<p>The centromere is essential for chromosome segregation and genome stability. It is the site of kinetochore assembly and chromosome attachment to the spindle microtubules, and it is important for chromosome movement during mitosis and meiosis. Normal human chromosomes have one centromere, but genome rearrangements that occur with instability, aging, and disease often result in chromosomes with two centromeres, called dicentrics. Nearly seventy-five years ago, Barbara McClintock demonstrated that dicentric chromosomes in plants are associated with instability through mitotic "breakage-fusion-bridge" cycles. However, human dicentrics are unusually stable due to the poorly understood phenomenon of centromere inactivation. Centromere inactivation has been primarily studied in patient-derived dicentrics, limiting the derivation of a molecular pathway. Key centromere and kinetochore proteins are not present at inactive centromeres, but beyond these observations, the process of centromere inactivation is unclear. Epigenetic and sequence-dependent factors are known to contribute to centromere specification, but requirements for centromere assembly, maintenance, and suppression remain obscure. The aims of this research were to (1) determine the mechanism(s) by which de novo dicentric chromosomes are stabilized, (2) ascertain the factors influencing the involvement of specific chromosomes in de novo fusions, and (3) establish the epigenomic, temporal, and mechanistic basis of centromere inactivation. To uncover the mechanistic foundations of these processes, we developed in vitro cell culture systems to study the formation and stabilization of de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. This finding is notable since the most prevalent rearrangement in humans involves the acrocentrics and is called Robertsonian translocation (ROB). In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the centromeric DNA array is reduced compared to the same array before dicentric formation. Many functional dicentrics persist for months after formation. Our results indicate that dicentric human chromosomes undergo alternative fates after formation across a broad temporal window. During transient telomere disruption, we observed a dramatic change in nucleolar appearance. Nucleolar proteins did not coalesce into condensed structures, but appeared dispersed throughout the nucleus. This surprising alteration in nucleolar organization and nuclear architecture suggests remodeling of the nucleolus and subsequent effects on nucleolar-associated chromosomes, such as the acrocentrics, could contribute to the high incidence of ROB formation. Further studies and development of additional cell culture systems will allow us to evaluate current models of centromere assembly and disassembly and the importance of chromatin organization to centromere function and genome architecture.</p> / Dissertation

L’érythropoïèse est le processus aboutissant à la production des hématies à partir d’une cellule souche hématopoïétique. La différenciation érythroïde implique des changements morphologiques en partie liés à la perte d’expression membranaire du récepteur à activité tyrosine kinase de classe III, c-Kit. En réponse à son ligand, le SCF, c-Kit est activé puis internalisé et dégradé par la voie du protéasome, via l’ubiquitine E3-ligase c-Cbl, ou par la voie lysosomale suite à une endocytose. Dans la première partie de ce travail, nous avons pu mettre en évidence qu’en absence de SCF et en réponse à un inhibiteur de tyrosine kinase, l’imatinib, les érythroblastes cultivés ex vivo perdent l’expression membranaire de c-Kit et accélèrent leur entrée en différenciation terminale. Au vu de ces observations, nous avons cherché à comprendre les mécanismes impliqués. Sur un modèle de cellules érytholeucémiques dépendantes de l’érythropoïétine, mais exprimant de manière endogène c-Kit, nous avons montré que l’imatinib induit une internalisation du récepteur ainsi que sa dégradation par la voie lysosomale et de manière indépendant de c-Cbl. De plus, nous avons montré que cet effet est réversible et que l’imatinib ne bloque pas la réexpression de c-Kit après son internalisation en réponse au SCF. Des marquages métaboliques ont permis de montrer que l’imatinib ne modifie ni la synthèse ni la maturation de c-Kit et que le profil phospho-tyrosine des cellules traitées à