Le génome eucaryote est contenu dans le noyau sous forme de chromatine, le nucléosome étant son unité de base. Le nucléosome est composé d'ADN enroulé autour d'un octamère d'histones. La chromatine permet l'empaquetage de l'ADN, mais module également diverses fonctions cellulaires telles que la transcription, la réplication et la réparation de l'ADN. Il existe différents types de dommages à l'ADN, les plus toxiques étant les cassures d'ADN double brins (DNA double-strand breaks, DSBs) qui si elles ne sont pas réparées, peuvent compromettre l'intégrité du génome. Les histones peuvent être l'objet de modifications post-traductionnelles qui sont essentielles pour réguler la chromatine. NuA4 est un complexe acétyltransférase qui a été bien décrit pour son rôle dans la transcription et la réparation de l'ADN. Au fil des années, diverses études ont montré que NuA4 acétyle les histones, cependant, de nouvelles études ont permis de mettre en évidence des cibles non-histones. Des précédentes études du laboratoire ont montrées comment NuA4 est recruté au site de dommages à l'ADN et comment il régule la réparation de l'ADN en acétylant les histones et les protéines de réparation. Dans ce travail, nous disséquons davantage le rôle de NuA4 dans la réparation des dommages à l'ADN. Ainsi, nous avons pu déterminer qu'il peut être recruté aux DSBs par un mécanisme alternatif reposant sur la protéine Lcd1ᴬᵀᴿᴵᴾ, indépendamment de Xrs2. Nous décrivons également deux nouvelles cibles acétylées par NuA4, Nej1 et Yku80, deux facteurs qui sont impliqués dans la réparation par jonctions d'extrémités non-homologue (non-homologous end-joining, NHEJ). De plus, nous avons établis qu'il existe une relation antagoniste entre NuA4 et les facteurs du NHEJ. L'acétylation de certains de ces facteurs favorise la réparation des DSBs par des voies de réparation qui reposent sur la résection des extrémités de la cassure. Cette régulation semble conservée au cours de l'évolution puisque le complexe mammifère TIP60 antagonise 53BP1 (levure Rad9) qui favorise la réparation par NHEJ et ainsi permet de réguler le choix de la voie de réparation. De plus, nous démontrons, que chez la levure, la queue N-terminale de l'histone H2A contient un site SQ qui est phosphorylé par Mec1ᴬᵀᴿ en présence de dommages à l'ADN. Nos données suggèrent que cette marque d'histone est nécessaire pour maintenir la fidélité de la résection de l'extrémité de l'ADN en modulant la liaison de Rad9⁵³ᴮᴾ¹. Nous supposons que cette phosphorylation agit de façon similaire à l'ubiquitination sur H2A chez les mammifères, mettant en évidence que des modifications d'histones différentes chez plusieurs organismes convergent pour effectuer une même fonction. Enfin, nous décrivons le rôle du domaine YEATS de la sous-unité Yaf9 partagée par les complexes SWR1 et NuA4. Nous montrons que ce domaine reconnaît la modification d'histone H3K27ac et est impliqué dans l'échange d'histone Htz1ᴴ²ᴬ·ᶻ. Ainsi cette modification est impliquée dans la régulation de la transcription et de la réparation des dommages à l'ADN. Dans l'ensemble, les résultats présentés dans cette thèse ajoutent des contributions importantes aux connaissances actuelles qui permettront de mieux comprendre le rôle de NuA4 et des modifications d'histones dans la réparation des dommages à l'ADN et dans le maintien de l'intégrité du génome. / The eukaryotic genome is packed in the nucleus in the form of chromatin, with the nucleosome being its basic unit. The nucleosome is composed of DNA wrapped around an octamer of histone proteins. Chromatin not only helps in the packing of DNA but also modulates various cellular functions such as transcription, replication and DNA repair. DNA damage manifests in various forms with DNA double-strand breaks (DSBs) being the most toxic which, if unrepaired, compromises genome integrity. Histones are decorated by various post-translational modifications that are essential for fine-tuning and regulation of chromatin. NuA4 is an acetyltransferase complex which has been