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Nature Reviews Genetics | AOP, published online 10 June 2014; doi:10.1038/nrg3743

Transcriptional outcome of telomere signalling Jing Ye1, Valérie M. Renault2, Karine Jamet2 and Eric Gilson2,3,4

Abstract | 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. DNA damage response (DDR). A pathway triggered by DNA lesions, in which phosphatidylinositol‑3 kinases (ATM, ATR or DNA‑PK) activate appropriate repair pathways and/or cell cycle arrest.

Senescence A permanent arrest in the cell cycle.

Emergency Department, Pôle Sino-Français de Recherches en Sciences du Vivant et Génomique, Shanghai Ruijin Hospital, Shanghai 200025, China. 2 Institute for Research on Cancer and Aging, Nice (IRCAN), Nice University, CNRS UMR7284/INSERM U1081, Faculty of Medicine, Nice 06107, France. 3 Department of Medical Genetics, Hopital de l’Archet 2, CHU de Nice, BP 3079, 06202 Nice cedex 3, France. 4 Pôle Sino-Français de Recherches en Sciences du Vivant et Génomique, Shanghai Ruijin Hospital, Shanghai 200025, China. Correspondence to E.G.  e-mail: [email protected] doi:10.1038/nrg3743 Published online 10 June 2014 1

The linear nature of eukaryotic chromosomes poses two serious threats to genome integrity. The first threat stems from DNA extremities, which can be misidentified as DNA damage by the DNA damage response (DDR) machinery, leading to cellular senescence, apoptosis or double-strand break (DSB) repair 1. At natural chromosome extremities — telomeres — DDR activation is circumvented by both chromatin organization and binding of specific capping factors. In a wide range of organisms, telomeres comprise short tandem repeats of DNA sequences (for example, TTAGGG in vertebrates). The key protein component required for telomere capping is the shelterin complex 2,3 which, in vertebrates, comprises six proteins: telomeric repeat-binding factor 1 (TRF1; also known as TERF1), TRF2 (also known as TERF2), RAP1 (also known as TERF2IP), TERF1‑interacting nuclear factor 2 (TIN2; also known as TINF2), TIN2‑interacting protein  1 (TPP1) and protection of telomeres protein 1 (POT1). TRF1 and TRF2 bind to telomeric DNA duplexes, whereas POT1 binds to single-stranded 3ʹ overhangs (FIG. 1a). Other organisms use various combinations of shelterin components; for example, only a distant RAP1 homologue known as Rap1 is found at the telomeres of budding yeast, where it binds to the telomeric DNA as an array of ~20 molecules4–6 (FIG. 1b). Rap1 has key roles at telomeres by recruiting Rap1‑interacting factor 1 (Rif1), Rif2 (REF. 7) and silent information regulator proteins (Sir proteins; that is, Sir2, Sir3 and Sir4)8. Telomere repeat-containing RNAs (TERRAs) are a group of long non-coding RNAs transcribed at telo­ meres9,10 and represent another conserved component of telomeric chromatin (BOX 1).

Telomeres are also nucleation sites for heterochromatin spreading over several kilobases into subtelomeres, which results in nearby promoter silencing — an epigenetic phenomenon known as the telomere position effect (TPE)11. The molecular mechanism of such heterochromatin formation has been investigated extensively in budding yeast (FIG. 1b): Rap1 recruits a set of Sir proteins at telomeres that trigger recurrent rounds of deacetylation of subtelomeric nucleosomes, which allows them to spread along chromatin with deacetylated histone tails8 (FIG. 1b). TPE was the first phenomenon that was proposed to link telomere structure to transcriptional regulation11. The second threat to genome integrity stems from the inability of the conventional replication machinery to fully replicate the extremities of parental DNA12. Telo­ merase, which is composed of a catalytic subunit telomer­ase reverse transcriptase (TERT) and an associated RNA template (TERC), can compensate for this inexorable replicative erosion. Telomerase extends the 3ʹ ends of chromosomes by reverse transcribing the template region of its tightly associated RNA moiety 13. Telomerase expression is required for unlimited proliferation of yeast, protozoa and most tumour cells, as well as for extended proliferation of stem cells; mutations that reduce telo­merase activity give rise to various human diseases caused by excessive telomere shortening 14. In addition to replicative erosion, telomere structure is sensitive to a wide range of endogenous and environmental factors, such as improper cell cycle progression through mitosis15, oxidative stress16, genotoxic stress17, alcohol, caffeine and heat shock18, stress hormones19 and

