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International audience; When telomeres shorten, they are thought to lose their protective caps at a critical short length, activating the DNA damage response and recruiting DNA damage repair activities that would degrade, fuse or recombine dysfunctional telomeres. However, the structure(s) of short and dysfunctional telomeres, which respectively trigger permanent replicative senescence and/or potentially promote genome instability, remain unclear.To define the structure of telomeres at the point of dysfunction and the fate of cells carrying them, we have developed a system called FinalCut in Saccharomyces cerevisiae to induce a single telomere of defined length in cells in which we can conditionally inactivate telomerase. This allows 1/ the structural analysis of the shortest telomere in cells, 2/ the monitoring of the pace and the number of consecutive cell cycles from telomerase inactivation to cell death with our microfluidic system, and 3/ the combination of our single cell - single telomere resolved data with a mathematical model of senescence in budding yeast.Our results show that a very small fraction of cells can enter senescence in a probabilistic manner, as cells proliferate in the absence of telomerase. However, for the large majority of cells, when the shortest telomere reaches a critical threshold length, cells immediately stop proliferation. This is consistent with the model that the first telomere reaching a short threshold length is sufficient to abruptly stop cell divisions in a large majority of cells. The structure of this critically short telomere will be discussed.

International audience; Telomeres ensure genome integrity and are maintained by telomerase. During replicative senescence, telomerase is inactivated, telomere sequences progressively shorten and set the limit for cell proliferation. When telomeres shorten, they are thought to lose their protective caps at a critical short length, which activates the DNA damage response and recruits DNA damage repair activities that would degrade, fuse, or recombine dysfunctional telomeres. However, the structure(s) of short and dysfunctional telomeres, which respectively trigger permanent replicative senescence or potentially promote genome instability, remain unclear.To define the structure of telomeres at the point of dysfunction and the fate of cells carrying them, we have developed in Saccharomyces cerevisiae a system called FinalCut to induce a single telomere of defined length in cells in which we can conditionally inactivate telomerase. This allows structural analysis of this telomere and, combined with the use of our microfluidic system to track consecutive cell cycles from telomerase inactivation to cell death, we can achieve single telomere and single cell resolution. Our results show that cells reach senescence earlier and more synchronously, as the shortest telomere is set at a shorter length. This is in agreement with the first telomere reaching a short length being sufficient to trigger senescence in budding yeast. Below a certain threshold, the shorter telomere becomes unstable and degrades. However, we can define a length for which a short telomere appears to be stable, and the cells stop dividing at the same time. Combined with a mathematical model of senescence, our results suggest that the probability of a telomere signaling senescence increases as it shortens, but at low frequencies. When a telomere reaches a critical length, it can be stably maintained in cells, while activating the DNA damage checkpoint, without causing obvious fusions or degradation. The structure of this critically short telomere will be discussed.

International audience; Progress in understanding telomere replication and its relationship to senescence has been hampered by the intrinsic variations in telomeres and the stochastic nature of senescence onset. The work in our lab relies on our ability to circumvent and experimentally dissect the causes of this heterogeneity. Our strategy is to manipulate and track single telomeres in individual cells and experimentally evaluate the effects on proliferation potential of single cell lines to build a mathematical model of senescence.We therefore set up a microfluidics-based live cell imaging assay to study replicative senescence in single Saccharomyces cerevisiae cell lineages after telomerase inactivation. We found that most lineages undergo an abrupt and irreversible transition consistent with a mathematical model in which the first telomere reaching a short critical length triggers the onset of senescence. Other cells exhibit transient checkpoint-dependent cell cycle delays followed by normal cell cycles before senescence. We have now shown that these two pathways to senescence correspond to two kinetically and mechanistically distinct, age-dependent processes underlying non- terminal and terminal senescence arrest.Taking advantage of our novel FinalCut system, we also investigated the proliferation potential of telomerase-negative cells with critically short telomeres of different defined lengths. Our results are consistent with a model whereby a small portion of cells may enter senescence in a probabilistic manner as the length of the shortest telomere decreases. Our results thus directly demonstrate that the length of the shortest telomere is a major determinant of entry into senescence, but a hidden parameter may also contribute to the onset of senescence upon telomerase inactivation. However, when the shortest telomere reaches a critical length, all cells immediately arrest. In addition, we found that at this critical length, the shortest telomere can be stably maintained without signs of fusions or degradations. This suggests that telomeres can be both critically short and functional with respect to telomere protection.

