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.

The exosome, an evolutionarily conserved protein complex with exoribonucleolytic activity, is one of the key players in mRNA quality control. Little is known about the functions of the exosome in metazoans. We have studied the role of the exosome in nuclear mRNA surveillance using Chironomus tentans and Drosophila melanogaster as model systems. Studies of the exosome subunits Rrp4 and Rrp6 revealed that both proteins are associated with transcribed genes and nascent pre-mRNPs in C. tentans. We have shown that several exosome subunits interact in vivo with the mRNA-binding protein Hrp59/hnRNP M, and that depleting Hrp59 in D. melanogaster S2 cells by RNAi leads to reduced levels of Rrp4 at the transcription sites. Our results on Rrp4 suggest a model for cotranscriptional quality control in which the exosome is constantly recruited to nascent mRNAs through interactions with specific hnRNP proteins. Moreover, we show that Rrp6 interacts with mRNPs in transit from the gene to the nuclear pore complex, where it is released during early stages of nucleo-cytoplasmic translocation. Furthermore, we show that Rrp6 is enriched in discrete nuclear bodies in the salivary glands of C. tentans and D. melanogaster. In C. tentans, the Rrp6-rich nuclear bodies colocalize with SUMO. We have also constructed D. melanogaster S2 cells expressing human b-globin genes, with either wild type of mutated splice sites, and we have studied the mechanisms by which the cells react to pre-mRNA processing defects. Our results indicate that two surveillance responses operate co-transcriptionally in S2 cells. One requires Rrp6 and retains defective mRNAs at the transcription site. The other one reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. These observations support the view that multiple mechanisms contribute to co-transcriptional surveillance in insects. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

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

PML et les corps nucléaires (CN) sont impliqués dans la défense antivirale. En effet, notre équipe a montré que la surexpression de PMLIII confère la résistance au virus de la stomatite vésiculaire, au virus de l'influenza, au virus foamy mais pas au virus de l’encéphalomyocardite (EMCV). J’ai montré dans mon travail de thèse que l’EMCV contrecarre le pouvoir antiviral de PMLIII en induisant sa dégradation par un processus dépendant du protéasome et de SUMO. Cependant, les cellules de souris invalidées pour PML sont plus sensibles à l’infection par l’EMCV que les cellules issues de souris parentales. Pour déterminer l’isoforme de PML responsable de cet effet antiviral, j’ai analysé l’effet des sept isoformes de PML (PMLI-VII) et j’ai montré que seule l’expression en stable de PMLIV confère la résistance à l’EMCV en séquestrant la polymérase virale 3Dpol au sein des CN PML. De plus la déplétion de PMLIV augmente la production de l’EMCV dans les cellules traitées par l’interféron. Ces données indiquent le mécanisme par lequel PML confère la résistance à l’EMCV et révèlent que PML est l’une des protéines médiatrices des effets anti-EMCV de l’interféron. / PML and nuclear bodies (NBs) are implicated in antiviral defense. Indeed, our team showed that overexpression of PMLIII confers resistance to vesicular stomatitis virus, influenza virus, foamy virus but not to encephalomyocarditis virus (EMCV). I have shown during my thesis that EMCV counteracts the antiviral effect of PMLIII by inducing its degradation in SUMO and proteasome-dependent way. However, cells derived from PML knockout mice are more susceptible to EMCV infection than wild-type cells. To determine the isoforme of PML implicated in this antiviral effect, I analysed the effect of the seven PML isoforms (PMLI-PMLVII) and I showed that only stable expression of PMLIV confers resistance to EMCV by sequestring the viral polymérase 3Dpol in PML Nbs. In addition, depletion of PMLIV boosted EMCV production in interferon-treated cells. These finding sindicate the mechanism by which PML confers resistance to EMCV and reveal a new pathway mediating the antiviral activity of interferon against EMCV.