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Natureof Reviews | Genetics Figure 1 | Telomere organization and signalling.  a | Mammalian telomeres contain tandem repeats TTAGGG DNA sequences and the shelterin complex, which comprises telomeric repeat-binding factor 1 (TRF1), TRF2, RAP1, TERF1‑interacting nuclear factor 2 (TIN2), TIN2‑interacting protein 1 (TPP1) and protection of telomeres protein 1 (POT1). For clarity, only one shelterin complex is shown at the junction between the duplex and the single-stranded part of telomeric DNA. Telomerase, telomere repeat-containing RNA (TERRA) and other levels of telomeric chromatin organization, such as telomere loops and nucleosomes, are not shown. b | Telomeres in budding yeast are bound by an array of Rap1 proteins that cover the duplex part of telomeric DNA and that recruits silent information regulator (Sir) proteins, which promote the spreading of heterochromatin in the subtelomeric region. c | Telomeres can be viewed as a receiver and/or transmitter ‘antenna’ that can integrate a wide range of endogenous and exogenous signals (grey boxes) by modifying their length and/or chromatin structure, which leads to changes in cell fate (red boxes).

Apoptosis A form of programmed cell death.

Extracellular stress response pathways Pathways that are triggered by extracellular stimuli and that involve mitogen-activated protein kinases such as JNK and p38 to activate an adaptive response programme, which maximizes cell cycle progression and survival.

psychological stress 20. Therefore, telomeres have emerged as important cell cycle regulators, stress sensors and lifespan predictors1,21 (FIG. 1c). This implies that the mechanisms involved in translating the signals that emanate from altered telomeres are of paramount importance to the ability of a cell to proliferate, differentiate and adapt to new environments. The current view is that damaged or critically short telomeres result in activation of the DDR machinery 22,23 (FIG. 1c). Although the gene expression changes that result from telomere dysfunction share features with the DDR pathway and extracellular stress response pathways, they cannot be completely explained by the activation of these processes24,25. Given these findings and the involvement of telomeric factors in transcriptional regulation throughout the genome well beyond the subtelomeric genes regulated by TPE26–31, there are likely to be mechanisms other than the DDR pathway, stress response pathways and TPE that can translate telomeric changes

into appropriate transcriptional responses. An emerging picture suggests that telomeres control the mobilization of their associated factors at extratelomeric sites where they can act as transcriptional regulators. In this Review, we cover a broad range of the transcriptional programmes controlled by telomeric factors, including those related to stress responses, metabolism, the ner­ vous system and immunity. We begin our discussion with budding yeast, in which the Rap1 protein acts genome wide to affect processes such as senescence by functioning as both a major telomere capping protein and a transcription factor. We then review several recent studies demonstrating that the shelterin complex and telomerase in mammals can also be localized outside telo­ meres, where they can control the expression of specific gene networks. These findings show that the targets of telo­mere signalling are more diverse than anticipated as a result of a combination of DDR activation and changes in the transcriptional properties of telomeric proteins.

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REVIEWS Box 1 | Telomere repeat-containing RNAs Telomere repeat-containing RNAs (TERRAs) are a group of long non-coding RNAs transcribed at the telomeres and a conserved component of telomeric chromatin9,10. Similar to other non-coding RNAs, TERRAs may facilitate heterochromatin formation79,137. Furthermore, TERRAs function at extratelomeric loci; they accumulate at close proximity to the inactive X chromosome in female mouse cell lines10, as well as near both X and Y chromosomes in mouse stem cells155. Interestingly, telomere shortening in telomerase-deficient mice impairs the association of TERRAs with the inactive X chromosome and activates an X chromosome-linked transgene139. Therefore, changes in the extratelomeric location of TERRAs could contribute to nuclear reorganization and transcriptional changes triggered by telomere shortening.

Finally, we discuss the implications of these findings in relation to the role of telomeres in tissue-specific physiology of ageing and age-related pathologies.

