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[
International Worm Meeting,
2021]
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[
International Worm Meeting,
2019]
Small RNA-mediated gene regulatory responses are inherited in Caenorhabditis elegans across multiple generations. In addition, exposure to a number of environmental challenges, such as viral infection, starvation, and heat induces heritable physiological responses in C. elegans. In certain cases, the inheritance of such responses can be linked to heritable small RNAs or chromatin modifications and was suggested to prepare the progeny for the environmental challenges that the ancestors met. A feed-forward loop between heritable small RNAs and 'repressive' histone marks maintains the heritable information in various organisms. Previously, we showed that MET-2, an H3K9-mono/di-methyltransferase, inhibits dsRNA-induced small RNA inheritance. Accordingly, induction of RNAi in
met-2 mutants leads to permanent silencing of the RNAi-targeted gene for many generations. Recently, we found an asymmetry in the function of the RNAi-induced H3K9me3. The H3K9-tri-methyltransferases SET-25 and SET-32 promote heritable silencing of the foreign gene gfp, but are dispensable for inheritance of silencing of the endogenous gene
oma-1. Genome-wide analysis of heritable endogenous-small interfering RNAs (endo-siRNAs) revealed that endo-siRNAs that depend on SET-25 and SET-32 target newly acquired and highly H3K9me3-marked genes. Here we will present new insights on epigenetic inheritance and the roles that heritable small RNAs and chromatin modifications play.
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[
International Worm Meeting,
2015]
The discovery of inherited epigenetic marks, and in particular - the inherited characteristics of the RNAi response - gave rise to a process of reconsidering the Lamarckian ideas of inheritance of acquired traits. Recent studies suggest that certain traits acquired by an animal during its lifetime may be transmitted to next generations. Specifically, new findings have demonstrated that severe L1 arrest of the worms leads to the generation of specific small-RNAs pattern antisense to endogenous genes. These patterns are inherited and therefore poses the potential of "memorizing" the ancestral environment and prepare the progeny for anticipated difficulties.Using a combination of deep-sequencing methods and phenotypic assays, our recent efforts suggest that inherited small RNAs indeed reflect the ancestral environment, and have a possible role in preparing the progeny for relevant hardships. In addition, we identify a set of genes which are regulated by environmentally induced inherited small-RNA patterns and examine their involvement in epigenetically-mediated adaptation.
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Seroussi, Uri, Bril, Roberta, Anava, Sarit, Rechavi, Oded, Gingold, Hila, Lev, Itamar
[
International Worm Meeting,
2017]
In recent years transgenerational inheritance of acquired traits has been described in various species. Both small interfering RNAs (siRNAs) and chromatin modifications have been implicated in the maintenance of such heritable responses. In plants and yeast, a self-reinforcing feedforward loop was described, where nuclear siRNAs direct histone methylation at pericentromeric regions and in return, the siRNA machinery is recruited to pericentromeric methylated histones to synthesize additional siRNAs. In Caenorhabditis elegans, double-stranded RNA-induced RNA interference (RNAi) can produce long-term heritable silencing responses that involve the production of "secondary" siRNAs and histone methylation of the targeted locus. Recently, we showed that MET-2, a histone-H3 Lysine-9 mono/di- methyltransferase, suppresses transgenerational RNAi inheritance indirectly by affecting small RNA biogenesis. The current work discusses how chromatin modifications affect small RNA biogenesis and the different requirements for heritable silencing of transgenes and endogenous genes.
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Zaidel-Bar, Ronen, Rechavi, Oded, Anava, Sarit, Gingold, Hila, Antonova, Olga, Agarwal, Priti
[
International Worm Meeting,
2021]
Cell motility is essential for the normal development and physiology of an organism. In C. elegans, the stereotypical U-shaped gonad is formed by the directional chemotactic movement of two somatic cells, known as Distal-Tip Cells (DTCs), whose movement is divided into three phases. In phase I (early L3 larval stage) they move along the ventral surface. Phase II (late L3 stage) involves two 90° turns, and finally in phase III (early L4 larval stage) the DTCs move along the dorsal surface towards the midbody of the worm. Migration halts during the late L4 stage. While multiple genetic screens have identified general regulators of gonad morphogenesis, a DTC-specific role has remained largely unexplored. To address this, we isolated GFP-labelled DTCs from different stages of development and performed RNA-sequencing, to characterize and compare the transcriptome of migratory DTCs (late L3 and early L4 stage) with non-migratory DTCs (late L4 and adult stage). We identified between 1700 to 3000 genes that are upregulated in each of the larval and adult stage DTC relative to other cell types. We confirmed the identification of the few DTC-specific transcripts known in the literature. The overlap between the genes we found upregulated in DTCs and the published germline-enriched genes (Reinke et al., 2004) is only 0.9 to 6.4%. Furthermore, we found that among the 99 genes identified in a genome-wide RNAi screen (Cram et al., 2006) to have a role in DTC migration, 43 genes are enriched in the migratory early L4 stage DTC transcriptome, while only 7 genes are present in the immobile adult stage DTCs. Taken together, it appears our dataset of cell-specific and stage-specific DTC transcripts is highly accurate. A bioinformatic functional analysis of the migratory DTC transcripts revealed enrichment of genes related to neuronal guidance, cytoskeleton, signaling, and membrane trafficking. Currently, we are performing a DTC-specific RNAi screen to identify the novel cell-autonomous regulators of DTC migration which in turn guides gonad morphogenesis. Identifying these regulators will help us to decipher the molecular mechanisms of DTC migration deployed in a three-dimensional microenvironment, which will eventually give insight into the mechanism of gonadogenesis.
