The genetic diseases Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) arise from accumulation of farnesylated prelamin A because of defects in the lamin A maturation pathway. Both of these diseases exhibit symptoms that can be viewed as accelerated aging. The mechanism by which accumulation of farnesylated prelamin A leads to these accelerated aging phenotypes is not understood. Here we present evidence that in HGPS and RD fibroblasts, DNA damage checkpoints are persistently activated because of the compromise in genomic integrity. Inactivation of checkpoint kinases Ataxia-telangiectasia-mutated (ATM) and ATR (ATM- and Rad3-related) in these patient cells can partially overcome their early replication arrest. Treatment of patient cells with a protein farnesyltransferase inhibitor (FTI) did not result in reduction of DNA double-strand breaks and damage checkpoint signaling, although the treatment significantly reversed the aberrant shape of their nuclei. This suggests that DNA damage accumulation and aberrant nuclear morphology are independent phenotypes arising from prelamin A accumulation in these progeroid syndromes. Since DNA damage accumulation is an important contributor to the symptoms of HGPS, our results call into question the possibility of treatment of HGPS with FTIs alone.

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Un lien étroit existe entre l’intégrité du génome et l’architecture nucléaire. Les lamines, composants majeurs de l’enveloppe nucléaire sont impliquées dans de nombreux processus nucléaires, tels que la réplication, la transcription et le maintien de l’architecture nucléaire. Il a notamment été rapporté que les lamines de type A sont impliquées dans la réparation des cassures double brin de l’ADN et la stabilité des télomères. Toutefois, peu d’études ont été réalisées sur les lamines de type B. Fait intéressant, il a été observé que l’accumulation de la lamine B1 est retrouvée dans différentes tumeurs. Cependant, les conséquences d’une dérégulation de cette lamine sur la stabilité du génome restent peu documentées.Au cours de ma thèse, je me suis intéressée à l’impact d’une dérégulation de la lamine B1 sur le maintien de la stabilité du génome, notamment sur la réparation des cassures double brin de l’ADN et la stabilité des télomères. Nous avons pu mettre en évidence que la surexpression de lamine B1 conduit à un défaut de réparation par NHEJ, associé à une diminution de recrutement de 53BP1 aux dommages radio-induits. Nous avons également démontré que la lamine B1 interagit directement avec 53BP1, protéine impliquée dans le choix de la voie de réparation, et que cette interaction est régulée en cas de dommages à l’ADN. En effet, la liaison entre ces deux protéines est rompue après dommages en condition endogène, ce qui n’est pas le cas après surexpression de la lamine B1. Ce défaut de recrutement de 53BP1 aux dommages pourrait rendre compte de la diminution de l’efficacité du NHEJ. De plus, j’ai pu identifier les domaines protéiques impliqués dans cette interaction. Il est intéressant de noter que la surexpression du domaine de la lamine B1 impliquée dans l’interaction mime la surexpression de la lamine B1 entière. Au contraire, la lamine B1 délétée de ce domaine n’a aucun impact sur le recrutement de 53BP1 et la persistance des dommages. Ces différentes données confortent notre hypothèse quant à la séquestration de 53BP1 après surexpression de lamine B1.En parallèle, nous avons pu démontrer que la surexpression de la lamine B1 entraine l’apparition de diplochromosomes concomitants à une sénescence accrue. Ce phénomène d’endoréplication peut être induit par des défauts télomériques, tels que des télomères dysfonctionnels ou déprotégés. De façon intéressante, mes données montrent que la surexpression de la lamine B1 entrainent des dommages télomériques. Nous avons également établit que la lamine B1 interagit avec TRF2, protéine du complexe « shelterin » permettant la protection des télomères contre la signalisation des dommages à l’ADN. La rétention putative de TRF2 par la lamine B1 pourrait être à l’origine des défauts télomériques observés après la surexpression de cette dernièreCette étude démontre de nouveaux rôles de la lamine B1 dans le maintien de la stabilité du génome, notamment à travers ses interactions avec deux protéines clefs dans la réparation des cassures double brin et la stabilité des télomères. Cela nous ouvre de nouvelles pistes de recherche qui permettront une meilleure compréhension des mécanismes moléculaires impliqués dans la tumorigenèse et en particulier sur le lien existant entre l’intégrité de l’architecture nucléaire et la stabilité du génome. / A close link exists between genome stability and nuclear architecture. Lamins, major component of the nuclear envelope, are involved in many nuclear processes, such as replication, transcription and nuclear architecture. It has been reported than lamins A/C are involved in double strand break repair and telomere stability. However, few studies have been conducted on B-type lamins. Interestingly, it was observed that the accumulation of lamin B1 is found in different tumors. Nevertheless, consequences of its deregulation on genome stability remain poorly documented.During my PhD, I analysed the impact of deregulation of lamin B1 on genome maintenance, including double-strand breaks repair and telomere stability. We were able to demonstrate that overexpression of lamin B1 leads to defect of NHEJ, associated with decrease of the 53BP1 recruitment to DNA damage. We have also shown that lamin B1 interacts directly with 53BP1, a protein involved in the choice of the repair pathway, and that this interaction is regulated upon DNA damage. Indeed, the association between these two proteins is disrupted after damage, in endogenous condition, in contrast this dissociation is not observed after lamin B1 overexpression. The defect of 53BP1 recruitment to DNA damage could account for the decrease in the NHEJ efficiency. Moreover, I have identify the protein domains involved in this interaction. It is interesting to note that overexpression of the interaction domain mimics the overexpression of the full lamin B1. Instead, lamin B1 deleted from this domain has no impact on 53BP1 recruitment and on DNA damage persistence. These data support our hypothesis about the sequestration 53BP1 after overexpression of lamin B1.In parallel, we have demonstrated that the lamin B1 overexpression causes the appearance of diplochromosomes concurrent to an increase of senescence. This phenomenon of endoreduplication can be induced by telomere defects such as dysfunctional or deprotected telomeres. Interestingly, I have observed that lamin B1 overexpression leads telomere damages. We also established that lamin B1 interacts with TRF2, a protein of "shelterin" complex involved in the protection against the DNA damage signaling at telomere. The putative retention TRF2 by lamin B1 could cause telomere defects observed after overexpression of the latter.This study identifies new roles of lamin B1 in maintaining genome stability, including through its interactions with two key proteins in the repair of double-strand breaks and stability of telomeres. This opens up new ways of research that will enable a better understanding of the molecular mechanisms involved in tumorigenesis and in particular on the relationship between the integrity of the nuclear architecture and genome stability.

