Distinct roles for yeast Stn1 in telomere capping and telomerase inhibition

The budding yeast Cdc13, Stn1 and Ten1 (CST) proteins are proposed to function as an RPA-like complex at telomeres that protects (‘caps'') chromosome ends and regulates their elongation by telomerase. We show that Stn1 has a critical function in both processes through the deployment of two separable domains. The N terminus of Stn1 interacts with Ten1 and carries out its essential capping function. The C terminus of Stn1 binds both Cdc13 and Pol12, and we present genetic data indicating that the Stn1–Cdc13 interaction is required to limit continuous telomerase action. Stn1 telomere association, similar to that of Cdc13, peaks during S phase. Significantly, the magnitude of Stn1 telomere binding is independent of telomere TG tract length, suggesting that the negative effect of Stn1 on telomerase action might be regulated by a modification of CST activity or structure in cis at individual telomeres. Genetic analysis suggests that the Tel1 kinase exerts an effect in parallel with the Stn1 C terminus to counteract its inhibition of telomerase. These data provide new insights into the coordination of telomere capping and telomerase regulation.     

Tpz1TPP1 SUMOylation reveals evolutionary conservation of SUMO‐dependent Stn1 telomere association

Elongation of the telomeric overhang by telomerase is counteracted by synthesis of the complementary strand by the CST complex, CTC1(Cdc13)/Stn1/Ten1. Interaction of budding yeast Stn1 with overhang‐binding Cdc13 is increased by Cdc13 SUMOylation. Human and fission yeast CST instead interact with overhang‐binding TPP1/POT1. We show that the fission yeast TPP1 ortholog, Tpz1, is SUMOylated. Tpz1 SUMOylation restricts telomere elongation and promotes Stn1/Ten1 telomere association, and a SUMO‐Tpz1 fusion protein has increased affinity for Stn1. Our data suggest that SUMO inhibits telomerase through stimulation of Stn1/Ten1 action by Tpz1, highlighting the evolutionary conservation of the regulation of CST function by SUMOylation.     

Human Stn1 protects telomere integrity by promoting efficient lagging-strand synthesis at telomeres and mediating C-strand fill-in

Telomere maintenance is critical for genome stability. The newly-identified Ctc1/Stn1/Ten1 complex is important for telomere maintenance, though its precise role is unclear. We report here that depletion of hStn1 induces catastrophic telomere shortening, DNA damage response, and early senescence in human somatic cells. These phenotypes are likely due to the essential role of hStn1 in promoting efficient replication of lagging-strand telomeric DNA. Downregulation of hStn1 accumulates single-stranded G-rich DNA specifically at lagging-strand telomeres, increases telomere fragility, hinders telomere DNA synthesis, as well as delays and compromises telomeric C-strand synthesis. We further show that hStn1 deficiency leads to persistent and elevated association of DNA polymerase α (polα) to telomeres, suggesting that hStn1 may modulate the DNA synthesis activity of polα rather than controlling the loading of polα to telomeres. Additionally, our data suggest that hStn1 is unlikely to be part of the telomere capping complex. We propose that the hStn1 assists DNA polymerases to efficiently duplicate lagging-strand telomeres in order to achieve complete synthesis of telomeric DNA, therefore preventing rapid telomere loss.     

Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

Modelling cellular senescence as a result of telomere state

Telomeres in mammalian cells end in large duplex T loops. These loops protect the single-strand overhangs from degradation and/or interactions with signalling proteins. This protection is sometimes referred to as capping. At each cell division, telomeres shorten and there is a general consensus that telomere shortening triggers cell cycle exit. However, the exact mechanism by which telomere shortening causes cell cycle arrest is not known. Mathematical models of telomere shortening have been developed to help us understand the processes involved. Until now most models have assumed that the trigger for cell cycle arrest is the first telomere or a group of telomeres reaching a critically short length. However, there is evidence that cells stop cycling over a wide range of telomere lengths. This suggests that telomere length per se may not in fact be the trigger for cellular senescence. In this paper we develop a model which examines the hypothesis that uncapping of a telomere is the main trigger. By letting the probability of uncapping depend upon telomere length, we show that the hypothesized model provides a good fit to experimental data.     