Extratelomeric life of telomeric factors Sequestration and dispersion of Rap1 and Sir proteins in budding yeast. As a consequence of telomere clustering at the nuclear periphery and of subtelomeric heterochromatin formation, a large amount of Rap1 and associated proteins are sequestered in subnuclear compartments known as telomere foci32 (FIG. 2). This led to the speculation that a decrease in the number of telomeric repeats in senescent cells might disperse the telomeric pool of Rap1 complexes in telomere foci to modulate gene expression at extratelomeric loci33–36. Initial support for this sequestration–dispersion model included a set of fluorescent microscopy analyses and chromatin immunoprecipitation (ChIP) studies, which led to the following findings. First, in response to accidental DSBs, Rap1, Sir3 and Sir4 are partially released from telomere foci and relocalize to the newly formed DSBs37–39. Second, telomere shortening leads to Sir2 release from telomere foci to the nucleolus, as well as to Sir3 dispersion throughout the nucleoplasm40. Finally, Sir proteins relocalize from telomere foci to the nucleolus during yeast replicative ageing 41. Rap1 dispersion in senescent yeast cells. Apart from the suppression of ribosomal DNA (rDNA) recombination by Sir2 when it relocalizes to the nucleolus in aged yeast 41 and the repression of non-telomeric gene expression in mutant yeast in which the association with the nuclear periphery of Sir proteins is disrupted42, little was known about the consequences of the nucleoplasmic dispersion of telomeric factors. This was recently addressed using genome-wide Rap1 ChIP to compare wild-type and senescent telomerase-deficient yeast 31. At senescence, Rap1 levels decrease at telomeres and Rap1 occupancy concomitantly increases at the extratelomeric sites, which are already bound in non-senescent cells; ~500 new Rap1‑binding sites also appear throughout the genome, and they are known as new Rap1 targets at senescence (NRTS) (FIG. 2). The fact that the binding of Rap1 to telomeric DNA is stabilized through a scaffold of weak protein interactions with Rif proteins43 suggests that the release of Rap1 from telomeres at senescence is facilitated at short telomeres by a length threshold effect that leads to rapid disassembly of telomeric Rap1–DNA

complexes. The senescence-specific binding of Rap1 at NRTS probably results from a combination of DNA sequences that show suboptimal affinity for Rap1 and unfavoured nucleosome formation. As an increased cellular concentration of Rap1 in non-senescent cells is sufficient to increase its occupancy at NRTS sites, it is unlikely that this binding is the result of other factors that are associated with telomere shortening, such as those of the DDR31. Importantly, Rap1 delocalization contributes to the transcriptomic changes seen in senescent cells. First, the decreased occupancy of Rap1 at telomeres correlates with increased subtelomeric gene expression, which is likely to result from TPE alleviation due to the release of Rap1 and Sir proteins from telomere foci. Second, Rap1 represses the expression of core histone genes31, which is a conserved feature of the gene expression changes in senescent cells44. This Rap1‑dependent repression is likely to be direct, as histone genes are often associated with NRTS. Third, with the exception of histone genes, NRTS gene expression is generally not strongly regulated during senescence, although a tendency towards upregulation is observed. The upregulation of NRTS genes at senescence is likely to result from Rap1 binding, as ectopic expression of Rap1 in non-senescent cells is sufficient to increase their expression31. Last, the increased occupancy of Rap1 proteins at its normal targets (that is, ribosome genes and glycolysis genes) is directly correlated with their downregulation31. It remains to be determined whether this Rap1 switch from an activator to a repressor is a result of histone downregulation, chromatin changes at these promoters due to increased Rap1 occupancy or both. Together with the transcriptional changes that occur in mutant yeast (in which the association with the nuclear periphery of Sir proteins has been disrupted42) and in the transcriptome of senescent cells24, these findings suggest that the release of Rap1 and Sir proteins from telomere foci is a coordinated genomic response to telomere shortening (FIG. 2). Once released, these proteins upregulate the expression of subtelomeric genes and control the expression of genes involved in pathways that are physiologically relevant to senescence, such as chromatin structure, metabolism and stress response24,31,42,45–47. Consistent with this hypothesis, a decrease in Rap1 expression delays the onset of senescence in telomerase-defective yeast cells31 despite its role in protecting telomeres from DDR activation. The fate of the telomere-associated Rif molecules in senescent cells remains elusive, and no extratelomeric binding of Rif1 has so far been reported48. It is worth noting that the Rap1‑dependent repression in senescent cells of both normal targets and core histone genes is independent of Sir proteins31, which suggests that the loci regulated by the Rap1 and Sir proteins released from telomeres are different. Consistent with this view, the targets of Sir3 upon its release from the nuclear periphery do not overlap with Rap1‑binding sites42. Another study using ChIP followed by high-throughput sequencing (ChIP–seq) reported Rap1‑independent recruitment of Sir3 at extratelomeric

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REVIEWS sites46. However, these ChIP–seq data may need reevaluation in light of a recent study, which showed that highly expressed genes can be enriched in nonspecific proteins, as detected by ChIP49.

NF‑κB signalling A pathway mediated by nuclear factor-κB (NF-κB) that regulates many physiological processes, including innate and adaptive immune responses, cell death and inflammation.

Mass action law A mathematical model based on the principle that the rate of a chemical reaction is proportional to the molecular concentrations of the reacting substances.

Wnt/β‑catenin signalling A fundamental and complex pathway that is required for metazoan development and tissue homeostasis; it is often dysregulated in cancer.