well described for its role in transcription and DNA repair. Over the years, various studies have shown that NuA4 acts through acetylation of histones, however, new studies have highlighted non-histone targets. Our previous studies have shown how NuA4 is recruited to the site of DNA damage and how it regulates DNA repair by acetylating histones and repair proteins. Here we further dissect the role of NuA4 in DNA repair and found that it can be recruited to DSBs by an alternative mechanism relying on Lcd1ᴬᵀᴿᴵᴾ, independently of Xrs2. We also describe two new targets of NuA4 acetyltransferase activity, Nej1 and Yku80, both factors involved in repair by non-homologous end-joining (NHEJ). In fact, we observe an antagonistic relationship between NuA4 and NHEJ factors, with acetylation of the latter favouring repair of DSBs by resection-based pathways. This regulation seems evolutionary conserved with the mammalian TIP60complex antagonising pro-NHEJ factor 53BP1 (yeast Rad9) to govern the choice of repair pathway. In line with this, we further show that yeast histone H2A N-terminal tail harbours an SQ-site which is phosphorylated by Mec1ᴬᵀᴿ upon DNA damage. Our data suggests this histone mark is required to maintain the fidelity of DNA end resection by modulating the binding of Rad9⁵³ᴮᴾ¹. We speculate that this phosphorylation acts similarly to ubiquitination of mammalian H2A tail, highlighting different histone modifications across organisms converging to achieve a similar function. Lastly, we describe the role of the YEATS domain found in the Yaf9 subunit shared by SWR1 and NuA4 complexes. We show that this domain recognizes H3K27ac and is involved in histone Htz1ᴴ²ᴬ·ᶻ exchange, thus implicating it in transcription and DNA repair. Al together, the results presented in this thesis make important contributions to better understand the intricate roles played by NuA4 and histone modifications in the repair of DNA to maintain genome integrity.

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La chromatine est une structure essentielle qui régit l'homéostasie cellulaire à travers diverses voies dépendantes de la molécule d'ADN telles que l'expression des gènes, la stabilité du génome, l'apoptose, etc. Ainsi, étudier ses mécanismes de régulation est un enjeu crucial pour comprendre le fonctionnement cellulaire. Un des facteurs clés de la modulation de la structure de la chromatine est le complexe acétyltransférase NuA4/TIP60. En effet, ce complexe multi-protéiques très conservé permet l'acétylation des histones, dont H4, et l'incorporation du variant H2A.Z. Il agit alors sur plusieurs mécanismes cellulaires tels que la réparation des cassures doubles brins d'ADN en favorisant la recombinaison homologue ou encore l'activation de la transcription. C'est pourquoi, il est primordial de comprendre en détails les fonctions de ce complexe. Mon projet de doctorat se découpe en deux parties selon les sous-unités du complexe NuA4/TIP60 étudiées. La première partie concerne la sous-unité MRG15, associée à de nombreux complexes, et donc impliquée dans plusieurs fonctions telles que la régulation de l'épissage ou encore la réparation de l'ADN. Combinant des méthodes d'édition du génome avec des purifications de complexes natif et de spectrométrie de masse, nous avons pu identifier un nouveau complexe, composé des sous-unités BRD8, MRGBP, MRG15/X ainsi qu'une nouvelle sous-unité, EP400NL, initialement décrite comme un pseudogène. Puis, par une technique de séquençage d'ARNs, nous montrons que ce complexe est important pour moduler l'expression de gènes spécifiques. De plus, nous avons étudié le mutant W78A/F105A de MRGBP qui perd l'interaction avec MRG15, et le mutant W172A/Y235A de MRG15 qui n'interagit plus avec les facteurs de réparations PALB2/BRCA2. De ce fait, ces mutants aideraient à la compréhension de la fonction de MRG15 lors de la réparation de l'ADN. La seconde partie de mon doctorat concerne la sous-unité MBTD1. En effet, nous nous sommes intéressés à la fusion entre MBTD1 et