The chromosomal passenger complex (CPC: Aurora B-INCENP-Survivin-Borealin) is a key regulator of cell division whose localisation at centromeres is required for stable kinetochore-microtubule attachments and proper chromosomal segregation (Ruchaud et al. 2007; Carmena et al. 2012; van der Waal et al. 2012). Shugoshin1 (hSgo1) (via Borealin) and Histone H3 (via Survivin) have been implicated in centromeric targeting of CPC (Wang et al. 2010; Jeyaprakash et al. 2011; Tsukahara et al. 2010; Kawashima et al. 2010). Although the Survivin-Histone H3 pathway has been extensively studied, the intermolecular interactions dictating CPC-hSgo1 interactions remain unclear. My PhD work focused on characterising the molecular framework of the CPC-hSgo1 interaction using biochemical, biophysical and structural biology methods. I optimised and improved human CPC and hSgo1 recombinant protein production in an E. coli system. Post optimisation, I used Size-Exclusion Chromatography to successfully reconstitute the CPC-hSgo1 complex in vitro and further confirmed that hSgo1 possessing no modification or extra amino acids on its N-terminus can interact with Survivin and Borealin-Survivin-INCENP1-57. This suggested that the hSgo1 N-terminal tail interaction with Survivin is crucial for CPC-hSgo1 interaction. Furthermore, I conducted calorimetric binding studies to molecularly dissect the individual contributions of CPC components and their domains towards CPC-hSgo1 interaction. Towards this aim, I expressed and purified different versions of CPC and analysed their binding energetics with hSgo1. The results from these experiments clearly suggested the contribution of Borealin and INCENP towards CPC-hSgo1 interaction.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21400号 / 生博第401号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松崎 文雄, 教授 石川 冬木, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Le noyau des cellules est une structure très organisée, dont l'organisation joue un rôle important dans la régulation de l'expression des gènes. La compréhension des mécanismes à l'origine de cette organisation est donc essentielle à la compréhension du fonctionnement des génomes. De nombreuses expériences conduites chez la souris ont montré que les régions centromériques (RC) des chromosomes jouent un rôle dans l'organisation du noyau. L'organisation spatiale des RCs humaines est beaucoup moins étudiée, principalement à cause de la complexité des séquences qui les composent, qui rend plus difficile leur détection. Nous avons développé des outils de traitement et d'analyse quantitative d'image, qui, combinés à des nouveaux marqueurs des RCs humaines, nous ont permis de mieux décrire deux aspects de leur organisation spatiale. D'une part nous avons montré qu'elles se positionnent préférentiellement en périphérie du noyau ou aux bords des nucléoles, avec des fréquences qui dépendent des chromosomes. D'autre part nous avons montré qu'elles s'agrègent dans le noyau pour former un compartiment d'hétérochromatine, qui présente des caractéristiques similaires à celui observé dans d'autres espèces telles que la souris. Ces deux aspects sont tous deux inter-dépendants et varient au cours du cycle cellulaire. Cette description nouvelle met sur la piste de mécanismes responsables de l'organisation particulière des RCs, qui pourront être étudiés grâce à la méthode d'analyse et aux observables que nous avons développées. L'étude de ces mécanismes permettra de mieux comprendre la fonction des RCs humaines dans l'organisation du noyau. / The cell nucleus is a highly organized structure, playing an important role in gene regulation. Understanding the underlying mechanisms is therefore essential for understanding genome function. Numerous studies conducted in mouse cells have shown that centromeric regions (RC) of chromosomes play a role in nuclear organization. The spatial organization of human RCs is less studied, mainly because of the complexity of the underlying DNA sequences that make them hard to detect. We have developed image processing and analysis tools, that, combined with new markers for human RCs, have allowed us to draw a better description of two features of their spatial organization. On the one hand, we have shown that they are preferentially located close to the nuclear periphery or nucleoli borders, with chromosome-dependent frequencies. On the other hand, we have shown that they cluster to form a heterochromatic compartment that displays similar properties as the one observed in other species such as mouse. Both features are inter-dependent, and vary throughout the cell-cycle. This new description puts on the track of mechanisms responsible for the peculiar organization of RCs. Those mechanisms could be studied using the methodology and the observables we have developed. The study of those mechanisms will provide a better understanding of human RC function in nuclear organization.

