Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

Cancer cells exhibit genomic instability and are commonly defective in DNA damage signalling and/or DNA repair. There are many types of DNA damage inducing agents such as mechanical stress on chromosomes during recombination, chemotherapeutics, ionising and ultraviolet radiation and endogenously produced free radicals. These genetic lesions pose a serious threat to the cell and evoke a rapid and intricate DNA damage response signalling pathway involving many transducer and effector pathways including cell cycle arrest, DNA repair, chromatin remodelling, and apoptotic pathways. Genetic mutations within genes in this pathway often lead to genomic instability and cancer. The main effectors of the DNA damage response are the protein kinases ATM and ATR which are rapidly activated in response to DNA damage induction and phosphorylate a large and diverse number of targets including the checkpoint kinases Chk1, and Chk2, the tumour suppressors p53 and BRCA1 and chromatin associated proteins such as H2AX. BRCA1 is a key transducer molecule within the DNA damage response. This is evident from its loss, which leads to defects in many damage response processes such as cell cycle arrest and DNA repair. BRCA1s binding partner BARD1 has also been implicated in the DNA damage response and recent reports indicate that these proteins co-operate in this pathway. This study utilises a multifaceted approach to further characterise the function of the BRCA1/BARD1 complex within the DNA damage response. Firstly we have used shRNA to deplete the BRCA1/BARD1 complex and have shown that the BRCA1/BARD1 complex is required for ATM/ATR dependent phosphorylation of p53Ser-15 in response to IR and UV induced DNA damage. In contrast, we have shown that the phosphorylation of a number of other ATM/ATR dependent targets including H2AX, Chk2, and c-jun do not require the BRCA1/BARD1 complex. The study has also revealed that the prior phosphorylation of BRCA1 at Ser-1423 and Ser-1524 is required for the phosphorylation of p53 at Ser-15. Furthermore, we have shown that these phosphorylation events are required for IR induced G1/S cell cycle arrest via transcriptional induction of the cyclin dependent kinase inhibitor p21. The second part of this study involved the characterisation of a putative BRCA1 interacting protein – The KRAB associated protein 1 (Kap1). During this study we have been unable to confirm Kap1 as a bona fide BRCA1 interactor, however we have identified a clear role for Kap1 in the DNA damage response pathway. Using Mass spectrometric phospho amino acid mapping we have identified a novel Chk2 dependent phosphorylation site, Ser-473, within Kap1. Furthermore, we have shown that this phosphorylation event may regulate Histone H3-Lys-9 acetylation after DNA damage possibly regulating chromatin relaxation. This study has also identified a number of novel Kap1 interacting proteins, which appear to be regulated by Kap1 phosphorylation at Ser-473. These interactors may play an important role in the regulation of chromatin modification and/or structure after DNA damage. By studying the role of BRCA1 in the DNA damage response pathway we have not only uncovered a novel scaffolding function for BRCA1 in the G1/S checkpoint but have also identified a novel protein, Kap1, acting within the DNA damage response pathway. This study has identified a role for Kap-1 in the regulation of chromatin structure in response to DNA damage via the ATM – Chk2 pathway.