le facteur d'épissage ZMYND11. Elle est retrouvée dans des cas de leucémies myéloïdes aigües (LMA). Nous avons montré que les mécanismes oncogéniques de cette fusion sont dépendants de la délocalisation sur les corps des gènes du complexes NuA4/TIP60 induisant une augmentation d'acétylation de H4. Ceci génère alors un défaut transcriptionnel dont une surexpression de l'oncogène Myc et des dérégulations d'épissage alternatif de transcrits spécifiques. De plus, nous avons mis en évidence les résidus essentiels pour l'interaction entre MBTD1 et la sous-unité EPC1, associant alors MBTD1 avec le reste du complexe NuA4/TIP60. Ensemble, ces résultats permettraient le développement de nouveaux outils thérapeutiques pouvant cibler MBTD1 afin de traiter les cas de LMA spécifiques. L'ensemble de ces travaux offre une meilleure compréhension des fonctions du complexe NuA4/TIP60 liées à ses différentes sous-unités, à la fois au niveau transcriptionnel mais également au niveau de la réparation de l'ADN. De plus, ils établissent les mécanismes oncogéniques associées à ce complexe. Ainsi, de futures stratégies thérapeutiques pourraient être développées. / Chromatin is an essential structure that governs cell homeostasis through various DNA molecule-dependent pathways such as gene expression, genome stability, apoptosis, etc. Thus, studying its regulatory mechanisms is a crucial issue in understanding cell functioning. One of the key factors in the modulation of chromatin structure is the acetyltransferase complex NuA4/TIP60. Indeed, this highly conserved multi-protein complex allows the acetylation of histones, including H4, and the incorporation of the H2A.Z variant. It then acts on several cellular mechanisms such as the repair of DNA double strand breaks by promoting homologous recombination or even the activation of transcription. This is why it is essential to understand in detail the functions of this complex. My doctoral project is divided into two parts according to the subunits of the NuA4/TIP60 complex studied. The first part concerns the MRG15 subunit, associated with many complexes and therefore involved in several functions such as the regulation of splicing or DNA repair. Combining genome editing methods with native complex purifications and mass spectrometry analysis, we were able to identify a new complex, composed of the BRD8, MRGBP, MRG15/X subunits as well as a new subunit, EP400NL, initially described as a pseudogene. Then, by an RNA sequencing technique, we show that this complex is important for modulating the expression of specific genes. In addition, we studied the W78A/F105A mutant of MRGBP which loses interaction with MRG15 and the W172A/Y235A mutant of MRG15 which loses its interaction with repair factors PALB2/BRCA2. Thus, these mutants would help to understand the function of MRG15 during DNA repair. The second part of my doctorate concerns the MBTD1 subunit. Indeed, we were interested in the fusion between MBTD1 and the splicing factor ZMYND11 found in cases of acute myeloid leukemia (AML). We then showed that the oncogenic mechanisms of this fusion are dependent on the delocalization of the NuA4/TIP60 complex on the bodies of the genes, leading to an increase of H4 acetylation. This generates a transcriptional alteration including overexpression of the Myc oncogene and deregulation of alternative splicing of specific transcripts. In addition, we have characterized the residues required for the interaction between MBTD1 and the EPC1 subunit, essential for the association of MBTD1 with the rest of the NuA4/TIP60 complex. Together, these results would allow the development of new therapeutic tools that can target MBTD1 to treat specific AML cases. All of this work provides a better understanding of the functions of the NuA4/TIP60 complex linked to its various subunits both at the transcriptional level but also in DNA repair. In addition, they establish the oncogenic mechanisms associated with this complex. Thus, future therapeutic strategies could be developed.

