Le programme Pyramide: un programme de prévention ciblée de l'affiliation aux gangs de rue s'adressant aux parents de préadolescents hébergés au Centre jeunesse de Montréal-Institut Universitaire

Rapport d'analyse d'intervention présenté à la Faculté des arts et sciences en vue de l'obtention du grade de Maîtrise ès sciences (M. Sc.) en psychoéducationLe projet d'intervention vise à prévenir l'affiliation aux gangs de rue chez les préadolescents hébergés en milieu substitut en améliorant les habiletés parentales de leurs parents: les renforcements positifs, la supervision, la discipline et la relation parent-préadolescent. Le programme de prévention développé s'inspire de l'Adolescent Transition Program (Claes & Poirier, 1995; Dishion & Kavanagh, 2003) et du Strengthening Families Program : For Parents and Youth 10-14 (Molgaard, Kumpfer & Fleming, 2007). Cinq parents participent au programme développé et implanté au Centre jeunesse de Montréal-Institut Universitaire, à l'unité l'Entreprise située sur le site du mont St-Antoine. Le devis de type expérimental utilisé est un protocole à cas unique de type A-B-A avec 14 points de mesure répétée. Les habiletés parentales sont évaluées chez trois parents, par les parents eux-mêmes et par leur préadolescent. Les résultats obtenus auprès des parents montrent, chez un parent, un accroissement léger des renforcements positifs des comportements adéquats de son préadolescent et une amélioration de la relation parent-préadolescent suite à l'introduction de l'intervention. Les résultats obtenus auprès des préadolescents montrent une amélioration de la discipline chez un parent suite à l'introduction de l'intervention; chez un autre parent, ils montrent une légère amélioration de la relation parent-préadolescent. Il y a incohérence entre les sources au niveau des améliorations observées

Identification du rôle et des modifications post-traductionnelles modulant l’export nucléaire de l’hélicase virale E1 au cours du cycle de réplication du virus du papillome humain