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Azmon, Eran, Star, Ekaterina, Anava, Sarit, Rechavi, Oded, Posner, Rachel, Hobert, Oliver, Gingold, Hila, Bracha, Shahar
[
International Worm Meeting,
2017]
Exposure of C. elegans to artificial dsRNA can trigger exogenous small RNA-mediated silencing that transmits from somatic cells to the germline, and persists for multiple generations. However, it is unknown whether endogenous types of small RNAs (miRNAs, piRNAs and endo-siRNAs) produce systematic responses and what type of effect they may have on gene regulation. Our recent results suggest that endogenous siRNAs act in a non-cell autonomous manner, orchestrating gene silencing between tissues. We will present a dissection of the major components of the RNAi pathway required for non-cell autonomous gene regulation by endo-siRNAs and provide evidence possibly linking the regulation of physiological phenotypes to small RNAs mobilizing between tissues.
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[
International Worm Meeting,
2021]
Invertebrate immune systems are most often characterized by a "lack of x" compared to mammalian immune systems (Pradeu & Du Pasquier, 2018). "Lacking" processes akin to clonal selection and expansion of B- and T-cells, C. elegans is believed not to display an adaptive immune response. Ideas and concepts guiding the description of immunity-related phenomena, such as self/nonself, biotic/abiotic, and innate/adaptive, are developed from a mammalian perspective. This perspective is, however, epistemically pernicious, as it excludes other possible instantiations of immune systems. Ideally, a framework that resists biases is also a framework that describes the processes studied more accurately (Longino, 2008). I will give a conceptual talk, discussing the possibility of reinterpreting C. elegans small RNA inheritance as a transgenerational adaptive immune system. Upon viral infection, C. elegans produces virus-derived RNAs (viRNAs), which immunize offspring against repeated infection (Rechavi et al., 2011). Similar heritable responses are launched against abiotic stressors (e.g., Houri-Ze'evi et al., 2016). I will discuss C. elegans small RNA inheritance as an intersection of genetic- and immune systems. Considering small RNA inheritance as part of the C. elegans immune system requires redefining immune systems in several ways. An immune system is, first and foremost, a system that immunizes. Any step further needs to avoid presupposing particularities based on what works in mammals. On the one hand, the differentiation between biotic and abiotic stressors needs to break down. On the other hand, the idea of self/nonself, organism/environment, needs to be reconsidered: Apart from small RNAs recognizing what is "foreign," other small RNAs transmit information on what is "self" (Rechavi, 2014). Inheriting self- and nonself-recognizing small RNAs results in an intricate equilibrium, shifted by environmental stimuli (Ishidate et al., 2018), and thus, what the organism will recognize as "self" and "nonself." The environment continuously "blends into" the organism. In conclusion, I will not only show how examining C. elegans small RNA inheritance helps overcome biased accounts of immune systems but also how it contributes to addressing current theoretical questions in immunology that debate the adequacy of self-nonself-, danger-, and continuity theory (Matzinger, 1994; Pradeu, 2012) as overarching theoretical frameworks.
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[
International Worm Meeting,
2017]
Long-term effects of extended starvation during L1 arrest have been shown to persist to the F3 generation, suggesting transgenerational epigenetic inheritance. Though effects on lifespan and gene expression have been reported (Rechavi et al), we have only observed relatively subtle transgenerational effects on L1 starvation survival and heat resistance (Jobson, Jordan et al). Furthermore, we found that a minority of starved worms transmitted these effects, requiring phenotype-based sorting of the starved population to detect transgenerational effects among their descendants. In contrast to L1 arrest, larvae can survive dauer arrest for months and the majority of C. elegans in the wild are found as dauers. Persistent effects of dauer arrest have been reported for adults (Hall et al), but transgenerational effects have not been reported. We reasoned that dauer arrest is likely a more ecologically relevant and robust model for transgenerational effects of starvation. Here, we used a liquid culture system carefully controlling worm density and food availability to cause virtually 100% of N2 larvae to enter dauer arrest. These worms remain dauers for months and become reproductive adults when allowed to recover on plates. By assessing various life-history traits, we found that worms subjected to long-term dauer arrest exhibit apparent fitness costs upon recovery, including maternal effects in the F1 generation. However, L1 starvation survival was increased population-wide in the F3 generation (without phenotypic sorting of ancestors), suggesting potentially adaptive transgenerational effects of long-term dauer arrest. This phenotypic effect depended on the length of time the P0 generation spent in dauer arrest, suggesting that dauer formation alone is not sufficient. Notably, the transgenerational effects occurred in the absence of detectable gene expression changes, as if the presumed epigenetic effect on gene expression is distributed over many genes with a small effect on each. This work suggests that experiencing a stress such as starvation can initially be costly, but future generations may exhibit increased fitness in particular circumstances. References: Hall, S.E. et al., A cellular memory of developmental history generates phenotypic diversity in C. elegans. Current Biology, 2010. 20(2): p. 149-155. Jobson, M.A., Jordan J.M. et al., Transgenerational Effects of Early Life Starvation on Growth, Reproduction, and Stress Resistance in Caenorhabditis elegans. Genetics, 2015. 201(1): p. 201-12. Rechavi, O. et al., Starvation-Induced Transgenerational Inheritance of Small RNAs in C. elegans. Cell, 2014. 158(2): p. 277-287.
