Telomeres can fold into t-loops that may result from the invasion of the 3' overhang into duplex DNA. Their formation is facilitated in vitro by the telomeric protein TRF2, but very little is known regarding the mechanisms involved. Here we reveal that TRF2 generates positive supercoiling and condenses DNA. Using a variety of TRF2 mutants, we demonstrate a strong correlation between this topological activity and the ability to stimulate strand invasion. We also report that these properties require the combination of the TRF-homology (TRFH) domain of TRF2 with either its N- or C-terminal DNA-binding domains. We propose that TRF2 complexes, by constraining DNA around themselves in a right-handed conformation, can induce untwisting of the neighboring DNA, thereby favoring strand invasion. Implications of this topological model in t-loop formation and telomere homeostasis are discussed.

No abstract

Human telomeres are protected from DNA damage by a nucleoprotein complex that includes the repeat-binding factor TRF2. Here, we report that TRF2 regulates the 5' exonuclease activity of its binding partner, Apollo, a member of the metallo-beta-lactamase family that is required for telomere integrity during S phase. TRF2 and Apollo also suppress damage to engineered interstitial telomere repeat tracts that were inserted far away from chromosome ends. Genetic data indicate that DNA topoisomerase 2alpha acts in the same pathway of telomere protection as TRF2 and Apollo. Moreover, TRF2, which binds preferentially to positively supercoiled DNA substrates, together with Apollo, negatively regulates the amount of TOP1, TOP2alpha, and TOP2beta at telomeres. Our data are consistent with a model in which TRF2 and Apollo relieve topological stress during telomere replication. Our work also suggests that cellular senescence may be caused by topological problems that occur during the replication of the inner portion of telomeres.

Cellular senescence is a mechanism that virtually irreversibly suppresses the proliferative capacity of cells in response to various stress signals. This includes the expression of activated oncogenes, which causes Oncogene-Induced Senescence (OIS). A body of evidence points to the involvement in OIS of chromatin reorganization, including the formation of senescence-associated heterochromatic foci (SAHF). The nuclear lamina (NL) is an important contributor to genome organization and has been implicated in cellular senescence and organismal aging. It interacts with multiple regions of the genome called lamina-associated domains (LADs). Some LADs are cell-type specific, whereas others are conserved between cell types and are referred to as constitutive LADs (cLADs). Here, we used DamID to investigate the changes in genome-NL interactions in a model of OIS triggered by the expression of the common BRAF oncogene. We found that OIS cells lose most of their cLADS, suggesting the loss of a specific mechanism that targets cLADs to the NL. In addition, multiple genes relocated to the NL. Unexpectedly, they were not repressed, implying the abrogation of the repressive activity of the NL during OIS. Finally, OIS cells displayed an increased association of telomeres with the NL. Our study reveals that senescent cells acquire a new type of LAD organization and suggests the existence of as yet unknown mechanisms that tether cLADs to the NL and repress gene expression at the NL.

Cellular senescence is a largely irreversible form of cell cycle arrest triggered by various types of damage and stress, including oncogene expression (termed oncogene-induced senescence or OIS). We and others have previously demonstrated that OIS occurs in human benign lesions, acting as a potent tumor suppressor mechanism. Numerous phenotypic changes occur during OIS, both in the cytoplasm and in the nucleus. These include the activation of autophagy, a catabolic process operating in the cytoplasm and downregulation of lamin B1, a component of the nuclear lamina. However, it is unknown whether these changes relate to each other. We discovered that cells entering BRAF(V600E)- or H-RAS(G12V)-induced senescence downregulate not only lamin B1 but also lamin A, as well as several other nuclear envelope (NE) proteins, resulting in an altered NE morphology. Depletion of LMNB1 or LMNA/C was sufficient to recapitulate some OIS features, including cell cycle exit and downregulation of NE proteins. We further found that the global loss of NE proteins is a consequence of their degradation by the autophagy machinery, which occurs concomitantly with autophagy induction and increased lysosomal content and activity. Our study therefore reveals a previously unknown connection between autophagy and the disruption of NE integrity during OIS.

We investigated UV-induced signalling in an ex vivo skin organ culture model using phospho-antibody array. Phosphorylation modulations were analysed in time-course experiments following exposure to solar-simulated UV and validated by Western blot analyses. We found that UV induced P-p38 and its substrates, P-ERK1/2 and P-AKT, which were previously shown to be upregulated by UV in cultured keratinocytes and in vivo human skin. This indicates that phospho-antibody array applied to ex vivo skin organ culture is a relevant experimental system to investigate signalling events following perturbations. As the identified proteins are components of pathways implicated in skin tumorigenesis, UV-exposed skin organ culture model could be used to investigate the effect on these pathways of NMSC cancer drug candidates. In addition, we found that phospho-HCK is induced upon UV exposure, producing a new candidate for future studies investigating its role in the skin response to UV and UV-induced carcinogenesis.

