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Linear unconstrained DNA cannot harbor supercoils since these supercoils can diffuse and be eliminated by free rotation of the DNA strands at the end of the molecule. Mammalian telomeres, despite constituting the ends of linear chromosomes, can hold supercoils and be subjected to topological stress. While negative supercoiling was previously observed, thus proving the existence of telomeric topological constraints, positive supercoils were never probed due to the lack of an appropriate tool. Indeed, the few tools available currently could only investigate unwound (Trioxsalen) or overwound (GapR) DNA topology (variations in twist) but not the variations in writhe (supercoils and plectonemes). To address this question, we have designed innovative tools aimed at analyzing both positive and negative DNA writhe in cells. Using them, we could observe the build-up of positive supercoils following replication stress and inhibition of Topoisomerase 2 on telomeres. TRF2 depletion caused both telomere relaxation and an increase in positive supercoils while the inhibition of Histone Deacetylase I and II by TSA only caused telomere relaxation. Moving outside telomeres, we also observed a build-up of positive supercoils on the FRA3B fragile site following replication stress, suggesting a topological model of DNA fragility for this site.

Telomeric repeat binding factor 2 (TRF2) binds to telomeres and protects chromosome ends against the DNA damage response and senescence. Although the expression of TRF2 is downregulated upon cellular senescence and in various aging tissues, including skeletal muscle tissues, very little is known about the contribution of this decline to aging. We previously showed that TRF2 loss in myofibers does not trigger telomere deprotection but mitochondrial dysfunction leading to an increased level of reactive oxygen species. We show here that this oxidative stress triggers the binding of FOXO3a to telomeres where it protects against ATM activation, revealing a previously unrecognized telomere protective function of FOXO3a, to the best of our knowledge. We further showed in transformed fibroblasts and myotubes that the telomere properties of FOXO3a are dependent on the C-terminal segment of its CR2 domain (CR2C) but independent of its Forkhead DNA binding domain and of its CR3 transactivation domain. We propose that these non-canonical properties of FOXO3a at telomeres play a role downstream of the mitochondrial signaling induced by TRF2 downregulation to regulate skeletal muscle homeostasis and aging., (© 2023. The Author(s).)

Aging is a continuous process leading to physiological deterioration with age. One of the factors contributing to aging is telomere shortening, causing alterations in the protein protective complex named shelterin and replicative senescence. Here, we address the question of the link between this telomere shortening and the transcriptional changes occurring in senescent cells. We found that in replicative senescent cells, the genes whose expression escaped repression are enriched in subtelomeres. The shelterin protein TRF2 and the nuclear lamina factor Lamin B1, both downregulated in senescent cells, are involved in the regulation of some but not all of these subtelomeric genes, suggesting complex mechanisms of transcriptional regulation. Indeed, the subtelomeres containing these derepressed genes are enriched in factors of polycomb repression (EZH2 and H3K27me3), insulation (CTCF and MAZ), and cohesion (RAD21 and SMC3) while being associated with the open A-type chromatin compartment. These findings unveil that the subtelomere transcriptome associated with senescence is determined in a chromosome-end-specific manner according to the type of higher-order chromatin structure., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Zebrafish is broadly used as a model organism in gene loss-of-function studies in vivo, but its employment in vitro is greatly limited by the lack of efficient gene knockdown approaches in zebrafish cell lines such as ZF4. In this article, we attempted to induce silencing of telomere associated genes in ZF4 by applying the frequently-used siRNA transfection technology and a novel moiety-linked morpholino (vivo-MO). By proceeding with integrated optimization of siRNAs transfection and vivo-MOs treatment, we compared five transfection reagents and vivo-MOs simultaneously to evaluate the efficiency of terfa silencing in ZF4. 48 h after siRNAs transfection, Lipofectamine™ 3000 and X-tremeGENE™ HP leaded to knockdown in 35% and 43% of terfa transcription, respectively, while vivo-MO-terfa modulated 58% down-expression of zfTRF2 in contrast to vivo-MO-ctrl 72 h after treatment. Further siRNAs transfection targeting telomere associated genes by X-tremeGENE™ HP showed silencing in 40-68% of these genes without significant cytotoxicity and off-target effect. Our results confirmed the feasibility of gene loss-of-function studies in a zebrafish cell line, offered a systematic optimizing strategy to employ gene silencing experiments, and presented Lipofectamine™ 3000, X-tremeGENE™ HP and vivo-morpholinos as candidate gene silencing approaches for zebrafish in vitro gene loss-of-function studies. Successfully knockdown of shelterin genes further opened a new field for telomeric study in zebrafish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Protecting telomere from the DNA damage response is essential to avoid the entry into cellular senescence and organismal aging. The progressive telomere DNA shortening in dividing somatic cells, programmed during development, leads to critically short telomeres that trigger replicative senescence and thereby contribute to aging. In several organisms, including mammals, telomeres are protected by a protein complex named Shelterin that counteract at various levels the DNA damage response at chromosome ends through the specific function of each of its subunits. The changes in Shelterin structure and function during development and aging is thus an intense area of research. Here, we review our knowledge on the existence of several Shelterin subcomplexes and the functional independence between them. This leads us to discuss the possibility that the multifunctionality of the Shelterin complex is determined by the formation of different subcomplexes whose composition may change during aging.

