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Le génome est sans cesse menacé dans sa structure par des stress génotoxiques d’origine endogène (stress oxydant, blocage de la réplication…) ou exogène (irradiations, produits chimiques, métaux lourds…). La voie de réponse aux dommages de l’ADN coordonne un réseau d’événements en cascades qui incluent les points de surveillance du cycle cellulaire, la réplication/réparation/recombinaison de l’ADN et la mort cellulaire programmée. Ces voies collaborent pour assurer la transmission fidèle du génome et empêcher la prolifération des cellules qui aurait accumulé des altérations génétiques. En général, des défauts dans une de ces voies entraînent un changement de sensibilité aux agents génotoxiques, une instabilité génétique et une prédisposition au cancer. La recombinaison homologue (RH) est une voie essentielle pour la réparation de l’ADN ; elle joue un rôle fondamental dans le maintien de la stabilité du génome. Le stress oxydant, résultant d’une augmentation de la concentration intracellulaire de ERO, est une des causes majeures de dommages aux lipides, aux protéines et à l’ADN et pour cela, il représente un défi pour la survie cellulaire et pour la stabilité du génome. Les ERO apparaissent physiologiquement lors de la respiration cellulaire ou résultent d’un stress environnemental, comme l’exposition aux radiations UV ou agents chimiques oxydants. Elles contribuent à certains processus comme la croissance cellulaire, l’activité et au repliement des protéines, la sénescence et la mort cellulaire programmée. Il est important de souligner qu’un état redox altéré est souvent associé à un fonctionnement anormal des cellules, comme il est observé pour les cellules cancéreuses et les cellules sénescentes. L’objectif de ce projet a été d’analyser l’interface entre les voies DDR et SAPK (Stress activated protein kinase) évolutivement conservées et d’en étudier les conséquences sur la stabilité du génome en utilisant comme organisme modèle la levure à fission. Nos résultats montrent que la voie SAPK joue un rôle sur la RH en promouvant la phosphorylation de Rad52, une protéine impliquée dans différentes sous-voies de la RH. Nous avons aussi montré que Rad52 est phosphorylée sur différents acides aminés, parmi lesquels certains sont les cibles de kinases inconnues qui n’ont aucun lien avec la voie SAPK. Nous avons observé que Rad52 est phosphorylée soit après un stress oxydant, soit dans des cellules génétiquement sujettes à des perturbations de la RH. Nous avons identifié deux sites de phosphorylation de la protéine Rad52, dont un seul est dépendant de la voie SAPK. En étudiant la phosphorylation de Rad52 dans les cellules invalidées pour la voie SAPK ou mutées pour un des sites de phosphorylation de Rad52, nous avons pu montrer que la RH est modulée par la voie SAPK même en absence de insulte externe. Notre travail ouvre le chemin vers une nouvelle compréhension des mécanismes fondamentaux du maintien de l’intégrité du génome. / Genomes are routinely submitted to injuries from either endogenous stress (oxidative stress, DNA replication block…) or from exogenous sources (radiations, chemicals, heavy metals…). The DNA damage response (DDR) coordinates a network of pathways including cell cycle checkpoints, DNA replication/repair/recombination, and programmed cell death, ensuring faithful genome transmission and preventing from the proliferation of cells bearing genetic alterations. Defect in one of these pathways generally results in altered sensitivity to genotoxins, genetic instability and cancer predisposition. Homologous recombination (HR) is an essential DNA repair pathway playing pivotal role to maintain genome stability.Oxidative stress, resulting from increased intracellular concentration of ROS, is one of the major causes of lipid, protein and DNA damage, and therefore a challenge for cell survival and genome stability. ROS are generated physiologically as by-products of cellular respiration, or as result of environmental stresses, such as exposure to solar UV radiations or to oxidant chemicals, and they actively participate in processes such as cellular growth, protein activity and folding, senescence and programmed cell death. It is noteworthy that an altered redox homeostasis is often associated to abnormally functioning cells, such as cancer and senescent cells. The aim of this project was to study the interface between two major evolutionarily conserved pathways, DDR and SAPK (Stress activated protein kinase) and the consequences on genome stability using fission yeast as a model organism. We report data showing that SAPK pathway impinges on HR by promoting phosphorylation of Rad52, a key protein involved in all sub-pathways of HR. We also revealed that Rad52 is phosphorylated at multiple different sites some of which are substrate for unidentified kinases unrelated to the SAPK pathway. Rad52 phosphorylation occurs either after oxidative stress or in cells genetically prone to HR perturbation. We identified two sites of phosphorylation, one of which is dependent on functional SAPK pathway. By studying Rad52 phosphorylation in cells mutated in the SAPK pathway or mutated at the Rad52 site of phosphorylation, we showed that HR is modulated by SAPK even in the absence of external insults. Our work pave the way to a novel understanding of fundamental mechanisms required for genome integrity maintenance.