The centromeres of eukaryotes have a region on which the kinetochore is assembled, flanked by heterochromatin which provides cohesion between the sister chromatids during cell division. When centromeric heterochromatin is lost chromosomes no longer segregate evenly into the daughter cells during cell division. In the fission yeast Schizosaccharomyces pombe (S. pombe) RNA interference (RNAi) is responsible for maintaining this heterochromatin. The pathway is part of a feedback loop whereby siRNAs generated from non-coding centromere transcripts are loaded into an Argonaute complex. The siRNAs guide the complex to the homologous centromere repeats in order to recruit Clr4 which modifies histone H3 with the heterochromatin mark H3K9me. A previous screen to find factors affecting centromere silencing isolated 13 loci termed centromere: suppressor of position-effect (csp) 1-13. Several csp mutants have been identified to be RNAi components. In this investigation the csp6 locus has been identified to be the Hsp70 gene ssa2+. It has been demonstrated that Argonaute proteins from plants and flies require Hsp70/90 chaperone activity for loading of siRNA. It therefore seems likely that Hsp70 may play a similar role in fission yeast. Genetic and biochemical techniques have been used in this study to investigate if the csp6 alleles are affecting siRNA loading in S. pombe. RNA Polymerase II (RNAPII) transcribes the pre-siRNA transcripts from the centromere repeats. csp3 was identified to be an allele of the RNAPII subunit rpb7+. rpb7-G150D was found to cause a silencing defect in the centromeric heterochromatin through a defect in transcription. Another RNAPII mutation, rpb2-m203, was found to have strong silencing defects caused by an unidentified non-transcriptional role in RNAi-mediated heterochromatin formation at the centromere. In order to gain more insight into the role of RNAPII in heterochromatin assembly I performed a screen in which the subunits rpb3 and rpb11 were subjected to random mutagenesis. Several mutants were isolated and characterisation of phenotypes regarding heterochromatin at the centromere has been carried out for nine of the mutants. As a result a novel phenomenon of RNAi-independent silencing at the centromere has been discovered.

The ultimate goal of mitosis is the equal distribution of chromosomes between the two daughter cells. One of the key players that ensures faithful chromosome segregation is the chromosomal passenger complex (CPC). CPC localisation to mitotic centromeres is complex, involving interactions with Shugoshin and binding to phosphorylated histone H3T3. It was recently reported that Heterochromatin Protein 1 (HP1) has a positive impact on CPC function during mitosis. The interaction between HP1 and the CPC appears to be perturbed in cancer-­‐derived cell lines, resulting in decreased HP1 levels at mitotic centromeres and may be a potential cause for increased chromosome mis-­‐segregation rates. In this study, I tethered HP1α to centromeres via the DNA-­‐binding domain CENP-­‐B. However, instead of improving the rate of chromosome mis-­‐segregation, HP1α tethering resulted in activity of the spindle assembly checkpoint and destabilisation of kinetochore-­‐microtubule attachments, most likely caused by the robust recruitment of the CPC. Tethered HP1α even traps the CPC at centromeres during mitotic exit, resulting in a catalytically active CPC throughout interphase. However, it was not clear whether endogenous HP1 contributes to CPC localisation and function prior to mitosis. Here I also describe a substantial interaction between endogenous HP1 and the CPC during the G2 stage of the cell cycle. The two isoforms HP1α and HP1γ contribute to the clustering of the CPC into active foci in G2 cells, a process that is independent of CDK1 kinase activity. Furthermore, the H3S10ph focus formation in the G2 phase appears to be independent of H3T3ph and H2AT120ph, the two histone marks that determine the CPC localisation in early mitosis. Together, my results indicate that HP1 contributes to CPC concentration and activation at pericentromeric heterochromatin in G2. This novel mode of CPC localisation occurs before the Aurora B-­‐driven methyl/phos switch releases HP1 from chromatin, which possibly enables the H3T3ph and H2AT120ph driven localisation of the CPC during mitosis.