Adaptative response to stress is a strategy conserved across evolution to promote survival. In this context, the groundbreaking findings of Miroslav Radman on the adaptative value of changing mutation rates opened new avenues in our understanding of stress response. Inspired by this work, we explore here the putative beneficial effects of changing the ends of eukaryotic chromosomes, the telomeres, in response to stress. We first summarize basic principles in telomere biology and then describe how various types of stress can alter telomere structure and functions. Finally, we discuss the hypothesis of stress-induced telomere signaling with hormetic effects.

Senescence is a cellular response to stress for both dividing and post-mitotic cells. Noteworthy, long-lived post-mitotic cells (collectively named LLPMCs), which can live for decades in the organism, can exhibit a distinct type of cellular aging characterized by a progressive functional decline not associated to an overt senescence phenotype. The age-related drivers of senescence and aging in LLPMCs remain largely unknown. There is evidence that an increased production of reactive oxygen species (ROS) due to dysfunctional mitochondria, coupled with an inherent inability of cellular-degradation mechanisms to remove damaged molecules, is responsible for senescence and aging in LLPMC. Although telomeric DNA shortening, by nature linked to cell division, is generally not considered as a driver of LLPMC aging and senescence, we discuss recent reports revealing the existence of age-related telomere changes in LLPMC. These findings reveal unexpected roles for telomeres in LLPMC function and invite us to consider the hypothesis of a complex telomere clock involved in both dividing and non-dividing cell aging., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Repressor/activator protein 1 (RAP1) is a highly evolutionarily conserved protein found at telomeres. Although yeast Rap1 is a key telomere capping protein preventing non-homologous end joining (NHEJ) and consequently telomere fusions, its role at mammalian telomeres in vivo is still controversial. Here, we demonstrate that RAP1 is required to protect telomeres in replicative senescent human cells. Downregulation of RAP1 in these cells, but not in young or dividing pre-senescent cells, leads to telomere uncapping and fusions. The anti-fusion effect of RAP1 was further explored in a HeLa cell line where RAP1 expression was depleted through an inducible CRISPR/Cas9 strategy. Depletion of RAP1 in these cells gives rise to telomere fusions only when telomerase is inhibited. We further show that the fusions triggered by RAP1 loss are dependent upon DNA ligase IV. We conclude that human RAP1 is specifically involved in protecting critically short telomeres. This has important implications for the functions of telomeres in senescent cells., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Telomeres arose from the need to stabilize natural chromosome ends, resulting in terminal chromatin structures with specific protective functions. Their constituent proteins also execute general functions within heterochromatin, mediating late replication and facilitating fork progression. Emerging insights into the mechanisms governing heterochromatin replication suggest telomeres and heterochromatin act in concert during development and aging. They also suggest a common evolutionary origin for these two chromosome regions that arose during eukaryogenesis.