This thesis describes the cloning and characterisation of a novel human gene and its protein products, which have been designated SAPK- and Ras-interacting protein (SinRIP). SinRIP shares identity with JC310, a partial human cDNA that was previously identified a candidate Ras-inhibitor (Colicelli et al., 1991, Proc Natl Acad Sci USA 88, p. 2913). In this study, it was shown that SinRIP is a member of an orthologous family of proteins that is conserved from yeast to mammals and contains proteins involved in Ras- and SAPK-mediated signalling pathways. Comparison of this family of proteins showed that human SinRIP contains a potential Ras-binding domain (RBD; residues 279-354), a PH-like domain (PHL; 376-487), and a highly conserved novel region designated the CRIM (134-265). Several other potential targeting sites, such as nuclear localisation signals and target sites for kinases, were identified within the SinRIP sequence. The human SinRIP gene is unusually large (>280 kbp) and is located on chromosome 9 at 9q34. SinRIP mRNA was detected in a wide variety of tissue-types and cell lines by RT-PCR, and the SinRIP sequences in the EST database were derived from an diverse array of tissues, suggesting a widespread or ubiquitous expression. Northern blot analysis revealed the highest levels in skeletal muscle and heart tissue. However, the steady-state levels of SinRIP mRNA vary greatly from cell to cell, and SinRIP expression is likely to be regulated at multiple post-transcriptional levels. It was shown that SinRIP mRNA is likely to be translated inefficiently by the normal cap-scanning mechanism, due to the presence of a GC-rich and structured 5’-UTR, which also contains upstream ORFs. Alternative polyadenylation signals in the SinRIP 3’-UTR can be used, resulting in the expression of short and long SinRIP mRNA isoforms. Several potential A/T-rich regulatory elements were also identified in SinRIP mRNA, which may target specific SinRIP mRNA isoforms for rapid degradation. Importantly, it was shown that SinRIP mRNA is alternatively spliced, resulting in the production of distinct SinRIP protein isoforms. Three isoforms, SinRIP2-4, were definitively identified by RT-PCR and full-length cloning. The SinRIP isoforms contain deletions in conserved regions, and are likely to have biochemical characteristics that are different to full-length SinRIP1. SinRIP2 is C-terminally truncated and lacks the PHL domain and part of the RBD, and relatively high levels of SinRIP2 expression arelikely to occur in kidneys. The RBD is disrupted in SinRIP3, but all other domains are intact, and RT-PCR analyses suggest that SinRIP3 is present in some cells at levels comparable to SinRIP1. A rabbit polyclonal antiserum against SinRIP was generated and detected endogenous SinRIP proteins. Using the anti-SinRIP antibody in immunoblots, multiple SinRIP isoforms were observed in most cell types. SinRIP1 and another endogenous SinRIP protein, likely to be SinRIP3, were detected in most cell lines, and appear to be are the major SinRIP proteins expressed in most cells. The subcellular localisation of both recombinant and endogenous SinRIP proteins was investigated by immunofluorescence assays and biochemical fractionation. Recombinant SinRIP1 protein was found in the cytoplasm and associated with the plasma membrane. In contrast, the SinRIP2 protein was predominantly nuclear, with only low-level cytoplasmic staining observed. The endogenous SinRIP proteins, likely to comprise these and other SinRIP isoforms, were found in both the nucleus and cytoplasm. SinRIP1 interacted with GTP-bound (active) Ras, but not GDP-bound (inactive) Ras, in an in vitro assay, and also co-localised with activated H- and K-Ras in cells. The binding profile observed is typical of Ras-effectors, and SinRIP did not inhibit signalling by the Ras proteins, suggesting that it is not likely to be a Ras-inhibitor. It was also shown that SinRIP1 and SinRIP2 both