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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nat Rev Mol Cell Biol. Author manuscript; available in PMC Sep 1, 2010.
Published in final edited form as:
PMCID: PMC2842081
NIHMSID: NIHMS176923
Telomeres: protecting chromosomes against genome instability
Roderick J. O’Sullivan and Jan Karlseder
The Salk Institute for Biological Studies, Molecular and Cell Biology Laboratory, 10010 North Torrey Pines Rd., La Jolla, CA 92037, USA
Please address correspondence to: Jan Karlseder, Ph. D., karlseder/at/salk.edu, phone: 858 453 4100 x1867, fax: 858 457 4765
The natural ends of linear chromosomes require unique genetic and structural adaptations to facilitate the protection of genetic material. This is achieved by the sequestration of the telomeric sequence into a protective nucleoprotein cap that masks the ends from constitutive exposure to the DNA damage response (DDR). When telomeres are unmasked, genome instability arises. Balancing capping requirements with telomere replication and the enzymatic processing steps obligatory for telomere function is a complex problem. Telomeric proteins and their interacting factors create an environment at chromosome ends that inhibits DNA repair there, however, the repair machinery is essential for proper telomere function.
Linear DNA fragments are toxic to mammalian cells and effective mechanisms evolved to deal with them, involving signalling cascades resulting in detection, enzymatic degradation or repair of the fragments, cell cycle arrest and/or cell death. Failure to appropriately respond to broken DNA can result in unequal distribution of genetic material during cell division, in genome instability and eventually in the development of malignancies.
The natural ends of linear chromosomes resemble DNA breaks, but are an exception, where repair would lead to deleterious chromosome fusions and therefore has to be avoided. This is accomplished by specialized ribonucleoprotein structures, termed telomeres. They are composed of long tracts of double stranded G rich repeats, which in humans extend for 9–15kb, but can be as long as 100kb in rodents. The actual end of the telomere is conspicuous by the presence of a 50–300nt protrusion of single stranded repeats from the 3’ end, termed the G-tail or G-overhang (Figure 1) 1. This G-overhang is presumably the result of highly regulated post-replicative 5’-3’ exonucleolytic resection of the C-rich strand.
Figure 1
Figure 1
The structure of human telomeres
In somatic cells that lack telomere length maintenance mechanisms the failure of lagging strand synthesis to fully replicate the parental strain 2, coupled with the processing required to generate the G-overhang result in the progressive removal of telomeric sequence in each round of replication. In stem cells, germ cells and lineage progenitor cells this telomere shortening is offset by the addition of newly synthesized repeats by the telomerase complex, which uses the 3’-OH of the G-overhang as its substrate (Box 1). It is not intuitive how the G-tail provides protection from the pathways that detect and process broken DNA, but it has been proposed that the 3’ G-overhang can be sequestered into a lasso like structure known as the T-Loop 3 (Figure 1). The closed configuration of the T-loop provides a protective cap that defines the natural end of the chromosome and masks the telomere from the DNA damage response (DDR) machinery (Fig 2). Therefore, the generation of the G overhang and the manipulation of the tail by telomerase in telomerase positive cells is an important point of convergence of end protection and telomere length maintenance mechanisms.
Box 1. The telomerase complex
The discovery by Greider and Blackburn that telomeric repeats are added de novo to the end of eukaryotic chromosomes by telomerase is one of the most important findings in the recent history of molecular biology 108. The telomerase complex was biochemically purified and consists of the catalytic reverse transcriptase, TERT (TElomerase Reverse Transcriptase), the RNA subunit TR (Telomerase template RNA) that provides the template for repeat synthesis at chromosome ends and Dyskerin, a key auxillary protein 109. This complex is assembled in Cajal Bodies within the nucleus and shuttled to telomeres by an accessory factor, TCAB1 (Telomerase CAjal Body protein 1) 31. The ATPases Pontin and Reptin sequester this immature complex into an active conformation, whereupon it associates with the terminal exposed 3’ hydroxyl group and initiates nucleotide addition at the chromosome ends 30. For many years, it was thought that telomerase preferentially elongated the shortest telomeres in the cell 110, 111, however, recent studies have suggested that telomerase is more promiscuous and randomly targets telomeres during S-phase in cancer cells 112. By doing so, telomerase counteracts the generational shortening of telomeres, maintaining telomere length and stability. This latter property is essential for highly proliferative cells such as stem cells and lineage progenitors, perhaps as a requisite for “stemness”. Mice lacking the RNA subunit display diminished stem cell compartments and reduced stem cell proliferation, differentiation and self-renewal 113. This manifests as a phenotype of accelerated ageing and atrophy of key tissues in those animals lacking functional telomerase 114.
Recently non-telomeric functions for mammalian TERT have been suggested, which include the regulation of global chromatin dynamics, stem cell proliferation and transcription of developmentally regulated genes 29, 115, 116.