In the context of tumorigenesis, telomere shortening is associated with apparent antagonistic outcomes: On one side, it favors cancer initiation through mechanisms involving genome instability, while on the other side, it prevents cancer progression, due to the activation of the DNA damage response (DDR) checkpoint behaving as a cell-intrinsic proliferation barrier. Consequently, telomerase, which can compensate for replicative erosion by adding telomeric DNA repeats at the chromosomal DNA extremities, is crucial for cancer progression and is upregulated in nearly 90% of human cancers. Therefore, telomeres are considered potential anti-cancer targets and, to date, most of the studies have focused on telomerase inhibition. However, the development of clinically efficient telomerase targeting therapies is still in its infancy. In this context, the findings reported in this issue of EMBO Molecular Medicine by Bejarano et al (2019) open new avenues for alternative telomere therapies., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this issue of Molecular Cell, Kim et al. (2017) have studied the structure and organization of the shelterin protein complex protecting telomeres in Schizosaccharomyces pombe and humans and discovered an allosteric structural transition that drives the formation of the shelterin complex and participates in telomere length regulation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Academic stress (AS) is one of the most important health problems experienced by students, but no biomarker of the potential psychological or physical problems associated with AS has yet been identified. As several cross-sectional studies have shown that psychiatric conditions accelerate aging and shorten telomere length (TL), we explored whether AS affected TL.Between June 2014 and July 2014, we recruited 200 junior high school students with imminent final examinations for participation in this study. The students were divided into three subgroups (mild, moderate, and severe anxiety) using the Sarason Test Anxiety Scale (TAS). Saliva samples were collected for TL measurement via quantitative polymerase chain reaction (qPCR).Students from both a specialized and a general school suffered from anxiety (p > 0.05). A total 35% had severe anxiety (score: 26.09±3.87), 33% had moderate anxiety (16.98±2.64), and 32% had mild anxiety (7.89±1.92). The TAS values differed significantly (p < 0.05) among the three subgroups, but the TLs of saliva cells differed only slightly (p > 0.05): 1.14±0.46 for those with severe anxiety, 1.02±0.40 for those with moderate anxiety, and 1.12±0.45 for those with mild anxiety.Previous reports have found that AS is very common in Asian adolescents. We found no immediate telomere shortening in adolescents with AS. Longitudinal observations are required to determine if TL is affected by AS.

The shelterin proteins protect telomeres against activation of the DNA damage checkpoints and recombinational repair. We show here that a dimer of the shelterin subunit TRF2 wraps ∼ 90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The increased level of chromosome instability in cancer cells is not only a driving force for oncogenesis but also can be the Achille's heel of the disease since many chemotherapies kill cells by inducing a nontolerable rate of DNA damage. A wealth of published evidence showed that telomere stability can be more affected than the bulk of the genome by several conventional antineoplastic drugs. In the present study, HT1080 cell lines compromised for either telomere repeats binding factor 2 (TRF2) or POT1 were treated with ICRF-193 (3 μM, 24 h) or bleomycin (1 μM, 24 h). DNA damage was assayed by combining telomeric DNA staining of a (CCCTAA)n PNA probe with immunofluorescence of 53BP1 to score the rate of telomere colocalization with 53BP1 foci. We found that ICRF-193, but not bleomycin, leads to DNA damage preferentially at telomeres, which can be rescued by TRF2 inhibition. POT1 inhibition exacerbates telomere dysfunction induced by ICRF-193. Thus, ICRF-193 induces damage at telomeres properly capped by TRF2 but not by POT1. These findings are expected to broaden our view on the mechanism by which conventional therapeutic molecules act to eliminate cancer cells and how to use TRF2 and POT1 levels as surrogate markers for anti-topoisomerase II sensitivity., (Copyright © 2015 the American Physiological Society.)