interact and colocalise with c-Jun NH2- terminal kinase (JNK). Both SinRIP proteins were able to recruit JNK to their respective sub-cellular compartments. These interactions suggest an adaptor role for SinRIP in the Ras and/or JNK pathways. In addition, Sam68 was isolated as a SinRIP-binding protein in a yeast two-hybrid screen. Sam68 was shown to colocalise with SinRIP2 and endogenous SinRIP proteins, but not SinRIP1. Further colocalisation studies showed that endogenous SinRIP proteins localise in nuclear structures that may be associated with pre-mRNA splicing. Likely functions for SinRIP, as indicated by experimental results and studies of the orthologues of SinRIP in other species, are discussed.

Stress activated protein kinase (SAPK) interacting protein 1 (Sin1) is a member of a recently characterized gene family, conserved from yeast to humans. The gene copy number is strictly conserved (one Sin1 gene per genome), and the protein may be expressed ubiquitously in mammalian tissues. The Sin1 family has been implicated in several different signal transduction pathways. Originally identified as a partial cDNA and candidate Ras inhibitor, recent functional studies have revealed interactions with an interferon (IFN) receptor subunit (IFNAR2), and the SAPK JNK. Interactions have also been described between the yeast orthologues and the phosphatidylinositol kinase TOR2. Collectively, these data suggest that Sin1 has an important cellular role, and this study has investigated possible functions for this protein. As human Sin1 proteins have no paralogues within the genome, secondary structure homology was used to identify major domains within the protein. Four major domains within human Sin1 were deduced: an N-terminal domain containing a functional nuclear localization signal, a functional nuclear export signal, and a coiledcoil region; the conserved region in the middle that is likely to be a ubiquitin-like β-grasp protein binding domain; a Ras binding domain; and a pleckstrin homology-like domain that targets Sin1 to the plasma membrane and lipid rafts in vivo. Full and partial length EGFP constructs were used to examine the localization of human Sin1, and several isoforms derived from alternative splicing. All isoforms localized to the nucleus and nucleolus. Beyond this, Sin1α and Sin1ϒ had cytoplasmic staining, while Sin1 and Sin1β were also found at the plasma membrane and lipid rafts. Both the N-terminal domain and the conserved region in the middle were found to contribute to nuclear localization. Comparative genomic analysis between human, mouse, rat, dog, and chicken Sin1 genes revealed a number of conserved intronic regions, and the putative functions of these were predicted. Additionally, a putative promoter module within a CpG island and encompassing the transcription start site was predicted in all species. The human CpG island was found to have promoter activity in HEK293 cells. Using bioinformatics, genes that may be co-regulated with Sin1 were identified. These genes contained the Sin1 promoter module, and were found to co-express in large scale gene expression studies. Most of these genes were directly involved in the cellular response to pathogen infection, suggesting a conserved role for Sin1 in this pathway. Key biochemical functions of the Sin1 proteins were also identified, including the ability of Sin1 proteins to form dimers, and the ability of over-expressed Sin1 to induce apoptosis (mediated through the conserved region in the middle). Additionally, endogenous Sin1 protein levels were found to change following serum deprivation and hypoosmotic stress. Together, these studies have provided significant insight into the cellular role of Sin1, suggesting a role in inducing apoptosis as part of the IFN response to viral infection. The biological significance of the Sin1 proteins is discussed in the context of their predicted functions and the evolution of the protein family.

