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Identification du chaperon d'histones Spt16 acteur essentiel des réarrangements du génome et méthylation des adénines chez Paramecium tetraurelia

Abstract : In eukaryotes, chromatin organization is required for the regulation of gene expression and genome stability. Ciliate provide excellent model to study mechanisms involved in maintain of genome integrity. Our study model, the unicellular eukaryote Paramecium tetraurelia, has the particularity to eliminate massively and reproducibly 30% of germinal DNA sequences during the development of the somatic macronucleus after sexual events. Those sequences are eliminated by a multi-step process involving small RNA-directed heterochromatin formation followed by DNA excision by the domesticated transposase Piggy Mac (Pgm) and DNA repair. Molecular mechanisms underlying the specific recognition of those germinal sequences in chromatin context and the precision of the excision, remain elusive. The histone chaperone Spt16, associated to its partner Pob3, is part of the heterodimeric complex FACT (FAcilitates Chromatin Transactions). FACT is implicated in many mechanisms involving DNA metabolism such as transcription, repair, replication or chromatin accessibility. In P. tetraurelia, we identified two homologous proteins to Spt16 and Pob3 expressed only during macronucleus development at the time when genome rearrangements occur. Spt16-1 and Pob3-1 fused to GFP are localized in developing macronuclei. We showed that Spt16-1 is required to obtain a viable sexual progeny. Genome re-sequencing after SPT16-1 inactivation showed that Spt16-1 was required for all DNA elimination events and leads to similar phenotypes and defects to those obtained after PGM inactivation. Spt16-1 acts downstream of small RNA-directed heterochromatin formation and upstream of Pgm. We showed that Spt16-1 was required for the correct localization of Pgm responsible for DNA double strand breaks in developing macronuclei. We proposed a model in which Spt16-1 mediates interaction between chromatin and excision machinery that facilitates access to DNA cleavage sites for the Pgm endonuclease. Adenine DNA methylation, well known in bacteria for its role in restriction modification system, has been described during the last two years in several eukaryotes but in low proportion in the genome. However, its role in eukaryotes remains elusive. Previous analyses by chromatography with radio labelled nucleotides detected around 2,5% of methylated adenine in P. tetraurelia but its precise localization and role have never been analyzed. It has been proposed that this modification could mark with precision the excision sites during genome rearrangements when part of the eliminated sequences carry AT boundaries. The abundance of methylated adenine makes of Paramecium an excellent model to study this modification. Combining immunofluorescence techniques, HPLC-MS and sequencing, we described the presence and the cellular localization during life cycle of 6mA in P. tetraurelia. Those approaches allowed us to show that methylated cytosine are absent in Paramecium. We showed that methylation is mainly found in the somatic genome and transiently in germinal genome. It appears during somatic macronucleus development when genome rearrangements occur. Those preliminary results will allow us to pursue the study of adenine methylation by identifying responsible enzymes and its role in the cell.
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Amandine Touzeau. Identification du chaperon d'histones Spt16 acteur essentiel des réarrangements du génome et méthylation des adénines chez Paramecium tetraurelia. Biologie cellulaire. Université Sorbonne Paris Cité, 2018. Français. ⟨NNT : 2018USPCC334⟩. ⟨tel-02988266⟩

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