Here, with the aim of obtaining insight into the intriguing selectivity of G-quadruplex (G4) ligands toward cancer compared to normal cells, a genetically controlled system of progressive transformation in human BJ fibroblasts was analyzed. Among the different comparative evaluations, we found a progressive increase of DNA damage response (DDR) markers throughout the genome from normal toward immortalized and transformed cells. More interestingly, sensitivity to G4 ligands strongly correlated with the presence of a basal level of DNA damage, including at the telomeres, where the chromosome ends were exposed to the DDR without concurrent induction of DNA repair activity, as revealed by the lack of 53BP1 recruitment and telomere aberrations. The link between telomere uncapping and the response to G4 stabilization was directly assessed by showing that a partial TRF2 depletion, causing a basal level of telomere localized DDR, rendered telomerized fibroblasts prone to G4-induced telomere damage and anti-proliferative defects. Taken together these data strongly indicate that the presence of a basal level of telomere-associated DDR is a determinant of susceptibility to G4 stabilization., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Telomeres protect chromosome ends from degradation and inappropriate DNA damage response activation through their association with specific factors. Interestingly, these telomeric factors are able to localize outside telomeric regions, where they can regulate the transcription of genes involved in metabolism, immunity and differentiation. These findings delineate a signalling pathway by which telomeric changes control the ability of their associated factors to regulate transcription. This mechanism is expected to enable a greater diversity of cellular responses that are adapted to specific cell types and telomeric changes, and may therefore represent a pivotal aspect of development, ageing and telomere-mediated diseases.

The stability of mammalian telomeres depends upon TRF2, which prevents inappropriate repair and checkpoint activation. By using a plasmid integration assay in yeasts carrying humanized telomeres, we demonstrated that TRF2 possesses the intrinsic property to both stimulate initial homologous recombination events and to prevent their resolution via its basic N-terminal domain. In human cells, we further showed that this TRF2 domain prevents telomere shortening mediated by the resolvase-associated protein SLX4 as well as GEN1 and MUS81, 2 different types of endonucleases with resolvase activities. We propose that various types of resolvase activities are kept in check by the basic N-terminal domain of TRF2 in order to favor an accurate repair of the stalled forks that occur during telomere replication.

The increased level of chromosome instability in cancer cells, leading to aneuploidy and gross chromosomal rearrangements, is not only a driving force for oncogenesis but also can be the Achille's heel of the disease since many chemotherapies (CT) kill cells by inducing a non-tolerable rate of DNA damage. A wealth of published evidence showed that telomere stability can be more affected than the bulk of the genome by several conventional antineoplasic drugs. These results raise the interesting possibility that CT with genotoxic drugs preferentially target telomeres. In agreement with this view, accelerated shortening of telomere length has been described in blood lineage cells following high-dose CT (stem cell transplantation) or non-myeloablative CT. However, almost nothing is known on the consequences of this shortening in terms of telomere stability, senescence and on the development of second cancers or post-treatment aging-like syndromes in cancer survivors (cognitive defect, fertility impairment, etc.). In this article, we propose: (1) telomeres of cancer cells are preferential genomic targets of chemotherapies altering chromosome maintenance; (2) telomere functional parameters can be a surrogate marker of chemotherapy sensitivity and toxicity; (3) the use of anti-telomere molecule could greatly enhance the sensitivity to standards chemotherapies.

Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Untransformed HTLV-1 positive CD4(+) cells from infected individuals are selected for expressing tax and displaying morphological features consistent with telomere dysfunctions. We show that in resting HTLV-1 positive CD4(+) cells cloned from patients, hTERT expression parallels tax expression and cell cycling. Upon activation, these cells dramatically augment tax expression, whereas their increase in telomerase activity is about 20 times lower than that of their uninfected counterpart. Activated HTLV-1 positive CD4(+) but not uninfected CD4(+) or CD8(+) clones also repress the transcription of TRF1, TPP1, TANK1, POT1, DNA-PKc and Ku80. Both infected and uninfected lymphocytes from infected individuals shared common telomere gene deregulations toward a pattern consistent with premature senescence. ATLL cells displayed the highest telomerase activity (TA) whereas recovered a telomere gene transcriptome close to that of normal CD4(+) cells. In conclusion HTLV-1-dependent telomere modulations seem involved in clonal expansion, immunosuppression, tumor initiation and progression., (Copyright © 2012 UICC.)

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.