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[
European Worm Meeting,
2006]
Eva Brozova1, Katerina Simeckova1, Zdenek Kostrouch2, Marta Kostrouchova1 In this study, we functionally characterized NHR-40, a member of an extended group of supplementary nuclear receptors (Robisnon-Rechavi et al, J. Mol. Evol. 2005, 60: 577-586). We show that
nhr-40 is expressed in 3 isoforms that are regulated by two promoters organized in tandem. The function of NHR-40 was studied by RNA interference, by overexpression of
nhr-40 and using a mutant strain, RB840, which carries a deletion in the first intron. We show that NHR-40 regulates embryonic and early larval development. This phenotype is connected with defective movement and muscle cell development. This adds NHR-40 to the growing list of nematode NHRs involved in the regulation of development.. Acknowledgement: We thank Drs. A. Fire for vectors and host used in RNAi, M.W. Krause for support and advice, The C.elegans Gene Knockout Consortium for preparation of the deletion strain RB840. The work was supported by grants 303/03/1115 and 301/ 05/0859 from the Czech Science Foundation and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.
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Larance, Mark, Bradshaw, Charles, Miska, Eric A., Di Domenico, Tomas, Akay, Alper, Lamond, Angus I., Allen, George, Medhi, Ragini
[
International Worm Meeting,
2015]
Endogenous small RNA pathways can induce transcriptional gene silencing in C. elegans. piRNAs bound to the PRG-1 PIWI protein recognise their target by sequence complementarity and lead to the recruitment of RNA-dependent RNA polymerases (RdRPs). RdRPs, in turn, generate secondary siRNAs that are antisense to the target RNA and are 22 nt long with a 5' G (22G-RNAs). Other endogenous small RNA pathways such as the 26G-RNA pathway use similar amplification steps leading to 22G-RNA synthesis. In the C. elegans germline, the amplified 22G-RNAs are loaded into the argonaute protein HRDE-1 which localises to the nucleus initiating the nuclear RNAi pathway. The nuclear RNAi pathway leads to the transcriptional silencing of target genes and requires multiple chromatin factors. Silenced genes accumulate the repressive chromatin mark H3K9me3 and, remarkably, silencing of genes can last for several generations (1-6). The majority of the data linking small RNA mediated transcriptional and heritable silencing is based on evidence from genetic experiments. In order to better understand the mechanism of transcriptional and heritable silencing, it is essential to understand how key proteins in this silencing pathway function. We identified the intron binding protein EMB-4/AQR as an interaction partner of HRDE-1 in our immunoprecipitation experiments coupled to SILAC (7) based quantitative proteomics. Similar to
hrde-1,
emb-4/AQR is required for piRNA-mediated transgene silencing and the recently described 22G-RNA spreading (8) is inhibited in
emb-4/AQR mutant animals. RNA-seq experiments revealed that
emb-4/AQR is required for the repression of a subset of piRNA targets including transposable elements. In addition, some transposable elements which are de-silenced in
emb-4/AQR mutants are not targeted by the piRNA pathway, indicating a more global role for
emb-4/AQR in transposable element regulation. We are currently elucidating the molecular mechanism by which
emb-4/AQR communicates with the nuclear RNAi machinery. 1. Ashe A et al. Cell. 150(1):88. (2012) 2. Buckley BA et al. Nature. 489(7416):447. (2012) 3. Luteijn MJ et al. EMBO J. 31(16):3422. (2012) 4. Shirayama M et al. Cell. 150(1):65. (2012) 5. Rechavi O et al. Cell. 158(2):277. (2014) 6. Han T et al. Proc Natl Acad Sci USA. 106(44):18674. (2009) 7. Larance M et al. Nat Meth. 8(10):849. (2011) 8. Sapetschnig A et al., PLoS Genet. 11(3):
e1005078. (2015).