Regulation of transcription by Stress Activated Protein Kinases (SAPKs) is an essential aspect for adaptation to extracellular stimuli. In mammals, the activation of the p38 SAPK results in the regulation of gene expression through the direct phosphorylation of several transcription factors. However, how p38 SAPK regulates the proper gene expression program of adaptation to stress as well as the basic mechanisms used by the SAPK remains uncharacterized. The results displayed in this manuscript show that the p38 SAPK plays a central role in the regulation of gene expression upon stress, as up to 80% of the upregulated genes are p38 SAPK dependent. Moreover, we also observed that a specific set of genes were upregulated in response to each specific stimuli, and just a small set of genes were commonly up-regulated by several stresses, which involves mainly transcription factors. In addition, we observed that, to proper regulate gene transcription, the p38 SAPK is recruited to stress-induced promoters via its interaction with transcription factors. Additionally, p38 activity allows the recruitment of RNA polymerase II and the MAPKK MKK6 to stress-responsive promoters. The presence of active p38 SAPK at open reading frames also suggests the involvement of the SAPK in elongation. Altogether, the results showed in this manuscript establish the p38 SAPK as an essential regulator in the transcriptional response to stress, as well as define new roles for p38 in the regulation of transcription in response to stress. / La regulación de la transcripción por las Proteínas Quinasas activadas por Estrés (SAPKs) es un aspecto esencial para la adaptación a los estímulos extracelulares. En mamíferos, la activación de la SAPK p38 da lugar a la regulación de la expresión génica a través de la fosforilación de varios factores de transcripción. Sin embargo, cómo p38 SAPK regula el programa de expresión génica de adaptación al estrés así como los mecanismos utilizados por la SAPK permanece sin caracterizar. Los resultados presentados en este manuscrito muestran que p38 SAPK juega un rol central en la regulación de la expresión génica en respuesta a estrés, ya que hasta el 80% de los genes inducidos son dependientes de p38 SAPK. También observamos que en respuesta a cada tipo de estrés se induce un grupo de genes específicos, y sólo hay una pequeña respuesta de genes comunes a los diferentes tipos de estrés la cual engloba principalmente factores de transcripción. Además, hemos observado que para regular la transcripción, p38 se recluta a los promotores de respuesta a estrés a través de su interacción con factores de transcripción. Asimismo, la actividad de p38 permite el reclutamiento de la RNA Polimerasa II y de la MAPKK MKK6 a los promotores inducidos por estrés. La presencia de p38 activa en las regiones codificantes sugiere su participación durante la elongación. En conjunto, los resultados mostrados en este manuscrito establecen a p38 como un regulador esencial de la transcripción en respuesta a estrés, así como definen nuevas funciones de p38 en la regulación de la transcripción en respuesta a estrés.