Luciferase reporter assays Assays commonly used to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest.

Extratelomeric binding of shelterin in mammals. Similar to the case in budding yeast, a wealth of data indicates that mammalian shelterin binds outside telomeres. Unlike budding yeast, mammalian RAP1 does not bind directly to telomeric DNA but binds through its interaction with the shelterin subunit TRF2, and these two proteins show significant colocalization at telo­ meres50–52. Although the vast majority of RAP1 binds to telomeres in a TRF2‑dependent manner, ChIP–seq analyses revealed that RAP1 can also be found at discrete chromatin sites throughout the genome of mouse embryonic fibroblasts53 and human cancer cells54. The extratelo­meric localization of RAP1 does not seem to be confined to the nucleus, as a substantial amount of RAP1 protein is found in the cytoplasm of many human cancer cells, where it has a role in NF‑kB signalling55–57. This cytoplasmic localization seems to be controlled by phosphatase of regenerating liver 3 (PRL3; also known as PTP4A3)57. There is also compelling evidence, including evidence from ChIP–seq analyses, that other shelterin subunits can bind outside telomeres54,58–61. The pattern of the extratelomeric binding sites of RAP1 and TRF2 between mouse and human cells shares remarkable features that are reminiscent of yeast Rap1 owing to the following reasons. First, RAP1- and TRF2‑binding sites are enriched in the subtelomeric region compared with the rest of the genome53,54,61, where they coincide with a high density of interstitial telomeric sequences (ITSs) that contain short stretches of telomeric DNA repeat sequences62 (FIG. 3a). Moreover, the preferential upregulation of subtelomeric genes in Rap1‑null mouse cells53 suggests that RAP1 is involved in the formation of subtelomeric heterochromatin. Second, if most of the internal binding sites of RAP1 or TRF2 coincide with ITSs, then only a subset of the ITSs predicted from genome sequence analyses are bound (FIG. 3a). A similar phenomenon is seen in budding yeast, in which only a portion of the high-affinity Rap1‑binding sites are occupied in vivo63. Third, RAP1 and TRF2 can bind to regions devoid of ITSs. Interestingly, some of these non-telomeric sites are enriched in satellite III repeat sequences that are mostly found in pericentromeric heterochromatin61. It is thus possible that RAP1 and TRF2 can be recruited to chromatin through non-canonical interactions with proteins and/or nucleic acids64–66 (FIG. 3a). In support of this view, different human cell lines show only partial overlap in their extratelomeric TRF2‑binding sites54,61, which may reflect cell type-specific chromatin determinants of TRF2 internal binding. This is reminiscent of the case in senescent yeast cells, in which Rap1 binds to NRTS, which do not contain consensus binding sites31. Moreover, in both mouse and human cells, RAP1 binds to some of its extratelomeric sites in a TRF2‑independent manner, which suggests the existence of distinct extratelomeric binding mechanisms for these two proteins53,54. Fourth, RAP1 and TRF2 seem to have much higher occupancy

Figure 2 | Subnuclear distribution of telomeric factors ▶ has a role in the transcriptional patterns of senescent yeast.  In budding yeast, Rap1, silent information regulator 2 (Sir2), Sir3 and Sir4 are highly concentrated in 3–7 perinuclear telomere foci. Consequently, lower concentrations of these proteins are found throughout the nucleoplasm, which precludes their action at certain weak binding sites. Upon telomere shortening in the absence of telomerase, delocalization of Rap1 and Sir proteins from telomere foci leads to their redistribution throughout the nucleus. This redistribution of Rap1 and Sir proteins modifies their activity at the sites where they were originally bound and allows them to regulate transcription at new sites, thereby contributing to the establishment of distinct transcriptional patterns that are associated with senescent yeast. Gene categories that show increased Rap1 occupancy in senescent cells include glycolysis and ribosome genes, as well as those corresponding to binding sites of new Rap1 targets at senescence (NRTS; that is, genes encoding histones and stress response proteins)31. For simplicity, the genes bound by Sir proteins after they are dispersed in the nucleoplasm are not indicated in the figure; these genes are different from those targeted by Rap1 (REFS 31,42,46). Only the nucleus but not the nucleolus is shown.