Figure 2
Figure 2
(A) Damage Sensing Pathways
Here we will discuss how functional telomeres prevent chromosomes from fusion, which factors contribute to chromosome end protection and how dysfunctional telomeres can be the source of genome instability and cancer.
The repetitive and GC rich nature of telomeric DNA endows it with the capability to form higher order DNA secondary structures, such as G-quadruplexes, which have been proposed to represent obstacles for the replication machinery 4, 5. TTAGGG repeats have also been identified as poor substrates for nucleosome assembly in vitro 6 and therefore telomeric chromatin is quite distinctive 7. Given these properties, telomeres are thought to resemble fragile sites and telomeric proteins aid the DNA replication machinery in accurate duplication of the chromosome end 8, 9. This is further substantiated by the observation that the complete replication of telomeric DNA tends to occur later than other chromosomal regions 10 (ROS and JK, unpublished).
Such are the unusual properties of telomeres, a bona fide telomeric protein complex has evolved. In mammals this complex is termed Shelterin and consists of six individual proteins, TRF1 (Telomeric Repeat binding Factor 1), TRF2, RAP1 (Repressor/Activator Protein 1), TIN2 (TRF1 INteracting protein 1), TPP1 (TINT1/PIP1/PTOP 1) and POT 1 (Protection Of Telomeres 1) 11 (Figure 1). The double stranded telomeric repeats are bound by TRF1 and TRF2 whereas POT1 attaches to the single stranded overhang. These DNA binding modules are bridged by TPP1 and TIN2 and are crucial for chromosome end protection and telomere length regulation. TRF1 and TRF2 are constitutively present at telomeres and the proportion of TRF1 and TRF2 loaded on telomeres is important for telomere length regulation. TRF1 has DNA remodelling activity 12, 13 and has recently been shown to promote efficient replication of telomeres 8, 9. TRF2 primarily functions in chromosome end protection by promoting topological changes in telomeric DNA 14, T-loop assembly 15, 16 and by suppression of ATM (Ataxia Telangiectasia Mutated)-dependent DDR and non-homologous end joining (NHEJ) (Fig 2) 17, 18. TRF2 also plays a role in chromatin assembly, indicated by the finding that TRF2 overexpression causes aberrant nucleosome spacing and decreases the abundance of the core histones H3 and H4 at chromosome ends 19. The function of RAP1 is more enigmatic. Unlike its homologue in Saccharomyces cerevisiae, mammalian RAP1 does not bind TTAGGG repeats and its telomeric localization is dependent on interaction with TRF2 20, but the factor has recently been implicated in the inhibition of NHEJ in vitro and in vivo 21, 22. POT1 contributes to telomere protection by binding to the overhang and by suppression of ATR (ATM Rad3 related protein)-dependant DDR pathways 17, 23. Also, the high specificity of POT1 for single stranded telomeric DNA leaves the possibility open that it might bind to the displaced G-strand in the T-Loop and “lock-in” the closed configuration of this structure (Figure 1). The loading of POT1 and TPP1 onto the overhang is also an important determinant of telomere length. Current models suggest that POT1 and TPP1 compete with telomerase for access to the overhang 23. A direct interaction between TPP1 and telomerase has been shown to bolster telomerase processivity 24, 25. However, increased loading of POT1 along the overhang seems to block telomerase accessibility to the 3’-OH substrate.
Taken together, the shelterin complex, while consisting of six proteins only, has an immensely complex role in telomere length regulation, protection from enzymatic attack, recruitment of required enzymatic activities and control of signalling cascades from the natural chromosome ends. While our understanding of the individual roles of shelterin components is growing fast, much remains to be discovered about the transcriptional, translational and post-translational regulation of these components and about the importance of shelterin stoichiometry upon cell cycle changes, DNA damage and differentiation.
In a recent study, the biochemical purification of telomeric proteome produced a listing of 210 proteins that interacted with and might influence telomeric structure 26. Many of these factors have previously been independently shown to localize to telomeres where they modulate higher order telomeric DNA and chromatin, unravel secondary DNA structures, promote resection and strand invasion of the overhang into the double stranded DNA and consequently aid in generation of the displacement loop (D-Loop) 27, 28. In addition, there is a growing number of proteins that localize to telomeres that are involved in the assembly and regulation of telomerase in cells where this complex is expressed 2931.
A recent addition to the telomeric RNP (RiboNucleoProtein complex) is TERRA (Telomeric Repeat containing RNA). For many years telomeres were viewed as transcriptionally inert. However, transcription of the C-strand of telomeres by RNAPII produces long UUAGGG containing transcripts that are regulated by the NMD (Nonsense Mediated Decay) pathway 3234. TERRA display a strong inverse correlation with telomerase activity 34, 35 and it is speculated that TERRA are non-coding structural RNAs that maintain higher order telomeric chromatin structures, either by forming highly stable DNA-RNA hybrids 34 or through interaction with TRF2 36, 37.