TRF1 and TRF2 are key proteins in human telomeres, which, despite their similarities, have different behaviors upon DNA binding. Previous work has shown that unlike TRF1, TRF2 condenses telomeric, thus creating consequential negative torsion on the adjacent DNA, a property that is thought to lead to the stimulation of single-strand invasion and was proposed to favor telomeric DNA looping. In this report, we show that these activities, originating from the central TRFH domain of TRF2, are also displayed by the TRFH domain of TRF1 but are repressed in the full-length protein by the presence of an acidic domain at the N-terminus. Strikingly, a similar repression is observed on TRF2 through the binding of a TERRA-like RNA molecule to the N-terminus of TRF2. Phylogenetic and biochemical studies suggest that the N-terminal domains of TRF proteins originate from a gradual extension of the coding sequences of a duplicated ancestral gene with a consequential progressive alteration of the biochemical properties of these proteins. Overall, these data suggest that the N-termini of TRF1 and TRF2 have evolved to finely regulate their ability to condense DNA.

Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization.

Telomeres containing vertebrate-type DNA repeats can be stably maintained in Saccharomyces cerevisiae cells. We show here that telomerase is required for growth of yeast cells containing these vertebrate-type telomeres. When present at the chromosome termini, these heterologous repeats elicit a DNA damage response and a certain deprotection of telomeres. The data also show that these phenotypes are due only to the terminal localization of the vertebrate repeats because if they are sandwiched between native yeast repeats, no phenotype is observed. Indeed and quite surprisingly, in this latter situation, telomeres are of virtually normal lengths, despite the presence of up to 50% of heterologous repeats. Furthermore, the presence of the distal vertebrate-type repeats can cause increased problems of the replication fork. These results show that in budding yeast the integrity of the 3' overhang is required for proper termination of telomere replication as well as protection.

Cells of B-cell chronic lymphocytic leukemia (B-CLL) are characterized by short telomeres despite a low proliferative index. Because telomere length has been reported to be a valuable prognosis criteria, there is a great interest in a deep understanding of the origin and consequences of telomere dysfunction in this pathology. Cases of chromosome fusion involving extremely short telomeres have been reported at advanced stage. In the present study, we address the question of the existence of early telomere dysfunction during the B-CLL time course. In a series restricted to 23 newly diagnosed Binet stage A CLL patients compared with 12 healthy donors, we found a significant increase in recruitment of DNA-damage factors to telomeres showing telomere dysfunction in the early stage of the disease. Remarkably, the presence of dysfunctional telomeres did not correlate with telomere shortening or chromatin marks deregulation but with a down-regulation of 2 shelterin genes: ACD (coding for TPP1; P = .0464) and TINF2 (coding for TIN2; P = .0177). We propose that telomeric deprotection in the early step of CLL is not merely the consequence of telomere shortening but also of shelterin alteration.

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.

Objective: Telomeres are protected by tightly regulated factors and elongated by telomerase. Short and/or deprotected chromosomes are recombinogenic and thereby cancer prone., Materials and Methods: Together with the quantification of telomerase activity (TA), measuring telomere length (TL) and expression of the genes that govern telomere protection and elongation are useful for assessing telomere homeostasis., Results: By these means we demonstrate that TL, hTERT, and TA are in the order acute myelogenous leukemia (AML) > T-cell acute lymphoblastic leukemia (T-ALL) > B-cell acute lymphoblastic leukemia (B-ALL) > T-ALL > AML, and B-ALL > AML > T-ALL. AML0 and AML3 display the lowest amounts of hTERT transcripts, and ALL and AML cells with cytogenetic abnormalities possess the shortest telomeres. hTERT expression includes phenotype-specific RNA maturation and correlates with TA but not with TL. A wide ratio of TA to hTERT expression between leukemia subtypes suggests phenotype-specific hTERT post-transcriptional deregulations. B- and T-ALL overexpress Ku70 and Pinx1, T-ALL PTOP and RAP1, and B-ALL TRF2, the expression of which is significantly higher in cases with abnormal karyotype. hTERT transcription and TL correlate with response to intensive chemotherapy, and hTERT and RAD50 are independent prognostic factors for survival., Conclusions: Each leukemia subtype possesses specific telomere dysregulations that rely on phenotype, karyotype, response to treatment, and survival., (Copyright © 2011 Published by Elsevier Inc.)

