2. Abstract Histone methylation plays an important role in almost all cellular processes and its homeostasis is maintained by histone methyltransferases and histone demethylases. Misregulation of histone methylation levels is associated with gene expression misregulation and consequently also with various developmental defects and diseases. In this thesis we focus on the lysine demethylases KDM2A and KDM2B and on their demethylation deficient isoforms KDM2A-SF and KDM2B-SF. The lysine specific demethylases KDM2A and KDM2B have been predominantly studied for their demethylation function on CpG island-rich gene promoters. However, KDM2A-SF and KDM2B-SF have not been studied in detail. Therefore, the main goal of this thesis was to characterize KDM2A-SF more in detail and to focus on the role that KDM2A/B-SF might potentially play in canonical Wnt signaling pathway. We found that the KDM2A-SF mRNA arises through the action of an alternative intronic promoter and not by alternative splicing. We showed that the KDM2A-SF start codon is located in the exon that corresponds to KDM2A exon 14 and we thus determined the exact amino acid sequence of the KDM2A-SF protein. Furthermore, using an isoform specific knockdown assay we showed that KDM2A-SF, unlike KDM2A-LF, forms distinct nuclear foci on pericentromeric...

2. Abstract Histone methylation plays an important role in almost all cellular processes and its homeostasis is maintained by histone methyltransferases and histone demethylases. Misregulation of histone methylation levels is associated with gene expression misregulation and consequently also with various developmental defects and diseases. In this thesis we focus on the lysine demethylases KDM2A and KDM2B and on their demethylation deficient isoforms KDM2A-SF and KDM2B-SF. The lysine specific demethylases KDM2A and KDM2B have been predominantly studied for their demethylation function on CpG island-rich gene promoters. However, KDM2A-SF and KDM2B-SF have not been studied in detail. Therefore, the main goal of this thesis was to characterize KDM2A-SF more in detail and to focus on the role that KDM2A/B-SF might potentially play in canonical Wnt signaling pathway. We found that the KDM2A-SF mRNA arises through the action of an alternative intronic promoter and not by alternative splicing. We showed that the KDM2A-SF start codon is located in the exon that corresponds to KDM2A exon 14 and we thus determined the exact amino acid sequence of the KDM2A-SF protein. Furthermore, using an isoform specific knockdown assay we showed that KDM2A-SF, unlike KDM2A-LF, forms distinct nuclear foci on pericentromeric...

Le rôle fonctionnel des transcrits de l’hétérochromatine péricentromérique reste à ce jour largement incompris chez les eucaryotes supérieurs. Néanmoins, il a été montré que ces transcrits sont soumis à un contrôle très précis, fonction du cycle cellulaire. La régulation de la transcription est fortement contrôlée par la structure de la chromatine qui peut être modifiée localement en changeant la composition biochimique du nucléosome, notamment par l’utilisation des variantes d’histones. L’objectif de ma thèse a été de mieux comprendre le rôle de la protéine chaperonne d’histone DAXX et de sa variante d’histone H3.3 dans la régulation de la transcription des séquences répétées péricentromériques. Par la méthode de purification TAP-TAG, les partenaires spécifiques de DAXX ont été identifiés à partir d’extraits solubles nucléaires de fibroblastes embryonnaires murins. Ces analyses ont mis en évidence que CAF-1, classiquement associé à H3.1, et les facteurs de remodelage de la chromatine ATRX et CHD4 interagissent spécifiquement avec DAXX. Le rôle de ces protéines dans le contrôle de la transcription de l’hétérochromatine péricentromérique a ensuite été mis en évidence par une approche combinant l’interférence ARN et la Q-PCR. Enfin, les résultats suggèrent fortement que ces mécanismes de régulation ont lieu au niveau des corps nucléaires PML. L’ensemble de ces données montre qu’il existe une régulation spatio-temporel très fine de la structure de la chromatine régulant la transcription de l’hétérochromatine péricentromérique. / The functional role of pericentromeric heterochromatin transcripts remains largely unknown in higher eukaryotes. Nevertheless, it has been shown that these transcripts are subject to very precise control, depending on the cell cycle. Regulation of transcription is tightly controlled by chromatin structure that can be modified locally by changing the biochemical composition of the nucleosome, including the use of histone variants. The aim of my thesis was to better understand the role of the histone chaperone protein DAXX and its histone variant H3.3 in the regulation of transcription of pericentromeric repeats. By the method of TAP-TAG purification, DAXX specific partners were identified from soluble nuclear extracts of murine embryonic fibroblasts. These analyzes revealed that CAF-1, classically associated with H3.1, and the chromatin remodeling factors, ATRX and CHD4, specifically interact with DAXX. The role of these proteins in the control of transcription of pericentromeric heterochromatin was then highlighted by an approach combining RNAi and Q-PCR. Finally, the results strongly suggest that these regulatory mechanisms take place at PML nuclear bodies. Taken together, these data show that there is a spatio-temporal regulation of the fine structure of chromatin regulates transcription of pericentromeric heterochromatin.