Taken together, it is becoming increasingly obvious that the six-protein telomeric core complex provides the basis for a highly regulated and sophisticated network of proteins and RNA, which responds to the ever-changing environment in cells during the life span of the organism.
The interactions of telomeric DNA with shelterin and telomerase are highly regulated and essential for chromosome end protection. Defects in shelterin components and telomerase directly and adversely affect telomere structure and length, and, as a result, inappropriate changes in telomere length and/or structure are commonly invoked as the primary triggers of telomere dysfunction. Here we are first examining the proposition that changes in telomere length lead to telomere dysfunction.
The role of telomere length in end protection
In somatic cells naturally lacking telomere length maintenance pathways, replication itself and the post replicative restoration of the protective cap at chromosome ends is accompanied by a net loss of 100 to 200bp of telomeric sequence in every cell division. The molecular basis for this DNA loss is due to the inabilities of conventional polymerases to fully replicate the parenting DNA by lagging strand synthesis (termed as the ‘end replication problem’) 2, combined with the requirement to enzymatically generate G tails at both leading and lagging strand replication products 3840. As a function of the rate of replication associated telomere shortening and initial telomere length a somatic cell can only undergo a defined number of doublings before telomeres become critically short, lose their protective properties, and send cells into a terminal arrest termed replicative senescence, or cell death. This mechanism limits the replicative lifespan of individual cells and likely of some cellular compartments in organisms and therefore represents a tumor suppressive pathway that prevents cells from becoming immortal 41, 42. Introduction of the catalytic subunit of telomerase into somatic cells is sufficient to counteract replicative telomere shortening 42, 43, however, as a consequence such cells are only rendered immortal, not transformed 44. Ectopic expression of the catalytic subunit of telomerase extended the life span of cancer resistant mice, demonstrating a role of the telomerase complex in organismal longevity 45. The fact that most cancers need to activate a telomere length maintenance pathway for survival emphasizes the attraction of this unifying principle as a potential target for cancer therapy, and a number of efforts are underway to develop in vivo inhibitors of telomerase.
The true length of telomeres that lose their protective function is rather unclear and subject to debate. Yeast cells carrying a single telomere devoid of any telomeric repeats are still capable of several divisions before cell cycle arrest, suggesting that telomere length is not the only determinant of proliferative potential 46. Telomeres in primary human fibroblasts that enter senescence are readily detectable by imaging techniques that lack the sensitivity of detecting less than 0.5kb of repeat sequence 47, and the bulk of telomeres as determined by southern analysis appears around 4kb 48. In primary mouse cells, where telomere lengths are far greater than that in human cells, replicative senescence is frequently observed after few population doublings in culture, suggesting the complete loss of telomeric sequence as trigger for the loss of proliferative potential as unlikely. At the same time ultra-short telomeres of ~13 repeats, termed T-stumps have been shown to efficiently protect chromosome ends when cellular proliferation bypasses p53 and p16 imposed checkpoints which sense critically short chromosome ends, and in cancer cells 49. Therefore, the hypothesis of critically short telomere length, a phrase borrowed from observations of yeast telomeres and defined by sequence loss, apparently does not apply in mammals, where the telomere dependent limitation of the proliferative life span is much more complex.
We suggest that senescence could be triggered by the accumulation of damage and stress in cells, resulting from additive signals of shorter telomeres, increased replicative stress in aging cells, reactive oxygen species, chromatin changes, degradation of the nuclear envelope and pore complexes as well as protein damage 5053. The more a single component contributes to the total, the less additional damage is required to exceed the critical threshold, illustrated by the fact that primary cells grown under low oxygen conditions undergo many more population doublings and enter senescence with shorter bulk telomeres.
The role of telomere uncapping in genome instability
Telomeres can lose their protective function when they shorten to a critical length, or when they fail to mask themselves from recognition by the DNA damage repair machinery, even in the presence of long stretches of TTAGGG repeats. The most striking and obvious example of acute telomere uncapping is illustrated by removal of TRF2 from telomeres, either by a dominant negative allele 18, 54, 55, or by targeted deletion of the gene in mouse 56, 57. Within a few cell divisions all chromosome ends are detected as DNA breaks and then fuse in an ATM pathway dependent manner, leading to the appearance of metaphase chromosomes as a ‘plate of spaghetti’ 56, 57 (Box 2). POT1, while strongly implicated in telomere protection in yeast 58, has a less pronounced capping phenotype in mammals. Mouse cells possess two POT1 proteins, POT1a and POT1b, suggesting recent expansion of the telomeric complex in rodents 59, 60, and POT1a is sufficient to repress damage signaling at telomeres. The subsequent processing of dysfunctional telomeres is virtually identical to the canonical DDR at intra-chromosomal break sites. The association of 53BP1 (p53 Binding Protein 1), a protein involved in the detection and processing of double strand breaks, with several telomeres creates large chromatin domains conducive for covalent fusion of chromosomes 61, 62. The build-up of DDR proteins as well as changes in local telomeric chromatin structure exemplified by γH2AX, a variant of histone H2A that localizes to sites of DNA damage, can be visualized by florescence-based techniques such as Telomere-dysfunction Induced Foci (TIFs) (Box 2) that are virtually identical to the foci detected at sites of breaks and repair following irradiation-induced DNA damage 63. TIFs are observed in almost all settings of engineered telomere dysfunction but also in cells that undergo replicative senescence, blurring the boundary between telomere uncapping and telomere length phenotypes.
