Variation génétique naturelle dans une phénotype multigénérationnelle chez Caenorhabditis elegans

par Marie Saglio

Projet de thèse en Génétique et génomique

Sous la direction de Marie-Anne Felix.

Thèses en préparation à l'Université Paris sciences et lettres , dans le cadre de École doctorale Complexité du vivant , en partenariat avec Institut de Biologie de l'École Normale Supérieure (laboratoire) et de École normale supérieure (Paris ; 1985-....) (établissement opérateur d'inscription) depuis le 01-09-2018 .


  • Résumé

    La lignée germinale des organismes multicellulaires est une lignée maintenue indéfiniment à travers les générations, un défaut de maintenance entrainant l'extinction de la lignée. Chez le nématode Caenorhabditis elegans, des mutants présentant un phénotype de mortalité de la lignée germinale (i.e. qui deviennent stériles après quelques générations) ont été isolés dans le fond génétique de référence N2 [1]. Les mutations affectent divers mécanismes, tel que la réparation de l'ADN. Une sous catégorie de mutants présente un phénotype sensible à la température, et les mutations impactent spécifiquement les 2° siARNs dans la lignée germinale. Leur effet sur la méthylation des histones, et de fait l'hérédité multigénérationnelle de l'interférence ARN est compromise [2]. En échantillonnant des nématodes C. elegans dans la nature, nous avons remarqué que de nombreuses souches isolées présentent un phénotype de mortalité de la lignée germinale sensible à la température (Mrt). En utilisant des approches de génétique quantitative, nous avons tout d'abord étudié les variations génétiques entre deux souches sauvages: MY10 (Mrt) et JU1395 (non Mrt). De plus, un GWAS a été effectué en utilisant 95 C. elegans sauvages et une forte association a été déterminé avec le polymorphisme sur le chromosome III. Enfin, certains microorganismes semblent influencer le phénotype Mrt. Le projet a trois axes: 1. Identifier le polymorphisme moléculaire à l'origine du pic d'association observer pour le chromosome III et expliquer ainsi les variations génétiques majeures observée pour ce phénotype dans la nature chez C. elegans. 2. Tester si la variation naturelle observée dans ce phénotype sensible à la température correspond à une variation naturelle dans la mémoire transgénérationnelle des effets de l'ARN interférence [3-5]. 3. Étudier les conditions environnementales qui ont une influence sur le phénotype Mrt et potentiellement leurs effets sur la transmission épigénétique des effets de l'ARN interférence [6-9]. Ce projet s'intéresse à un phénotype qui présente de la variation génétique naturelle pour différent mode d'hérédité. Au delà de la variation de la séquence de l'ADN, différents modes d'hérédité ont été observé chez différents organismes [2,10]. Cependant, leur importance dans la nature et leur impact en évolution restent à éclaircir. 1. Smelick C, Ahmed S (2005) Achieving immortality in the C. elegans germline. Ageing Res Rev. 4, 67-82. 2. Rechavi O, Lev I (2017) Principles of transgenerational small RNA inheritance in Caenorhabditis elegans. Curr Biol 27, R720-R730. 3. Ashe A, et al. (2012) piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans. Cell 150, 88-99. 4. Spracklin G et al (2017) The RNAi inheritance machinery of Caenorhabditis elegans. Genetics 206, 1403-16. 5. Lev I et al (2017) MET-2-dependent H3K9 methylation suppresses transgenerational small RNA inheritance. Curr Biol 27, 1138-47. 6. Watson et al. (2014) Interspecies systems biology uncovers metabolites affecting C. elegans gene expression and life history traits. Cell 56, 759-70. 7. Chaudhari SN, et al. (2016) Bacterial folates provide an exogenous signal for C. elegans germline stem cell proliferation. Dev Cell 38, 33-46. 8. Lin CJ, Wang MC (2017) Microbial metabolites regulate host lipid metabolism through NR5A-Hedgehog signalling. Nat Cell Biol 19, 550-57. 9. Gracida X, Eckmann CR (2013) Fertility and germline stem cell maintenance under different diets requires nhr-114/HNF4 in C. elegans. Curr Biol 23, 607-13. 10. Heard E, Martienssen RA (2014) Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95-109

  • Titre traduit

    Natural genetic variation in a multigenerational phenotype in Caenorhabditis elegans