at telomeres than at internal genomic sites, and increased TRF2 expression leads to enhanced occupancy of ITSs or can even trigger TRF2 binding to ITSs that were not bound in control cells28,54,67, which indicates that telo­ meres bind to TRF2 with a much higher affinity than putative extratelomeric sites. The higher specificity of TRF2–RAP1 for telomeric DNA than TRF2 alone66 and the existence of different pools of telomere-bound TRF2 molecules in vivo 68 suggest that, similar to the yeast Rap1–Rif–DNA complexes43, TRF2 is stabilized at telomeres through a network of protein interactions. Thus, according to the mass action law, slight variation in TRF2 concentration in cells is expected to have a greater impact on extratelomeric DNA binding than on telomere binding. This is reminiscent of the case in yeast, in which a twofold increase in Rap1 concentration is enough to reveal NRTS binding 31. TRF2 expression is modulated by the canonical Wnt/β‑catenin signalling pathway in human cancer cells and in mouse intestinal tissues; β‑catenin-dependent upregulation of TRF2 leads to its increased occupancy at ITSs, which suggests that TRF2 binding at extratelomeric sites can be a developmentally regulated process67. Transcriptional properties of mammalian shelterin. Extratelomeric binding sites of RAP1 and TRF2 are found more frequently within or in close proximity to genes than would be expected by chance (FIG. 3b), which suggests a role for these factors in regulating gene expression. Several lines of evidence support this view. First, luciferase reporter assays with constructs that contain internal binding sites for RAP1, together with comparison of wild-type and Rap1‑null mouse embryonic fibroblasts, reveal that mouse RAP1 can act as a transactivator. Second, internal RAP1‑binding sites are found at a significant frequency in differentially regulated

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genes in Rap1‑null cells30,53. Third, RAP1 and TRF2 can regulate the transcription of the genes to which they are bound28,54,69. Fourth, increased TRF2 dosage leads to increased occupancy at and expression of HS3ST4, which encodes a neuronal isoform of heparan sulphate (glucosamine) 3‑O‑sulphotransferase28. So far, no evidence has been found for the binding of mammalian RIF1 to RAP1 or to normal telomeres70. Rather than functioning as an authentic telomeric protein, mammalian RIF1 seems to be a key factor in DSB repair 71 and replication timing 72. Mouse RIF1 was recently reported to indirectly regulate telomere length of embryonic stem cells (ESCs) by reducing the expression of zinc-finger and SCAN domain-containing protein 4 (ZSCAN4), which controls telomere recombination73. Important questions that warrant further study include how RAP1 and TRF2 control the expression of their target genes, and whether these mechanisms involve other shelterin components. This could be related to the effects of TRF2 on chromatin remodelling 74,75, or through direct interactions between RAP1 and TRF2 with chromatin regulators76–78 or between TRF2 and the non-coding  TERRA79 (BOX 1). Overall, conserved properties of shelterin proteins include the ability to bind to selected locations throughout the genome and to act as general transcriptional regulators. As detailed below, mammalian RAP1 and TRF2 coordinate important cell type-specific transcriptional programmes.

Shelterin components regulate gene networks RAP1 regulates metabolism and immunity-related gene networks. In budding yeast, Rap1 has long been known to have a central role in metabolism by controlling the transcription of genes encoding ribosomal protein subunits and glycolytic enzymes63. In response to glucose depletion, yeast Rap1 is required for the expression of genes involved in alternative energy production80. The link between Rap1 and metabolism seems to be evolutionarily conserved, as mice lacking the Rap1 gene are viable but develop obesity, glucose intolerance and hepatic steatosis29,30. These signs of metabolic disorders are more severe in females than in males. However, in contrast to yeast, no overt signs of telomere damage or DDR activation are observed in Rap1‑null cells, which indicates that RAP1 is not essential for telomere stability in mice, nor is the metabolic phenotype merely a result of telomere dysfunction. Altered expression of numerous key metabolism genes is observed in Rap1‑null mouse embryonic fibroblasts and in Rap1‑null mouse liver, and has a substantial impact on the regulation of energy homeostasis and mitochondrial function. The keys genes include those involved in insulin secretion, caloric restriction pathways, fatty acid metabolism, androgen and oestrogen metabolism, steroid biosynthesis, pyruvate metabolism, oxidative phosphorylation and peroxisome proliferatoractivated receptor (PPAR) signalling 29,30,53. This effect is especially evident on the PPARα–PGC1α (PPAR-γ coactivator 1α) pathway, which has essential roles in the regulation of energy metabolism, development and cancer by controlling biogenesis, antioxidation and oxidative

