Les corps nucléaires PML (CNs) sont des structures sphériques organisées par la protéine PML où, de nombreuses protéines impliquées dans divers processus biologiques tels que la sénescence, la mort cellulaire ou la défense virale, y sont recrutées. Ces protéines ont été identifiées individuellement dans des études spécifiques mais aucune étude n’avait permis la purification des CNs et l’établissement de liste des partenaires de PML de façon systématique. La protéine PML est sensible à l’oxydation et le stress oxydant promeut son assemblage en CNs permettant le recrutement de l’enzyme E2 clé de la conjugaison, UBC9. Les interférons (IFNs), cytokines aux propriétés antivirales induisent l’expression de PML. L’arsenic et les IFNs augmentent l’assemblage des CNs.Dans cette étude nous avons exploré, in vivo, le rôle de PML dans la sumoylation en réponse au stress en utilisant deux modèles de souris et un modèle cellulaire, les cellules souches embryonnaires de souris (mESC), exprimant des versions taguées de SUMO1 ou SUMO2. Nous avons montré que PML augmente rapidement la sumoylation dans les foies de souris KI His6-HA-SUMO1 en réponse aux traitements arsenic/IFN. De façon similaire, dans la leucémie aiguë promyélocytaire (LAP), où les CNs sont désorganisés, l’arsenic promeut la reformation des CNs et la conjugaison par SUMO. L’analyse par spectrométrie de masse quantitative des conjugués His10-SUMO2 a permis de mettre en évidence une liste de protéines sumoylées en réponse au traitement thérapeutique à l’arsenic. Parmi ces protéines, TRIM28/KAP1 et d’autres protéines appartenant au même complexe régulant la chromatine ont été identifiées. TRIM28, qui joue un rôle clé dans la répression des rétrovirus endogènes (ERV) dans les mESC, est sumoylée de façon dépendante de PML dans ces cellules. Les CNs régulent également l’ubiquitinylation et la dégradation dépendante du protéasome, de certaines de ces protéines. Enfin, nous avons montré que les CNs favorisent la formation de chaînes SUMO2/3, expliquant le rôle des CNs dans la dégradation des protéines. Cette étude nous a permis de mettre en évidence que les CNs sont des compartiments nucléaires qui contrôlent la conjugaison de diverses protéines dans des conditions physiologiques de stress cellulaire, montrant pour la première fois leur activité de contrôle de MPT in vivo. / PML drives assembly of PMLNuclear Bodies (NBs) where it recruits hundreds of serendipitously-identified proteins, among which the key UBC9 E2-sumoylation enzyme. Interferons (through transcriptional PML induction) and arsenic or oxidative stress (through PML aggregation) dramatically enhance NB assembly. Here we directly investigated any role for PML in stress-responsive sumoylation in vivo. We demonstrate that PML very rapidly promotes arsenic/interferon-responsive sumoylation in mouse liver or mouse embryonic stem cells. Similarly, in Acute Promyelocytic Leukemia (APL), where PML NBs are disorganized in the basal state, arsenic therapy promoted NB-reformation and broad SUMO-conjugation. Label free quantitative proteomic analysis of His10-SUMO2-conjugates revealed a comprehensive list of therapy-responsive sumoylated proteins, among which TRIM28 and other proteins belonging to the same epigenetic complex. PML NBs-regulated sumoylation also drives ubiquitination and proteasome-dependent degradation of some targets. Finally, by expressing conjugation-resistant SUMO2, we demonstrate that PML NBs promotes processive SUMO2/3 chain elongation, thus explaining PML role in partner degradation. Collectively, our findings highlight the key activity of NBs in stress-regulated sumoylation/degradation in vivo.

Epstein-Barr virus (EBV) latent infection is associated with lymphoid and epithelial tumours, including nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). Since EBNA1 protein is expressed in all EBV tumours, I explored whether EBNA1 alters the cellular environment in ways that would contribute to the development of these epithelial tumours. I have shown that EBNA1 disrupts nuclear bodies (NBs) formed by the PML tumor suppressor and degrades PML proteins in a proteasome dependent manner in NPC and GC cell lines. I have verified the role of EBNA1 in disrupting PML NBs through overexpression and silencing of EBNA1 and shown that EBNA1 alone is sufficient to mediate these effects. Using EBNA1 mutants I found that USP7 and protein kinase CK2 (two enzymes that negatively regulate PML NBs) are important for EBNA1-mediated disruption of PML NBs. Furthermore, I have shown that EBNA1 localizes to PML NBs, and interacts with PML IV, which mediates the enrichment of USP7 and CK2β with PML NBs and increases CK2 phosphorylation of PML proteins, a known prerequisite for PML degradation. Consequently, functions downstream of PML were impaired in the presence of EBNA1. In particular, cells expressing EBNA1 had decreased levels of p53acetylation, p21 and apoptosis in response to DNA damage. Furthermore, DNA repair was markedly impaired in these cells, despite the fact that they survived better after induction of DNA damage than cells lacking EBNA1. In keeping with these observations, immunohistochemistry staining of GC biopsies showed that EBV-positive GC biopsies had lower PML staining compared to EBV-negative samples. These results show that EBNA1 directly affects host cell processes that would be expected to promote malignant transformation. Additionally, I have shown that EBNA1's ability to disrupt PML NBs is important for reactivation of EBV from latency; hence, is required for efficient spread of EBV from host to host.