International audience; Telomeres ensure genome integrity and are maintained by telomerase. During replicative senescence, telomerase is inactivated, telomere sequences progressively shorten, and set the limit for cell proliferation. As telomeres shorten, they are thought to lose their protective caps at a critical short length, activating the DNA damage response and recruiting DNA damage repair activities that would degrade, fuse, or recombine dysfunctional telomeres. However, the structure(s) of short and dysfunctional telomeres, which respectively trigger permanent replicative senescence or potentially promote genome instability, remain unclear. To define the structure of telomeres at the point of dysfunction and the fate of cells carrying them, we developed a system called FinalCut to induce a single telomere of defined length in cells in which we can conditionally inactivate telomerase. This allows structural analysis of this telomere, and combined with the use of our microfluidic system to track consecutive cell cycles from telomerase inactivation to cell death, we can achieve single telomere and single cell resolution. Our results show that the loss of telomere stability is progressive as the telomere shortens, and that telomeres of a given length can be protected or unprotected in separate cells. We conclude that telomere length is not the sole determinant of the switch between functional and dysfunctional telomeres. We therefore propose that the minimum length of telomeres to maintain cell viability follows a probabilistic, rather than deterministic, model.

Gli HPV ad “alto rischio” rappresentano l’agente eziologico del carcinoma cervicale e, in particolare, HPV-16 è quello maggiormente riscontrato. L’oncoproteina E7, insieme ad E6, è la proteina virale più importante per l’insorgenza del tumore. Nel laboratorio dove è stata svolta questa tesi, è stata identificata una proteina cellulare, glutatione S-transferasi P1- 1 (GSTP1), in grado di interagire con E7 di HPV-16. È stato generato un modello molecolare tridimensionale dell’interazione tra queste due proteine, che ha permesso di ingegnerizzare una proteina E7 HPV-16 mutata fortemente deficitaria nella possibilità di legare GSTP1. L’espressione ectopica di E7 HPV-16 in cheratinociti umani immortalizzati (HaCaT) induce grandi cambiamenti nell’equilibrio redox cellulare. Il ruolo determinante di E7 nell’interazione con GSTP1 sembra essere quello di proteggere GSTP1 dall’ossidazione, che causerebbe una inattivazione della proteina cellulare. In particolare, E7 impedisce la formazione dello stato ossidato del monomero GSTP1. Nella forma monomerica ridotta, GSTP1 inibisce l’attivazione della cascata pro-apoptotica regolata da JNK1 in quanto non consente la fosforilazione di JNK1. Utilizzando tale meccanismo, E7 aumenta la sopravvivenza cellulare. Utilizzando la linea cellulare MCF-7, che non produce GSTP1 endogena, e siRNA, specifici per GSTP1, nelle HaCaT infettate con E7, è stato scoperto un ruolo cruciale di GSTP1 nel mediare l’azione di E7 HPV-16 in tale aumento della sopravvivenza cellulare. I dati ottenuti con la proteina virale mutata sono stati una valida conferma di questa ipotesi, che fornisce un’ulteriore tassello nei meccanismi molecolari indotti da E7 nella trasformazione della cellula infettata. Questo studio pone le basi per un possibile impiego di molecole in grado di interferire con il legame tra E7 HPV-16 e GSTP1 e, quindi, minimizzare l’aumentata sopravvivenza cellulare nelle cellule umane tumorali che esprimono E7 HPV-16. Human Papillomavirus (HPV)-16 is a paradigm for “high-risk” HPVs, which are the causative agents of virtually all cervical carcinomas. The viral oncoprotein E7, along with E6, is the key factor for the transformation of the infected cell. By direct protein-protein interaction, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular target of the E7 oncoprotein from HPV-16. After a molecular mapping of the HPV-16 E7 amino acids involved, we generated a three-dimensional molecular model of the interaction between the oncoprotein and GSTP1, which allowed us to engineer a HPV-16 E7 mutant molecule strongly impaired in its ability to bind GSTP1. Exogenous HPV-16 E7 expression in HaCaT human immortalized keratinocytes induced dramatic changes in the intracellular redox equilibrium. Nevertheless, in this context, HPV-16 E7 appeared to protect GSTP1 from oxidative inactivation, increasing the amount of reduced GSTP1, which, as a monomer, inhibits pro-apoptotic JNK phosphorylation, enhancing the survival ability of the cell. Using the GSTP1-deficient MCF-7 cancer cell line as well as siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing the wild-type or the mutant oncoprotein, we underscored a pivotal role for GSTP1 in the pro-survival program elicited by HPV-16 E7, providing further evidence of the transforming abilities of this oncoprotein. This study sets the groundwork for devising unique molecular tools to interfere with the interaction between HPV-16 E7 and GSTP1 and to minimize the survival of HPV-16 E7- expressing human cancer cells. Dottorato di ricerca in Genetica e biologia cellulare