The linear nature of eukaryotic chromosomes necessitates protection of their physical ends, the telomeres, because the DNA-repair machinery can misconstrue the ends as double-stranded DNA breaks. Thus, protection is crucial for avoiding an unwarranted DNA-damage response that could have catastrophic ramifications for the integrity and stability of the linear genome. In this Commentary, we attempt to define what is currently understood by the term `telomere protection'. Delineating the defining boundaries of chromosome-end protection is important now more than ever, as it is becoming increasingly evident that, although unwanted DNA repair at telomeres must be avoided at all costs, the molecular players involved in recognition, signaling and repair of DNA damage might also serve to protect telomeres.

Introduction

Telomeres consist of a repetitive non-coding sequence that terminates in a single-stranded overhang at the end of linear chromosomes in eukaryotes and some prokaryotes. Both the overhang and the upstream double-stranded region serve as binding sites for a specialized network of proteins that act in concert to ensure protection of the chromosome end. This protection is crucial for the integrity of the entire genome.

Early on, it was noted that a normal cell could undergo only a limited number of cell divisions before entering into a permanent state of growth arrest (Hayflick and Moorhead, 1961), termed senescence, which is now understood to be a tumor-suppressive mechanism. It was later revealed that this growth limit imposed on the cell is determined by the length and integrity of its telomeric DNA. Today, what constitutes a protected telomere is still not entirely understood. How does the telomeric end cooperate with and, at the same time, avoid the DNA repair machinery? To what extent is telomere protection tampered with in immortalized cells that have activated telomere-maintenance pathways [namely alternative lengthening of telomeres (ALT)] (Bryan et al., 1997) or the pathway mediated by the ribonucleoprotein enzyme telomerase (Greider and Blackburn, 1985)?

Two key observations made about the chromosome end are that telomeres are not blunt-ended (Makarov et al., 1997), and that they take up a higher-order conformation known as the telomeric loop or T-loop (Griffith et al., 1999). This structural organization, first observed a decade ago, is molded by shelterin proteins and allows for sequestration of the chromosome terminus from unwarranted repair by DNA-damage machinery, aberrant homologous recombination and unwanted access by telomerase (de Lange, 2004; de Lange, 2005). More recent developments have also suggested that chromatin is another determinant of chromosome-end protection. Thus, it has become increasingly evident that telomere protection is ensured at structural, proteomic, signaling and epigenetic levels. In this Commentary, we summarize novel findings pertaining to these multiple layers of telomere protection.

Structural organization of the chromosome end

The overhang: a role in protection

A signature feature of the telomeric end is its guanine (G)-rich composition in the 5′-3′ orientation and the protrusion of a single-stranded (ss) DNA tail, which is often called the G-overhang. The presence of the terminal overhang was first noted in ciliated protozoa as short 12-16-nucleotide tails (Henderson and Blackburn, 1989; Klobutcher et al., 1981; Pluta et al., 1982) and later confirmed to be present in all species examined (Hemann and Greider, 1999; Jacob et al., 2003; Riha et al., 2000; Wellinger et al., 1993) as well as at the majority (>80%) of human telomeres (Makarov et al., 1997; McElligott and Wellinger, 1997; Wright et al., 1997). Collectively, these data suggest that the telomeric G-overhang is a conserved and, most likely, functionally significant structural property of telomeres. Despite the universal prevalence of the G-overhang, the molecular details of how it is generated and regulated have yet to be determined.

The G-overhang in yeast and vertebrates is bound by a telomere-specific ssDNA-binding protein, known as protection of telomeres 1 (POT1) (Baumann and Cech, 2001). In addition to the G-overhang, there is now also evidence for a cytosine (C)-rich counterpart: in the nematode Caenorhabditis elegans, it has been found that G-rich and C-rich telomeric tails are present in equal abundance (Raices et al., 2008). Although it is not clear whether the G- and C-rich overhangs can co-exist at alternative ends of the same chromosome, Raices and colleagues have identified two C. elegans homologs of the human POT1 protein (hPOT1), CeOB1 and CeOB2, that specifically interact with either the G- or C-rich overhang, respectively.

The G-overhang is thought to play key roles at the chromosome end, in addition to its role as a binding site for POT1. First, it is the natural substrate for telomerase, which carries out de novo synthesis of telomeric DNA by using the overhang as its primer (Box 1). Second, it might serve as the necessary structural motif for the recombination-based mechanism of telomere maintenance, termed ALT (Box 1). Finally, the presence of the telomeric overhang is needed for the formation of a higher-order lasso-like structure at the chromosome end, referred to as the T-loop (Griffith et al., 1999) (Fig. 1).

