Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.

Overall, this study enhances our understanding of the role of c-Myc activation in chromosome 11 repositioning in mouse PreB v-abl/myc cells and a possible interaction between telomeres, TRF2, and lamin A/C underlying this phenomenon. Additionally, the importance of human 17q25.3 is confirmed as a potential region involved in NSCLC tumorigenesis. A utilizationof the 3D telomeric organization profiles is demonstrated a tendency to categorize NSCLC patients into different prognostic subgroups, underscoring a potential future value of its clinical application. / February 2017

While a large body of work has focused on the transcriptional regulation of cellular identity, the role of the mechanical properties of cells and the importance of their physical interactions with the local environment remains less well understood. In this project, we explored the impact of cytoskeleton-generated forces exerted on the nucleus in the context of early embryonic stem (ES) cell fate decisions. We chose to perturb force generating components in the cytoskeleton – notably the molecular motor non-muscle myosin II - and key structural and chromatin binding proteins in the nuclear envelope, notably, the lamins (LMNA), Lamin B receptor (LBR) and components of the LINC complex (nesprins/KASH). The structural proteins in the nuclear envelope regulate both the mechanical response of the nucleus to force and the stabilization of peripheral heterochromatin (repressed genes). Our hypothesis is that reducing forces transmitted directly to chromatin or increasing tethering of peripheral heterochromatin to the nuclear envelope would restrict access to lineage specific genes sequestered at the nuclear lamina and thereby either impair, or delay, differentiation. We found phenotypes in the capacity of mouse ES cells to specify to the neural lineage following our perturbations: overexpression of LMNA, LBR and KASH proteins resulted in a significant fraction of cells that did not express the neuroectoderm marker Sox1 after four days of differentiation, while inhibiting non-muscle myosin II delayed Sox1 expression in the entire population. Overexpression of LMNA and LBR did not affect the ability of the cells to exit the naive pluripotent state, which raises the possibility that the perturbations are halting the cells in a formative phase prior to lineage specification. Future work will focus on looking at genome-wide transcriptional changes accompanying differentiation combined with an analysis of spatial information of differentially regulated genes.

De nombreuses études montrent un rôle important de l'architecture du noyau sur la stabilité du génome. Les lamines sont les constituants majeurs de l’enveloppe nucléaire et sont impliquées dans de nombreux processus, notamment, la régulation génique, la réplication et le maintien de la structure du noyau. Il en existe 2 types, les lamines A/C et les lamines B. Certaines mutations des lamines A/C sont à l’origine de syndromes progéroïdes, classés jusqu’à présents en deux catégories : ceux associés à une dérégulation des lamines (laminopathies) et ceux associés à un défaut de réparation de l’ADN, dont l’Ataxie Télangiectasie (A-T). Il est proposé que le vieillissement prématuré observé dans les laminopathies est dû à un défaut de réparation de l’ADN, qui serait alors la voie commune d’induction de sénescence des syndromes progéroïdes. Ceci est appuyé par le fait que de plus en plus de données associent les mutations des lamines A/C à des défauts de réparation de l’ADN. La mise en évidence, par notre laboratoire d’une accumulation de lamine B1 dans A-T et dans deux autres syndromes progéroïdes, pose la question de l’impact de la surexpression de la lamine B1 sur la réparation de l’ADN, d’autant plus que de plus en plus de données associent une augmentation de la lamine B1 à de nombreux cancers, bien que le mécanisme moléculaire ne soit pas connu. Au cours de ma thèse, j’ai donc pu montrer, notamment à l’aide de substrats intra-Chromosomiques, qu’une surexpression de lamine B1 entraînait un défaut de réparation des cassures double-Brin par NHEJ associé à un défaut de recrutement de 53BP1 à la cassure. La mise en évidence d’une interaction entre 53BP1 et la lamine