Box 2. Cytological representation of telomere dysfunction
Covalent fusions are efficiently detected by spreading of metaphase chromosomes and marking of telomeres by fluorescent in situ hybridization (A). This allows for the distinction of fusions in the presence (left panel) and the absence (center panel) of telomeric repeats. The right panel represents a metaphase where all chromosomes have been fused due to deletion of TRF2 (image kindly provided by E. Lazzerini Denchi). DNA has been stained in red, and telomeric repeats in green.
Telomeres that are detected as damage can be visualized as telomere dysfunction induced foci (TIFs) (B). Cells are simultaneously stained with antibodies against factors that localize to sites of damage and with probes recognizing telomeric DNA. While the chance for telomeric colocalization with intra-chromosomal damage sites is slim, the merged images clearly designate telomeres that bind damage factors, and therefore can be considered as recognized by the damage machinery. Here the DNA of an interphase nucleus has been stained in the blue channel, telomeres in green, and γH2AX (upper panel) or 53BP1 (lower panel) in red. Yellow dots (indicated by white arrows) in the merge represent the colocalization of γH2AX or 53BP1 with telomeres.
ALT associated PML bodies (APBs) (C) are structures in cells that employ the recombination based ALT (Alternative Lengthening of Telomeres) mechanism for telomere length regulation 117, 118. They represent loci that contain telomeric DNA, telomeric proteins and factors involved in DNA metabolism. Here the DNA of an interphase nucleus has been stained in blue, PML in green and TRF2 as marker for telomeres in red. The merge of all 3 colors is on the right. It has been proposed that APBs represent the sites of telomeric recombination, however, they could also simply serve as storage pools for proteins required for ALT and ALT by-products.
Recently, the accumulation of damage signals within cells has been shown to be required for senescence entry and that DDR components function to suppress the detrimental outcomes that arise from replicative stress 64, 65. Therefore, the changes in telomeric chromatin and loading of DDR proteins on several telomeres could provide the impetus to exit the cell cycle and induce apoptosis or senescence, depending on cell type. How are these telomeres selected? Cell cycle dependent chromatin dynamics or cell cycle dependent phosphorylation of DDR proteins might influence the choice and timing of these events and addressing this question will be complex but essential in the future.
Many of the phenotypes observed in engineered and acute human and mouse models of telomere dysfunction only manifest in circumstances where p53 and/or pRb pathways are absent or suppressed, allowing cell growth in the presence of DNA damage signals, emphasizing the role of these essential tumor suppressor pathways (Fig 2) in detecting damaged chromosome ends. At the same the involvement of p53 and pRB suggests that dysfunctional telomeres signal in the same way as intra-chromosomal breaks. In addition, contrary to primary human cells, primary mouse fibroblasts display a significant level of telomerase activity, allowing the speculation that extension of the 3’overhang by telomerase has a role in signaling from mouse telomeres, whereas the same effects would not arise in human somatic cells.
Dysfunctional telomeres are also potential substrates for homologous recombination (HR) (Fig 2). Expression of TRF2 lacking the amino-terminal basic domain results in massive loss of telomeric sequence and the formation of extrachromosomal circular arrays of dsTTAGGG repeats, termed t-circles 66, suggesting that this domain is involved in protecting telomeres against inappropriate HR events, potentially by binding to telomeric Holliday junctions and directly inhibiting resolvase activity 67. The basic TRF2 domain also directly interacts with WRN, a RecQ helicase missing in patients suffering from Werner Syndrome (WS). Therefore, it has been hypothesized that strand invasion that would otherwise be promoted by WRN cannot occur. The exposed G-overhang then engages in rampant HR resulting in sister-telomere loss (STL), sister chromatid exchange (SCE) and other gross chromosomal aberrations. Finally, the recently identified TERRA have been shown to interact with the basic domain of TRF2 36 and suppression of TERRA leads to changes in heterochromatin, however the molecular pathways are not known. This lends support to the speculation that TERRA might function in the suppression of HR by forming stable higher order structures at telomeres. Solving the enigma of TERRA will undoubtedly prove challenging, judging from the difficulties encountered in analyzing other non-coding RNAs such as Xist (X inactive specific transcript), a transcript that plays a major role in the X chromosome inactivation process.