The T-loop

T-loops were first recognized in mouse and human telomere restriction fragments with the use of electron microscopy (Griffith et al., 1999). They were thought to arise as a result of the invasion of the G-overhang into the upstream double-stranded region of the telomere, whereby the overhang pairs with the opposite strand, giving rise to a smaller displacement loop (D-loop) of the G-rich strand. This DNA-protein-mediated structural organization is presumed to sequester the otherwise exposed telomeric overhang, hence deterring recognition of the chromosome end as a double-strand break and limiting the unwarranted access of telomerase to its substrate. POT1 is believed to stabilize the base of the T-loop through its interaction with the D-loop. In C. elegans, either CeOB1 or CeOB2 appear to reside at the base of T-loops (Raices et al., 2008). Furthermore, there is evidence from in vitro invasion assays that artificially constructed 3′ G-rich and 5′ C-rich overhangs of vertebrate telomeric sequences can invade double-stranded DNA with equal efficiency, showing no preference for the polarity of the DNA (Verdun and Karlseder, 2006). This allows for the possibility that 5′ C-rich overhangs might also participate in T-loop formation and hence play a role in telomere protection.

Box 1. Telomere length maintenance pathways

The telomerase complex

Telomerase is a ribonucleoprotein (RNP) enzyme that catalyzes de novo synthesis of telomeric DNA by reverse transcription. The reverse transcriptase (TERT) protein, in association with the telomerase RNA (TR), constitutes the catalytic core of telomerase, which is sufficient for enzyme activity in vitro (Collins and Gandhi, 1998; Weinrich et al., 1997). Purification and mass spectrometric analysis have revealed that hTERT, hTR and dyskerin (57 kDa; an RNA-binding protein) are the principal components of the active human telomerase complex (Cohen et al., 2007). Telomerase Cajal body protein 1 (TCAB1) was demonstrated to mediate the passage of hTR through Cajal bodies (sub-nuclear structures implicated in RNP maturation) and ultimate delivery of the fully assembled holoenzyme to telomeres (Venteicher et al., 2009).

The first step in the mechanism of telomere synthesis by telomerase is the alignment of the 3′ end of the G-overhang with the 3′ end of the RNA template (Fig. 3A), whereupon telomerase catalyzes the addition of deoxyribonucleotides to the substrate until the 5′ terminus of the template is reached. Subsequently, the DNA product is repositioned so that a second round of synthesis can take place. Multiple rounds of extension and re-alignment, otherwise known as `translocation', can occur without dissociation of the enzyme.

ALT

Cells that use the ALT pathway exhibit several features: heterogeneity and dynamic fluctuations in telomere length, extrachromosomal telomeric repeats (ECTRs) in the form of double-stranded circles, single-strand linear DNA, low-mobility branched DNA (Nabetani and Ishikawa, 2009) and a high frequency of telomere sister-chromatid exchange (t-SCE) events (Londono-Vallejo et al., 2004). Another characteristic feature is the presence of ALT-associated promyelocytic leukaemia (PML) bodies (APBs) (Yeager et al., 1999), which are sub-nuclear loci that are enriched in telomeric DNA and a growing list of proteins involved in telomere protection, DNA synthesis and recombination (Henson et al., 2002). New additions include topoisomerase IIIα (Temime-Smaali et al., 2008), the MUS81 endonuclease (Zeng et al., 2009), the structural maintenance of chromosomes 5 and 6 (SMC5/6) complex (Potts and Yu, 2007) and the heterochromatin-associated protein HP1 (Jiang et al., 2009). The involvement of APBs in the ALT mechanism remains a matter of conjecture. It has been proposed that APBs might actively participate in the recombination process; alternatively, it has also been speculated that they are passive storage depots for the macromolecules needed for ALT or for the byproducts of ALT.

ALT involves homologous-recombination-driven replication of telomeric DNA. Recombination can be either inter- or intramolecular (Fig. 3B). The former was originally demonstrated when telomeres tagged with a selection marker could pass on their tag to neighboring telomeres with increasing passage. No tag-movement was observed in telomerase-positive cells (Dunham et al., 2000). Evidence for intra-telomeric recombination has now been presented, indicating that an individual telomere can use itself to generate DNA (Muntoni et al., 2009). ECTR has been postulated to serve as a template for DNA synthesis via rolling-circle replication (Henson et al., 2002).

Fig. 1.

Schematic representation of the chromosome end decorated with members of the shelterin complex. (A) Linear (top) and folded T-loop (bottom) structural states of the telomere are depicted. Note that in the `open' linear conformation, POT1 alone might be sufficient to confer 3′ overhang protection. (B) An anti-parallel intramolecular G-quadruplex either alone (top) or in the context of a T-loop (bottom) might perform a capping role to protect the chromosome terminus from degradation by nucleases or extension by telomerase or ALT.

Fig. 1.