La latence post-intégrative du VIH-1 génère des réservoirs qui empêchent l'éradication du virus avec les thérapies actuelles. La compréhension des mécanismes moléculaires de la latence servirait à l'identification de nouvelles cibles thérapeutiques et le développement de molécules de type LRA (Latency Reversing Agent) permettant la guérison fonctionnelle des patients et un arrêt des traitements. Nous avons démontré que la combinaison de deux types différents de LRAs à base de composés libérant P-TEFb (JQ1, I-BET, I-BET151 et HMBA) ou des agonistes de PKC (prostratine, bryostatine-1 et Ing-B) conduit à une robuste activation synergique de la production du VIH-1 dans divers modèles cellulaires de latence post-intégrative et dans des réservoirs de lymphocytes T CD4+ primaires. Le répresseur transcriptionnel CTIP2 recrute des complexes multienzymatiques pour favoriser l'établissement et la persistance de la latence du VIH-1 dans les cellules microgliales, le principal réservoir viral du système nerveux central. De plus, CTIP2 s’associe au complexe 7SK snRNP pour inhiber P-TEFb, un facteur d’élongation indispensable à l’expression du VIH-1 et à la réactivation des provirus latents. Des expériences d’immunoprécipitations de CTIP2, couplées à la spectrométrie de masse, nous ont permis d’identifier près de 900 partenaires protéiques de CTIP2. Parmi ces nouveaux partenaires, nous avons identifié la SUMO E3 ligase KAP1. Nous montrons que KAP1 réprime les phases précoce et tardive Tat dépendante de la transcription du VIH-1. KAP1 induit une dégradation de Tat, qui est sensible aux modulations de la voie SUMO. En effet, la sumoylation favorise l'association de Tat avec KAP1, de même que sa dégradation. Globalement, nos résultats suggèrent que KAP1 contribue à l'établissement et à la persistance des réservoirs latents du VIH-1. Cibler les voies SUMO constituerait un nouveau champ d'investigation dans le cadre du développement de nouvelles classes de LRAs. / The HIV-1 post-integration latency generates reservoirs that prevent the eradication of the virus with the current therapies. The understanding of the molecular mechanisms of this latency enable the identification of new therapeutic targets and the development of LRA (Latency Reversing Agent) for functional cure and treatments interruption. We have demonstrated that the combination of two different LRAs based on P-TEFb releasing compounds (JQ1, I-BET, I- BET151 and HMBA) or PKC agonists (prostratin, bryostatin-1 and Ing-B) leads to robust synergistic activation of HIV-1 production in various cellular models of post-integration latency and in primary CD4+ T cells reservoirs. The transcriptional repressor CTIP2 recruits multienzymatic complexes to promote the establishment and persistence of HIV-1 latency in microglial cells, the main viral reservoir of the central nervous system. Furthermore, CTIP2 binds to the 7SK snRNP complex to inhibit P-TEFb, an elongation factor essential for HIV-1 expression and reactivation of latent proviruses. Immunoprecipitation experiments of CTIP2 coupled to mass spectrometry allowed us to identify almost 900 proteins partners of CTIP2. Among these new partners, we have identified the E3 SUMO ligase KAP1. We found that KAP1 contributes to HIV-1 gene silencing by repressing the initiation and the Tat–dependent steps of the viral gene transcription. KAP1 induces Tat degradation via a SUMO-sensitive pathway. Indeed, favoring the sumoylation promotes Tat association with KAP1 and the resulted Tat degradation. Altogether, our results suggest that KAP1 contributes to the establishment and the persistence of the latently infected HIV-1 reservoirs. Moreover, these results suggest that targeting the SUMO pathways may be a new field of investigation to develop new classes of LRAs for cure strategies.

Nous avons identifié une association poly(ADP-ribose) (PAR)-dépendante entre PARP1 et UHRF1. UHRF1 est PARylé par PARP1 et lie le PAR de façon non covalente. L’absence de PARP1 (i) perturbe l’association de UHRF1 et DNMT1, (ii) induit une ubiquitination excessive de DNMT1 par UHRF1 favorisant sa dégradation au cours du cycle, (iii) favorise la transcription des régions de l’hétérochromatine péricentrique (pHC) (iv) et perturbe la localisation de la marque répressive H4K20me3 au niveau des foyers de l’pHC. Dans un deuxième temps, nous avons étudié le rôle de l’association KAP1-HP1 dans la réponse cellulaire aux dommages. L’interaction entre ces deux partenaires est essentielle pour le recrutement de KAP1 sur les sites de cassures. Après induction de cassures, l’absence d’interaction induit un délai dans la réparation des cassures double-brins et une diminution de la survie cellulaire. Une analyse détaillée suggère une déficience du mécanisme de réparation par recombinaison homologue. / We identified a poly(ADP-ribose) (PAR)-dependent interaction between PARP1 and UHRF1. UHRF1 is PARylated by PARP1 and binds PAR in a non-covalent way. The absence of PARP1 (i) impairs the UHRF1/DNMT1 interaction, (ii) induces excessive UHRF1-mediated ubiquitination of DNMT1 promoting its degradation during the cell cycle, (iii) increases the transcription of pericentric heterochromatin (pHC) regions (iv) and impairs the localization of the repressive histone mark H4K20me3 on pHC. In a second project we studied the role of the KAP1/HP1 interaction in response to DNA damage. The interaction between the two partners is essential for KAP1 recruitment to DNA damage sites. The absence of the interaction, after damage, induces a delay of the double strand break repair kinetics and decreases the cell survival rate. A more detailed analysis suggests a deficiency of the homologous recombination repair pathway.