l’imatinib est globalement inchangé. Enfin, nous avons montré que la fixation de l’imatinib à la poche catalytique de c-Kit est indispensable à son internalisation, et par conséquent à sa dégradation. Il apparait donc que l’imatinib lève l’auto-inhibition de c-Kit, qui semble nécessaire pour son maintien à la membrane. Dans la seconde partie de ce travail, nous nous sommes intéressés aux changements morphologiques subis par les nucléoles, lieu de la biogenèse des ribosomes, au cours de différenciation des érythroblastes. L’étude de la taille et du potentiel prolifératif des cellules, ainsi que l’analyse morphologique des nucléoles, nous a permis de confirmer que la réduction de taille des cellules est contemporaine d’un ralentissement de leur prolifération ainsi que de la réduction du volume et de la surface du composé granulaire (CG), « matrice » du nucléole. En microscopie électronique, nous montrons la persistance des CG en fin de maturation. Enfin, nous avons également étudié l’évolution des nucléoles dans un contexte pathologique de syndromes myélodysplasiques de faible risque, qui se caractérisent par une hématopoïèse inefficace. Nous observons que les cellules pathologiques immatures ont des CG plus volumineux que les cellules normales immatures, et qu’au cours de la différenciation, la morphologie des nucléoles est identique entre les cellules normales et pathologiques. En conclusion, ce travail a permis de décrire 1) le mécanisme d’internalisation d’un récepteur à activité tyrosine kinase de classe III, c-Kit par l’imatinib et 2) la morphologie du nucléole au cours de la différenciation érythroïde normale et pathologique des syndromes myélodysplasiques de faible risque. / Erythropoiesis is the process leading to the production of red blood cells from hematopoietic stem cell. The erythroid differentiation involves morphological cell changes, in part related to the loss of membrane expression of the type III receptor tyrosine kinase, c-Kit. In response to its ligand SCF, c-Kit is activated, then internalized and degraded by the proteasome pathway via the E3 ubiquitin ligase c-Cbl, or by the lysosomal pathway, after endocytosis. In the first part of this work, we demonstrated that in the absence of SCF and in response to tyrosine kinase inhibitor, imatinib, erythroblasts cultured ex vivo, lose membrane expression of c-Kit and accelerate their terminal differentiation. In view of these observations, we sought to understand the mechanisms involved. On an erythropoietin dependent cell line expressing c-Kit at the membrane, we showed that imatinib induces receptor internalization and degradation by the lysosomal pathway, independently of c -Cbl. Furthermore, we showed that this effect is reversible and that imatinib does not block the c-Kit re-expression after its internalization, in response to SCF. Metabolic labelling showed that imatinib does not alter synthesis or maturation of c -Kit and that the phospho-tyrosine profile of cells treated with imatinib is generally unchanged. Finally, we showed that the binding of imatinib to the catalytic pocket of c-Kit is essential for its internalization, and therefore its degradation. So, it appears that imatinib removes c-Kit self-inhibition, which seems necessary to its retention at the membrane. In the second part of this work, we studied the morphological changes of nucleoli, the site of ribosome biogenesis, during erythroid differentiation. We showed that the reduction of cell size takes place at the same time than reduction of cell proliferation and reduction of surface and volume of the Granular Compound (GC), the “matrix” of the nucleolus. Moreover, we showed by electronic microscopy, the persistence of GC at the end of maturation. Finally, we also studied the evolution of nucleoli in a pathological context of low risk myelodysplastic syndromes, which are characterized by ineffective hematopoiesis. We observed that immature pathological cells have larger GC than immature normal cells, but that during differentiation, the morphology of nucleoli is identical between normal and pathological cells. In conclusion, this work has allowed us to describe 1) the mechanisms of internalization of a class III receptor tyrosine kinase, c-Kit by imatinib and 2) the morphology of the nucleolus during normal and pathological low risk myelodysplastic syndromes of erythroid differentiation.

