Régulation épigénétique du développement : Déméthylation active de l’ADN et Méthylation de l’ARN chez l’huître Crassostrea gigas, un modèle distanT
To characterize and understand the epigenetic mechanisms of development, their evolution and their transgenerational implications in a distant model submitted to a changing environment using high throughput sequencing (hMeDIP-, MeRIP-, RNA-seq...) and functional studies (CRISPR/Cas9...).
Epigenetic mechanisms are crucial regulators of vertebrate and insect development. Indeed, DNA(1) and RNA(2,3) methylation control the pre and post-transcriptional regulation of gene expression thereby the differentiation of Celle from a single genome as well as the transmission of differentiated phenotypes through cell divisions (2,4,5). Because the environment strongly influences epigenetic mechanisms, they have been widely involved in the transmission of life traits through generations. For example, caste differentiation in the honeybee relies on the distinct methylation of a subset of genes depending on the larval food supply (6). In the zebrafish, the methylation of RNAs, that are accumulated during oogenesis, triggers their degradation in the embryo and thus the activation of the zygotic genome (7,8). Nevertheless, those mechanisms remain widely unknown in distant organisms, questioning their origin, evolution and functions. Besides, our recent findings demonstrate the importance of DNA methylation in the development of the oyster C. gigas (9,10), a mollusk of developmental, evolutionary, ecological and economic interests. Furthermore, importantly regarding the global change and pollution contexts, embryotoxic effects of environmental stressors during the gametogenesis are mediated by epigenetic modifications (11). However, the underlying mechanisms such as the control of DNA methylation dynamics as well as the role of RNA methylation remain elusive, especially regarding the potential influence of the environment on the transgenerational inheritance of life traits.
This project aims at characterizing the roles of active DNA demethylation and RNA methylation in oyster development. For tis purpose, the developmental kinetics of 5-hydroxymethylcytosine (DNA) and of 6-methyladenosine (RNA) will be characterized (ELISA, F-DotBlot). iN parallel, the mRNA expression of the putative actors of those mechanisms will be studied in early life stages (in toto hybridization, RT-qPCR and in silico genomic as well as transcriptomic database mining ). The gene targets of those marks will be determined during the embryogenesis using immunoprecipitation and sequencing (hMeDIP-seq, MeRIP-seq). Knock out approaches using genome edition (microinjection of CRISPR/Cas9 constructs) followed by phenotype investigations (mutation characterization, molecular markers, morphological studies), will be developed in order to study the function of the genes related to active DNA active demethylation (DAD) and RNA. Those studies will be also led regarding determining environmental factors such as the presence of contaminants and toxic algae.
These results will constitute a pioneer and important work at the fundamental level. Indeed, to date to our knowledge, no study focuses on DAD or RNA methylation in a non-insect invertebrate. In addition, the expected results will participate to a better understanding of the evolution of the epigenetic mechanisms of development and of their regulation, and will lead to better consider the extent of environmental constraints in this context. Besides, the success of a CRISPR/Cas9 methodology in bivalves in vivo would constitute an world premiere. Taken together, these results should contribute to solve significant scientific and technical issues and open numerous research perspectives.