Schematic representation of the chromosome end decorated with members of the shelterin complex. (A) Linear (top) and folded T-loop (bottom) structural states of the telomere are depicted. Note that in the `open' linear conformation, POT1 alone might be sufficient to confer 3′ overhang protection. (B) An anti-parallel intramolecular G-quadruplex either alone (top) or in the context of a T-loop (bottom) might perform a capping role to protect the chromosome terminus from degradation by nucleases or extension by telomerase or ALT.

G-quadruplexes: an alternative to the T-loop

A non-linear loop-like configuration has been observed at chromosome termini (Fig. 1), but it is not clear what proportion of chromosome ends adopt this structure or whether T-loops are the only structural state that chromosome ends can adopt. This raises the possibility that G-quadruplex folding of the G-overhang serves as an alternative structural motif for telomere protection.

Any DNA that is G-rich in composition has the potential to assemble into a G-quadruplex in optimal ionic conditions (Box 2, Fig. 2). This structure forms readily in vitro using oligonucleotides of telomeric sequence, and is stabilized by monovalent cations such as sodium and potassium, both of which are abundant inside the cell. For decades it has been presumed that G-quadruplexes have a physiologically significant role at the telomeres; however, providing evidence for their existence in vivo has remained challenging in most organisms (Oganesian and Bryan, 2007). Nonetheless, there is now convincing evidence for the in vivo existence of an intramolecular G-quadruplex in the somatic macronuclei of the ciliated protozoan Stylonichia lemnae (Paeschke et al., 2005; Schaffitzel et al., 2001). Synthetically constructed G-quadruplex-specific antibodies have been used to detect this structure in immunofluorescence experiments. Positive staining was not observed when S. lemnae telomere-binding proteins (αTBP and βTBP) were depleted using RNA interference, indicating that these proteins are needed for G-quadruplex formation in vivo. Furthermore, cell-cycle-dependent cyclin-dependent kinase 2 (Cdk2)-mediated phosphorylation of the C-terminal portion of βTBP was required for the unfolding of the G-quadruplex because inhibition of this phosphorylation in vivo resulted in positive staining for G-quadruplex DNA at the replication band of S. lemnae macronuclei, a region that was previously devoid of G-quadruplex signals. This indicated that G-quadruplex DNA must be resolved during telomere replication and that this resolution event is mediated by βTBP phosphorylation (Paeschke et al., 2005).

On the basis of these findings, it can be surmised that, at least at telomeres in ciliates, G-quadruplex DNA performs a capping role. It is conceivable that this might also be the case at some human telomeres. In support of this possibility, it has been shown that hPOT1 can destabilize an intramolecular telomeric G-quadruplex, thereby allowing for its extension by recombinant human telomerase in vitro (Zaug et al., 2005). Destabilization of the structure is achieved by passive trapping of its dissociated state in an equilibrium population. The ability of hPOT1 to favor an open state of the G-quadruplex indicates that this structure, when in a folded and/or closed conformation, might serve in an alternative strategy to protect the telomeric terminus from degradation or inappropriate elongation by telomerase.

Werner syndrome protein (WRN), a RecQ helicase with the ability to unfold G-quadruplexes, has been proposed to play a role in telomere replication (Chang et al., 2004; Crabbe et al., 2004), strengthening the likelihood that higher-order G-rich structures play a pivotal role at chromosome ends. Therefore, the terminal overhang and its participation in higher-order structure formation ensure telomere protection at a structural level. However, assembly of the T-loop and, potentially, of G-quadruplex DNA is unlikely to occur in the absence of proteins that catalyze and chaperone these conformational changes.

Box 2. Brief overview of G-quadruplexes and their polymorphic nature The core unit of G-quadruplex DNA is the G-quartet (Fig. 2A), a planar arrangement in which each guanosine residue serves as both the donor and acceptor in a Hoogsteen G-G base pair, and the cavity that is formed at the core is the binding site for monovalent cations that serve to stabilize this structure (Williamson et al., 1989). In vitro, multiple layers of G-quartets stack to form G-quadruplexes, in which one or more DNA strands assemble together in either intra- or intermolecular configuration (Fig. 2B). Both intra- and intermolecular G-quadruplex conformations can be further subdivided into parallel and anti-parallel categories, depending on the orientation of the DNA strands (Keniry, 2000). Hybrid conformations of mixed polarities have also been reported. Therefore, G-quadruplexes – especially those assembled from the human telomeric sequence – exhibit extensive structural polymorphism (Oganesian and Bryan, 2007).