Understanding the role of microtubule-associated proteins is the key to understand the complex mechanisms regulating microtubule dynamics. This study employs the model system Dictyostelium discoideum to elucidate the role of the microtubule-associated protein TACC (Transforming acidic coiled-coil) in promoting microtubule growth and stability. Dictyostelium TACC was localized at the centrosome throughout the entire cell cycle. The protein was also detected at microtubule plus ends, however, unexpectedly only during interphase but not during mitosis. The same cell cycle-dependent localization pattern was observed for CP224, the Dictyostelium XMAP215 homologue. These ubiquitous MAPs have been found to interact with TACC proteins directly and are known to act as microtubule polymerases and nucleators. This work shows for the first time in vivo that both a TACC and XMAP215 family protein can differentially localize to microtubule plus ends during interphase and mitosis. RNAi knockdown mutants revealed that TACC promotes microtubule growth during interphase and is essential for proper formation of astral microtubules in mitosis. In many organisms, impaired microtubule stability upon TACC depletion was explained by the failure to efficiently recruit the TACC-binding XMAP215 protein to centrosomes or spindle poles. By contrast, fluorescence recovery after photobleaching (FRAP) analyses conducted in this study demonstrate that in Dictyostelium recruitment of CP224 to centrosomes or spindle poles is not perturbed in the absence of TACC. Instead, CP224 could no longer be detected at the tips of microtubules in TACC mutant cells. This finding demonstrates for the first time in vivo that a TACC protein is essential for the association of an XMAP215 protein with microtubule plus ends. The GFP-TACC strains generated in this work also turned out to be a valuable tool to study the unusual microtubule dynamics in Dictyostelium. Here, microtubules exhibit a high degree of lateral bending movements but, in contrast most other organisms, they do not obviously undergo any growth or shrinkage events during interphase. Despite of that they are affected by microtubuledepolymerizing drugs such as thiabendazole or nocodazol which are thought to act solely on dynamic microtubules. Employing 5D-fluorescence live cell microscopy and FRAP analyses this study suggests Dictyostelium microtubules to be dynamic only in the periphery, while they are stable at the centrosome. In the recent years, the identification of yet unknown components of the Dictyostelium centrosome has made tremendous progress. A proteomic approach previously conducted by our group disclosed several uncharacterized candidate proteins, which remained to be verified as genuine centrosomal components. The second part of this study focuses on the investigation of three such candidate proteins, Cenp68, CP103 and the putative spindle assembly checkpoint protein Mad1. While a GFP-CP103 fusion protein could clearly be localized to isolated centrosomes that are free of microtubules, Cenp68 and Mad1 were found to associate with the centromeres and kinetochores, respectively. The investigation of Cenp68 included the generation of a polyclonal anti-Cenp68 antibody, the screening for interacting proteins and the generation of knockout mutants which, however, did not display any obvious phenotype. Yet, Cenp68 has turned out as a very useful marker to study centromere dynamics during the entire cell cycle. During mitosis, GFP-Mad1 localization strongly resembled the behavior of other Mad1 proteins, suggesting the existence of a yet uncharacterized spindle assembly checkpoint in Dictyostelium. / Die Kenntnis der Funktion von Mikrotubuli-assoziierenden Proteinen (MAPs) ist von grundlegender Bedeutung für das Verständnis der Mikrotubuli-Dynamik und deren Regulation. Im Rahmen dieser Arbeit wurde die Rolle des Mikrotubuli-assoziierenden Proteins TACC (Transforming acidic coiled-coil), welches in vielen Organismen an der Stabilisierung und dem Wachstum von Mikrotubuli beteiligt ist, im Modellorganismus Dictyostelium discoideum untersucht. Das Dictyostelium TACC Protein konnte während des gesamten Zellzyklus am Centrosom nachgewiesen werden. Darüber hinaus wurde es an den Mikrotubuli-Plus-Enden vorgefunden, überraschenderweise jedoch ausschließlich während der Interphase. Die gleiche Zellzyklusabhängige Lokalisation wurde für CP224 beobachtet, einem Homologen der XMAP215 Proteine in Dictyostelium. Diese ubiquitären MAPs sind konservierte, direkte Interaktionspartner der TACC Proteine und spielen eine zentrale Rolle bei der Nukleation und der Polymerisation von Mikrotubuli. Durch diese Arbeit konnte erstmals in vivo gezeigt werden, dass TACC und XMAP215 Proteine während der Interphase und Mitose unterschiedlich stark mit Mikrotubuli-Plus-Enden assoziiert sein können. Durch Untersuchungen an Knockdown-Mutanten wurde ersichtlich, dass Dictyostelium TACC eine Rolle beim Mikrotubuli-Wachstum während der Interphase spielt und über weite Strecken der Mitose essentiell für die Ausbildung von astralen Mikrotubuli ist. In anderen Organismen konnte als Ursache instabiler Mikrotubuli