Philosophiae Doctor - PhD / Histological observations revealed that oral epithelium originated from a single ectodermal layer. As the ectoderm grew so it differentiated into squamous epithelium. The first features of squamous differentiation were noticed at 8 weeks in utero in areas where keratinized mucosae were developing, and these were the changing of cuboidal to cylindrical basal cells and the subsequent growth of prickle cells from these cylindrical basal cells. The prickle cells merged with the existing primitive cells and at no stage could a separate squamous epithelial layer I such as the stratum tritermedium of the epidermis I be observed inside the mouth. At 12 weeks in utero squamous differentiation had reached a stage where acidophilic layers appeared in certain regions on the epithelial layer. The time of appearance of these layers varied from case to case. At this stage most of the primitive characteristics had disappeared from the keratinizing epithelium. Unlike the periderm of the skin which was shed into the amniotic fluid, shedding of primitive epithelial cells from the keratinizing squamous epithelium was not noticeable. Thence, the growth of keratinizing epithelium was followed by an increase of acidophilic layers, the appearance of keratohyaline granules in cells and, in some instances, full keratinization. The latter I however I was almost exclusively confined to the vermilion border of the lip. The squamous epithelium of the lining mucosa, which is unkeratinized I developed at a much slower tempo. It retained its cuboidal-shaped basal cells and the primitive features of the overlying cells were lost only at about 4- 5 months in utero I when squamous differentiation set in. At no stage was the squamous differentiation a prominent feature. At junctions between keratinized and unkeratinized epithelia and epidermis the epithelium exhibited features of both types of epithelia that were being joined. This was especially noticeable at the junction between vermilion epithelium and epidermis, where part of the vermilion epithelium displayed a prominent intermediate type of layer. Similarly, acidophilic layers of keratinizing epithelium merged imperceptibly with the walls of cells of unkeratinizing epithelium, creating a small region of an unkeratinizing type of epithelium with keratinized cells. Thus the development of the oral epithelium is through differentiation and renewal of epithelial cells: the ectodermal layer developes into an epithelial layer which is recognised by its squamous appearance. The subsequent growth is by constant renewal of this differentiated epithelium. The pattern of epithelial development I the appearance of the junctional epithelia and the manner in which acidophilic layers merge with unkeratinized epithelial cells I indicate a unity between these epithelia. According to these developmental features, the epithelium of the mouth and epidermis can be classified into less differentiated and better differentiated, but with a commonbackground for these epithelia. When the formation and the established appearance of keratin in the mouth and on the skin was compared histologically I ultrastructurally and histochemically I a unity between these features became apparent. Ultrastructurally it appeared that keratin consisted basically of 2 cytoplasmic constituents: tonofilaments and a fine granular substance. The tonofilaments were gathered at first into bundles and then broken up into finer tonofibrils. These finer fibrils mixed with a granular ground substance to form a homogenous granular filamentous material. This product can be regarded as a pre-keratin. With the addition of a keratohyaline layer to the process I keratin was formed, Apart from the keratohyaline granules several additional changes took place in cells concerned in this process I whether keratin was formed or not. These changes were flattening of cells, extensive interdigitation between cell walls, disappearance of micro-villi I loss of structure in desmosomes I thickening of cell walls and the disappearance of glycogen from cells. Some of these features were displayed in each of the types of epithelium examined here.

