In eukaryotic cells, the genome is packed into chromatin, a hierarchically organized complex composed of DNA and histone and nonhistone proteins. In this thesis we have addressed the role of chromatin in cellular response to DNA damage (DDR) using various methodologies encompassing functional genomics and proteomics. First, we analyzed histone post-translational modifications (PTM) in the context of specific kind of DNA lesions (ICL-Interstrand Crosslinks) in Fanconi anemia using quantitative proteomics methodology, SILAC (Stable Isotope Labeling of Amino acids during Cell Culture). Using mass spectrometry (MS), we have successfully identified and quantified a number of histone PTM marks in histone H3 and H4, mainly acetylations and methylations,which have shown dependence upon functional FA-pathway. As a next step, we applied a functional genomics approach to study DDR in FA cells. In this analysis we first monitored the expression profile of histone modifying enzymes related to histone acetylations and methylations. Our results suggest some correlations between histone PTMs and gene expression of histone modifying enzymes, although conclusive evidence warrants further investigations. Next, we analyzed the total transcriptome after DNA damage induction in FA mutant and wild type cells. We also included in this analysis IR irradiation, in an attempt to dissociate more generic DDR from more specific changes that are associated with the role of FA pathway to the DNA ICLs. By performing a factorial interaction analysis, we were able to isolate the part of transcriptional response to DNA damage that was requiring functional FA pathway, as well as the genes that were sensitized to DNA damage by the inactivation of FA pathway. In the final part of the thesis, we attempted to solve one of the limitations that we encountered in the histone PTM analysis. The current approaches used to study histone PTMs from particular loci involves classical chromatin immunoprecipitation, which due to involvement of formaldehyde crosslinking render the protein part mostly unavailable for MS-based proteomics. We have proposed a novel methodology, which is based upon the biotin tagging of histones proximal to a protein of interest and subsequent purification of nucleosomes carrying the tagged histone. This methodology does not involve any crosslinking, enabling us to purify histones from specific loci, and subject them to large scale MS-based histone PTM analysis. A time dimension can also be added to our approach, as we can follow the modification status of particular fraction of histones once they get biotinylated. Another advantage is the use of alternate variant histones, which allows us to study the PTM profile of different functional states of chromatin. This methodology certainly has an edge on current techniques to study histone PTMs pattern associated with a particular protein of interest or with particular chromatin state.

Le génôme des cellules eucaryotes est condensé au sein d'une structure complexe hiérarchiquement organisée : la chromatine. La chromatine est composée d'ADN, de protéines histone et non-histone. Cette thèse a pour but d'étudier le rôle de la chromatine dans la réponse cellulaire aux dommages de l'ADN (DDR) par les méthodologies de génomique fonctionnelle et de protéomique. Nous avons tout d'abord analysé les modifications post-traductionnelles (PTM) des histones dans le cadre des pontages inter-brins (ou "Interstrand Crosslinks", ICL), type particulier de lésions de l'ADN, en choisissant le modèle de l'Anémie de Fanconi (FA). Ceci a été réalisé grâce aux techniques de protéomique quantitative SILAC (Stable Isotope Labeling of Amino acid during Cell culture) et de spectrométrie de masse (MS). Nous avons ainsi réussi à identifier et à quantifier de nombreuses PTMs dans les histones H3 et H4, et à démontrer que certaines de ces PTM sont dépendantes d'une voie fonctionnelle de la signalisation de FA. Nous avons également approfondi l'étude des DDR dans les cellules de FA par une approche de génomique fonctionnelle. Pour cela, nous avons analysé le profil l'expression d'enzymes associées à l'acétylation et à la méthylation des histones. Nos résultats suggèrent l'existence de corrélations entre le profil d'expression de ces enzymes et les PTMs des histones. Des études complémentaires sont nécessaires en vue de confirmer ces corrélations. Nous avons également comparé le transcriptome de deux lignées cellulaires de FA (mutée en FANCC et corrigée en FANCC) après induction de dommages à l'ADN. Afin de différencier les changements spécifiquement associés à la voie de signalisation de FA en réponse aux ICL de l'ADN des réponses plus