Telomerase abrogates aneuploidy‐induced telomere replication stress,senescence and cell depletion

The causal role of aneuploidy in cancer initiation remains under debate since mutations of euploidy‐controlling genes reduce cell fitness but aneuploidy strongly associates with human cancers. Telomerase activation allows immortal growth by stabilizing telomere length, but its role in aneuploidy survival has not been characterized. Here, we analyze the response of primary human cells and murine hematopoietic stem cells (HSCs) to aneuploidy induction and the role of telomeres and the telomerase in this process. The study shows that aneuploidy induces replication stress at telomeres leading to telomeric DNA damage and p53 activation. This results in p53/Rb‐dependent, premature senescence of human fibroblast, and in the depletion of hematopoietic cells in telomerase‐deficient mice. Endogenous telomerase expression in HSCs and enforced expression of telomerase in human fibroblasts are sufficient to abrogate aneuploidy‐induced replication stress at telomeres and the consequent induction of premature senescence and hematopoietic cell depletion. Together, these results identify telomerase as an aneuploidy survival factor in mammalian cells based on its capacity to alleviate telomere replication stress in response to aneuploidy induction.     

Lifestyle impacts on the aging‐associated expression of biomarkers of DNA damage and telomere dysfunction in human blood

Cellular aging is characterized by telomere shortening, which can lead to uncapping of chromosome ends (telomere dysfunction) and activation of DNA damage responses. There is some evidence that DNA damage accumulates during human aging and that lifestyle factors contribute to the accumulation of DNA damage. Recent studies have identified a set of serum markers that are induced by telomere dysfunction and DNA damage, and these markers showed an increased expression in blood during human aging. Here, we investigated the influence of lifestyle factors (such as exercise, smoking, body mass) on the aging‐associated expression of serum markers of DNA damage (CRAMP, EF‐1α, stathmin, n‐acetyl‐glucosaminidase and chitinase) in comparison with other described markers of cellular aging (p16^INK4a upregulation and telomere shortening) in human peripheral blood. The study shows that lifestyle factors have an age‐independent impact on the expression level of biomarkers of DNA damage. Smoking and increased body mass indices were associated with elevated levels of biomarkers of DNA damage independent of the age of the individuals. In contrast, exercise was associated with an age‐independent reduction in the expression of biomarkers of DNA damage in human blood. The expression of biomarkers of DNA damage correlated positively with p16^INK4a expression and negatively with telomere length in peripheral blood T‐lymphocytes. Together, these data provide experimental evidence that both aging and lifestyle impact on the accumulation of DNA damage during human aging.     

Stn1-Ten1 and Taz1 independently promote replication of subtelomeric fragile sequences in fission yeast

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The association between stressors and telomeres in non‐human vertebrates: a meta‐analysis

Animal response to stressors such as harsh environmental conditions and demanding biological processes requires energy generated through increased mitochondrial activity. This results in the production of reactive oxygen species (ROS). In vitro and some in vivo studies suggest that oxidative damage of DNA caused by ROS is responsible for telomere shortening. Since telomere length is correlated with survival in many vertebrates, telomere loss is hypothesised to trigger cellular ageing and/ or to reflect the harshness of the environment an individual has experienced. To improve our understanding of stress‐induced telomere dynamics in non‐human vertebrates, we analysed 109 relevant studies in a meta‐analytical framework. Overall, the exposure to possible stressors was associated with shorter telomeres or higher telomere shortening rate (average effect size = ?0.16 ± 0.03). This relationship was consistent for all phylogenetic classes and for all a priori‐selected stressor categories. It was stronger in the case of pathogen infection, competition, reproductive effort and high activity level, which emphasises their importance in explaining intraspecific telomere length variability and, potentially, lifespan variability. Interestingly, the association between stressor exposure and telomeres in one hand, and oxidative stress in the other hand, covaried, suggesting the implication of oxidative stress in telomere dynamics.     