It is assumed that dysfunctional telomeres are recognized as damage in the same ways the cell detects intrachromosomeal double stranded breaks (Fig 2). However, many factors involved in damage recognition can be found at functional telomeres, suggesting a dual role for the damage machinery in chromosome end protection and detection of uncapped telomeres.
Interactions of telomeres with DNA damage machineries
One of the major functions of telomeres is to shield the natural chromosome ends from inappropriate repair and to distinguish them from intrachromosomal double stranded breaks. It was therefore surprising and seemed paradoxical that many proteins that have a major role in the detection of DNA damage as well as in signaling to DNA damage cascades localize to functional telomeres. The yeast MRX complex (consisting of Mre11, Rad50 and XRN2), as well as the ATM and ATR related kinases TEL1 and MEC1 are all involved in the early phases of detection of DNA damage, they are all found at yeast telomeres, and their deletions lead to telomere length and telomere deprotection phenotypes 6871. Similarly human cells derived from patients suffering from Nijmegen Breakage Syndrome or Ataxia Telangiectasia display accelerated telomere shortening 72, 73, but the pathways that lead to elevated levels of TTAGGG repeat loss are not clear. Also, similarly to yeast, the mammalian MRN complex, consisting of MRE11, RAD50 and NBS1 (the homolog of yeast XRN2), ATM and ATR can be detected at telomeres, suggesting that the DNA damage machinery has a pivotal role at functional and dysfunctional telomeres 74, 75.
TRF2 and POT1 have major roles in the interaction of telomeres with the DNA damage signaling machinery. Human telomeres that lose telomeric TRF2 succumb to ATM and p53 dependent death in cells that are competent for apoptosis 55. At the same time, TRF2 seems to be capable of directly inhibiting ATM signaling at chromosome ends, by generating an environment where the capability of ATM to autophosphorylate and self-activate is compromised 76, 77. These preliminary findings suggested a complex role for shelterin components in suppressing ATM and ATR dependent damaging pathways (Fig 2), which was recently molecularly defined by the targeted deletion of TRF2 and POT1 in mice. Analysis of mice lacking combinations of TRF2, POT1, ATM and ATR revealed independently controlled pathways 17. TRF2 and POT1 were shown to act separately, where TRF2 repressed ATM and POT1 prevented activation of the ATR signaling cascade. When ATM or ATR signaling was inhibited, dysfunctional chromosome ends were not detected as damage, and also evaded NHEJ dependent fusion 17. Activation of ATM by TRF2 was dependent on the MRN complex, and it is possible that TRF2 inhibits MRN activation at functional telomeres by inhibiting it to bind to the very end of telomeres 62. At them same time the MRN complex has been suggested to be required for proper processing of telomeres after replication and in processing of dysfunctional telomeres prior to fusion, emphasizing the multiple roles this complex plays at telomeres 62, 75, 78 (Figure 3).
Figure 3
Figure 3
Telomeres as the cause of genome instability
Getting ever closer to the primary signal event that initiates detection of dysfunctional telomeres by the damage machinery it was discovered that binding of 53BP1 to uncapped telomeres is an essential event for NHEJ dependent end to end fusion 61. Dysfunctional telomeres bound to 53BP1 were found to be significantly more mobile than such telomeres without 53BP1, suggesting that altering telomere mobility enhanced the chances of finding a partner telomere for fusion, facilitating NHEJ repair reactions that involve distant sites 61.
Taken together, it is becoming increasingly clear that the interactions of functional and dysfunctional telomeres with the damage machineries are highly complex and that many levels of interaction exist. Functional telomeres require the damage machinery for efficient replication of TTAGGG repeats, processing of the ends after replication and for formation of a protective cap at chromosome ends. Dysfunctional telomeres require the same machinery for detection of uncapped telomeres, for processing of those uncapped telomeres for repair and for mobility to enhance the efficiency of repair. Adding to the complexity are individual and specific interactions of shelterin components with the signaling molecules in the damage repair cascades, and at this point the field has not even scratched the surface of post transcription modifications that are expected to also play a role in protection and signaling from telomeres.
Amplification of the signal
While it is becoming clearer how telomeres interact with the damage machinery and how uncapped telomeres initiate signaling cascades, it is not known how the localized damage signal at chromosome ends gets translated to a response affecting the whole cell. ATM comes to mind as a molecule capable of amplifying damage signals, as it has been suggested that localized damage leads to phosphorylation of most of the ATM molecules in the cell, potentially via changes in chromatin structure 79 (Box 3). However, the specialized nature of the telomeric environment, where ATM is directly suppressed locally argues against this possibility 76, 77, supported by the finding that local ATM activation can occur at telomeres in the absence of a response that spreads throughout the nucleus 75.