in TACC Mutanten häufig unzureichendes Rekrutieren des jeweiligen XMAP215 Proteins an das Centrosom ausgemacht werden. Um entsprechende Auswirkungen auf die Lokalisation von CP224 durch den Knockdown von TACC in Dictyostelium zu untersuchen, wurden Fluorescence Recovery after Photobleaching (FRAP) Experimente durchgeführt. Diese ergaben, dass CP224 auch in Abwesenheit von TACC in vollem Umfang an die Centrosomen und Spindelpole rekrutiert wird. Anders als im Wildtyp, konnte in TACC Mutanten allerdings kein CP224 an den Mikrotubuli-Plus-Enden nachgewiesen werden. Somit konnte erstmals in vivo gezeigt werden, dass ein TACC Protein essentiell für die Assoziation eines XMAP215 Proteins mit den Mikrotubuli-Plus-Enden ist. Im Laufe der genannten Experimente stellte sich heraus, dass sich die GFP-TACC Stämme aufgrund ihrer markierten Plus-Enden sehr gut für Untersuchungen zur ungewöhnlichen Mikrotubuli-Dynamik in Dictyostelium eignen. Zwar weisen Mikrotubuli hier über die gesamte Länge ausgeprägte Krümmungs- und Seitwärtsbewegungen auf, es können jedoch im Vergleich zu anderen Organismen während der Interphase kaum Wachstums- oder Verkürzungsvorgänge beobachtet werden. Dennoch können Dictyostelium Mikrotubuli unter Verwendung von Agenzien wie Thiabendazol oder Nocodazol, welche ausschließlich auf dynamische Mikrotubuli wirken, signifikant verkürzt werden. Durch FRAP Experimente und Einsatz von 5D Fluoreszenz-Mikroskopie an lebenden Zellen konnte in dieser Arbeit erstmalig nachgewiesen werden, dass Dictyostelium Mikrotubuli nur in der Zellperipherie, nicht aber im pericentrosomalen Bereich dynamisch sind. Die Identifikation bislang unbekannter Bestandteile des Dictyostelium Centrosoms erfuhr in den vergangenen Jahren große Fortschritte. Ein von unserer Gruppe durchgeführter Proteomics-Ansatz brachte eine Vielzahl potentiell centrosomaler Proteine zu Tage, von welchen bereits viele am Centrosom nachgewiesen werden konnten. Der zweite Teil dieser Arbeit befasst sich mit der Charakterisierung dreier noch unbekannter Proteine aus dem Proteomics-Ansatz, Cenp68, CP103 und dem Dictyostelium Homologen des Spindle Assembly Checkpunkt Proteins Mad1. Hierbei zeigte sich, dass lediglich CP103 Bestandteil isolierter, Mikrotubuli-freier Centrosomen ist, während Cenp68 an die Centromere und Mad1 an die Kinetochoren lokalisieren. Die Charakterisierung von Cenp68 umfasste außerdem die Herstellung eines polyklonalen anti-Cenp68 Antikörpers, das Suchen nach Interaktionspartnern und die Erzeugung eines Cenp68 Knockout-Stammes. Letzterer wies jedoch keinen offensichtlichen Phänotyp auf. Das Verhalten des Dictyostelium Mad1 Proteins während der Mitose stimmte in großen Teilen mit dem anderer Mad1 Proteine überein, was auf die Existenz eines bislang unerforschten Spindle Assembly Chekpunkts in Dictyostelium hinweisen könnte.

Centromeres are responsible for sister-chromatid cohesion, kinetochore formation, and accurate transmission of chromosomes. Rice provides an excellent model for organizational and functional studies of centromeres since several of its chromosomes contain limited amounts of satellite and other repetitive sequences in their centromeres. To facilitate molecular characterization of the centromeres, we screened several BIBAC and BAC libraries of japonica and indica rice, using several centromere-specific repeat elements as probes. The positive clones were identified, fingerprinted and integrated into our whole genome physical map databases of the two rice subspecies. BAC/BIBACbased physical maps were constructed for the centromeric regions of the subspecies. To determine whether the genomic organization of the centromeres has changed since the cultivated rice split from its progenitor and to identify the sequences potentially playing an important role in centromere functions, we constructed a large-insert BIBAC library for the wild progenitor of Asian cultivated rice, O. rufipogon. The library contains 24,192 clones, has an average insert size of 163 kb, and covers 5 x haploid genome of wild rice. We screened the wild rice library with two centromere 8-specific overgo probes designed from the sequences flanking centromere 8 of japonica rice. A BIBACbased map was constructed for wild rice centromere 8. Two of the clones, B43P04 and B15E04, were found to span the entire region of the wild rice centromere and thus selected for sequencing the centromere. By sequencing the B43P09 clone, a 95% genomic sequence of the long arm side of wild rice centromere 8 was obtained. Comparative analysis revealed that the centromeric regions of wild rice have a similar gene content to japonica rice, but the centromeric regions of japonica rice have undergone chromosomal rearrangements at both large scale and nucleotide levels. In addition, although the 155-bp satellite repeats showed dramatic changes at the middle region, they are conserved at the 5' and 3' ends of satellite monomers, suggesting that those regions might have important functional roles for centromeres. These results provide not only new insights into genomic organization and evolution, but also a platform for functional analysis of plant centromeres.

