Le régulateur transcriptionnel BAP1 est une déubiquitinase nucléaire (DUB) dont le substrat est l’histone H2A modifiée par monoubiquitination au niveau des residus lysines 118 et 119 (K118/K119). Depuis les dernières années, BAP1 emerge comme un gene suppresseur de tumeur majeur. En effet, BAP1 est inactivé dans un plethore de maladies humaines héréditaires et sporadiques. Cependant, malgré l’accumulation significative des connaissances concernant l’occurrence, la pénétrance et l’impact des défauts de BAP1 sur le développement de cancers, ses mécanismes d’action et de régulation restent très peu compris. Cette étude est dédiée à la caractérisation moléculaire et fonctionnelle du complexe multi-protéique de BAP1 et se présente parmi les premiers travaux décrivant sa régulation par des modifications post-traductionnelles. D’abord, nous avons défini la composition du corps du complexe BAP1 ainsi que ses principaux partenaires d’interaction. Ensuite, nous nous sommes spécifiquement intéressés a investiguer d’avantage deux principaux aspects de la régulation de BAP1. Nous avons d’abord décrit l’inter-régulation entre deux composantes majeures du complexe BAP1, soit HCF-1 et OGT. D’une manière très intéressante, nous avons trouvé que le cofacteur HCF-1 est un important régulateur des niveaux protéiques d’OGT. En retour, OGT est requise pour la maturation protéolytique de HCF-1 en promouvant sa protéolyse par O-GlcNAcylation, un processus de régulation très important pour le bon fonctionnement de HCF-1. D’autre part, nous avons découvert un mécanisme unique de régulation de BAP1 médiée par l’ubiquitine ligase atypique UBE2O. en effet, UBE2O se caractérise par le fait qu’il s’agit aussi bien d’une ubiquitine conjuratrice et d’une ubiquitine ligase. UBE2O, multi-monoubiquitine BAP1 au niveau de son domaine NLS et promeut son exclusion du noyau, le séquestrant ainsi dans le cytoplasme. De façon importante, nos travaux ont permis de mettre de l’emphase sur le rôle de l’activité auto-catalytique de chacune de ces enzymes, soit l’activité d’auto-déubiquitination de BAP1 qui est requise pour la maintenance de sa localisation nucléaire ainsi que l’activité d’auto-ubiquitination d’UBE2O impliquée dans son transport nucléo-cytoplasmique. De manière significative, nous avons trouvé que des défauts au niveau de l’auto-déubiquitination de BAP1 due à des mutations associées à certains cancers indiquent l’importance d’une propre regulation de cette déubiquitinase pour les processus associés à la suppression de tumeurs. / BAP1 is a nuclear deubiquitinating enzyme (DUB) that acts as a transcription regulator and a DUB of nucleosomal histone H2AK119. In the recent years, it has become clear that BAP1 is a major tumor suppressor, inactivated in a plethora of hereditary and sporadic human malignancies. Although, we now accumulated a significant body of knowledge in respect to the occurrence, penetrance and impact of BAP1 disruption in cancer, its mechanism of action and regulation remained poorly defined. This work is dedicated to the biochemical and functional characterization of the BAP1 multiprotein complex and presents one of the first cases regarding its regulation by post-translational modifications. First, we defined the initial composition of the BAP1 complex and its main interacting components. Second, we specifically focused on two aspects of BAP1 regulation. We described the cross regulation between the two major components of the complex namely HCF-1 and OGT. We found that HCF-1 is important for the maintenance of the cellular levels of OGT. OGT, in turn, is required for the proper maturation of HCF-1 by promoting O-GlcNAcylation-mediated limited proteolysis of its precursor. Third, we discovered an intricate regulatory mechanism of BAP1 mediated by the atypical ubiquitin ligase UBE2O. UBE2O multi-monoubiquitinates BAP1 on its NLS and promotes its exclusion from the nucleus. Importantly, our work emphasises the role of the autocatalytic activity of both enzymes namely the auto-deubiquitination activity of BAP1, required for the maintenance of nuclear BAP1 and the auto-ubiquitination of UBE2O implicated in its nucleocytoplasmic transport. Significantly, we found that auto-deubiquitination of BAP1 is disrupted by cancer-associated mutations, indicating the involvement of this process in tumor suppression.

The ability of plants to sense and respond to environmental and endogenous signals is essential to their growth and development. As part of these diverse cellular functions, ubiquitin-mediated proteolysis has emerged to be an important process involved in how plant signalling pathways can be regulated in response to such cues. Of the three enzymes involved in linking ubiquitin to protein targets, E3 ubiquitin ligases are of interest as they confer substrate specificity during this ubiquitination process. The overall focal point of this research is on plant U-box (PUB) E3 ubiquitin ligases, a family that has undergone a large gene expansion possibly attributable to the regulation of biological processes unique to the plant life cycle. In Arabidopsis there are 64 predicted PUBs, many for which biological roles have yet to be determined. And as research continues to uncover PUB functions, the functional diversity in the gene family will likely expand. Specifically the focus of this research is on characterizing two ARM repeat-containing PUBs – AtPUB18 and AtPUB19. General analysis of pub18 and pub19 T-DNA insertion lines for growth defects did not yield distinct altered phenotypes. Closer inspection of selected lines showed independent gene assortment phenotypes that, with further inordinately convoluted pursuit, proved to have an AtPUB18/19-unrelated outcome. The availability of Arabidopsis microarray databases provided exploratory expression profiling as a starting point to elucidate PUB function. AtPUB19 and closely related AtPUB18 are notable for their increased expression during abiotic stresses. While condition-directed germination assays showed a decreased sensitivity to salt and ABA for pub18 pub19 double insertion lines, no related change in susceptibility to these or other abiotic stress treatments were seen with condition-directed root growth assays. Thus, this preliminary work has begun to reveal insight into the complex abiotic stress-related roles AtPUB18 and AtPUB19 have during mediation of environmental stress acclimation in Arabidopsis.