Box 3. Mammalian Telomeric Chromatin
In human chromatin, 147bp of DNA are wrapped around nucleosomes, comprised of histones H3, H4, H2A and H2B 119. The amino terminal tails of histones are subject to an elaborate system of post-translational modification (PTM), namely through the addition and removal of acetyl, methyl, phospho and ubiquitin groups. These are viewed as epigenetic marks and function by extending the informational capacity of cells 120. The distribution of these marks at diverse regions across the genome results in the simplified characterization of regions as being either heterochromatic or euchromatic.
G1 phase telomeric chromatin bears hallmarks of constitutive heterochromatin, which are H3K9me3, H4K20me3 and HP1 binding 36, 121 (top panel, right). Subtelomeric chromatin is distinctive by a regular nucleosomal distribution, harbors extensive DNA methylation 122 and histone PTMs distinct from those at telomeres (top panel, left). The replication of DNA during S-phase coincides with the disruption and restoration of the parental chromatin identity. Newly synthesized histones are acetylated at H4 lysines 5, 12 and 16 and H3 lysines 9 and 56 123. The removal of these acetyl groups at telomeres by SIRT proteins appears to be important for regulating the association of accessory proteins to telomeres, as exhibited by the relationship between H3K9ac, SIRT6 and WRN function 124 (middle panel).
Changes in chromatin structure have been shown to occur at dysfunctional telomeres 7. These are often similar to those exhibited at sites of DNA damage, such as phosphorylation of H2AX, changes H4K20me2 and recruitment of 53BP1, implying a general “epigenetic” stress response (lower panel). However, it is still unclear whether changes seen at dysfunctional telomeres are proactive or merely responsive to changes in telomeric architecture. Nuclear reprogramming also leads to dramatic changes in telomeric chromatin and telomere length, emphasizing the dynamic and developmentally regulated nature of chromosome ends 125.
Nonetheless, it is likely that chromatin plays a role in signal amplification, as changes in chromatin structure can efficiently spread through the nucleus and could be established and re-established rapidly in every cell cycle. Telomeric and subtelomeric chromatin in mice displays marks of heterochromatin, particularly trimethylation of histone H3 Lys9 (H3K9) and H4K20 (Box 3). Increasing evidence suggests that histone and DNA methylation and the enzymes involved in these events play a key role in regulating telomere length, and these events are also likely to play a role in signaling from dysfunctional telomeres 7, 8083 (Box 3).
To date little is known about global chromatin changes upon replication associated telomere shortening or telomere dysfunction, and it is not clear yet whether the long telomeres in mice directly compare to short human telomeres. It will be exciting to see whether signaling from telomeres is involved in global changes in chromatin and histone modifications, and whether this is the mechanism that translates localized damage signals to a nucleus-wide level.
In 1938 Barbara McClintock noticed that chromosomes in plants that had previously been irradiated with x-rays engaged in spontaneous chromosome breakage-fusion-bridge cycles, laying the foundation for the hypothesis that aberrantly fused chromosomes would break in the subsequent cell division, leading the unequal and random distribution of genetic material into the daughter cells 84 (Figure 3). Dysfunctional telomeres that fail to be distinguished from broken DNA lend themselves perfectly to the hypothesis that loss of chromosome end protection could lead to genome instability via McClintock’s breakage-fusion-bridge cycles. This hypothesis was formally proven in mice, when targeted deletion of the RNA subunit of telomerase rendered the complex inactive for telomere length maintenance 85. As a consequence telomeres shortened progressively by approximately 5kb per generation and after four generations telomeres lacking TTAGGG signals were detected. Loss of telomeric sequences led to chromosome end-to end fusions, chromosomal abnormalities and aneuploidy, formally supporting the suggestion that loss of chromosome end protection can be the basis for genome instability in mammals.
However, these data originally seemed inconsistent with the finding that telomerase is found activated in the majority of human tumors 86 and with the finding that cells that fail to maintain their telomeres succumb to replicative senescence or cell death. Again, the p53 dependent signaling pathway emerged as a key player, when it was discovered that the deletion of p53 attenuated the adverse effects of telomere dysfunction in late generations of mice lacking an active telomerase complex. Consequently it was suggested that the loss of telomerase and p53 cooperate to promote cellular transformation 87. Telomere attrition in a mouse model lacking p53 similarly led to breakage-fusion-bridge cycle based genome instability and to a massive increase in non-reciprocal translocations, resulting in the development of epithelial cancers, formally linking telomere dysfunction with genome instability and malignancy 88 (Figure 3). Eventually a model emerged that recognizes and emphasizes the dual role that telomerase plays 89: When telomerase is activated in cells that entered breakage-fusion cycles and have lost tumor suppressive pathways such as p53 and pRb, telomeres are stabilized and immortality in the presence of genome instability is promoted, pushing cells towards malignancy. On the other hand, when telomerase is activated before telomeres become critically short, telomeres never reach a state where they are processed by the NHEJ machinery and fused. In this case telomerase acts to suppress the accumulation of chromosomal aberrations and genome instability, and, while controversial, it has been argued that bursts of telomerase might be useful to counteract transformation and cancer development.