B1, rompue après dommages permet de suggérer un nouveau rôle de la lamine B1 comme réservoir de 53BP1, régulant son recrutement aux cassures. De plus, d’autres résultats suggèrent que la lamine B1 agirait également au niveau de la signalisation du dommage en altérant l’activation d’ATM par un mécanisme qu’il reste à caractériser. L’ensemble de ces résultats montrent un nouveau rôle très important de la lamine B1 dans la signalisation des dommages et la régulation du recrutement des protéines de réparation, ouvrant la voie à une meilleure compréhension de l’implication de la lamine B1 dans la sénescence et le cancer. / Many studies show an important role of nuclear shape on genome stability. Lamins are the major components of the nuclear envelope and are implicated in numerous processes like gene regulation, DNA replication and the maintenance of nuclear structure. There are 2 types of lamins : lamin A/C and lamin B. Some mutations of lamin A/C cause progeroid syndromes, which are classified untill now in two categories : those due to lamins deregulation and those due to DNA repair defects, including Ataxia Telangiectasia (A-T). Accelerated aging observed in laminopathies is proposed to be due to a DNA repair defect, which would be the common pathway leading to senescence in progeroid syndromes. This is supported by many data linking lamin A mutations to DNA repair defects. Our laboratory reported that lamin B1 accumulates in A-T and Fanconi and another study showed also an accumulation in Werner syndrome, which is another progeroïd syndrome. This discovery raises a question about the impact of lamin B1 overexpression on DNA repair, especially as more and more data show an increase of lamin B1 in several cancers, although the molecular mechanism is still unclear. During my thesis, I showed, in particular with intrachromosomal substrates, that lamin B1 overexpression leads to an NHEJ double-Strand break (DSB) repair defect associated with a defect of 53BP1 recruitment to the break. The discovery of an interaction between 53BP1 and lamin B1, which is broken after damage, suggests a new role of lamin B1 as a « reservoir » of 53BP1, regulating its recruitment to the break. In addition, we obtained results suggesting that lamin B1 could also act in the DSB signalisation pathway by affecting ATM activation through a mechanism that we still have to characterize.All together, these datas show a new important role of lamin B1 in DSB signalisation and in the regulation of the recruitment of repair proteins, paving the way to a better understanding of the implication of lamin B1 in senescence and cancer.

No description available.

A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.

La surexpression de la lamine B1, un composant majeur de l'enveloppe nucléaire, a été rapportée dans diverses tumeurs. Cependant, les causes et les conséquences de cette augmentation sur la stabilité du génome n'ont pas été étudiées à ce jour. En effet, l'instabilité du génome est considérée comme une caractéristique majeure des cellules cancéreuses. Pour assurer le maintien de la stabilité du génome, les cellules ont développé de multiples et complexes mécanismes parmi lesquels les voies de réparation de l'ADN et la gestion du stress réplicatif sont essentielles. Au cours de ma thèse, l'impact de l'augmentation de niveau de lamine B1 sur la stabilité du génome, en particulier sur la réparation de cassure double-brin (CDB) et sur le contrôle du stress réplicatif a été étudié. En effet, nous montrons qu'une augmentation de la lamine B1 entraîne une accumulation de CDB et leur persistance en réponse à l'irradiation (foyers γH2AX), en plus d'une sensibilité accrue à l'irradiation (formation de colonies et cassures chromosomiques). Les cellules surexprimant la lamine B1 montrent également des défauts de recrutement de 53BP1 aux sites de dommages d’ADN, couplés à une diminution de l'efficacité de la réparation de CDB par NHEJ (Non-Homologous End-Joining). De plus, nous avons identifié une interaction directe entre la lamine B1 et 53BP1 régulant le recrutement de ce dernier aux CDB. Nos résultats supportent un modèle dans lequel l'augmentation de la lamine B1 conduit à la séquestration de 53BP1, modifiant ainsi son recrutement aux CDBs. En parallèle, nous montrons que