La forme du noyau peut varier significativement au cours du développement ou lors de processus pathologiques en raison des forces mécaniques émanant du microenvironnement ou générées par le cytosquelette. L’impact de la morphologie nucléaire sur la machinerie transcriptionnelle n’est cependant pas connu. En utilisant plusieurs approches afin de manipuler la morphologie nucléaire, nous avons observé que des changements de forme de l’enveloppe nucléaire régulent l’activité de AP1 et TEAD. Nous avons montré que l’aplatissement du noyau augmente la phosphorylation de c-Jun et la translocation de YAP, conduisant à une augmentation de la transcription des gènes cibles de AP1 et TEAD. Nous avons également observé que l’aplatissement du noyau se produit au cours du cycle cellulaire et favorise la prolifération via l’activation de TEAD et AP1 qui stimulent la progression de la phase G1 à la phase S. / .The shape of the cell nucleus can vary considerably during developmental and pathological processes as a consequence of the mechanical forces emanating from the microenvironment or generated by the cytoskeleton. However the impact of nuclear morphology on the transcriptional machinery is not known. Using a combination of tools to manipulate the nuclear morphology, we observed that changes in nuclear shape regulate the activity of AP1 and TEAD. We showed that nuclear flattening increases c-Jun phosphorylation and YAP nuclear translocation, leading to transcriptional induction of AP1 and TEAD-target genes. Surprisingly, we found that nuclear compression is necessary and sufficient to mediate c-Jun and YAP activation in response to cell- generated contractility or cell spreading. We additionally observed that nuclear flattening occurs during the cell cycle and promotes proliferation via TEAD and AP1- dependent G1 to S progression.

The A-type lamins are intermediate filament proteins that constitute a major part of the eukaryotic nuclear lamina—a tough, polymerized, mesh lining of the inner nuclear membrane, providing shape and structural integrity to the nucleus. Lamin A (LA) filaments also permeate the nucleoplasm, providing additional structural support, but also scaffolding numerous tethered molecules to stabilize, organize, and facilitate molecular interactions to accomplish critical functions of cellular metabolism. Over the past 2 decades, much attention has been focused on roles of LA in maintenance of nuclear structural integrity. Only since the late 1990s have scientists discovered the devastating effects of LA gene (LMNA) mutations, as they have associated hundreds of LMNA mutations to a large group of diseases, called laminopathies, with a broad spectrum of phenotypes, ranging from skeletal, muscular, and neurological defects, to defective lipid storage, to accelerated aging phenotypes in diseases called progerias. Recent advances demonstrate LA regulatory functions include cell signaling, cell cycle regulation, transcription, chromatin organization, viral egress, and DNA damage repair. Amidst the flurry of fascinating research, only recently have researchers begun to focus attention on the different isoforms that exist for LA, a precursor form among them. LA is initially synthesized as Prelamin A (PreA), and undergoes a series of modifications that truncate the protein to produce “mature” LA. Existence of the precursor form, and its complex maturation pathway, have puzzled researchers since their realization. With a pattern of expression related to cell cycle phase, we hypothesized a role for PreA in cell cycle control. To investigate, we have performed array studies to assess gene expression effects at the levels of transcript expression, protein expression, and phosphorylation modification status. Here, we present evidence for a PreA-mediated program of cell cycle regulatory gene and protein expression modulation. Implicated pathways include RB-E2F, p53, p27Kip1, FoxOs, p300, and the Cyclins, with additional evidence indicating a role for the Pin1 prolyl isomerase in mediating PreA regulation of the cell cycle.