Le premier membre de la famille des rétrovirus humains HTLV (Virus T-lymphotropique Humain), HTLV-1, a été découvert en 1980 et l’on estime aujourd’hui à plus de 10 millions le nombre d’individus infectés à travers le monde. Après une période de latence d’environ 40 ans, 5% des individus infectés développent des leucémies, des lymphomes adultes de lymphocytes T (ATLL) ou encore une myélopathie associée à HTLV-1/ paraparésie spastique tropicale (HAM/TSP). L’apparition de la maladie serait en grande partie orchestrée par deux protéines virales, soit Tax et HTLV-1 bZIP factor (HBZ). L’expression du génome viral se fait à partir d’un transcrit sens de pleine longueur suite à un épissage alternatif, à l’exception du gène HBZ. HBZ est produite à partir d’un transcrit antisens initié dans la séquence terminale longue répétée (LTR)’3. Elle a été décrite comme étant capable de réguler négativement la transcription virale dépendante de Tax en se dimérisant avec des facteurs de transcription cellulaires tels que CREB-2 et certains membres de la famille Jun. HBZ a aussi un pouvoir prolifératif et bien que nous ne sachions toujours pas le mécanisme moléculaire menant à l’oncogenèse par HBZ, nous savons qu’elle module une multitude de voies de transduction de signaux, dont AP-1. Nous avons récemment mis en évidence un transcrit antisens nommé Antisense Protein of HTLV-2 (APH-2) chez HTLV-2 qui n’est associé qu’à une myélopathie apparentée au HAM/TSP. Ce n’est qu’en 2005 que HTLV-3 et HTLV-4 se sont rajoutés au groupe HTLV. Cependant, aucune corrélation avec le développement d’une quelconque maladie n’a été montrée jusqu’à ce jour. Le premier volet de ce projet de doctorat avait pour objectif de détecter et caractériser les transcrits antisens produits par HTLV-3 et HTLV-4 et d’étudier les protéines traduites à partir de ces transcrits pour ainsi évaluer leurs similitudes et/ou différences avec HBZ et APH-2. Nos études de localisation cellulaire réalisées par microscopie confocale ont montré que APH-3 et APH-4 sont des protéines nucléaires, se retrouvant sous la forme de granules et, dans le cas d’APH-3, partiellement cytoplasmique. Ces granules co-localisent en partie avec HBZ. Les analyses à l’aide d’un gène rapporteur luciférase contenant le LTR 5’ de HTLV-1 ont montré que APH-3 et APH-4 peuvent aussi inhiber la transactivation du LTR 5’ par Tax. Aussi, des études faisant appel au gène rapporteur précédé d’un promoteur de collagénase (site AP-1), ont montré que ces deux protéines, contrairement à HBZ, activent la transcription dépendante de tous les membres des facteurs de transcription de la famille Jun. De plus, les mutants ont montré que le motif fermeture éclair (LZ) atypique de ces protéines est impliqué dans cette régulation. En effet, APH-3 et APH-4 modulent la voie Jun-dépendante en se dimérisant via leur LZ atypique avec la famille Jun et semblent activer la voie par un mécanisme ne faisant pas par d’un domaine activateur autonome. Dans un deuxième volet, nous avions comme objectif d’approfondir nos connaissances sur la localisation nucléolaire de HBZ. Lors de nos analyses, nous avons identifié deux nouveaux partenaires d’interaction, B23 et la nucléoline, qui semblent être associés à sa localisation nucléolaire. En effet, ces interactions sont plus fortes suivant une délétion des domaines AD et bZIP de HBZ qui dans ce cas est localisée strictement au nucléole. De plus, bien que APH-3 et APH-4 puissent se localiser aux nucléoles, HBZ est la seule protéine traduite à partir d’un transcrit antisens pouvant interagir avec B23. Finalement, ces travaux ont clairement mis en évidence que HTLV-3 et HTLV-4 permettent la production de transcrits antisens comme chez d’autres rétrovirus. Les protéines traduites à partir de ces transcrits antisens jouent d’importants rôles dans la réplication rétrovirale mais semblent avoir des fonctions différentes de celles de HBZ au niveau de la régulation de la transcription de la voie Jun. HBZ semble aussi jouer un rôle unique dans