les cellules surexprimant la lamine B1 présentent des signes accrus de stress réplicatif tels que l'accumulation de foyers spontanés de p-RPA, l'augmentation des figures radiales lors du traitement par mitomycine C, et une sensibilité accrue au traitement par camptothécine. Nous avons en outre cherché à identifier les causes de l'augmentation du stress réplicatif dans ces cellules, et les conséquences potentielles, en particulier sur l'induction de phénotypes inflammatoires. En fait, nous montrons que la surexpression de la lamine B1 conduit à une diminution de l'efficacité de la réparation de CDB par la recombinaison homologue, couplée à un défaut de formation de foyers BRCA1 après irradiation. De plus, nous avons obtenu des données préliminaires suggérant une induction de l'inflammation lors de la surexpression de la lamine B1. En résumé, ce travail de Thèse a permis d’identifier un nouveau mécanisme régulant le recrutement de 53BP1 aux CDB par son interaction avec la lamine B1, et souligne le rôle de l'augmentation de la lamine B1 dans la promotion de l'instabilité génomique au moins partiellement par des défauts de réparation de CDB et une augmentation de stress réplicatif. Après confirmation de l'induction de phénotypes inflammatoires, nous aurions identifié des rôles de l'augmentation de la lamine B1 dans la promotion de deux caractéristiques majeures du cancer - l'instabilité génomique et l'inflammation - favorisant ainsi le rôle de la lamine B1 dans le développement tumoral et proposant cette dernière comme une cible thérapeutique antitumorale potentielle. / The overexpression of lamin B1, a major component of nuclear envelope, has been reported in various tumors. However, the causes and consequences of this increase on the genome stability have not been studied to date. Indeed, genome instability is considered a major hallmark of cancer cells. To ensure the maintenance of genome stability, cells have developed multiple complex mechanisms among which pathways of DNA repair and replication stress management are essential. Therefore, during my thesis the impact of an increased lamin B1 level on genome stability, in particular on double-strand break (DSB) repair and on the control of replication stress was studied. Indeed, we show that increased lamin B1 leads to an accumulation of DSBs and their persistence in response to irradiation (γH2AX foci), in addition to an increased sensitivity to irradiation (colony formation and chromosomal breaks). Lamin B1 overexpressing cells also show defects in the recruitment of 53BP1 to damage sites, coupled to a decreased efficiency of DSB repair by Non-Homologous End-Joining. Moreover, we identified a direct interaction between lamin B1 and 53BP1 regulating the latter’s recruitment to DSBs. Our results support a model where increased lamin B1 leads to the sequestration of 53BP1, thereby altering its recruitment to DSBs. In parallel, we show that cells overexpressing lamin B1 display increased signs of replication stress such as accumulation of spontaneous p-RPA foci, increased radial chromosomes upon mitomycin C treatment, and enhanced sensitivity to treatment with camptothecin. We further aimed to identify the causes of the increased replication stress in these cells, in addition to the potential consequences, in particular on the induction of inflammatory phenotypes. In fact, we show that lamin B1 overexpression leads to a decreased efficiency of DSB repair by Homologous Recombination, coupled to a defect in irradiation-induced BRCA1 foci formation. In addition, we obtained preliminary data suggesting a possible induction of inflammation upon lamin B1 overexpression. Altogether, this work identifies a novel mechanism regulating the recruitment of 53BP1 to damage sites through its interaction with lamin B1, and highlights the role of increased lamin B1 in promoting genome instability at least partially through defective DSB repair and increased replication stress. Upon confirming the induction of inflammatory phenotypes, we would have identified roles of increased lamin B1 in promoting two major hallmarks of cancer – genomic instability and inflammation - thereby favorizing a role for lamin B1 in tumor development and proposing the latter as a potential anti-tumor therapeutic target.