La sénescence cellulaire est définie comme un arrêt permanent dans le cycle, généralement à la transition G1/S, dans lequel les cellules sont réfractaires à toute stimulation mitogénique [1]. Après un nombre défini de divisions, les cellules primaires humaines entrent en sénescence, suggé-rant qu'un mécanisme « compte » le nombre de divisions. L'érosion télomérique, rythmée par le nombre de divisions, est le principal mécanisme responsable de cette forme de sénescence, appelée sénescence réplicative. Néanmoins, d'autres voies, généralement liées à la réponse à un stress exogène, peuvent déclencher un arrêt cellulaire similaire à celui de la sénescence réplicative. C'est ce que l'on appelle la sénescence prématurée, qui peut être déclen-chée par une activation mitogénique supraphysiologique, en réponse par exemple à l'expression d'un oncogène, par un stress génotoxique ou par une modification de la chromatine, par exemple par des agents déméthylants. Tous les types de sénescence cellulaire ont en commun des voies de signalisation et des caractéristiques morphologiques. Ainsi, l'activation de p53 apparaît très importante pour l'entrée en sénescence. Il est bien documenté que la mort cellulaire peut servir à limiter la prolifération tumorale [2]. Plus récemment, une série d'articles a montré que la sénescence, lorsqu'elle est induite par des oncogènes, constitue aussi une barrière très efficace contre la formation de tumeurs malignes [3]. Dans ce contexte, deux articles viennent de démontrer que, chez la souris, la perte excessive d'ADN télomérique bloque le développement tumoral en induisant la sénes-cence réplicative [4,5]. Ces résultats confirment le rôle de la sénescence dans la suppression tumorale, indiquent que le dysfonctionnement télomérique peut forcer les cellules cancéreuses à rentrer en sénescence et attestent de l'intérêt pour des stratégies thérapeutiques anti-cancéreuses qui cibleraient la télomérase et les télomères. Structure et fonctions des télomèresLes télomères préservent l'intégrité du matériel génétique au cours du cycle cellulaire et participent à l'architecture fonctionnelle des chromosomes. L'ADN télomérique est de nature répétée. Il est raccourci à chaque division cellulaire car la réplication des extrémités d'une molécule d'ADN linéaire est incomplète [6] (Figure 1). L'érosion réplicative des télomères peut être compensée par l'action de la télo-mérase, une transcriptase inverse spécialisée qui se sert d'un ARN matrice spécifique pour allonger le brin 3' des > Dans les cellules somatiques humaines, les télomères raccourcissent au rythme des divisions cellulaires, jusqu'à l'apparition de télomères dysfonctionnels qui induisent la sénescence ou l'apoptose selon le type cellulaire. Deux études viennent d'être publiées montrant qu'un dysfonctionnement télomérique chez la souris supprime la formation de cancers en déclenchant la sénescence. Ces résultats renforcent la notion que la sénescence est un mécanisme qui limite la prolifération tumorale et suggèrent de nouvelles stratégies thérapeut...

Cellular senescence is a stable form of cell cycle arrest triggered by a variety of damages and stresses including oncogene expression (oncogene-induced senescence or OIS). We and others previously demonstrated that OIS occurs in human benign lesions, acting as a potent tumor suppressor mechanism preventing cancer progression. Numerous phenotypic changes occur during OIS both in the cytoplasm and in the nucleus. These include the activation of autophagy, a catabolic process operating in the cytoplasm, and downregulation of lamin B1, a component of the nuclear lamina. However, it is unknown whether these changes relate to each other. We discovered that cells entering BRAFV600E- or H-RASV12-induced senescence downregulate not only lamin B1 but also lamin A, as well as several other nuclear envelope (NE) proteins. This depletion was associated with an altered NE morphology. We further found that the global loss of NE proteins is a consequence of their degradation by the autophagy machinery. This occurs concomitantly with autophagy induction and increased lysosomal content and activity. Our study reveals for the first time to our knowledge, a connection between autophagy and the disruption of nuclear envelope integrity during OIS. Note: This abstract was not presented at the meeting. Citation Format: Christelle Lenain, Olga Gusyatiner, Sirith Douma, Daniel S. Peeper. Autophagy-mediated degradation of nuclear envelope proteins during oncogene-induced senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1266. doi:10.1158/1538-7445.AM2015-1266