Telomere driven genome instability can also ensue in the presence of long stretches of double stranded telomeric repeats. When TRF2 was removed from telomeres by expression of a dominant negative allele, chromosome ends fused displaying long stretches of TTAGGG repeats at the fusion sites 54 (Box 2). When cells continued to cycle, the chromosomes entered fusion-breakage cycles resulting in non-reciprocal translocations and genome instability, demonstrating that a telomere capping dysfunction can lead to the same outcome as telomere dysfunction due to catastrophic loss of telomeric repeats 18. One would expect that partial or complete loss of TRF2 therefore could be implicated in cancer formation, however few evidence for this hypothesis has been found so far. TRF1 and TRF2 have been found up regulated in gastric carcinoma and during hepatocarcinogenesis, however, it is not intuitively obvious how overexpression of these proteins might contribute to cancer formation 90, 91. When TRF2 was overexpressed in basal and stem cells of the epidermis skin cancer levels increased in mice, again suggesting that destabilization of the shelterin complex by overexpression or removal of individual complex members can lead to malignancy 92, 93.
Telomeres, due to their G rich and repetitive nature, represent obstacles to the replication fork and require telomeric proteins for efficient duplication, as has been originally observed in Schizosaccharomyces pombe 94. Targeted deletion of TRF1 in mice revealed that telomeres represented fragile sites and replication forks stalled without the aid of this telomeric factor 8. It remains to be established whether TRF1 driven replication defects directly contribute to genome instability, but it has been noted that mice that harbor a deletion of TRF1 in the stratified epithelia display epithelia dysplasia and develop squamous cell carcinomas in a p53 negative background 9.
The RecQ helicase WRN also contributes to efficient telomere replication. Over-expression of a dominant negative allele of WRN led to the occasional loss of telomeres generated by the lagging-strand machinery, a phenotype that was counteracted by telomerase 47. Accordingly, targeted deletion of WRN in the mouse only lead to phenotypes that resemble the human Werner Syndrome when telomerase was co-deleted 95. However, in the case of WRN deletion there is little doubt that telomere driven genome instability ensues, as cells deficient for telomerase and WRN accumulate chromosomal aberrations and elevated recombination rates between telomeres of sister chromatids 96, and the genomic instability observed in human Werner Syndrome cells was directly dependent on telomere function 97.
Taken together there is no doubt that telomere dysfunction, be it due to loss of telomeric sequences, loss of telomere capping or end protection and due to telomere replication problems can lead to genome instability and cancer. It remains to be investigated whether mutations or aberrant expression patterns of shelterin components and shelterin associating factors play direct roles in tumorigenesis in humans, and whether such proteins lend themselves as targets for cancer therapy.
While the link between telomeres, telomere dysfunction, telomere replication and cancer has been well established, there are no telomere- or telomerase based therapies so far. Small molecule telomerase inhibitors, while working well in vitro and in tissue culture, still await to pass clinical trials and to be established in standard care. Similarly, efforts to generate telomerase based cancer vaccines so far have failed to lead to viable therapies. While the approaches are promising and telomere length maintenance and telomere function make for unifying cancer targets, only time and effort will tell whether such approaches are crowned by success.
Recently telomere function has been directly implicated in two additional diseases, Dyskeratosis Congenita (DC) and Idiopathic Pulmonary Fibrosis (IPF). DC is an inherited disease marked by bone marrow failure, abnormal skin pigmentation, nail dystrophy and leucoplakia 98, 99. X-linked recessive DC is caused by mutations in dyskerin, a protein that associates with a subgroup of small nucleolar RNAs, but also with the RNA component of telomerase, TERC, an association that stabilizes the telomerase complex 100. Autosomal dominant DC has been linked to mutations in TERC itself 101, in the catalytic telomerase subunit TERT 102, and also in the shelterin component TIN2 103, 104. One unifying feature in DC is short telomeres, and the importance of limiting telomere length has been emphasized by the observation that mice that suffer from extensive telomere shortening due to the lack of Pot1b suffer from clear signs of DC 105.