New anti-telomere strategies represent important goals for the development of selective cancer therapies. In this study, we reported that uncapped telomeres, resulting from pharmacological stabilization of quadruplex DNA by RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate), trigger specific recruitment and activation of poly-adenosine diphosphate (ADP) ribose polymerase I (PARP1) at the telomeres, forming several ADP-ribose polymers that co-localize with the telomeric repeat binding factor 1 protein and are inhibited by selective PARP(s) inhibitors or PARP1-specific small interfering RNAs. The knockdown of PARP1 prevents repairing of RHPS4-induced telomere DNA breaks, leading to increases in chromosome abnormalities and eventually to the inhibition of tumor cell growth both in vitro and in xenografts. More interestingly, the integration of a TOPO1 inhibitor on the combination treatment proved to have a high therapeutic efficacy ensuing a complete regression of the tumor as well as a significant increase in overall survival and cure of mice even when treatments started at a very late stage of tumor growth. Overall, this work reveals the unexplored link between the PARP1 and G-quadruplex ligands and demonstrates the excellent efficacy of a multi-component strategy based on the use of PARP inhibitors in telomere-based therapy.

Telomeres are nucleoprotein structures that protect the ends of human chromosomes through the formation of a 'cap', thus preventing exonucleolytic degradation, inter- and intra-chromosomal fusion, and subsequent chromosomal instability. During aging, telomere shortening correlates with tissue dysfunction and loss of renewal capacity. In human cancer, telomere dysfunction is involved in early chromosome instability, long-term cellular proliferation, and possibly other processes related to cell survival and microenvironment. Telomeres constitute an attractive target for the development of novel small-molecule anti-cancer drugs. In particular, individual protein components of the core telomere higher-order chromatin structure (known as the telosome or 'shelterin' complex) are promising candidate targets for cancer therapy.

A major issue in telomere research is to understand how the integrity of chromosome ends is controlled. Although several nucleoprotein complexes have been described at the telomeres of different organisms, it is still unclear how they confer a structural identity to chromosome ends in order to mask them from DNA repair and to ensure their proper replication. In this review, we describe how telomeric nucleoprotein complexes are structured, comparing different organisms and trying to link these structures to telomere biology. It emerges that telomeres are formed by a complex and specific network of interactions between DNA, RNA and proteins. The fact that these interactions and associated activities are reinforcing each other might help to guaranty the robustness of telomeric functions across the cell cycle and in the event of cellular perturbations. We propose that telomeric nucleoprotein complexes orient cell fate through dynamic transitions in their structures and their organization., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Telomere protection in budding yeast requires the heterotrimer named CST (for Cdc13-Stn1-Ten1). Recent data show that CST components are conserved and required for telomere stability in a wide range of eukaryotes, even those utilizing the shelterin complex to protect their telomeres. A common function of these proteins might be to stimulate priming at the C-strand gap that remains after telomerase elongation, replication termination, and terminal processing. In light of the budding yeast situation, another conserved function of CST might well be the regulation of telomerase. The cohabitation at telomeres of CST and shelterin components highlights the complexity of telomere biology., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo's conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/Apollo(-/-)ATM(-/-) MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5'-3' exonuclease function of SNM1B/Apollo in the generation of 3' single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway.

Telomeres, the nucleoprotein complexes located at the ends of chromosomes, are involved in chromosome protection and genome stability. Telomeric repeat binding factor 1 (TRF1) and telomeric repeat binding factor 2 (TRF2) are the two telomeric proteins that bind to duplex telomeric DNA through interactions between their C-terminal domain and several guanines of the telomeric tract. Since the antitumour drug cisplatin binds preferentially to two adjacent guanines, we have investigated whether cisplatin adducts could affect the binding of TRF1 and TRF2 to telomeric DNA and the property of TRF2 to stimulate telomeric invasion, a process that is thought to participate in the formation of the t-loop. We show that the binding of TRF1 and TRF2 to telomeric sequences selectively modified by one GG chelate of cisplatin is markedly affected by cisplatin but that the effect is more drastic for TRF2 than for TRF1 (3-5-fold more sensitivity for TRF2 than for TRF1). We also report that platinum adducts cause a decrease in TRF2-dependent stimulation of telomeric invasion in vitro. Finally, in accordance with in vitro data, analysis of telomeric composition after cisplatin treatment reveals that 60% of TRF2 dissociate from telomeres.