Epstein-Barr virus (EBV) latent infection is associated with lymphoid and epithelial tumours, including nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). Since EBNA1 protein is expressed in all EBV tumours, I explored whether EBNA1 alters the cellular environment in ways that would contribute to the development of these epithelial tumours. I have shown that EBNA1 disrupts nuclear bodies (NBs) formed by the PML tumor suppressor and degrades PML proteins in a proteasome dependent manner in NPC and GC cell lines. I have verified the role of EBNA1 in disrupting PML NBs through overexpression and silencing of EBNA1 and shown that EBNA1 alone is sufficient to mediate these effects. Using EBNA1 mutants I found that USP7 and protein kinase CK2 (two enzymes that negatively regulate PML NBs) are important for EBNA1-mediated disruption of PML NBs. Furthermore, I have shown that EBNA1 localizes to PML NBs, and interacts with PML IV, which mediates the enrichment of USP7 and CK2β with PML NBs and increases CK2 phosphorylation of PML proteins, a known prerequisite for PML degradation. Consequently, functions downstream of PML were impaired in the presence of EBNA1. In particular, cells expressing EBNA1 had decreased levels of p53acetylation, p21 and apoptosis in response to DNA damage. Furthermore, DNA repair was markedly impaired in these cells, despite the fact that they survived better after induction of DNA damage than cells lacking EBNA1. In keeping with these observations, immunohistochemistry staining of GC biopsies showed that EBV-positive GC biopsies had lower PML staining compared to EBV-negative samples. These results show that EBNA1 directly affects host cell processes that would be expected to promote malignant transformation. Additionally, I have shown that EBNA1's ability to disrupt PML NBs is important for reactivation of EBV from latency; hence, is required for efficient spread of EBV from host to host.

Loss of the promyelocytic leukemia (PML) protein is associated with genomic instability/cancer. There are several isoforms of the PML protein that localize in PML nuclear bodies (PML NBs). How each individual isoform contributes to the functions of PML NBs is unknown. The objective of this study was to identify and characterize PML isoform-I (PML-I) specific protein-protein interactions. Using yeast two-hybrid screens, several interacting partners of PML-I were identified that play roles in translational regulation, including eukaryotic initiation factor 3 subunit K (eIF3K). Our studies demonstrated that eIF3K interacts with PML-I in vitro and in vivo. Through its interaction with eIF3K, overexpression of PML-I resulted in the concomitant increase in eIF3K protein levels in mammalian cells. This suggests that PML-I may be involved in regulating eIF3K protein translation or stability, which in turn could affect translation of specific mRNAs or global translation in cancer cells with reduced expression of PML-I.

Us3, a serine/threonine kinase encoded by all alphaherpesviruses, plays diverse roles during virus infection. Recently, work done in our laboratory determined that Us3 orthologues from herpes simplex type 2 (HSV-2) and pseudorabies virus (PRV) are capable of disrupting promyelocytic leukaemia (PML) protein nuclear bodies (-NBs). PML-NBs are discrete, dynamic nuclear bodies named for PML, their essential structural component and one that plays a key role in diverse cellular processes, including transcriptional regulation, apoptosis, and cellular antiviral defense. In infected cells, PML-NBs exert transcriptional silencing on the viral genome to prevent viral gene expression and virus replication. Based on this finding, my studies were aimed to understand the mechanism and physiological function of Us3-mediated PML-NB disruption. The degradation of one or more cellular proteins seems necessary for this Us3 activity, as the proteasome inhibitor, MG132, dramatically reduced Us3-mediated PML-NB disruption. The target of this proteasome activity is not likely PML protein, as Us3 expression did not lead to detectable PML protein degradation. Nonetheless, the involvement of proteasome activity suggests that Us3 may utilize the host ubiquitylation pathway to disrupt PML-NBs. Supporting this hypothesis, PRV and HSV-2 Us3 orthologues were shown to interact with KLHL21, a substrate adaptor protein for cullin-3 ubiquitin ligase. PRV and HSV-2 Us3 were re-localized to PML-NBs when co-expressed with KLHL21, and knock-down of KLHL21 prevented Us3-mediated PML-NB disruption. Taken together, these findings suggest that Us3-KLHL21 complex recruits the cullin-3 ubiquitin ligase to PML-NBs, where subsequent ubiquitylation of unknown target(s) leads to PML-NB disassembly. Since it is well established that PML is an important antiviral effector induced by interferon (IFN), Us3 may contribute to viral resistance to IFN by disrupting PML-NBs. Favoring this hypothesis, virus yield and viral gene transcription were dramatically reduced in IFN-exposed cells in the absence of Us3. These reductions were associated with an increased number of PML-NBs in the absence of Us3, and were partially recovered in cells knocked down for PML. Therefore, by disrupting PML-NBs, Us3 may alleviate IFN-induced, host-mediated transcriptional silencing of the viral genome, allowing efficient viral gene transcription and replication in cells exposed to IFN. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2014-04-28 16:36:54.079