IPF is a lung disorder marked by progressive scarring, leading to destruction of lung architecture with a frequently fatal outcome. The discovery that short telomeres correlated with the disease eventually led to the finding that heterozygous mutations in TERT or TERC can be the cause of the disease 106, 107. It is not obvious how short telomeres, usually associated with limited proliferation potential of stem cells or other rapidly dividing tissues can lead to a scarring phenotype in lungs only, but it will be exciting to learn about the implicated pathways in the future.
Telomere biology has come far in the recent 70 years, from the observation by McClintock that chromosomes need protection, to the award of the 2009 Nobel price in Physiology and Medicine to Elizabeth Blackburn, Carol Greider and Jack Szostak for the discovery of telomerase and the effects of telomere shortening on cells. Telomere function has been implicated in the replicative aging process and shown to play a major role in the establishment of genome instability in cancer development. The understanding of mammalian telomeric proteins has progressed from the idea that they were simply covering the telomeric repeats, to the telomeric core complex shelterin, whose members regulate telomere length, individual signaling cascades at telomeres, aid in telomere processing, interact with the DNA damage machineries in many ways and attract many other factors to telomeres transiently. Telomeric proteins and telomere length regulation have been implicated in stem cell management and various diseases.
However, some major questions remain: It is not clear what the primary signal is that detects critically short or uncapped telomeres. It is not known how the signal gets translated and amplified throughout the nucleus, and whether this cascade plays a role in organismal aging and disease. It remains to be discovered how functional telomeres change throughout the lifespan of an organism, and whether telomeric proteins are modified during the cell cycle or during aging. It has not been understood how mutations in individual telomeric proteins impact on diseases such as DC and IPF. And finally, while the connection between telomere shortening, telomere dysfunction and cancer is now abundantly clear, this knowledge has not yet translated to disease management.
Online summary
  • Telomeric proteins control telomere length and telomere integrity. The six bona fide telomeric binding proteins form shelterin, a complex that maintains chromosome end integrity.
  • Telomere dysfunction can be caused by loss of telomeric repeats or by loss of protective features, both of which are essential for telomere function.
  • Functional telomeres interact with the DNA damage machinery, but the machinery is prevented form processing these ends. Dysfunctional telomeres are recognized as damage and repaired.
  • Repair of dysfunctional telomeres by fusion propels cells into breakage-fusion-bridge cycles, resulting in unequal distribution of genetic material into daughter cells, and hence, genome instability.
  • Telomere dysfunction and the failure to maintain telomere length is emerging as the cause for a number of diseases syndromes.
Acknowledgements
ROS is supported by the George E. Hewitt Foundation for Medical Research and JK acknowledges support by the NIH (R01 GM069525 and R01 AG025837). We thank A. Cesare, D. Lackner, C. Naeger and L. Oganesian for images.
Glossary terms
G-quadruplexG-quadruplexes consist of G quartets, where guanosine residues serve as donors and acceptors in a G-G base pair, a structure that represents an obstacle to the moving replication fork.
Fragile siteA fragile site is a location in the chromosome where breaks occur frequently.
Displacement loopA displacement loop is a single stranded DNA loop, resulting from the invasion and pairing of a DNA end into homologous double stranded sequences.
Replicative senescenceReplicative senescence is a permanently differentiated state that cells enter when their telomeres become critically short, or a threshold of DNA damage is exceeded.
Homologous recombination (HR)HR is a repair pathway where homologous sequences align and genetic information is copied from one DNA strand to the other.
Werner Syndrome (WS)WS is an inherited genetic disease characterized by premature aging symptoms and the early onset of cancer.
Sister-telomere loss (STL)STL is defined by the loss of telomeric sequences from a single sister chromatid, while the other telomere stays intact.
Sister chromatid exchange (SCE)SCE is the HR based exchange of DNA strands between sister chromatids.
Non-reciprocal translocationA non reciprocal translocation is defined by the transfer of genetic information from one non homologous chromosome to another.
Nijmegen Breakage SyndromeNijmegen breakage syndrome, is a rare syndrome characterized by chromosomal instability, as a result of mutations in the NBS1 gene.
Ataxia Telangiectasia (AT)AT is a rare inherited disease, characterized by neurodegeneration, cancer susceptibility and radiation sensitivity, caused by mutations in the AT kinase gene.

Biographies
Jan Karlseder was educated in Austria and got his Ph. D. from the University of Vienna. He then worked as a postdoc in Titia de Lange’s laboratory at the Rockefeller University, before joining the Salk Institute as faculty in 2002. The Karlseder laboratory is interested in telomere metabolism and aging, using mammalian cells and nematodes as model systems.
Rodderick J. O’Sullivan achieved his BA at the Trinity College in Dublin, Ireland, and got his Ph. D. at the Institute of Molecular Pathology in Vienna, Austria. During his doctoral thesis he explored chromatin modifications in mammalian cells. He joined the Karlseder laboratory as a postdoc in 2007 and is exploring epigenetic changes during replicative aging.
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