The ability of plants to sense and respond to environmental and endogenous signals is essential to their growth and development. As part of these diverse cellular functions, ubiquitin-mediated proteolysis has emerged to be an important process involved in how plant signalling pathways can be regulated in response to such cues. Of the three enzymes involved in linking ubiquitin to protein targets, E3 ubiquitin ligases are of interest as they confer substrate specificity during this ubiquitination process. The overall focal point of this research is on plant U-box (PUB) E3 ubiquitin ligases, a family that has undergone a large gene expansion possibly attributable to the regulation of biological processes unique to the plant life cycle. In Arabidopsis there are 64 predicted PUBs, many for which biological roles have yet to be determined. And as research continues to uncover PUB functions, the functional diversity in the gene family will likely expand. Specifically the focus of this research is on characterizing two ARM repeat-containing PUBs – AtPUB18 and AtPUB19. General analysis of pub18 and pub19 T-DNA insertion lines for growth defects did not yield distinct altered phenotypes. Closer inspection of selected lines showed independent gene assortment phenotypes that, with further inordinately convoluted pursuit, proved to have an AtPUB18/19-unrelated outcome. The availability of Arabidopsis microarray databases provided exploratory expression profiling as a starting point to elucidate PUB function. AtPUB19 and closely related AtPUB18 are notable for their increased expression during abiotic stresses. While condition-directed germination assays showed a decreased sensitivity to salt and ABA for pub18 pub19 double insertion lines, no related change in susceptibility to these or other abiotic stress treatments were seen with condition-directed root growth assays. Thus, this preliminary work has begun to reveal insight into the complex abiotic stress-related roles AtPUB18 and AtPUB19 have during mediation of environmental stress acclimation in Arabidopsis.

Germ-line mutations in the breast cancer susceptibility gene BRCA1 strongly increase the risk of developing breast and ovarian cancer in women. Different hypothesis have been proposed to explain this tissue specificity. One of the most argued hypothesis is the one that proposes a link between BRCA1 and ovarian hormones' action. Much data have been published in the last years pointing to an important role of progesterone receptor (PR) in inducing normal mammary development and also breast cancer formation. This study aimed to search for a functional relationship between BRCA1 and PR in breast cancer cells. We have found that BRCA1 inhibits the transcriptional activity of PR. We have investigated in more detail the mechanism of this effect. BRCA1 and PR interact in vivo in a ligand-independent fashion. Most importantly, BRCA1 alters the ligand-independent and dependent degradation of PR protein through its ubiquitination and this might have a direct effect on the level of PR recruitment on regulated promoters. BRCA1 is recruited to the hormone-responsive regions of PR-target genes and affects the presence of histone deacetylase activity and the level of monoubiquitinated histone H2A, linking BRCA1 action with chromatin status. These findings support a connection between BRCA1, the principal tumour suppressor responsible for familial breast cancer, and the progesterone receptor transcriptional activity. This relationship can be hypothesized to be reflected in the BRCA1-related breast tumourigenesis. / Mutaciones germinales en el gen breast cancer susceptibility gene BRCA1 aumentan altamente el riesgo de padecer cáncer de mama y ovario en mujeres. Se han propuesto diferentes hipótesis para explicar esta especificidad de tejido. Una de las hipótesis más argumentadas es la que propone una relación entre BRCA1 y la acción de las hormonas ováricas. En los últimos años se han publicado numerosos datos señalando al papel esencial del receptor de progesterona (PR) en la inducción del desarrollo normal de la mama y en la formación del cáncer de mama. Este estudio pretendía buscar una relación funcional entre BRCA1 y PR en células de cáncer de mama. Hemos demostrado que BRCA1 inhibe la actividad transcripcional de PR. Hemos investigado en más detalle el mecanismo de este efecto. BRCA1 y PR interaccionan in vivo de una manera independiente de ligando. Y lo que es más, BRCA1 altera la degradación independiente y dependiente de ligando de PR a través de su ubiquitinización y esto podría tener un efecto directo en el nivel de reclutamiento de PR en promotores regulados. BRCA1 es reclutado a las regiones de respuesta a hormona de genes diana de PR y afecta la presencia de actividad histona desacetilasa y el nivel de histona H2A monoubiquitinada, estableciendo un enlace entre la acción de BRCA1 y el estado de la cromatina. Estos hallazgos apoyan una conexión entre BRCA1, el principal supresor de tumor responsable del cáncer de mama hereditario, y la actividad transcripcional del receptor de progesterona. Se puede hipotetizar que esta relación se ve reflejada en el proceso de tumorigénesis BRCA1-dependiente.