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Figure 3 | Extratelomeric binding of mammalian RAP1 and TRF2.  a | The general distribution Nature patternReviews of RAP1 |and Genetics telomeric repeat-binding factor 2 (TRF2) on the chromosome — based on a set of chromatin immunoprecipitation followed by sequencing (ChIP–seq) analyses in mouse embryonic fibroblasts53 and in human cancer cells54,61 — is shown. The densities of RAP1- and TRF2‑binding sites are higher at subtelomeres than in the rest of the genome, and these binding sites are coincident with frequent interstitial telomeric sequences (ITSs) found in these regions. Thus, RAP1 and TRF2 are recruited to subtelomeric regions primarily through the preferential binding of TRF2 to telomeric repeats. A lower density of ITSs is observed in the remainder of the genome, and only a limited subset of them are bound by either RAP1 or TRF2. Indeed, RAP1 and TRF2 do not necessarily colocalize, and they can associate with DNA sequences that have no clear telomeric repeats. The mode of association between RAP1 and TRF2 at these non-canonical sites is unknown. b | The genome is segmented into genic regions (which are defined as sequences that extend the transcribed regions from –50,000 bp before the transcription start sites (TSSs) to +50,000 bp after the stop codons) and extragenic regions. There is a significant enrichment (P 2,000 discrete chromatin regions, which suggests that telomerase acts as a transcription factor throughout the genome95. It remains unclear how telo­ merase acts at these sites and in transcription. A link has been proposed between TERT, the chromatin remodelling factor BRG1 and the regulation of gene targets of the Wnt/β‑catenin signalling pathway 96–99. Moreover, alternatively spliced variants of TERT that cannot elongate telo­ meres but that are able to stimulate Wnt signalling can increase human cell proliferation100. However, the exact contribution of telomerase to the Wnt signalling pathway is unclear, as its influence is not consistently observed in different experimental settings101–104. Nevertheless, the potential importance of a direct link between telomerase and Wnt signalling warrants further investigation. TERT also binds to the p65 subunit of NF‑κB, which leads to enhanced transcription of a subset of NF‑κB target genes105. The finding that telomerase expression is partly controlled by the Wnt/β‑catenin106 and NF‑κB107 signalling pathways raises the interesting possibility of a positive regulatory loop through which Wnt/β‑catenin and NF‑κB signalling could be amplified through telo­merase. Further highlighting a close link between telo­meres and inflammation, a high level of interleukin‑6 (IL-6) expression in transformed cells can protect telo­meres from DDR28, and TERT can regulate expression of metalloproteinases independently of telomerase activity through NF‑κB‑dependent transcription28,108. Telomere–transcription coupling All of these findings raise the question of whether the transcriptional properties of telomeric factors are part of

a general mechanism by which cells adequately respond to telomere modifications. Three non-exclusive mechanisms (described below) can be envisaged to explain how telo­meres signal their changes through the regulation of the transcriptional properties of their constituents (FIG. 5). Post-translational modifications. Different types of posttranslational modifications can control the stability and cellular localization of telomeric factors, as well as the ability of these factors to bind to DNA. For example, the Mec1 kinase (which is the yeast homologue of the mammalian serine/threonine protein kinase ATR) is required for Rap1 redistribution in budding yeast, thereby contributing to the transcriptional changes that accompany senescence31, whereas Sir3 and Sir4 phosphorylation modulates Sir protein sequestration at subtelomeres47. Regarding the mammalian shelterin complex, TRF1 and TRF2 are phosphorylated in vivo by several kinases, thereby regulating their expression and binding to DNA109. Similarly, poly(ADP-ribosyl)ation of TRF1 by tankyrase 1 (TNKS1) and TNKS2 causes TRF1 release from telomeres110,111, which renders TRF1 susceptible to proteasome-dependent degradation112. The poly(ADP-ribose) polymerases PARP1 and PARP2 can poly(ADP-ribosyl)ate TRF2 and thereby reduce TRF2 binding to telomeric DNA113,114. A p53‑dependent ubiquitylation of TRF2 through the E3 ubiquitin ligase activity of SIAH1 occurs at the onset of replicative senescence, which leads to its degradation115. This change in TRF2 stability is likely to contribute to telomere signalling at senescence by affecting both the capping and the extratelomeric functions of TRF2. This mechanism seems to be highly regulated, as it can be counteracted by the p300‑mediated acetylation of TRF2 (REF. 116). Overall, it emerges that post-translational modifications of telomeric factors are important mechanisms to couple their extratelomeric functions to various signalling pathways, including those triggered by telomere dysfunction, such as the DDR pathway (FIG. 5a). Looping. In many organisms, chromosome extremities can form back-folding or looped structures that result from long-range interactions within the telomeric DNA (which are called telomere loops117), between telomeres and subtelomeres118,119, or between telomeres and internal genomic loci120–123. In yeast, the terminal loops that involve subtelomeres are thought to control TPE118,119. It remains to be determined whether the loops formed between telomeres and internal genomic loci can regulate transcription in a telomere-dependent manner (FIG. 5b). One candidate gene that might be regulated by such a mechanism is ISG15 ubiquitin-like modifier (ISG15) in humans. The expression of this gene is regulated by telomere length independently of DDR25,27, and ISG15 is localized ~1 Mb from the nearest telomere. In addition, several genes that lie between ISG15 and the telomere are not regulated by telomere length; thus, it seems unlikely that ISG15 could be suppressed by the continuous spreading of silent chromatin that emanates from the proximal telomere. Rather, it may correspond to a particular type of telomeric silencing that is