La ségrégation fidèle des chromosomes est dirigée par le centromère, un locus chromosomique spécialisé qui est requis pour l’assemblage des kinetochores actifs. Les centromères sont marqués épigénétiquement par la présence d’un nucléosome unique qui contient un variant centromérique de l’histone H3 appelé Centromere protein A (CENP-A). Une question fondamentale est comment CENP-A est spécifiquement déposé aux centromères. L’objectif de ma thèse a été d’identifier les facteurs spécifiques de la déposition de CENP-A. Pour identifier les facteurs spécifiques impliqués dans la déposition de CENP-A aux centromères, j’ai utilisé la méthode de purification TAP-TAG à partir d’une fraction nucléaire soluble de cellules HeLa exprimant stablement une copie ectopique de CENP-A (e-CENP-A). J’ai ainsi pu identifié la protéine Holliday Junction Recognition protein (HJURP). En utilisant un siRNA spécifique de HJURP, j’ai montré que la localisation et la déposition de CENP-A étaient fortement affectées. La protéine recombinante HJURP lie de manière stoechiométrique le tétramère CENP-A/H4 mais il ne lie pas le tétramère H3/H4. La liaison se fait grâce à un petit domaine conservé en position N-terminal de HJURP, dénommé CBD (CENP-A binding domain). De plus, j’ai pu mettre en évidence in vitro que HJURP facilitait la déposition du tétramère CENP-A/H4 sur de l’ADN satellite. L’ensemble de mes résultats démontre très clairement que HJURP est la principale chaperone responsable de la déposition de CENP-A aux centromères. / Centromere is a specialized chromosomal locus, where kinetochore assembles, which is required for correct chromosome segregation during cell division. In higher eukaryotes, centromere specification is independent of the DNA sequence and is determined epigenetically by the presence of a unique nucleosome that contains a histone H3 variant, called CENP-A. A fundamental question in centromere biology is that how CENP-A is specifically delivered to and maintained on centromeres. The aim of my thesis was to identify specific chaperone in human, responsible for CENP-A loading to centromeres, by using biochemical and proteomic strategies. To identify CENP-A deposition machinery, I purified the prenucleosomal CENP-A complex from HeLa cells stably expressing epitope tagged CENP-A. By mass spectrometry analysis of proteins present in CENP-A and H3.1 complex, I found HJURP uniquely in CENP-A prenucleosomal complex. Down regulation of HJURP by specific siRNA strongly diminished centromeric localization of CENP-A. Bacteriallyexpressed HJURP specifically binds to the CATD domain of CENP-A, via a highly conserved Nterminal domain, called CBD. Finally, I showed that HJURP is able to facilitate the efficient deposition of CENP-A/H4 tetramer on naked DNA. Taken together, my data demonstrate that HJURP is a key chaperone responsible for the targeting and deposition of newly synthesized CENPA at centromeres.

