The methyltranferases Suv39h1 and Suv39h2 place the trimethyl mark on lysine 9 of histone H3 (H3K9me3). Heterochromatin Protein 1 (HP1) interacts with H3K9 through its chromodomain (Nielsen et al., 2001). Kap-1, another heterochromatin associated protein interacts with HP1, and together they help to form the compact heterochromatin structure. Without these methyltransferases, cells have less heterochromatin and increased genomic instability (Peters et al., 2001). Previous work in our lab (unpublished) showed that cells lacking these enzymes were more sensitive to ionizing radiation than wild type cells. This indicated a defect in double strand break repair. We wanted to ask if these cells were also more sensitive to methylating agents that cause damage that is repaired through the BER pathway. We found that cells lacking the Suv39h2 methyltransferase treated with MMS, a methylating agent, were more resistant to methylation damage than the wild type. We also wanted to ask whether Kap-1, a heterochromatin associated protein that interacts with the Suv39h1/2 methyltransferases, was phosphorylated after MMS treatment. We found ATM dependent phosphorylation of Kap-1, indicating that cells had relaxed chromatin after treatment with MMS. We hypothesize that this relaxation of chromatin gives the cells resistance to MMS as it allows for easier repair without the need for chromatin remodeling.

Chez l'homme, l'épissage alternatif (EA) affecte presque tous les gènes permettant de générer de vastes répertoires d'ARN et de protéines. L'épissage est un processus hautement régulé qui s'effectue principalement lorsque l'ARN est en cours de synthèse sur la chromatine. Beaucoup d'études suggèrent que la chromatine et ses marques épigénétiques influencent les décisions d'épissage au locus correspondant. A l'inverse, d'autres données laissent penser que l'épissage peut moduler les marques épigénétiques. Au cours de ma thèse, j'ai étudié différentes voies de couplage entre l'épissage et la chromatine. D'une part, j'ai exploré l'impact de la méthylation de l'ADN sur la régulation de l'épissage. J'ai montré que les enzymes qui méthylent l'ADN ont un effet global sur l'épissage d'exons enrichis en méthylation. Mes données suggèrent que les protéines qui lient la méthylation de l'ADN sont impliquées dans cette régulation. D'autre part, j'ai exploré les conséquences de l'EA sur la régulation de la chromatine en étudiant son impact de deux histones-methyltransferases (HMTase) : G9A et SUV39H2 dont les gènes génèrent des transcrits alternatifs. Tous les transcrits variants codent pour des protéines. La conservation des variants d'épissage de G9A dans des espèces et l'absence de différences dans leur activité HMTase, nous amènent à proposer que l'EA est associé à une fonction non liée aux histones. A l'inverse, les isoformes de SUV39H2 exhibent des activités HMTases différentes et régulent l'expression de gènes cibles différents. Ensemble, nos résultats apportent de nouvelles connexions dans le couplage épissage-chromatine et supporte un modèle où ces derniers s'auto-influencent. / In humans, alternative splicing affects almost all genes in the genome and generates extensive repertoires of RNAs and proteins. Splicing is a highly regulated process which occurs primarily when the RNA is being synthesized on chromatin. Many studies suggest that chromatin and epigenetic marks influence splicing choices to the corresponding locus. Conversely, other data suggest that splicing can modulate epigenetic marks. During my thesis, I studied different ways of crosstalk between splicing and chromatin. First, I investigated the effect of DNA methylation on splicing regulation. I have shown that the enzymes that methylate DNA have an overall effect on the splicing of exons with enriched methylation. My data suggest that proteins which bind to methylated DNA are involved in this regulation. On the other hand, I explored the impact of alternative splicing on chromatin regulation studying its impact on the expression and activity of both histone methyltransferases (HMTase): SUV39H2 and G9A. G9A and SUV39H2 generate variants transcripts whose expression is regulated according to tissues. All variants transcripts encode proteins. Conservation of G9A splice variants in species and no differences in their HMTase activity, lead us to propose that G9A alternative splicing is associated with a non-histone function. Conversely, SUV39H2 isoforms exhibit different HMTases activities, and regulate the expression of different target genes. All our results provide new connections in chromatin - splicing coupling and support a model in which they harbor self-influence.