le nucléole en ciblant les protéines nucléolaires de la cellule. Ces études démontrent que les protéines produites à partir de transcrits antisens chez les rétrovirus HTLV partagent plusieurs ressemblances, mais démontrent aussi des différences. Ainsi, les APH pourraient, en tant qu’outil comparatif, aider à mieux cibler les mécanismes moléculaires importants utilisés par HBZ pour induire la pathogénèse associée à une infection par HTLV. / The first human T-cell lymphotropic virus (HTLV) family member was discovered in 1980 and it is estimated that approximately 10 million people are infected with HTLV-1 worldwide. After about 40 years, 5% of infected individuals will develop an adult T-cell leukemia/lymphoma (ATLL) while another 4% will develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is believed that two viral proteins, Tax and HBZ, together orchestrate the oncogenic process. The viral proteins are expressed from an alternatively spliced sense transcript except for the HBZ gene. HBZ is translated from an antisense transcript initiated in the long terminal repeat (LTR)’3. This viral protein is capable of inhibiting Tax transactivation of the LTR5’ by dimerizing with cellular transcription factors such as CREB-2 and c-Jun. HBZ also has proliferating capacities and while the molecular mechanisms leading to the disease still need to be elucidated, it is well known that HBZ can modulate a multitude of signal transduction pathways like AP-1. We have recently discovered an antisense transcript termed Antisense Protein of HTLV-2 (APH-2) produced in HTLV-2. HTLV-2 is only associated to myelopathies resembling HAM/TSP. HTLV-3 and HTLV-4 were discovered in 2005 and have not been associated with any type of disease thus far. The first goal of this PhD project was hence to detect and characterize the antisense transcripts produced in HTLV-3 and HTLV-4, to study the functions of these translated proteins and to evaluate their similarities and/or differences shared with HBZ and APH-2. Our localization studies using confocal microscopy demonstrated that APH-3 and APH-4 are found in the nucleus as speckles, and for APH-3, also partially cytoplasmic. These two proteins can also partially colocalize with HBZ. Using a luciferase reporter plasmid bearing the HTLV-1 LTR5’, we demonstrated that APH-3 and APH-4 could inhibit Tax transactivation of the LTR5’. We also used a luciferase reporter plasmid bearing the collagenase promoter, which bears an AP-1 site, and demonstrated that both viral proteins could activate transcription in the presence of any of the Jun family of transcription factors. We generated several mutants and the atypical leucine zipper (LZ) found in APH-3 and APH-4 is crucial for this regulation. In fact, APH-3 and APH-4 using their atypical LZ dimerize with Jun family members and activate this pathway using a mechanism other than an autonomous activation domain. Our next goal was to investigate the significance of the HBZ nucleolar localization. During this project, we identified two new interacting partners, B23 and nucleolin, which seem to be associated with its nucleolar localization. In fact, these interactions are stronger when HBZ is deleted of its AD and bZIP domains and hence when HBZ demonstrates a stronger nucleolar distribution. Moreover, while APH-3 and APH-4 are also found in the nucleolus, HBZ is the only antisense protein able to interact with B23. Finally, this work clearly demonstrates that HTLV-3 and HTLV-4 can produce an antisense transcript alike other retroviruses. The encoded proteins play an important role in retroviral replication and seem to regulate Jun-dependant transcription differently than HBZ. HBZ also seems to have a unique role in the nucleoli by targeting specific cellular nucleolar proteins. Similarities but also differences are shared between the antisense proteins. Thus, the APH proteins represent a good comparative tool in order to better understand the molecular mechanisms involved in HTLV induced diseases.