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Figure 5 | Coordination of telomeric state with transcriptional regulation.  Several non-exclusive mechanisms Nature Reviews | Genetics can couple telomere integrity to the ability of telomeric factors to control transcription. a | Post-translational modifications (denoted by dashed lines) of telomeric factors — for example, as a consequence of DNA damage response (DDR) activation — can directly or indirectly modify their ability to bind to and regulate the transcription of extratelomeric loci. b | During telomere dysfunction, large terminal loops can be disturbed, which leads to changes in the expression of distantly located genes. c | Telomere dysfunction can disperse telomeric factors that are normally sequestered at telomere foci, as well as heterochromatic factors.

propagated in a discontinuous manner through a series of subtelomeric silencers that act as relay elements124 and/or through its interaction with the telomere, thereby forming a large subtelomeric loop. Subnuclear compartmentalization of telomeric factors. Telomeres are not randomly distributed within the nuclear space. For example, telomere foci in budding yeast are primarily associated with the nuclear envelope32, whereas mammalian telomeres and shelterin proteins are associated with the nuclear matrix and nuclear envelope125–128. Therefore, changes in the subnuclear environment of telomeric proteins can be a mechanism that couples telomere dysfunction to transcriptional regulation. Indeed, in budding yeast, the assembly and disassembly of telomere foci regulate the ability of Rap1 and Sir proteins to bind to extratelomeric sites, which provides a way to regulate genome-wide transcription31,42 (FIG. 2). This can occur in response to telomeric changes or to stress conditions that modulate TPE31,37–39,129–131, telo­mere clustering 42,132 and their anchoring at the nuclear periphery 121,133,134 (FIG. 5c). It is expected that such a subnuclear compartmentalization mechanism is a widespread phenomenon by which the transcriptional activity of telomeric proteins is

regulated given the following reasons. First, other unicellular organisms — such as Candida spp., Trypanosoma spp. and Plasmodium spp. — seem to use telomere clustering and its associated heterochromatin to regulate gene expression networks, which improves survival in adverse situations32. Second, subtelomeric heterochromatin in mammalian cells can be regulated by telomere length135–140 and by TRF1 dosage141. Third, heterochromatic proteins, such as HP1, are enriched at telomeres and subtelomeres of Drosophila melanogaster 142 and mammals141. Fourth, replicative senescence triggers changes in the nuclear localization of methylated TRF2 that correlate with misshapen nuclei and abnormal nuclear matrix organization143. Finally, the human shelterin component TRF2 can relocalize from telomeres to extratelomeric sites of DNA damage in response to ultraviolet irradiation144.

Biomedical implications As discussed above, each telomeric protein may act as a transcriptional regulator to control specific gene networks in response to telomeric changes. Consequently, the mechanisms by which telomere structure affects cell fate may be more diverse than previously expected, and specific adaptations may be associated with different cell

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REVIEWS

DDR

Telomeric proteins

DDR transcriptional targets

Telomere-component-specific transcriptional regulatory networks

Tissue-specific telomere signalling

Figure 6 | Model of telomere signalling that links DDR activation and transcriptional Nature Reviews | Genetics regulation by telomeric proteins.  Accumulating evidence implicates telomeric factors in the regulation of diverse transcriptional responses, which suggests a model for telomere signalling that couples DNA damage response (DDR) to telomere-dependent gene regulation through the ability of telomeric factors to regulate transcription genome wide. An important feature of this model is that each telomeric protein that acts as a transcriptional regulator controls specific gene networks (indicated by various grey arrows). Examples of telomere-dependent targets include neuronal genes regulated by telomeric repeat-binding factor 2 (TRF2), metabolic genes regulated by RAP1 and inflammatory genes regulated by telomerase reverse transcriptase (TERT). Some telomere-dependent genes can also be regulated by DDR, such as the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1A) that is regulated by both RAP1 and p53. This modulates the ability of telomeric proteins to act as transcriptional regulators and is expected to enable greater diversity of cellular responses that are adapted to specific cell types and telomeric changes.