Introdução: As Distrofias Musculares Congênitas (DMCs) são um grupo clínica e geneticamente heterogêneo de doenças musculares que se manifestam ao nascimento ou no primeiro ano de vida, sendo caracterizadas por hipotonia, fraqueza muscular, retardo do desenvolvimento motor e retrações fibrotendíneas. O músculo esquelético apresenta-se distrófico, mas sem alterações estruturais específicas. Em quase metade dos casos a doença é causada pela deficiência da laminina alfa;-2 (merosina). Outras deficiências proteicas descritas incluem: colágeno VI, selenoproteína N1, várias glicosiltransferases responsáveis pela glicosilação da alfa- distroglicana e lamina A/C. Vários genes já foram identificados. Objetivo: o objetivo deste estudo foi a caracterização clínica, histológica e molecular das crianças com DMC por deficiência de lamina A/C. Método: Foram incluídos 13 pacientes com diagnóstico clínico e histológico de DMC, com expressão muscular normal para distrofina, sarcoglicanas, merosina, colágeno 6 e disferlina. Os pacientes foram reavaliados segundo protocolo clínico e neurológico. As biópsias musculares realizadas previamente foram revisadas e o estudo das mutações no gene da lamina A/C foi realizado através de sequenciamento de toda região codificadora do gene. Resultados: Identificamos mutações em 30,7% dos pacientes (quatro casos) com fenótipo clínico de DMC por deficiência de lamina A/C. Todas as mutações encontradas (p.E358K, p.R249W, e p.N39S) ocorreram em heterozigose e de novo e já haviam sido descritas na literatura em pacientes com distrofias musculares. Em geral, estes pacientes apresentavam um grave comprometimento motor com o característico aspecto de cabeça caída, com início dos sintomas nos primeiros dois anos de vida. A CPK estava elevada entre 2 a 6 vezes o padrão superior da normalidade. O padrão histológico variou desde um músculo levemente até gravemente distrófico. Curiosamente, no estudo histológico do músculo, um dos pacientes apresentou agregados intracitoplasmáticos. Um outro paciente apresentava associadamente alterações neurogênicas ao estudo eletroneuromiográfico. Em todos os casos observamos complicações respiratórias, cardíacas e distúrbios de deglutição. Houve um caso de morte súbita, provavelmente em decorrência de arritmia cardíaca. Conclusões: A correlação genótipo-fenótipo permanece difícil, mas todos os casos apresentaram sinal da cabeça caída, comprometimento respiratório, cardíaco e biópsia muscular distrófica. A ampliação do conhecimento clínico e histológico pode orientar o diagnóstico e direcionar para o estudo molecular adequado, além de permitir o diagnóstico precoce das complicações, tão frequentes na DMC por deficiência de lamina A/C. Os exons 1, 4 e 6 são os mais frequentemente mutados e devem ser pesquisados inicialmente. Esta série de casos contribui também por demonstrar a distribuição universal da doença / Background: The Congenital Muscular Dystrophies (CMD) are a clinically and genetically heterogeneous group of myopathies characterized by muscle hypotonia, delayed motor development and early onset of progressive muscle weakness with dystrophic pattern on muscle biopsy. The clinical course is broadly variable and can comprise the involvement of the brain and eyes. Almost half of the cases is caused by deficiency of laminin-alfa 2 (merosin). Other protein deficiencies described include: collagen VI, selenoprotein N1, several glycosyltransferases responsible for glycosylation of alfa-dystroglycan and lamin A/C. Several genes have been identified and the increased knowledge of new clinical and histological forms of CMD can guide diagnosis and direct appropriate molecular studies. LMNA-related CMD is often characterized by muscle weakness and a dropped head developed in the early years of life. Regarding lamin A/C deficiency, the immunohistochemical findings can be normal, probably because the protein change is functional only; this makes diagnosis using muscle samples more difficult. Objectives: The aim of this study was to characterize the clinical, histological and molecular aspects in patients with CMD related to deficiency of lamin A/C. Methods: thirteen children with clinical and histological diagnosis of CMD with normal muscle expression for dystrophin, merosin, collagen 6, sarcoglycans and dysferlin were included in this study. The LMNA gene was sequenced after amplification of all coding exons. In addition, the muscle