1 is mediated by LIN-10/MINT1. (1) Hart, A.C. et al. (1995). Nature 378:82-85. (2) Maricq, A.V. et al. (1995). Nature 378:78-81. (3) Rongo, C. et al. (1998). Cell 94:751-759. (4) Burbea, M. et al. (2002). Neuron 35:107-120. (5) Dhavan, R. and Tsai, L. H. (2001). Nat Rev Mol Cell Biol 2:749-759.

Chou, H. et al. (2017) International Worm Meeting "High-Throughput smFISH Analysis to Measure Natural Genetic Variation at Early Developmental Stages."

Deb, D., Paaby, A, Chou, H.

[International Worm Meeting, 2017]

The development of C. elegans is precise and stereotyped, including patterns of cell division in early embryogenesis. Nevertheless, natural genetic variation in wild-type isolates can cause dramatic differences in phenotype following single-gene perturbations, indicating that different wild-type genotypes harbor functional variation in critical gene networks1. These same wild isolates also show extreme variation in the efficacy of germline RNAi1. What are the genetic, molecular and cellular mechanisms that govern these differences? And how do they evolve when stabilizing selection ensures that phenotypic development remains stable and stereotyped? Here we use single-molecule FISH to quantitatively measure the gene expression at specific locations and time points in early development. By characterizing the temporal and spatial heterogeneities of mRNA transcript numbers in the first few cell divisions, we can connect sub-cellular phenotypes to known variations in early embryonic pathway function and germline RNAi. We use a high-throughput, semi-automated pipeline to acquire precise transcript counts at precisely staged embryos, including implementation of the machine learning spot-counting software Aro2. Despite near-invariant cell division phenotypes, wild isolates show significant differences in transcript abundance for critical embryonic genes. These differences in gene expression do not fully explain differences in embryonic lethality following gene knockdown, as neither wild-type gene expression nor transcript abundance following RNAi correlates perfectly with patterns of embryonic lethality. Notably, we observe significant difference in transcript abundance variance following RNAi among wild-type isolates, suggesting inefficiency of RNAi may be controlled by stochastic thresholds. Currently, we are scaling up the experiments using a microfluidic chip specifically designed for worm embryos in order to test hypotheses with high statistical rigor. References: 1- Wild worm embryogenesis harbors ubiquitous polygenic modifier variation. A.B. Paaby, A.G. White, D.D. Riccardi, K.C. Gunsalus, F. Piano, M.V. Rockman. Elife (2015), p. 4 2- Aro: a machine learning approach to identifying single molecules and estimating classification error in fluorescence microscopy images. A.C. Wu and S.A. Rifkin. BMC Bioinformatics (2015), 16:102

Zhang, Donglei et al. (2009) International Worm Meeting "THE ROLE OF RAB SMALL GTPASES IN GLUTAMATE RECEPTOR TRAFFICKING."

Rongo, Christopher, Zhang, Donglei, Isack, Nora, Glodowski, Doreen

[International Worm Meeting, 2009]

Regulated endocytosis and trafficking of AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, the specific combinations of clathrin-dependent and -independent mechanisms that mediate AMPAR trafficking in vivo have not been fully characterized. To better understand AMPAR trafficking, we have been examining the trafficking of the AMPAR GLR-1 in C. elegans (1). GLR-1 is localized on synaptic membranes, where it regulates reversals of locomotion in a simple behavioral circuit (2). We previously identified two genes that regulate GLR-1 membrane recycling: RAB-10 and LIN-10 (2, 3). Animals lacking RAB-10, a small GTPase required for endocytic recycling of intestinal cargo, or LIN-10, a PDZ-domain containing protein, share the same phenotype: GLR-1 accumulates in large, internalized accretions and animals display a decreased frequency of reversals (3, 4). Interestingly, reducing clathrin-dependent endocytosis specifically suppresses the lin-10 mutant phenotype, whereas reducing clathrin-independent endocytosis specifically suppresses the rab-10 mutant phenotype. Thus, we hypothesize that LIN-10 and RAB-10 recycle AMPARs from intracellular endosomal compartments to synapses along distinct pathways. Moreover, we suspect that another Rab protein, analogous to RAB-10, functions in the LIN-10 pathway. We have taken two approaches to identify additional cellular factors involved in LIN-10-mediated trafficking of GLR-1. First, we have performed a yeast 2-hybrid screen using LIN-10 as bait. We are characterizing the functions of the proteins identified in the screen to determine if they regulate AMPAR trafficking. Second, we are testing all of the known Rab genes in the genome for a role in GLR-1 trafficking, either by analyzing previously identified mutations or by generating mutant transgenes that express GDP- or GTP-locked versions of the Rabs. Our long-term goal is to further define the regulatory pathways involved in the movement and localization of AMPARs at synapses in vivo. 1. Hart, A.C. et al., Nature 378, 82-85 (1995) and Maricq, A.V. et al., Nature 378, 78-81 (1995). 2. Rongo, C. et al., Cell 94, 751-759 (1998). 3. Glodowski, D. et al., Mol Biol Cell 18, 4387-96 (2007). 4. Chen et al., Mol Biol Cell 17, 1286-97 (2006).