Natural killer cells Cytotoxic cells that are crucial for eliminating infected, damaged or cancerous cells.

types. A tissue-specific response to telomere dysfunction might provide an explanation for the range of telomererelated pathologies14. For example, mice with short telo­ meres show glucose intolerance and insulin secretion defects as a result of gene expression changes in pancreatic β‑cells145. Given that telomere shortening leads to decreased TRF2 level through p53 activation and that TRF2 contributes to RAP1 stability 56,146, telomere shortening is expected to affect the level and cellular localization of RAP1. Moreover, as Pgc1a expression is inhibited by activated p53 (REF. 147), a decrease in RAP1 function is expected to act together with p53 to downregulate Pgc1a transcription upon telomere shortening. A loss of RAP1 transcriptional activity may therefore provide an explanation for the implication of telomere length in the pathogenesis of diabetes145,148. Given that the cellular concentrations of telomerase and shelterin components modulate their extratelomeric functions and that their expression is altered in cancers, the transcriptional activities of telomeric components could contribute to the transcriptomic changes that accompany malignant transformation. Indeed, there is compelling evidence that the ability of telomerase

to lengthen telomeric DNA may not account for all of its cellular effects, especially those promoting proliferation, preventing apoptosis and favouring malignant transformation (reviewed in REFS. 26,149). Although still speculative, some of these functions are proposed to rely on extratelomeric roles of telomerase in transcriptional regulation, particularly for genes targeted by the Wnt and NF‑κB signalling pathways97,105. Additionally, TERT can form a complex with BRG1 and nucleostemin — a GTP-binding protein essential to drive transcriptional programmes that are relevant for the maintenance of cancer stem cells99. TRF2 overexpression prevents the elimination of cancer cells by natural killer cells independently of its role in telomere capping and DDR28. One mechanism by which TRF2 controls natural killer cell biology in cancer cells is to upregulate HS3ST4 transcription28. This regulation is accompanied by increased binding of TRF2 at an ITS located within the HS3ST4 gene28. These findings have therapeutic implications for the use of telomerase and the shelterin complex as multihit therapeutic targets, as they act on both telomeric and extratelomeric mechanisms of tissue homeostasis and tumour control. In particular, blocking non-canonical functions of telomeric proteins should be considered as a viable option, as these may not have secondary pro-ageing effects due to telomere uncapping.

Conclusions Collectively, all of the findings presented here indicate that key components of the telomere maintenance pathways in budding yeast and mammals exert both pleiotropic and genome-wide effects on transcriptional regulation. In budding yeast, this dual role of telomeric proteins shapes the transcriptional pattern of replicative senescent cells31, whereas telomere shortening in human cells influences the transcription of extratelomeric genes in a DDR-independent manner 25. These data suggest that the multifunctionality of telomeric proteins is not simply a meaningless side effect of evolution but is instead an important mechanism of telomere signalling by which cells can translate changes in their telomeres into an appropriate physiological response (FIG. 6). Further investigations of how telomere-mediated transcriptional regulation operates may provide greater insights into the consequences of telomeric changes. The role of DDR in the ability of telomeric proteins to regulate transcription remains unclear. There are examples in which transcriptional regulation mediated by telomeric proteins operates separately from DDR25,28,29 whereas, in other cases, DDR and stress response mechanisms are able to control both the release of telomeric proteins and their ability to regulate transcription31,37,38,115. Additional work will be necessary to fully delineate the role of DDR in telomere-mediated transcriptional regulation. Overall, the links that have been established between telomere maintenance and transcription predict the following. First, the consequences of telomeric changes on cell fate may be much more diverse and tissue specific than expected. Second, limited telomeric changes below

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REVIEWS the threshold that is necessary to activate DDR may influence cell fate and progenitor or stem cell functions through subtle changes in the transcriptional activity of telomeric proteins. Finally, telomere biology may have an important role in non-dividing cells by controlling their differentiation and adaptation to environmental changes. We believe that the mechanisms of telomere signalling through transcriptional control reviewed here highlight the necessity of tissue homeostasis to rely on ‘fine-tuned’ coordination between telomeric state and various other cellular processes. For example, in addition to their

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Acknowledgements

The authors thank the discussions and suggestions of all the members of the E.G. team, as well as M. Shkreli, V. Géli, P. Lopez and E. van Obberghen. They are also grateful to the anonymous reviewers for helpful suggestions. The E.G. team is supported by La Ligue Nationale contre le Cancer (‘Equipe labellisée’), the French Institut National du Cancer (pro‑ grammes TELOFUN and TELOCHROM), the French National Research Agency (ANR) (programmes TELOREP and INNATELO) and the Investments for the Future LABEX SIGNALIFE (programme reference # ANR‑11‑LABX‑0028‑01). They apologize for all the important works that have not been cited owing to space limitation.

Competing interests statement

The authors declare no competing interests.

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