biopsies were revised. Results: In 30.7% (four cases) of our patients with typical clinical phenotype of lamin A/C deficiency were detected mutations on LMNA gene and all of them presented dropped-head syndrome, restrictive ventilator insufficiency, cardiac changes, increased serum CPK level and myopathic/dystrophic aspect on muscle biopsy. Two of the patients had normal motor development milestones in the first months of life and subsequently developed cervical and limb weakness. The other two patients presented a more severe motor involvement and failure to walk. One patient showed associated peripheral neuropathy. Curiously one case had myofibrillar aggregates on muscle biopsy. All mutations (p.E358K, p.R249W and p.N39S) were heterozygous and de novo and had been previously described in patients with muscular dystrophy. Conclusion: Genotype/phenotype correlation in CMD remains difficult. However patients with LMNA mutation and CMD seems to have a more homogeneous phenotype characterized by dropped head, severe motor disability, and cardiac and pulmonary involvement. Mutations on exons 1, 4 and 6 should be tested first. This case series also contributes for showing the universal distribution of the disease

Introdução: As Distrofias Musculares Congênitas (DMCs) são um grupo clínica e geneticamente heterogêneo de doenças musculares que se manifestam ao nascimento ou no primeiro ano de vida, sendo caracterizadas por hipotonia, fraqueza muscular, retardo do desenvolvimento motor e retrações fibrotendíneas. O músculo esquelético apresenta-se distrófico, mas sem alterações estruturais específicas. Em quase metade dos casos a doença é causada pela deficiência da laminina alfa;-2 (merosina). Outras deficiências proteicas descritas incluem: colágeno VI, selenoproteína N1, várias glicosiltransferases responsáveis pela glicosilação da alfa- distroglicana e lamina A/C. Vários genes já foram identificados. Objetivo: o objetivo deste estudo foi a caracterização clínica, histológica e molecular das crianças com DMC por deficiência de lamina A/C. Método: Foram incluídos 13 pacientes com diagnóstico clínico e histológico de DMC, com expressão muscular normal para distrofina, sarcoglicanas, merosina, colágeno 6 e disferlina. Os pacientes foram reavaliados segundo protocolo clínico e neurológico. As biópsias musculares realizadas previamente foram revisadas e o estudo das mutações no gene da lamina A/C foi realizado através de sequenciamento de toda região codificadora do gene. Resultados: Identificamos mutações em 30,7% dos pacientes (quatro casos) com fenótipo clínico de DMC por deficiência de lamina A/C. Todas as mutações encontradas (p.E358K, p.R249W, e p.N39S) ocorreram em heterozigose e de novo e já haviam sido descritas na literatura em pacientes com distrofias musculares. Em geral, estes pacientes apresentavam um grave comprometimento motor com o característico aspecto de cabeça caída, com início dos sintomas nos primeiros dois anos de vida. A CPK estava elevada entre 2 a 6 vezes o padrão superior da normalidade. O padrão histológico variou desde um músculo levemente até gravemente distrófico. Curiosamente, no estudo histológico do músculo, um dos pacientes apresentou agregados intracitoplasmáticos. Um outro paciente apresentava associadamente alterações neurogênicas ao estudo eletroneuromiográfico. Em todos os casos observamos complicações respiratórias, cardíacas e distúrbios de deglutição. Houve um caso de morte súbita, provavelmente em decorrência de arritmia cardíaca. Conclusões: A correlação genótipo-fenótipo permanece difícil, mas todos os casos apresentaram sinal da cabeça caída, comprometimento respiratório, cardíaco e biópsia muscular distrófica. A ampliação do conhecimento clínico e histológico pode orientar o diagnóstico e direcionar para o estudo molecular adequado, além de permitir o diagnóstico precoce das complicações, tão frequentes na DMC por deficiência de lamina A/C. Os exons 1, 4 e 6 são os mais frequentemente mutados e devem ser pesquisados inicialmente. Esta série de casos contribui também por demonstrar a distribuição universal da doença / Background: The Congenital Muscular Dystrophies (CMD) are a clinically and genetically heterogeneous group of myopathies characterized by muscle hypotonia, delayed motor development and early onset of progressive muscle weakness with dystrophic pattern on muscle biopsy. The clinical course is broadly variable and can