Oxidative stress‐induced renal telomere shortening as a mechanism of cyclosporine‐induced nephrotoxicity

Due to the association of oxidative stress and telomere shortening, it was aimed in the present study to investigate the possibility whether cyclosporine‐A exerts its nephrotoxic side effects via induction of oxidative stress‐induced renal telomere shortening and senescent phenotype in renal tissues of rats. Renal oxidative stress markers, 8‐hydroxydeoxyguanosine, malondialdehyde, and protein carbonyl groups were measured by standard methods. Telomere length and telomerase activity were also evaluated in kidney tissue samples. Results showed that cyclosporine‐A treatment significantly (P < 0.05) enhanced renal malondialdehyde, 8‐hydroxydeoxyguanosine, and protein carbonyl groups levels, decreased renal telomere length, and deteriorated renal function compared with the controls. Renal telomerase activity was not affected by cyclosporine‐A. Renal telomere length could be considered as an important parameter of both oxidative stress and kidney function. Telomere shortening and accelerated kidney aging may be caused by cyclosporine‐induced oxidative stress, indicating the potential mechanism of cyclosporine‐induced nephrotoxicity.     

Ionizing radiation‐induced long‐term expression of senescence markers in mice is independent of p53 and immune status

Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR‐induced senescence markers could persist long‐term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16^INK4a expression, two hallmarks of cellular senescence and aging. BrdU‐labeling experiments revealed that IR‐induced damaged cells are preferentially eliminated, at least partially, in a tissue‐dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild‐type and Rag2^?/? γC^?/? mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long‐term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.     

Inhibition of thioredoxin reductase 1 by caveolin 1 promotes stress‐induced premature senescence

Thioredoxin reductase 1 (TrxR1) is an important antioxidant enzyme that controls cellular redox homeostasis. By using a proteomic‐based approach, here we identify TrxR1 as a caveolar membrane‐resident protein. We show that caveolin 1, the structural protein component of caveolae, is a TrxR1‐binding protein by demonstrating that the scaffolding domain of caveolin 1 (amino acids 82–101) binds directly to the caveolin‐binding motif (CBM) of TrxR1 (amino acids 454–463). We also show that overexpression of caveolin 1 inhibits TrxR activity, whereas a lack of caveolin 1 activates TrxR, both in vitro and in vivo. Expression of a peptide corresponding to the caveolin 1 scaffolding domain is sufficient to inhibit TrxR activity. A TrxR1 mutant lacking the CBM, which fails to localize to caveolae and bind to caveolin 1, is constitutively active and inhibits oxidative‐stress‐mediated activation of the p53/p21^Waf1/Cip1 pathway and induction of premature senescence. Finally, we show that caveolin 1 expression inhibits TrxR1‐mediated cell transformation. Thus, caveolin 1 links free radicals to activation of the p53/p21^Waf1/Cip1 pathway and induction of cellular senescence by acting as an endogenous inhibitor of TrxR1.     

Vitamin C alleviates the senescence of periodontal ligament stem cells through inhibition of Notch3 during long‐term culture

Periodontal ligament stem cells (PDLSCs), as potential “seed cells” for periodontal tissue repair and regeneration, require to be expanded in vitro for a large scale. Senescence of PDLSCs occurred during long‐term culture may compromise the therapeutic effects of PDLSCs. Medium supplements may be useful in antisenescence. However, the effects and mechanisms of vitamin C (Vc) treatment on PDLSCs during long‐term culture are still unclear. In this study, we identified that Vc‐treated PDLSCs cells maintained a slender morphology, higher growth rate and migration capacity, stemness, and osteogenic differentiation capability during a long‐term culture. Moreover, we also identified that Notch3 was significantly upregulated during the cell senescence, and Vc treatment alleviated the senescence of PDLSCs through inhibition of Notch3 during long‐term culture. In summary, Vc treatment suppressed PDLSCs senescence by reducing the expression of Notch3 and might be a simple and useful strategy to inhibit cellular senescence during the cell long‐term culture.     