Les virus du papillome humain (VPH) sont de petits virus à ADN double brin infectant les épithéliums de la peau et des muqueuses. La réplication nécessaire au maintien de leur génome dans les cellules infectées dépend des protéines virales E1 et E2. Au cours de la réplication, E1 est recrutée à l’origine de réplication par E2 afin d’être assemblée en doubles hexamères capables de dérouler l’ADN. E1 contient un domaine C-terminal responsable de l’activité ATPase/hélicase, un domaine central de liaison à l’origine et une région N-terminale régulant la réplication in vivo. Cette région contient des signaux de localisation et d’export nucléaire qui modulent le transport intracellulaire de E1. Chez le virus du papillome bovin (VPB), il a été proposé que ce transport est régulé par la sumoylation de E1. Finalement, la région N-terminale de E1 contient un motif de liaison aux cyclines permettant son interaction avec la cycline E/A-Cdk2. La phosphorylation de E1 par cette dernière régule différemment l’export nucléaire des protéines E1 du VPB et du VPH. Dans la première partie de cette étude, nous avons démontré que bien que la protéine E1 des VPH interagit avec Ubc9, l’enzyme de conjugaison de la voie de sumoylation, cette voie n’est pas requise pour son accumulation au noyau. Dans la seconde partie, nous avons déterminé que l’accumulation nucléaire de E1 est plutôt régulée pas sa phosphorylation. En fait, nous avons démontré que l’export nucléaire de E1 est inhibé par la phosphorylation de sérines conservées de la région N-terminale de E1 par Cdk2. Puis, nous avons établi que l’export nucléaire de E1 n’est pas nécessaire à l’amplification du génome dans les kératinocytes différenciés mais qu’il est requis pour le maintien du génome dans les kératinocytes non différenciés. En particulier, nous avons découvert que l’accumulation nucléaire de E1 inhibe la prolifération cellulaire en induisant un arrêt du cycle cellulaire en phase S et que cet effet anti-prolifératif est contrecarrée par l’export de E1 au cytoplasme. Dans la troisième partie de cette étude, nous avons démontré que l’arrêt cellulaire induit par E1 dépend de sa liaison à l’ADN et à l’ATP, et qu’il est accompagné par l’activation de la voie de réponse aux dommages à l’ADN dépendante de ATM (Ataxia Telangiectasia Mutated). Ces deux événements semblent toutefois distincts puisque la formation d’un complexe E1-E2 réduit l’activation de la voie de réponse aux dommages par E1 sans toutefois prévenir l’arrêt de cycle cellulaire. Finalement, nous avons démontré que la réplication transitoire de l’ADN viral peut avoir lieu dans des cellules arrêtées en phase S, indépendamment de l’activation de la voie de réponse aux dommages à l’ADN et de la kinase ATM. Globalement, nos résultats démontrent que l’export nucléaire de E1 est régulé par sa phosphorylation et non par sa sumoylation. Ils démontrent également que l’export nucléaire de E1 est essentiel au maintien du génome dans les kératinocytes, possiblement parce qu’il prévient l’inhibition de la prolifération cellulaire et l’activation de la voie de réponse aux dommages à l’ADN en limitant l’accumulation de E1 au noyau.Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect the differentiating epithelium of the skin and the mucosa. HPV rely on two viral proteins, E1 and E2, to replicate and maintain their genome in the nucleus of infected cells. During replication, the E1 helicase is recruited to the origin of replication by E2 and is assembled into a double-hexamer that unwinds DNA ahead of the replication fork. E1 is comprised of a C-terminal enzymatic domain with ATPase/helicase activity, a central origin-binding domain and a N-terminal regulatory region that is required for viral DNA replication in vivo. The latter region of E1 contains a nuclear localization signal and a nuclear export signal that regulate its shuttling between the nucleus and cytoplasm. For bovine papillomavirus (BPV) E1, this shuttling was suggested to be controlled by the sumoylation of E1. In addition to the NES and NLS, the N-terminal region of E1 contains a conserved cyclin-binding motif that is required for the interaction of E1 with cyclin E/A-Cdk2. Cdk2 phosphorylation of E1 has been reported to control the nuclear export of E1 from BPV and HPV, albeit differently. In the first part of this study, we showed that although HPV E1 interacts with Ubc9, the conjugating enzyme of the sumoylation pathway, this pathway is not required for its accumulation in the nucleus. In the second part, we found that the nuclear accumulation of E1 is, instead, regulated by phosphorylation. Specifically, we found that Cdk2-dependent phosphorylation of conserved serines in the E1 N-terminal region inhibits the nuclear export of HPV E1. Furthermore, we reported that nuclear export is not essential to amplify the viral genome in differentiating keratinocytes but that it is required for its long-term maintenance in undifferentiated keratinocytes. Importantly, we found that the nuclear accumulation of E1 induces a S-phase arrest that is detrimental to cellular proliferation and that this anti-proliferative effect can be counteracted by the export of E1 from the nucleus to the cytoplasm. In the last part of this study, we showed that this arrest is dependent on the DNA- and ATP-binding activities of E1. Furthermore, we found that the cell cycle arrest induced by E1 is accompanied by the activation of a DNA damage response (DDR) dependent on the ATM (Ataxia Telangiectasia Mutated) pathway. However, these two events seem to be distinct since complex formation with E2 reduces the ability of E1 to induce a DDR but does not prevent cell cycle arrest. Importantly, we demonstrated that transient viral DNA replication still occurs in S-phase arrested cells, independently of the induction of a DDR and of the ATM kinase. Collectively, these data indicate that nuclear export of E1 is regulated by phosphorylation and not by sumoylation. They also revealed that nuclear export of E1 is essential for maintenance of the viral episome in keratinocytes, at least in part to limit its nuclear accumulation and prevent its detrimental effect on cellular proliferation and induction of a DDR