générales aux dommages de l'ADN, nous avons inclus des cellules traitées par rayonnement ionisant. En réalisant une analyse d'interactions factorielles, nous avons pu identifier une réponse transcriptionnelle aux dommages de l'ADN nécessitant une voie fonctionnelle de la signalisation de FA. Nous avons également tenté de pallier aux limitations rencontrées dans l'analyse des PTMs des histones. En effet, les PTMs des histones que nous avons identifiées représentent l'ensemble des modifications, c'est-à-dire les PTMs concernant les histones se trouvant immédiatement à proximité du site du dommage et en relation directe avec celui-ci, et les PTMs se trouvant à distance du dommage et pouvant ne pas être en relation directe avec celui-ci. Les approches courantes pour identifier les PTMs se trouvant à des loci particuliers sont basées sur l'immunoprécipitation classique de la chromatine où l'utilisation de formaldéhyde altère les protéines, ce qui en rend impossible l'analyse par MS. Nous avons proposé une nouvelle méthodologie basée sur la biotinylation expérimentale d'histones situées à proximité d'une protéine particulière, suivie de la purification des nucléosomes contenant ces histones biotinylées. Contrairement àl'immunoprécipiatation classique de la chromatine, cette méthode n'induit pas d'altération des protéines, permettant ainsi de purifier les histones à partir d'un locus spécifique et d'analyser à grande échelle leurs PTMs par MS. Cette approche permet aussi de suivre dans le temps les PTMs d'une fraction des histones juste après leur biotinylation. Enfin, elle présente l'avantage de pouvoir étudier le profil des PTMs de différents états fonctionnels de la chromatine grâce à l'utilisation de variants d'histones. / In eukaryotic cells, the genome is packed into chromatin, a hierarchically organized complex composed of DNA and histone and nonhistone proteins. In this thesis we have addressed the role of chromatin in cellular response to DNA damage (DDR) using various methodologies encompassing functional genomics and proteomics. First, we analyzed histone post-translational modifications (PTM) in the context of specific kind of DNA lesions (ICL-Interstrand Crosslinks) in Fanconi anemia using quantitative proteomics methodology, SILAC (Stable Isotope Labeling of Amino acids during Cell Culture). Using mass spectrometry (MS), we have successfully identified and quantified a number of histone PTM marks in histone H3 and H4, mainly acetylations and methylations,which have shown dependence upon functional FA-pathway. As a next step, we applied a functional genomics approach to study DDR in FA cells. In this analysis we first monitored the expression profile of histone modifying enzymes related to histone acetylations and methylations. Our results suggest some correlations between histone PTMs and gene expression of histone modifying enzymes, although conclusive evidence warrants further investigations. Next, we analyzed the total transcriptome after DNA damage induction in FA mutant and wild type cells. We also included in this analysis IR irradiation, in an attempt to dissociate more generic DDR from more specific changes that are associated with the role of FA pathway to the DNA ICLs. By performing a factorial interaction analysis, we were able to isolate the part of transcriptional response to DNA damage that was requiring functional FA pathway, as well as the genes that were sensitized to DNA damage by the inactivation of FA pathway. In the final part of the thesis, we attempted to solve one of the limitations that we encountered in the histone PTM analysis. The current approaches used to study histone PTMs from particular loci involves classical chromatin immunoprecipitation, which due to involvement of formaldehyde crosslinking render the protein part mostly unavailable for MS-based proteomics. We have proposed a novel methodology, which is based upon the biotin tagging of histones proximal to a protein of interest and subsequent purification of nucleosomes carrying the tagged histone. This methodology does not involve any crosslinking, enabling us to purify histones from specific loci, and subject them to large scale MS-based histone PTM analysis. A time dimension can also be added to our approach, as we can follow the modification status of particular fraction of histones once they get biotinylated. Another advantage is the use of alternate variant histones, which allows us to study the PTM profile of different functional states of chromatin. This methodology certainly has an edge on current techniques to study histone PTMs pattern associated with a particular protein of interest or with particular chromatin state.