Pluripotenz wird durch viele Stammzell-spezifische Transkriptionsfaktoren wie Oct3/4, Nanog und Sox2 sowie deren Funktion in ihrem regulatorischen Netzwerk etabliert und aufrechterhalten. Viele Studien haben gezeigt, wie diese Pluripotenz-assoziierten Faktoren ihre Zielgene regulieren. Dies geschieht durch die Interaktion mit bekannten und unbekannten Interaktionspartnern. In der vorliegenden Arbeit haben wir Zfp819 als einen neuen Pluripotenz-assoziierten Faktor beschrieben und dessen Funktion in pluripotenten Stammzellen untersucht. Im ersten Teil der vorliegenden Arbeit haben wir zwei cDNA-Banken für Yeast two Hybdrid (Y2H)-Assays aus unterschiedlichen pluripotenten Stammzelltypen generiert. Dies hatte zum Ziel, potentielle Interaktionspartner eines Kandidatenproteins zu identifizieren um dadurch Eindrücke über die Funktion des Proteins zu gewinnen. Für die Identifizierung von potentiellen Interaktionspartnern von Zfp819 haben wir die cDNA-Bank aus embryonalen Stammzellen benutzt. Wir konnten 17 putative Interaktionspartner identifizieren und daraus ein hypothetisches „Interaktom“ von Zfp819 generieren. Die Einordnung der putativen Interaktionspartner nach ihrer Gen-Ontologie (GO) ließ vermuten, dass Zfp819 eine Rolle in der Regulation der Transkription, der Aufrechterhaltung der genetischen Integrität und im Zellzyklus bzw. bei der Apoptose spielt. Im zweiten Teil der vorliegenden Arbeit wurde die sehr intensive Expression von Zfp819 in undifferenzierten pluripotenten Zelllinien gezeigt. Desweiteren konnte die Promotorregion von Zfp819 identifiziert werden, und es wurde gezeigt, dass diese mit epigenetischen Mustern ausgestattet ist. Zusätzlich konnten wir Regionen im Zfp819-Gen identifizieren, die für die nukleäre Lokalisation von Zfp819 verantwortlich sind. Desweiteren konnten wir zeigen, dass Zfp819 in der transkriptionellen Repression von spezifischen endogenen, retroviralen Elementen (ERVs) in pluripotenten Zellen eine Rolle spielt. Durch zelluläre und biochemische Studien konnten wir zeigen, dass Zfp819 mit vielen Proteinen interagiert (z.B. Kap1 und Chd4), welche für die Aufrechterhaltung der genomischen Integrität von Bedeutung sind. Tatsächlich resultierte der Verlust von Zfp819 in embryonalen Stammzellen in einer erhöhten Anfälligkeit für DNA-Schäden und in einer verminderten DNA-Reparatur. Zusammenfassend lassen die Identifizierung der Interaktionspartner sowie die Ergebnisse der molekularen und der funktionellen Studien vermuten, dass Zfp819 durch die Unterdrückung von ausgewählten ERVs eine Rolle in der Regulation der genomischen Stabilität von pluripotenten Zellen spielt.