Two redundant ubiquitin-dependent pathways of BRCA1 localization to DNA damage sites

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.Genome Instability and Cance

Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin

Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity

ATM localization and heterochromatin repair depend on Direct Interaction of the 53BP1-BRCT2 domain with γH2AX

53BP1 plays multiple roles in mammalian DNA damage repair, mediating pathway choice and facilitating DNA double-strand break repair in heterochromatin. Although it possesses a C-terminal BRCT2 domain, commonly involved in phospho-peptide binding in other proteins, initial recruitment of 53BP1 to sites of DNA damage depends on interaction with histone post-translational modifications-H4K20me2 and H2AK13/K15ub-downstream of the early γH2AX phosphorylation mark of DNA damage. We now show that, contrary to current models, the 53BP1-BRCT2 domain binds γH2AX directly, providing a third post-translational mark regulating 53BP1 function. We find that the interaction of 53BP1 with γH2AX is required for sustaining the 53BP1-dependent focal concentration of activated ATM that facilitates repair of DNA double-strand breaks in heterochromatin in G1

Integrating DNA damage repair with the cell cycle

Abstract DNA is labile and constantly subject to damage. In addition to external mutagens, DNA is continuously damaged by the aqueous environment, cellular metabolites and is prone to strand breakage during replication. Cell duplication is orchestrated by the cell division cycle and specific DNA structures are processed differently depending on where in the cell cycle they are detected. This is often because a specific structure is physiological in one context, for example during DNA replication, while indicating a potentially pathological event in another, such as interphase or mitosis. Thus, contextualising the biochemical entity with respect to cell cycle progression provides information necessary to appropriately regulate DNA processing activities. We review the links between DNA repair and cell cycle context, drawing together recent advances

Cryo-electron microscopy of chromatin biology

The basic unit of chromatin, the nucleosome core particle (NCP), controls how DNA in eukaryotic cells is compacted, replicated and read. Since its discovery, biochemists have sought to understand how this protein-DNA complex can help to control so many diverse tasks. Recent electron-microscopy (EM) studies on NCP-containing assemblies have helped to describe important chromatin transactions at a molecular level. With the implementation of recent technical advances in single-particle EM, our understanding of how nucleosomes are recognized and read looks to take a leap forward. In this review, the authors highlight recent advances in the architectural understanding of chromatin biology elucidated by EM

Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation

Human papillomaviruses recruit a host of DNA damage response factors to their viral genome to facilitate homologous recombination replication in association with the viral replication factors E1 and E2. We previously demonstrated that SIRT1 deacetylation of WRN promotes recruitment of WRN to E1-E2 replicating DNA and that WRN regulates both the levels and fidelity of E1-E2 replication. The deacetylation of WRN by SIRT1 results in an active protein able to complex with replicating DNA, but a protein that is less stable. Here, we demonstrate an inverse correlation between SIRT1 and WRN in CIN cervical lesions compared to normal control tissue, supporting our model of SIRT1 deacetylation destabilizing WRN protein. We CRISPR/Cas9 edited N/Tert-1 and N/Tert-1+HPV16 cells to knock out WRN protein expression and subjected the cells to organotypic raft cultures. In N/Tert-1 cells without WRN expression, there was enhanced basal cell proliferation, DNA damage, and thickening of the differentiated epithelium. In N/Tert-1+HPV16 cells, there was enhanced basal cell proliferation, increased DNA damage throughout the epithelium, and increased viral DNA replication. Overall, the results demonstrate that the expression of WRN is required to control the proliferation of N/Tert-1 cells and controls the HPV16 life cycle in these cells. This complements our previous data demonstrating that WRN controls the levels and fidelity of HPV16 E1-E2 DNA replication. The results describe a new role for WRN, a tumor suppressor, in controlling keratinocyte differentiation and the HPV16 life cycle