La protéine p53 est surtout étudiée pour sa capacité à empêcher la prolifération de cellules dont le génome est endommagé. Toutefois, en analysant les souris p53Δ31/Δ31 qui expriment une protéine p53 hyperactive, notre laboratoire a découvert un rôle inattendu de p53 dans la régulation du métabolisme des télomères. Ces souris modélisent la dyskératose congénitale (DC), un syndrome d’insuffisance médullaire héréditaire (SIMH) causé par un dysfonctionnement télomérique. Mon projet de thèse a combiné les analyses de modèles murins et de lignées cellulaires humaines afin d’approfondir l'étude de cette nouvelle fonction de p53 et de mieux comprendre le rôle de la voie p53 dans le développement des SIMH.En poursuivant notre analyse du modèle murin p53Δ31, nous avons montré que les cellules p53Δ31/Δ31 présentent une diminution d’expression de 12 gènes mutés dans l’anémie de Fanconi (AF), un autre SIMH étroitement lié à la DC. De plus, ces cellules montrent une capacité réduite à réparer les ponts inter-brins de l’ADN, une caractéristique typique de l’AF. Notre étude suggère que l’activation soutenue de p53 pourrait ainsi contribuer aux similitudes cliniques entre ces deux syndromes. Par ailleurs, nous avons identifié par quel mécanisme p53 régule négativement le métabolisme des télomères et la voie Fanconi de réparation de l’ADN. Ce mécanisme de régulation indirect, via p21 et E2F4, est très conservé entre la souris et l’Homme. De fait, les cellules p53Δ31/Δ31 constituent un outil puissant pour identifier de nouvelles cibles de p53 réprimées par ce mécanisme. Ainsi, elles nous ont permis de montrer que deux gènes essentiels pour la structure des centromères sont réprimés par p53.L’activation de p53 est clairement impliquée dans les étapes ultimes d’une insuffisance médullaire, mais le rôle d’une suractivation de p53 dans l’initiation d’un SIMH chez l'Homme reste, à ce jour, controversé. En effet, une mutation inactivatrice de la ribonucléase PARN a été trouvée chez des patients atteints de DC. Il a été proposé que cette protéine déstabilise l’ARNm de p53 tout en favorisant la maturation de l’ARN de la télomérase, si bien que les conséquences d’une inactivation de PARN restent mal comprises. Au cours de ma thèse, j'ai également étudié de nouveaux modèles murins et établi des modèles cellulaires humains pour déterminer l’implication d’une activation de p53 dans l’initiation d’un SIMH chez l’homme. Les résultats en cours, particulièrement prometteurs, devraient notamment déterminer l’impact d’une déficience en Parn sur la voie de régulation de p53.En conclusion, nous avons montré qu’une activation soutenue de p53 conduit à des phénotypes principalement associés aux SIMH. Par ailleurs, nous avons montré que p53 réprime de nombreux gènes impliqués dans la maintenance du génome, ce qui peut sembler surprenant au regard du concept de « gardien du génome » communément attribué à p53, et nous a amené à rediscuter ce concept. Ainsi, les résultats obtenus pendant ma thèse devraient permettre de mieux comprendre les mécanismes impliqués dans l’insuffisance médullaire, la suppression tumorale et le vieillissement. / The p53 protein is mostly studied for its capacity to prevent the proliferation of cells with damaged genome. However, while studying p53Δ31/Δ31 mice that express a hyperactive p53 protein, our laboratory uncovered an unexpected role of p53 in the regulation of telomere metabolism. These mice model dyskeratosis congenita (DC), an inherited bone marrow failure syndrome (IBMFS) caused by defects in telomere maintenance. My PhD project combined analyses of mouse models and human cell lines to extend the study of this new function for p53 and to understand the role of the p53 pathway in the development of IBMFS.By extending our analysis of the p53Δ31 mouse model, we revealed that the p53Δ31/Δ31 cells exhibit decreased expression levels for 12 genes mutated in Fanconi anemia (FA), another IBMFS closely related to DC. Furthermore, these cells display a reduced capacity to repair DNA inter-strand crosslinks, a typical feature of FA cells. Our study suggests that a sustained activation of p53 might actually contribute to the clinical overlap between both syndromes. Importantly, we identified the mechanism used by p53 to downregulate telomere metabolism and the FA DNA repair pathway. This regulatory mechanism is indirect, via p21 and E2F4, and largely conserved between mice and humans. In fact, the p53Δ31/Δ31 cells constitute a powerful tool to find p53 target genes downregulated through this regulatory pathway. Accordingly, they were useful to show that two genes essential for centromere structure are also downregulated by p53.p53 activation is clearly involved in the final stages of bone marrow failure, but to-date, the role of p53 hyperactivation in the initiation of IBMFS remains controversial. Indeed, an inactivating mutation of the PARN gene, encoding a ribonuclease, was found in patients with DC. This protein was proposed to destabilize the p53 mRNA while promoting the maturation of the telomerase RNA, so that the consequences of PARN inactivation remain poorly understood. During my thesis, I also studied new mouse models and established human cellular models to determine the contribution of p53 activation in initiating IBMFS in humans. The current results, which are particularly promising, should notably determine the impact of Parn deficiency on the p53 regulatory pathway.In conclusion, we demonstrated that a sustained activation of p53 leads to phenotypes mainly associated with IBMFS. In addition, we showed that p53 suppresses many genes involved in genome maintenance, which may seem surprising in view of the concept of "guardian of the genome" commonly attributed to p53, leading us to revisit this concept. Hence, the results obtained during my thesis should help to better understand the mechanisms involved in bone marrow failure, tumor suppression and aging.