The promyelocytic leukemia protein (PML) is a tumour suppressor. It has been reported that PML interaction with the p53 protein is involved in the activation of cell cycle checkpoints and, when persistent, may lead to the premature onset of cellular senescence. Cellular senescence is a state of permanent cell growth arrest that is associated with characteristic morphological and metabolic changes and persistent DNA damage signalling. Importantly, PML nuclear bodies coassociate with persistent DNA damage foci in senescent cells; however, the role of this interaction is still obscure. My goal was to characterize the role of PML in DNA damage response (DDR) and the induction of premature cellular senescence after genotoxic stress, namely X-radiation, using both siRNA-mediated PML knock down (PML KD) and complete PML knock out (PML KO) in human cells. The dynamics of DNA damage foci, levels of various proteins involved in DDR, and proliferation rate were measured in both PML KD and KO cells. No significant changes in the formation of DNA damage foci, activated DDR (p53 and Chk2), activated p21CIP1/WAF1 cyclin-dependent kinase inhibitor, senescent morphology, and SA-β-galactosidase activity in PML KO cells were observed. However, PML KO cells displayed higher levels of retinoblastoma protein (Rb) and...

Le rôle fonctionnel des transcrits de l’hétérochromatine péricentromérique reste à ce jour largement incompris chez les eucaryotes supérieurs. Néanmoins, il a été montré que ces transcrits sont soumis à un contrôle très précis, fonction du cycle cellulaire. La régulation de la transcription est fortement contrôlée par la structure de la chromatine qui peut être modifiée localement en changeant la composition biochimique du nucléosome, notamment par l’utilisation des variantes d’histones. L’objectif de ma thèse a été de mieux comprendre le rôle de la protéine chaperonne d’histone DAXX et de sa variante d’histone H3.3 dans la régulation de la transcription des séquences répétées péricentromériques. Par la méthode de purification TAP-TAG, les partenaires spécifiques de DAXX ont été identifiés à partir d’extraits solubles nucléaires de fibroblastes embryonnaires murins. Ces analyses ont mis en évidence que CAF-1, classiquement associé à H3.1, et les facteurs de remodelage de la chromatine ATRX et CHD4 interagissent spécifiquement avec DAXX. Le rôle de ces protéines dans le contrôle de la transcription de l’hétérochromatine péricentromérique a ensuite été mis en évidence par une approche combinant l’interférence ARN et la Q-PCR. Enfin, les résultats suggèrent fortement que ces mécanismes de régulation ont lieu au niveau des corps nucléaires PML. L’ensemble de ces données montre qu’il existe une régulation spatio-temporel très fine de la structure de la chromatine régulant la transcription de l’hétérochromatine péricentromérique. / The functional role of pericentromeric heterochromatin transcripts remains largely unknown in higher eukaryotes. Nevertheless, it has been shown that these transcripts are subject to very precise control, depending on the cell cycle. Regulation of transcription is tightly controlled by chromatin structure that can be modified locally by changing the biochemical composition of the nucleosome, including the use of histone variants. The aim of my thesis was to better understand the role of the histone chaperone protein DAXX and its histone variant H3.3 in the regulation of transcription of pericentromeric repeats. By the method of TAP-TAG purification, DAXX specific partners were identified from soluble nuclear extracts of murine embryonic fibroblasts. These analyzes revealed that CAF-1, classically associated with H3.1, and the chromatin remodeling factors, ATRX and CHD4, specifically interact with DAXX. The role of these proteins in the control of transcription of pericentromeric heterochromatin was then highlighted by an approach combining RNAi and Q-PCR. Finally, the results strongly suggest that these regulatory mechanisms take place at PML nuclear bodies. Taken together, these data show that there is a spatio-temporal regulation of the fine structure of chromatin regulates transcription of pericentromeric heterochromatin.

No description available.