comprise the involvement of the brain and eyes. Almost half of the cases is caused by deficiency of laminin-alfa 2 (merosin). Other protein deficiencies described include: collagen VI, selenoprotein N1, several glycosyltransferases responsible for glycosylation of alfa-dystroglycan and lamin A/C. Several genes have been identified and the increased knowledge of new clinical and histological forms of CMD can guide diagnosis and direct appropriate molecular studies. LMNA-related CMD is often characterized by muscle weakness and a dropped head developed in the early years of life. Regarding lamin A/C deficiency, the immunohistochemical findings can be normal, probably because the protein change is functional only; this makes diagnosis using muscle samples more difficult. Objectives: The aim of this study was to characterize the clinical, histological and molecular aspects in patients with CMD related to deficiency of lamin A/C. Methods: thirteen children with clinical and histological diagnosis of CMD with normal muscle expression for dystrophin, merosin, collagen 6, sarcoglycans and dysferlin were included in this study. The LMNA gene was sequenced after amplification of all coding exons. In addition, the muscle biopsies were revised. Results: In 30.7% (four cases) of our patients with typical clinical phenotype of lamin A/C deficiency were detected mutations on LMNA gene and all of them presented dropped-head syndrome, restrictive ventilator insufficiency, cardiac changes, increased serum CPK level and myopathic/dystrophic aspect on muscle biopsy. Two of the patients had normal motor development milestones in the first months of life and subsequently developed cervical and limb weakness. The other two patients presented a more severe motor involvement and failure to walk. One patient showed associated peripheral neuropathy. Curiously one case had myofibrillar aggregates on muscle biopsy. All mutations (p.E358K, p.R249W and p.N39S) were heterozygous and de novo and had been previously described in patients with muscular dystrophy. Conclusion: Genotype/phenotype correlation in CMD remains difficult. However patients with LMNA mutation and CMD seems to have a more homogeneous phenotype characterized by dropped head, severe motor disability, and cardiac and pulmonary involvement. Mutations on exons 1, 4 and 6 should be tested first. This case series also contributes for showing the universal distribution of the disease

Failure to process prelamin A, by the enzyme ZMPSTE24, leads to the build up of farnesylated prelamin A, which has been implicated in causing the symptoms experienced in laminopathies and HIV therapy. A common feature to these conditions is the development of an irregular nuclear boundary, often including deep invaginations that form a nucleoplasmic reticulum. Additionally, dysregulated lipid synthesis is frequently associated with improper lamin A processing and I set out to address the molecular mechanisms behind these two features that could explain lipoatrophy experienced in patients. By using siRNA targeted against Zmpste24 I utilised an array of biochemical, molecular and imaging techniques to uncover a mechanism that leads to the production of a nucleoplasmic reticulum that was dependent on both the farnesylated tail of prelamin A and the phosphatidylcholine synthesising enzyme CCTα. The morphology of this structure consisted of an invagination of both the inner and outer nuclear membranes with a cytoplasmic core or just invagination of the inner nuclear membrane. Serial section dual axis electron tomography provided a new insight into the ultrastructural changes at the nuclear periphery that revealed novel structural features. The dysregulation of lipid synthesis was assessed by investigating the effects farnesylated prelamin A has on the distribution and dynamics of the transcription factor SREBP-1 and assessment of the downstream consequences this has on its targets that regulate adipocyte differentiation potential. Finally, the metabolomic profile of an HIV protease inhibitor that leads to prelamin A build up was generated and revealed increases in lipolysis, glycolysis and mediators of inflammation. The research presented offers a new insight into the development of a convoluted nuclear boundary and nucleoplasmic reticulum, in the context of lamin A mutants and how dysregulated lipid synthesis, caused by farnesylated prelamin A, leads to lipoatrophy.