Ghose, Piya et al. (2009) International Worm Meeting "The Effect of Hypoxia on Neuronal Necrosis and Glutamate Receptor Trafficking."

Rongo, Christopher, Ghose, Piya, Park, Eun Chan

[International Worm Meeting, 2009]

Neurons in the brain are sensitive to oxygen levels, undergoing necrosis within minutes after hypoxia (e.g., oxygen deprivation during ischemic stroke). This neuronal toxicity is mediated in part by the excitatory neurotransmitter glutamate, which is released at high levels during hypoxia, and the ionotropic glutamate receptors (GluRs) that are trafficked to postsynaptic membranes. However, the mechanistic relationship between hypoxia, GluR function and subcellular trafficking, and excitotoxic necrosis has not been fully elucidated. Here we examine the effects of hypoxia and mutations in hypoxia response factors on necrosis and GluR trafficking in C. elegans. In C. elegans, an excitotoxic-like necrosis can be modeled in vivo by expressing a constitutively active G alpha S subunit in neurons. Activated G alpha S modulates the activity of yet unknown ion channels, resulting in the necrosis of a subset of neurons that express it (1). The resulting intermediate necrosis phenotype affords us the ability to screen for factors that either enhance or suppress neuronal necrosis, including hypoxia. The C. elegans GluR GLR-1 is expressed in these same neurons, where it functions to mediate locomotion reversal behavior (2,3) and is trafficked to synaptic connections (4). We find that hypoxic treatment enhances the necrosis caused by activated G alpha S expression, and alters the trafficking of GLR-1 in these same neurons. We anticipate that our studies will further elucidate the mechanisms of neuronal damage after hypoxia. By identifying the molecular mechanisms that regulate GluR signaling and necrosis in response to hypoxia, we hope to provide useful therapeutic targets for the prevention of nervous system damage after hypoxia exposure.(*co-first author) References 1. Berger, A.J. et al., G alphas-induced neurodegeneration in Caenorhabditis elegans. J Neurosci. 18,2871-80 (1995). 2. Hart, A.C. et al., Synaptic code for sensory modalities revealed by C. elegans GLR-1 glutamate receptor. Nature. 378,82-5 (1995). 3. Maricq, A.V. et al., Mechanosensory signalling in C. elegans mediated by the GLR-1 glutamate receptor. Nature. 378,78-81 (1995). 4. Rongo, C. et al., LIN-10 is a shared component of the polarized protein localization pathways in neurons and epithelia. Cell 94,51-759, 1998.

Alexander G. Eberhardt et al. (2007) International Worm Meeting "Sexual reproduction in the free-living generation of the parasitic nematode Strongyloides papillosus."

Werner E. Mayer, Adrian Streit, Linda Nemetschke, Alexander G. Eberhardt

[International Worm Meeting, 2007]

The nematode genus Strongyloides consists of parasites that live as parthenogenetic females in the small intestines of their hosts. In addition to producing parasitic offspring, they can also form a facultative free-living generation with males and females. In most Strongyloides species the progeny of these free-living adults is uniformously female and infective. Based on cytological observations in multiple species several authors1,2,3 have proposed that males do not contribute genetically to the progeny of the free-living generation but that the sperm is merely required to induce parthenogenetic development of the oocyte (pseudogamy). In contrast to these findings, Viney and colleagues4 have found that genetic markers can be passed on to the next generation in S. ratti. We are analyzing the mode of reproduction of the free-living generation of S. papillosus, a parasite of ruminants, cytologically and genetically. We have shown that also in S. papillosus males do contribute genetic material to the next generation5. While some genetic markers are inherited in a manner that is consistent with standard, mendelian, autosomal inheritance, others behave differently in that heterozygous males pass on preferentially or exclusively only one of their two alleles. We are currently testing the hypothesis that this is the consequence of the particular mode of sex determination in S. papillosus. In contrast to most other Strongyloides species that employ an XX/XO sex determining system, in S. papillosus males have a chromosome pair where one of the homologous chromosomes is intact and the other one lacks a large portion as the result of a sex specific chromatin diminution event6. In the process of our work we have noticed that the taxon S. papillosus, most probably, does not reflect a true biological species, but comprises of at least two relatively closely related but reproductively isolated species that can occur as mixed infections in the same host individual. We are currently analyzing the distribution of these two species in local sheep and cattle populations. 1Nigon, V., Roman, E., 1952 Bull biol Fr Belg 86:404-448. 2Bolla, R.I., Roberts, L.S., 1968 J Parasitol 54:849-855. 3Triantaphyllou, A.C., Moncol, D.J., 1977 J Parasitol 63:961-973. 4Viney, M.E., Matthews, B.E., Walliker, D., 1993 Proc R Soc Lond B Biol Sci 254:213-219. 5Eberhardt, A.G., Mayer, W.E., Streit, A., 2007 Int J Parasitol., in press. 6Albertson, D.G., Nwaorgu, O.C., Sulston, J.E., 1979 Chromosoma 75, 75-87.