  • Résumé

    The germline of multicellular organisms is a cellular lineage that is maintained indefinitely along organismal generations. Lack of proper germline maintenance results in lineage extinction. In the nematode Caenorhabditis elegans, mutants that display a mortal germline phenotype (i.e. that become sterile after several generations) have been isolated in the reference genetic background N2 [1]. The mutations affect a diversity of processes, including DNA repair; a subclass is temperature-sensitive and specifically affects the synthesis and nuclear transfer of 2° siRNAs in the germline, their effect on histone methylation, and thereby multigenerational inheritance of RNAi silencing effects [2]. By sampling isolates of C. elegans from the wild, we noticed that, surprisingly, many displayed a temperature-sensitive mortal germline (Mrt) phenotype. Each shows a characteristic mean number of generations until sterility at a given temperature. For example, the half-life of lineages of the wild isolate JU775 is of 4 generations at 25°C, 5 at 23°C, and 10 at 21.5°C, in standard laboratory conditions, feeding on E. coli OP50. Using a quantitative genetic approach, we first examined the genetic variation underlying the variation in the Mrt phenotype between isolates MY10 (Mrt) and JU1395 (non-Mrt). The strongest locus affecting the mortal germline phenotype was identified to be an indel in the set-24 gene, encoding a SET- and SPK-domain protein. The set-24 deletion in MY10 is a rare allele (Frézal et al. submitted). We then also performed a whole-genome association study using 95 C. elegans wild isolates and found a strong association with polymorphisms on chromosome III. Finally, we found that some micro-organisms such as microsporidia suppress the Mrt phenotype. The present project has three aims: 1. Identify the molecular polymorphism(s) underlying the association peak on chr. III explaining a significant part of the natural variation in the mortal germline phenotype in C. elegans. We will use genetic approaches to confirm and narrow down the genetic interval through introgression and recombinant mapping on chromosome III using isolates JU775 (Mrt) and JU1171 (non-Mrt). Candidate polymorphisms including will be tested using various reverse genetic methods including CRISPR-mediated genomic modifications. 2. Test whether the natural variation in the heat-sensitive mortal germline phenotype corresponds to natural variation in transgenerational memory of RNA interference effects. We will test transgenerational memory of wild isolates using an assay that starts with RNAi silencing of a germline-expressed pie-1::GFP transgene introduced into diverse wild genetic backgrounds and then records the number of generations during which the sGFP remains silenced [3-5]. We may also follow endogenous gene silencing. We expect that, as seen in different mutants [5], variation may occur in both directions compared to the reference N2 strain, from no memory to a longer memory of silencing. 3. Study the environmental conditions affecting the mortal germline phenotype and possibly the epigenetic inheritance of RNAi effects. Microsporidia as well as some bacteria partially suppress the heat-sensitive Mrt phenotype of some wild isolates (G. Zhang, L. Frézal & M.-A. Félix). Even the E. coli strain that is used for inducing RNAi in C. elegans (HT115) has an effect compared to the standard E. coli food source (OP50). We will investigate further the specificity of this suppression. We will test whether compounds known to differ among E. coli strains and to affect C. elegans (methionine, folate, vitamin B12, tryptophan; [6-9]) may be at stake here. We will also test whether the micro-organisms and/or the compounds that suppress the Mrt phenotype also affect the epigenetic inheritance of RNAi effects as tested in the second aim. More broadly, the project addresses natural genetic variation in the use of different modes if inheritance. In addition to DNA sequence variation, other modes of inheritance have been uncovered in different organisms [2,10] yet it remains unclear whether these alternative heredity modes play an important role in nature and impact evolution. 1. Smelick C, Ahmed S (2005) Achieving immortality in the C. elegans germline. Ageing Res Rev. 4, 67-82. 2. Rechavi O, Lev I (2017) Principles of transgenerational small RNA inheritance in Caenorhabditis elegans. Curr Biol 27, R720-R730. 3. Ashe A, et al. (2012) piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans. Cell 150, 88-99. 4. Spracklin G et al (2017) The RNAi inheritance machinery of Caenorhabditis elegans. Genetics 206, 1403-16. 5. Lev I et al (2017) MET-2-dependent H3K9 methylation suppresses transgenerational small RNA inheritance. Curr Biol 27, 1138-47. 6. Watson et al. (2014) Interspecies systems biology uncovers metabolites affecting C. elegans gene expression and life history traits. Cell 56, 759-70. 7. Chaudhari SN, et al. (2016) Bacterial folates provide an exogenous signal for C. elegans germline stem cell proliferation. Dev Cell 38, 33-46. 8. Lin CJ, Wang MC (2017) Microbial metabolites regulate host lipid metabolism through NR5A-Hedgehog signalling. Nat Cell Biol 19, 550-57. 9. Gracida X, Eckmann CR (2013) Fertility and germline stem cell maintenance under different diets requires nhr-114/HNF4 in C. elegans. Curr Biol 23, 607-13. 10. Heard E, Martienssen RA (2014) Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95-109