Made available in DSpace on 2015-03-26T13:42:30Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2281509 bytes, checksum: 8e44475ccbcd12f91301ea5e4fda8830 (MD5) Previous issue date: 2013-03-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Plants are engaged in a continuous co-evolutionary struggle for dominance with their pathogens and the outcomes of these interactions are of particular importance to human activities, as they can have dramatic effects on agricultural systems (Dodds & Rathjen, 2010). Recently, the convergence of molecular studies of plant immunity and pathogen infection strategies is revealing an integrated picture of the plant pathogen interaction (Mukhtar et al., 2011) in which the pathogen effectors interaction converge onto highly connected subgroups of proteins, named hubs. A well-defined hub form plant immune system network corresponds to CSN5A protein, a catalytic subunit of the COP9 signalosome acting as a key regulator in several basic cellular processes. Consistent with the prediction that different effectors from different pathogens target similar connections in plant-pathogen interaction network, it has been shown, independently, that the protein C2 from geminivirus, a DNA virus that infects a wide variety of agronomic crops, interacts to CNS5A (Lozano- Duran et al, 2011). Additionally, it was shown that NIG and the immune receptor NIK, both targets of geminivírus NSP, interact to CSN5A (Machado, 2011; Mukhtar et al., 2011). Based on this information, it is expected that the hub CNS5A is a functional element in the geminivirus-host interaction network. Recently, it was reported that NIG, a cellular partner of CSN5A, also interacts with a unknown function protein, encoded by the locus AT2G41020 in yeast (Machado, 2011). As a possible component from geminivirus-host interaction network converging to CSN5A, AT2G41020 may interact directly or indirectly with virulence factors in defense response or compatibility. The objectives of this research involved biochemical characterization of the protein encoded by the locus AT2G4102, designated AtWWP1, and identification of its possible interactions with viral proteins and host factors. In silico analysis of tWWP1 predicted structure revealed the presence of two WW domains, and a C-terminal domain highly conserved between homologous in plant and animals. Furthermore, it has been shown that AtWWP1 is a nuclear protein capable of forming nuclear bodies via the conserved C-terminal domain. Coimmunoprecipitation and BiFC assays demonstrated that AtWWP1 interacts in vivo with the cytoplasmic protein NIG, redirecting it to nuclear bodies. In order to explore the formative activity of nuclear bodies AtWWP1, the interaction between AtWWP1 and a second protein partner AtMBD2 (methyl CG binding domaincontaining protein) was characterized in vivo. The ability to form nuclear bodies as interaction with AtMBD2 were mapped AtWWP1 occuring via its domain and C-terminal conserved, substantiating the argument that this region of AtWWP1 is responsible for the formation of nuclear bodies. Colocalization assays have shown that nuclear bodies contained in AtWWP1 are distinct from those formed by proteins involved in RNA splicing, but colocalized with nuclear bodies containing CDKC2. Furthermore, it was demonstrated that AtWWP1 does not bind to RNA, but exhibits a binding activity to DNA. These characteristics imply that AtWWP1 should be involved with basic nuclear functions. As a component of a functional hub in geminivirus-host interaction network, it is important to assess whether the viral infection would affect the nuclear bodies formed by AtWWP1. / Estudos moleculares envolvendo o sistema imune de plantas e a infecção por patógenos revelaram um panomara integrado de interações plantapatógeno em que as interações dos efetores de virulência convergem para subconjuntos de proteínas do hospedeiro altamente interconectadas e designadas hubs. Um hub funcional e bem definido do sistema imune de plantas corresponde a interconexões convergentes para a proteína CNS5A que constitui a subunidade catalítica do complexo COP9 signalosome, um regulador chave de diversos processos celulares básicos. Consistente com a previsão de que efetores de diferentes patógenos devem alvejar similares conexões na rede de interações planta-patógeno, foi demonstrado, independentemente, que a proteína C2 de geminivírus, um vírus de DNA que infecta uma grande variedade de culturas agronômicas, interage com a proteína CNS5A. Além disso, foi também demonstrado que tanto a proteína NIG, quanto o receptor imune NIK, ambos alvos da proteína NSP de geminivírus, também interagem com CNS5A. Baseado nestas informações, prevê-se que a interconexão (hub) representada por CNS5A seja um elemento funcional na interação geminivírus-hospedeiro. Recentemente, foi identificado que, além de interagir com CSN5A, a proteína NIG também interage com uma proteína de função desconhecida codificada pelo locus AT2G41020, em leveduras . Como possível componente da rede de interações geminivírus-hospedeiro que converge em CNS5A, AT2G41020 pode interagir direta ou indiretamente com fatores de virulência em resposta de defesa ou de compatibilidade. Sendo assim, os objetivos principais dessa investigação envolveram caracterização bioquímica da proteína codificada pelo locus AT2G41020 e identificação de possíveis interações com proteínas virais e fatores do hospedeiro. Análise in silico da estrutura predita da proteína codificada pelo lócus At2G41020, designada AtWWP1, revelou a presença de dois domínios WW e um domínio C-terminal altamente conservado entre proteínas homólogas de espécies vegetais e animais. Além disso, foi demonstrado que a proteína AtWWP1 é uma proteína nuclear capaz de formar corpos subnucleares via o domínio C-terminal conservado. Ensaios de coimunoprecipitação e BiFC demonstraram que AtWWP1 interage in vivo com a proteína citoplasmática NIG promovendo o seu redirecionamento para corpos nucleares. Com a finalidade de explorar a atividade formadora de corpos nucleares de AtWWP1, a interação entre AtWWP1 e uma segunda proteína parceira AtMBD2 (proteína contendo um domínio de interação com CG metilado) foi caracterizada in vivo. Tanto a capacidade de formar corpos nucleares quanto a interação com AtMBD2 foram mapeadas em AtWWP1 e ocorrem via seu domíno C-terminal conservado, substanciando o argumento de que esta região de AtWWP1 é responsável pela formação de corpos subnucleares. Ensaios de co-localização demonstraram que os corpos nucleares contidos em AtWWP1 são distintos daqueles formados por proteínas envolvidas em splicing do RNA; porém co-localizam com corpos nucleares contendo CDKC2. Além disso, foi demonstrado que AtWWP1 não liga a RNA, mas exibe uma atividade de ligação ao DNA. Estas características implicam que AtWWP1 deve estar envolvida com funções nucleares básicas. Como componente de um hub funcional na interação geminivírus-hospedeiro, torna-se relevante avaliar se a infecção viral afetaria os corpos nucleares formados por AtWWP1.