<p dir="ltr">Premature aging disorders such as mandibuloacral dysplasia disorder (MAD) can be caused by improper maturation of nuclear scaffolding protein lamin A from its precursor prelamin A. ZMPSTE24 is responsible for both the earlier C-terminal CAAX cleavage and a subsequent N-terminal upstream cleavage during the posttranslational processing of prelamin A to lamin A. Although ZMPSTE24’s structure and function are well characterized, the role of the 4 apparent openings into its hollow inner chamber remains unknown. We hypothesize that one of these entrances, portal 1, is the initial substrate entry point based on its proximity to the zinc-coordinating active site. Unfortunately, ZMPSTE24 is difficult to express and purify. Fortunately, the yeast homolog, Ste24, not only shares many structural and functional similarities to ZMPSTE24 but is also much easier to express and purify in an active state. Therefore, we will use Ste24 and its substrate <b>a</b>-factor as a model system for ZMPSTE24 and its substrate prelamin A to deduce whether portal 1 acts as the primary substrate entry point. We examined portal 1’s function in primary substrate entry by observing how the incubation of portal 1 mutants engineered with cysteine residues around the portal with cysteine-reactive bismaleimide crosslinkers affects the activity of the C-terminal CAAX cleavage. If crosslinking of the cysteine residues occludes the portal, we hypothesize that activity will decrease because substrate cannot enter. The cysteine-less enzyme Ste24 (QA), which cannot react with the crosslinkers, was engineered by mutagenesis to contain 1 or 2 new cysteines at specific positions around this portal. We hypothesize that portal 1 occlusion with cysteine reactive bismaleimide crosslinkers will inactivate the enzyme by preventing substrate entry. We monitored changes in CAAX cleavage activity with a radioactive endoprotease-coupled CAAX assay.</p><p dir="ltr">In crude membranes derived from yeast expressing QA Ste24, activity was not inhibited in the presence of either BMH or BMOE crosslinker. For the single cysteine-containing mutants M210C, T267C, I307C, and V311C, each crosslinker similarly decreased activity over 50%. For the double cysteine-containing mutants M210C-I307C, T267C-I307C, and T267C-V311C, we found between 20-60% decreased activity in the presence of the crosslinker which has a length most similar to the distance between the two cysteines. These results closely reflect previous data and further suggest that CAAX activity of the enzyme may be decreased due to the occlusion of the primary entry site, portal 1.</p><p dir="ltr">With purified QA Ste24, changes in activity were less apparent. Activity for purified QA was not decreased in the presence of either crosslinker. Single cysteine-containing mutants did not show decreased activity in the presence of either crosslinker. Unlike what was observed in crude membrane preparations, the double-cysteine containing mutants exhibited minimal decrease in activity in the presence of the crosslinker that has a length most similar to the distance between the two cysteines.</p><p dir="ltr">In crude membrane preparations, the cysteine-containing QA Ste24 mutants have diminished activity in the presence of crosslinkers. This may be due to the occlusion of the primary entry point, portal 1. However, we recognize the possibility that the decrease in activity was the result of the occlusion of the exit portal site. It is imperative that further experiments confirm that exit portal occlusion is not occurring. For purified cysteine-containing QA Ste24 mutants, the negligible decrease in activity suggests either that the portal 1 is not the primary substrate entry point or that the conditions of the assay were not optimized to generate inhibition. For example, the concentration of crosslinker was not sufficient in the presence of excess lipid and the reconstitution mixture sequestered the crosslinker. Further optimization of the reaction conditions is warranted. The cysteine-containing QA Ste24 mutants must be assessed for free thiols to determine how successful the reaction with the crosslinkers is. A more developed understanding of how all four portals function in the Ste24 CAAX processing of <b>a</b>-factor will be very insightful towards the mechanism of ZMPSTE24 in lamin A CAAX processing and may catalyze new targets of study for ZMPSTE24-related diseases like MAD-B.</p>

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13568号 / 論医博第2294号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 森田 智視, 教授 石見 拓, 教授 佐藤 俊哉 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

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.

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.