DNA-dependent protein kinase (DNA-PK) is a double-strand breaks repair complex, the subunits of which (KU and DNA-PKcs) are paradoxically present at mammalian telomeres. Telomere fusion has been reported in cells lacking these proteins, raising two questions: how is DNA-PK prevented from initiating classical ligase IV (LIG4)-dependent non-homologous end-joining (C-NHEJ) at telomeres and how is the backup end-joining (EJ) activity (B-NHEJ) that operates at telomeres under conditions of C-NHEJ deficiency controlled? To address these questions, we have investigated EJ using plasmid substrates bearing double-stranded telomeric tracks and human cell extracts with variable C-NHEJ or B-NHEJ activity. We found that (1) TRF2/RAP1 prevents C-NHEJ-mediated end fusion at the initial DNA-PK end binding and activation step and (2) DNA-PK counteracts a potent LIG4-independent EJ mechanism. Thus, telomeres are protected against EJ by a lock with two bolts. These results account for observations with mammalian models and underline the importance of alternative non-classical EJ pathways for telomere fusions in cells.

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of beta-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4-associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm-specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing., Competing Interests: The authors have declared that no competing interests exist.

The Nobel Assembly at Karolinska Institute has decided to award The Nobel Prize in Physiology or Medicine 2009 jointly to Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak for the discovery of "how chromosomes are protected by telomeres and the enzyme telomerase". This discovery had major impacts within the scientific community and led to intense research in this field. All the studies performed are now the bases for future investigations and stimulate the development of potential new therapies., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

Telomerase, the key enzyme essential for the maintenance of eukaryotic chromosome ends, contains a reverse transcriptase and an RNA that provides the template for the synthesis of telomeric repeats. Here, we characterize the telomerase subunits in the hemiascomycete yeast Candida glabrata. We propose a secondary structure model for the telomerase RNA that is the largest described to date. Telomerase deletion mutants show a progressive shortening of telomeres and a modest loss of viability. Frequent post-senescence survivors emerge that possess long telomeric repeat tracts. We suggest that the high telomere length heterogeneity accounts for this distinct senescence phenotype.

Functional telomeres are required to maintain the replicative ability of cancer cells and represent putative targets for G-quadruplex (G4) ligands. Here, we show that the pentacyclic acridinium salt RHPS4, one of the most effective and selective G4 ligands, triggers damages in cells traversing S phase by interfering with telomere replication. Indeed, we found that RHPS4 markedly reduced BrdU incorporation at telomeres and altered the dynamic association of the telomeric proteins TRF1, TRF2 and POT1, leading to chromosome aberrations such as telomere fusions and telomere doublets. Analysis of the molecular damage pathway revealed that RHPS4 induced an ATR-dependent ATM signaling that plays a functional role in the cellular response to RHPS4 treatment. We propose that RHPS4, by stabilizing G4 DNA at telomeres, impairs fork progression and/or telomere processing resulting in telomere dysfunction and activation of a replication stress response pathway. The detailed understanding of the molecular mode of action of this class of compounds makes them attractive tools to understand telomere biology and provides the basis for a rational use of G4 ligands for the therapy of cancer.

The localization of genes within the nuclear space is of paramount importance for proper genome functions. However, very little is known on the cis-acting elements determining subnuclear positioning of chromosome segments. We show here that the D4Z4 human subtelomeric repeat localizes a telomere at the nuclear periphery. This perinuclear activity lies within an 80 bp sequence included within a region known to interact with CTCF and A-type Lamins. We further show that a reduced level of either CTCF or A-type Lamins suppresses the perinuclear activities of D4Z4 and that an array of multimerized D4Z4 sequence, which has lost its ability to bind CTCF and A-type Lamins, is not localized at the periphery. Overall, these findings reveal the existence of an 80 bp D4Z4 sequence that is sufficient to position an adjacent telomere to the nuclear periphery in a CTCF and A-type lamins-dependent manner. Strikingly, this sequence includes a 30 bp GA-rich motif, which binds CTCF and is present at several locations in the human genome.