En el llevat Saccharomyces cerevisiae els canvis en les condicions osmòtiques del medi extracel.lular són sensades per la MAP cinasa Hog1, la qual permet dur a terme l'adaptació cel.lular mitjançant la modulació de l'expressió gènica, de la traducció i de la progressió del cicle cel.lular. A l'inici d'aquest projecte de tesi, els mecanismes pels quals Hog1 controla l'expressió gènica no eren del tot coneguts. El nostre objectiu va ser caracteritzar el mecanisme molecular pel qual Hog1 modula la transcripció en resposta a estrès osmòtic. Hem aconseguit demostrar que el reclutament de Hog1 als promotors sensibles a estrès osmòtic per part del factor de transcripció és essencial per al reclutament i activació de la RNA polimerasa II, mecanisme que podria estar conservat en les cèl.lules eucariotes. També hem identificat noves activitats remodeladores de cromatina implicades en la resposta gènica a osmoestrès mediada per Hog1. Vàrem realitzar un cribatge genètic per identificar mutacions que provoquessin osmosensibilitat i una reducció en l'expressió de gens de resposta a estrès osmòtic. Aquest cribatge ens va permetre identificar nous reguladors de la transcripció mediada per osmoestrès: la histona deacetilasa Rpd3 i els complexes SAGA i mediador. Els nostres resultats permeten, doncs, definir un important paper per a Rpd3, SAGA i mediador en la inducció gènica mediada per Hog1, i han estat importants per assolir una millor visió de com les cinases activades per estrès regulen la iniciació de la transcripció. / In Saccharomyces cerevisiae, changes in the extracellular osmotic conditions are sensed by the HOG MAPK pathway, which elicits the program for cell adaptation, including modulation of gene expression, translation and cell-cycle progression. At the beginning of this PhD Project, the mechanisms by which Hog1 was controlling gene transcription were not completely understood. Our main objective was to characterize the molecular mechanisms by which the Hog1 MAPK modulates transcription upon osmostress. We have shown that anchoring of Hog1 to osmoresponsive promoters by the transcription factor is essential for recruitment and activation of RNA polymerase II, a mechanism that might be conserved among eukaryotic cells. In addition, we identified novel chromatin modifying and remodelling activities involved in the Hog1-mediated osmostress gene expression. We performed a genome-wide genetic screening searching for mutations that render cells osmosensitive and displayed reduced expression of osmoresponsive genes. Rpd3 histone deacetylase, SAGA and Mediator complexes were identified as novel regulators of osmostress-mediated transcription. Thus, our results define a major role for Rpd3, SAGA and Mediator in the Hog1-mediated osmostress gene induction, and have been important to achieve a better view of how a SAPK regulates transcription initiation.

La adaptación de la levadura Saccharomyces cerevisiae a condiciones de alta osmolaridad está mediada por la vía de HOG ((high-osmolarity glycerol). La activación de esta vía induce una serie de respuestas que van a permitir la supervivencia celular en respuesta a estrés. La regulación génica constituye una respuesta clave para dicha supervivencia. Se han descrito cinco factores de transcripción regulados por Hog1 en respuesta a estrés osmótico. Sin embargo, éstos no pueden explicar la totalidad de los genes regulados por la MAPK Hog1. En el presente trabajo describimos cómo el complejo transcripcional formado por las proteínas Rtg1 y Rtg3 regula, a través de la quinasa Hog1, la expresión de un conjunto específico de genes. Hog1 fosforila Rtg1 y Rtg3, aunque ninguna de estas fosforilaciones son esenciales para regulación transcripcional en respuesta a estrés. Este trabajo también muestra cómo la deleción de proteínas RTG provoca osmosensibilidad celular, lo que indica que la integridad de la vía de RTG es esencial para la supervivencia celular frente a un estrés osmótico. / In Saccharomyces cerevisiae the adaptation to high osmolarity is mediated by the HOG (high-osmolarity glycerol) pathway, which elicits different cellular responses required for cell survival upon osmostress. Regulation of gene expression is a major adaptative response required for cell survival in response to osmotic stress. At least five transcription factors have been reported to be controlled by the Hog1 MAPK. However, they cannot account for the regulation of all of the genes under the control of the Hog1 MAPK. Here we show that the Rtg1/3 transcriptional complex regulates the expression of specific genes upon osmostress in a Hog1-dependent manner. Hog1 phosphorylates both Rtg1 and Rtg3 proteins. However, none of these phosphorylations are essential for the transcriptional regulation upon osmostress. Here we also show that the deletion of RTG proteins leads to osmosensitivity at high osmolarity, suggesting that the RTG-pathway integrity is essential for cell survival upon stress.

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.