Thesis advisor: Anthony T. Annunziato / The project I am presenting aimed to: 1. Elucidate the pattern of post- translational modification on the different variants of newly synthesized histones H3 in mammalian cells; 2. Reveal whether the acetylation of residue K56 on newly synthesized H3 histones plays a role in the incorporation of the histone into chromatin in mammalian cells; and 3. Determine whether the acetylation of residue K56 on newly synthesized H3 histones plays a role in the incorporation of the histone specifically in replicating chromatin in mammalian cells. The experiments to answer these questions were performed using HEK293 cells with inducible expression of FLAG-histones, enabling us to control the synthesis of new histones of interest and to detect and analyze their presence and relative levels in the cells. The results suggest that the acetylation of lysine 56 on histone H3 may play a positive role in the incorporation of the histone into new chromatin, and lack of acetylation may be reducing the efficiency of incorporation compared to acetylated histones. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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1. The work described in this thesis forms part of an investigation of eukaryotic gene control. The system studied was the avian erythroid cell series since it is possible to isolate pure populations of the various cell types which have well-defined biochemical activities. These cells contain an unusual tissue - specific histone H5, which may be involved in the progressive repression of transcription observed as these cells differentiate. Although the gene controlling function of this histone must be at a very gross level, this represents a unique opportunity to investigate one facet of gene control. Probably the most sensitive technique is to assay for specific messenger RNA and gene sequences by hybridisation to an appropriate probe. The aim of this thesis was to prepare such a probe from H5 mRNA and to use it to calculate the reiteration frequency of the H5 gene in the chicken genome. 2. The cells employed were chicken reticulocytes since the only histone made in these cells is H5. Experiments were conducted which demonstrated that H5 mRNA is probably a minor species compared to globin mRNA in these cells. Furthermore, calculations indicate that the two mRNAs are probably of similar molecular weight which may complicate the isolation of H5 mRNA. As a result globin mRNA was first purified and characterised. Properties which may have proved useful in the separation of this mRNA from H5 mRNA are discussed. The globin mRNA was used to optimise techniques for the in vitro translation and identification of chicken mRNAs. This was considered necessary as mRNAs from different sources vary in the conditions required for optimal translation and it was reasoned that mRNAs from the same cell would have similar optima. 3. Total polysomal RNA was fractionated on the basis of size and poly A content. Although large amounts of globin mRNA were present, H5 mRNA could only be detected in the non - poly A containing RNA. Even in this fraction however, there was still a large excess of globin mRNA which was difficult to remove due to the demonstrated similarity of their molecular weights. 4. Since it had proved impossible to isolate the H5 mRNA by conventional techniques, immunological methods of isolating the polysomes producing H5 were investigated. Using immunoadsorbents, mRNA was prepared in small amounts which programmed the synthesis in vitro of more than 70 % H5. The yield and specificity were improved by modifying the procedure to indirect immunoprecipitation followed by oligo ( dT ) - cellulose chromatography. The resulting mRNA programmes the synthesis in vitro of more than 90 % H5. The chemical purity of the mRNA is discussed. 5. The immunologically prepared H5 mRNA was not copied into cDNA by RNA - dependent DNA - polymerase. Since this was probably due to the lack of a 3 ' poly A tract on the mRNA, an enzyme was purified and characterised which would add such a tract. The enzymically modified mRNA could then be copied into cDNA of high specific activity. 6. The H5 cDNA was characterised in terms of size and fidelity of copying. By hybridisation analysis it was demonstrated that the amount of contaminating rRNA and globin mRNA complementary sequences present in the cDNA was insignificant. The complexity of the cDNA was shown to be of the same size as the H5 mRNA and will back hybridise to this mRNA to greater than 75 %. These results are discussed to demonstrate that the cDNA is a faithful copy of H5 mRNA. The possible uses of the resulting probe are also discussed. 7. The H5 cDNA was employed to quantify the number of H5 genes in the chicken genome. The significance of this result is discussed in terms of the known reiteration and organisation of histone genes in other species, and the possible role of H5 as a gene control agent. / Thesis (Ph.D.)--Department of Biochemistry, 1975.

Thesis (Ph. D)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Aug 16

Thèse de doctorat : Sciences du vivant. Aspects cellulaires et moléculaires de la biologie : Strasbourg 1 : 2008. / Titre provenant de l'écran-titre. Bibliogr. 47 p.

Reproduction de : Thèse de doctorat : Médecine. Cancérologie, Génétique, Hématologie, Immunologie : Nantes : 2008. / Bibliogr.