The ends of linear eukaryotic chromosomes are protected by telomeres, which serve to ensure proper chromosome replication and to prevent spurious recombination at chromosome ends. In this study, we show by single cell analysis that in the absence of telomerase, a single short telomere is sufficient to induce the recruitment of checkpoint and recombination proteins. Notably, a DNA damage response at eroded telomeres starts many generations before senescence and is characterized by the recruitment of Cdc13 (cell division cycle 13), replication protein A, DNA damage checkpoint proteins and the DNA repair protein Rad52 into a single focus. Moreover, we show that eroded telomeres, although remaining at the nuclear periphery, move to the nuclear pore complex. Our results link the DNA damage response at eroded telomeres to changes in subnuclear localization and suggest the existence of collapsed replication forks at eroded telomeres.

Telomeres protect chromosome ends from fusion and degradation. In the absence of a specific telomere elongation mechanism, their DNA shortens progressively with every round of replication, leading to replicative senescence. Here, we show that telomerase-deficient cells bearing a single, very short telomere senesce earlier, demonstrating that the length of the shortest telomere is a major determinant of the onset of senescence. We further show that Mec1p-ATR specifically recognizes the single, very short telomere causing the accelerated senescence. Strikingly, before entering senescence, cells divide for several generations despite complete erosion of their shortened telomeres. This pre-senescence growth requires RAD52 (radiation sensitive) and MMS1 (methyl methane sulfonate sensitive), and there is no evidence for major inter-telomeric recombination. We propose that, in the absence of telomerase, a very short telomere is first maintained in a pre-signalling state by a RAD52-MMS1-dependent pathway and then switches to a signalling state leading to senescence through a Mec1p-dependent checkpoint.

The ability of the telomeric DNA-binding protein, TRF2, to stimulate t-loop formation while preventing t-loop deletion is believed to be crucial to maintain telomere integrity in mammals. However, little is known on the molecular mechanisms behind these properties of TRF2. In this report, we show that TRF2 greatly increases the rate of Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities, including the newly identified human GEN1 protein. By using potassium permanganate probing and differential scanning calorimetry, we reveal that the basic domain of TRF2 induces structural changes to the junction. We propose that TRF2 contributes to t-loop stabilisation by stimulating HJ formation and by preventing resolvase cleavage. These findings provide novel insights into the interplay between telomere protection and homologous recombination and suggest a general model in which TRF2 maintains telomere integrity by controlling the turnover of HJ at t-loops and at regressed replication forks.

Normal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length. In agreement with the fact that telomere uncapping triggers lymphocyte senescence, we observed an increase in gamma-H2AX and 53BP1 foci as well as in the percentage of cells exhibiting DNA damage foci in telomeres. Such a DNA damage response may be related to the continuous increase of p16(ink4a) upon cell stimulation and cell aging. Remarkably, at each stimulation, the expression of shelterin genes, such as hTRF1, hTANK1, hTIN2, hPOT1 and hRAP1, was decreased. We propose that telomere dysfunction during lymphocyte senescence caused by iterative stimulations does not only result from an excessive telomere shortening, but also from a decrease in shelterin content. These observations may be relevant for T-cell biology and aging.

In this work we report that budding yeasts carrying human-type telomeric repeats at their chromosome termini show a chronic activation of the Rad53-dependent DNA damage checkpoint pathway and a G2/M cell cycle delay. Furthermore, in the absence of either TEL1/ATM or MEC1/ATR genes, which encodes phosphatidylinositol 3-kinase-related kinases (PIKKs), we detected telomere fusions, whose appearance correlates with a reduced cell viability and a high rate of genome instability. Based on sequence analysis, telomere fusions occurred primarily between ultrashort telomeres. Microcolony formation assays argue against the possibility that fusion-containing cells are eliminated by PIKK-dependent signalling. These findings reveal that humanized telomeres in yeast cells are sensed as a chronically damaged DNA but do not greatly impair cell viability as long as the cells have a functional DNA damage checkpoint.