En dégradant la majorité des protéines cellulaires, le protéasome se positionne comme un régulateur clé du protéome, vis-à-vis duquel la plupart des tumeurs présentent une forte addiction en raison du débalancement protéique qui les caractérise. Quoique son inhibition se soit avérée être une bonne stratégie anticancer, elle est demeurée limitée aux cancers sanguins. Malheureusement, leur traitement devient tôt ou tard compromis par la résistance cellulaire. Raison pour laquelle l'élucidation du mécanisme de mort en jeu pourrait permettre de mieux cerner cette résistance, ce qui constituerait les fondements pour un traitement plus efficace. L'un des événements les plus spectaculaires et les plus précoces à se manifester dans le cadre de ce traitement, est la déubiquitination massive de l'histone H2A sur la lysine (K) 119. Une corrélation positive plutôt paradoxale entre cet événement, qui est associé à l'expression génique, et la sensibilité cellulaire à l'inhibition du protéasome, a été remarquée. Cela a mené à s'intéresser à sa signification biologique. Des cellules cancéreuses et primaires ont servi de systèmes d'étude protéomique par immunobuvardage et par immunofluorescence, pour analyser l'état de la chromatine et la distribution spatio-temporelle des histones durant l'inhibition du protéasome. Des inhibiteurs chimiques, des ARN interférents et des vecteurs d'expression ont été utilisés à cette fin. Un impressionnant phénomène survenant à la suite de l'inhibition du protéasome a été révélé. En effet, une baisse drastique du niveau d'histones sur la chromatine s'opère simultanément à la déubiquitination de H2A-ub (K119). Le protéasome étant inhibé, celles-ci, et possiblement les histones synthétisées en phase S, subiraient une translocation irréversible dans les nucléoles, et ce avant le déclenchement de l'apoptose. Ce phénomène est reproduit par divers inhibiteurs du protéasome et par siRNA, et il survient autant dans des cellules cancéreuses que primaires, mais pas dans les cellules résistantes, qui ne démontrent d'ailleurs pas de déubiquitination de H2A-ub (K119). Par ailleurs, il a été montré que la surexpression d'histones exogènes mène à leur translocation nucléolaire, et que la combinaison de l'inhibition du protéasome à cette surexpression pourrait être léthale. Quoique majoritairement préliminaires, les résultats révèleraient un surprenant mécanisme de régulation post-traductionnelle des histones endogènes, qui seraient séquestrées dans les nucléoles lorsqu'elles ne sont pas incorporées à la chromatine. En effet, l'inhibition du protéasome occasionne une importante perturbation de la chromatine pendant plusieurs heures. En raison de la cytotoxicité intrinsèque des histones libres et de leur abondance dans les cellules, celles-ci pourraient bien être à l'origine de la mort induite par l'inhibition du protéasome. Enfin, en sa qualité de senseur majeur de stress, le nucléole pourrait bien être le point de départ de la signalisation menant à la mort. / By degrading most of cellular proteins, the proteasome is positioned as a key regulator of the proteome, against which most tumors have a strong addiction, due to the protein imbalance that characterizes them. Although its inhibition has been shown to be a good anticancer strategy, it is still limited to blood cancers. Unfortunately, their treatment sooner or later becomes compromised by cellular resistance. This is why the elucidation of the mechanism of death involved could allow a better understanding of this resistance, which would in turn constitute the basis for a more effective treatment One of the most spectacular and early events to manifest during this treatment is the massive deubiquitylation of histone H2A on lysine (K) 119. A rather paradoxical positive correlation between this event, which is associated with gene expression, and cellular sensitivity to proteasome inhibition, has been noticed. This led to an interest in its biological significance. Cancer and primary cells have been used as systems for proteomic study by immunoblot and immunofluorescence, to analyze chromatin status and the spatio-temporal distribution of histones during proteasome inhibition. Chemical inhibitors, interfering RNAs and expression vectors have been used for this purpose. An impressive phenomenon occurring during the proteasome inhibition has been revealed. Indeed, a drastic drop in histones level on chromatin occurs simultaneously with the deubiquitylation of H2A-ub (K119). The proteasome being inhibited, these, and possibly histones synthesized in S phase, would undergo an irreversible translocation in the nucleoli before the onset of apoptosis. This phenomenon is replicated by various proteasome inhibitors and siRNA, and occurs in both cancer and primary cells, but not in resistant cells, which do not demonstrate deubiquitination of H2A-ub (K119). Furthermore, overexpression of exogenous histones has been shown to lead to their nucleolar translocation, and it is thought that the combination of proteasome inhibition with this overexpression could be lethal. Although mostly preliminary, the results would reveal a surprising mechanism of post-translational regulation of endogenous histones, which would be sequestered in nucleoli when not incorporated into chromatin. Indeed, inhibition of the proteasome causes a significant disruption of the chromatin for several hours. Due to the intrinsic cytotoxicity of free histones and to their great cellular abundance, these may well be the cause of the death induced by proteasome inhibition. Finally, as a major stress sensor, the nucleolus could be the starting point of the death signaling.