人端粒保护蛋白1(hPOT1)保护和调节端粒长度机制

人端粒保护蛋白1(humanprotectionoftelomeres1,hPOT1)是一种端粒单链DNA结合蛋白,与端粒单链TTAGGG重复序列特异性结合。hPOT1蛋白分子有其特有的结构,其与TTAGGG重复单链序列的结合具有独特的分子机制。hPOT1与其他重要的端粒结合蛋白、端粒酶等相互作用,共同完成端粒保护和端粒长度调节。     

Translational switch for long‐term maintenance of synaptic plasticity

Memory can last a lifetime, yet synaptic contacts that contribute to the storage of memory are composed of proteins that have much shorter lifetimes. A physiological model of memory formation, long‐term potentiation (LTP), has a late protein‐synthesis‐dependent phase (L‐LTP) that can last for many hours in slices or even for days in vivo. Could the activity‐dependent synthesis of new proteins account for the persistence of L‐LTP and memory? Here, we examine the proposal that a self‐sustaining regulation of translation can form a bistable switch that can persistently regulate the on‐site synthesis of plasticity‐related proteins. We show that an αCaMKII–CPEB1 molecular pair can operate as a bistable switch. Our results imply that L‐LTP should produce an increase in the total amount of αCaMKII at potentiated synapses. This study also proposes an explanation for why the application of protein synthesis and αCaMKII inhibitors at the induction and maintenance phases of L‐LTP result in very different outcomes.     

Rap1‐mediated nucleosome displacement can regulate gene expression in senescent cells without impacting the pace of senescence

Cell senescence is accompanied, and in part mediated, by changes in chromatin, including histone losses, but underlying mechanisms are not well understood. We reported previously that during yeast cell senescence driven by telomere shortening, the telomeric protein Rap1 plays a major role in reprogramming gene expression by relocalizing hundreds of new target genes (called NRTS, for n ew R ap1 t argets at s enescence) to the promoters. This leads to two types of histone loss: Rap1 lowers histone level globally by repressing histone gene expression, and it also causes local nucleosome displacement at the promoters of upregulated NRTS. Here, we present evidence of direct binding between Rap1 and histone H3/H4 heterotetramers, and map amino acids involved in the interaction within the Rap1 SANT domain to amino acids 392–394 (SHY). Introduction of a point mutation within the native RAP1 locus that converts these residues to alanines (RAP1^SHY), and thus disrupts Rap1‐H3/H4 interaction, does not interfere with Rap1 relocalization to NRTS at senescence, but prevents full nucleosome displacement and gene upregulation, indicating direct Rap1‐H3/H4 contacts are involved in nucleosome displacement. Consistent with this, the histone H3/H4 chaperone Asf1 is similarly unnecessary for Rap1 localization to NRTS but is required for full Rap1‐mediated nucleosome displacement and gene activation. Remarkably, RAP1^SHY does not affect the pace of senescence‐related cell cycle arrest, indicating that some changes in gene expression at senescence are not coupled to this arrest.     

Longitudinal analysis of somatic and germ‐cell telomere dynamics in outbred mice

Although telomere length (TL) shortens with age in most tissues, an age‐related increase in length has been described in sperm through a mechanism that is not yet fully understood. Changes in TL with age in the same individual have not been explored. This longitudinal study examines TL dynamics in somatic tissue and gametes during an entire lifespan in an outbred mouse population (from 8 to up to 114 weeks of age). Our findings indicate a reduced life expectancy in males compared to females (84.75 ± 9.23 vs. 113.16 ± 0.20 weeks) and significant variability in TL dynamics between individuals. While with aging, a clear reduction in TL was produced in somatic cells and oocytes, telomeres in sperm cells significantly lengthened. Finally, we found evidence indicating that telomere elongation in sperm during aging may be dependent on different mechanisms, such as the survival of spermatogonia with longer telomeres and the alternative lengthening of telomeres mechanism in meiotic and postmeiotic spermatogenic cells.