Intramolecular G-quadruplex folding at the chromosome end has been demonstrated to inhibit recombinant telomerase of human and ciliate species (Oganesian et al., 2006; Zahler et al., 1991; Zaug et al., 2005). On the basis of these findings, a substantial effort has been made to identify synthetic and natural compounds that would `lock' telomeric DNA in a G-quadruplex conformation and thereby impede telomere elongation in vivo. Although this is a plausible anti-cancer strategy, many uncertainties remain (Oganesian and Bryan, 2007). For example, the presence of G-quadruplex DNA and its precise conformation and prevalence in vivo have yet to be elucidated. Furthermore, not all G-quadruplex conformations inhibit telomerase activity in vitro (Oganesian et al., 2007; Oganesian et al., 2006).

Fig. 2.

G-quadruplex heterogeneity. (A) Chemical structure of the G-quartet, the base unit of a G-quadruplex, with a monovalent cation at its core (indicated by the red +). (B) Multiple G-quartets stack on top of each other to form a G-quadruplex. A single DNA strand can fold upon itself to form an intramolecular G-quadruplex (I-III), whereas two or four DNA strands can assemble into dimeric (IV) or tetrameric (V) intermolecular G-quadruplexes, respectively. The arrows indicate the parallel (II, V) or anti-parallel (I, III, IV) orientation of DNA strands, introducing a further source of heterogeneity. Parallel and anti-parallel hybrid conformations have also been reported (III).

Fig. 2.

G-quadruplex heterogeneity. (A) Chemical structure of the G-quartet, the base unit of a G-quadruplex, with a monovalent cation at its core (indicated by the red +). (B) Multiple G-quartets stack on top of each other to form a G-quadruplex. A single DNA strand can fold upon itself to form an intramolecular G-quadruplex (I-III), whereas two or four DNA strands can assemble into dimeric (IV) or tetrameric (V) intermolecular G-quadruplexes, respectively. The arrows indicate the parallel (II, V) or anti-parallel (I, III, IV) orientation of DNA strands, introducing a further source of heterogeneity. Parallel and anti-parallel hybrid conformations have also been reported (III).

Protein players in end protection

Shelterin complex: an overview

Chromosome-end protection is mediated by shelterin (de Lange, 2005), a six-protein conglomerate (Fig. 1). Only three of the six shelterin members maintain direct contact with telomeric DNA: telomere-repeat-binding factor 1 (TRF1) and TRF2 specifically recognize the double-stranded portion of telomeric DNA, and POT1 interacts with the G-overhang. TRF1-interacting nuclear factor 2 (TIN2) and TPP1 are intermediary proteins. TIN2 mediates an interaction between TRF1-TRF2 and TPP1-POT1, providing a link between the subcomplexes that interact with the double- and single-stranded DNA, respectively; it also tethers TRF1 to TRF2. TPP1 is in direct contact with POT1 (Houghtaling et al., 2004; Liu et al., 2004b; Ye et al., 2004b) and TIN2. The sixth protein, repressor activator protein 1 (RAP1), interacts exclusively with TRF2 (Li et al., 2000). Although all six shelterin subunits can assemble into a single soluble complex (Liu et al., 2004a; Ye et al., 2004a), the presence of shelterin subcomplexes containing either TRF1 or TRF2 in association with other subunits has also been suggested (Kim et al., 2008b; Liu et al., 2004a; Mattern et al., 2004; Ye et al., 2004a). However, the stoichiometry with which shelterin or its derivative subcomplexes interact with telomeric DNA remains unknown.

So how does this elaborate network of telomeric proteins `shelter' the chromosome end?

TRF1 and TRF2: genome gatekeepers and telomeric architects

Consistent with the essential roles of TRF1 and TRF2 in chromosome-end protection, homozygous inactivation of either gene results in early embryonic lethality in mice (Celli and de Lange, 2005; Karlseder et al., 2003). TRF2 is one of the most well-studied telomeric proteins and, arguably, is the most versatile in fulfilling its protective role. The importance of TRF2 in telomere protection is unveiled through conditional deletion of the gene, or functional inhibition of the protein through the expression of a dominant-negative form (TRF2ΔBΔM) that lacks its basic (B) and DNA-binding (Myb) domains. These disruptions in TRF2 expression or function elicit a DNA-damage response that is marked by the telomere dysfunction-induced nuclear foci (TIFs) γ-H2AX (a phosphorylated form of the histone variant H2AX that normally accumulates at double-stranded DNA breaks) and 53BP1 (p53-binding protein 1, which is responsible for mediating the transduction of a DNA-damage response to downstream effectors) (Takai et al., 2003), by significant loss of the G-overhang and by DNA-ligase-IV-dependent chromosome end-to-end fusions (Smogorzewska et al., 2002; van Steensel et al., 1998). This response suggests that TRF2 is involved in inhibiting the non-homologous end-joining (NHEJ) pathway of DNA repair. In accordance with this idea, TRF2 maintains a direct interaction with ataxia telangiectasia mutated (ATM) kinase, a key DNA-damage signal transducer. This interaction occurs through a domain that modulates the autophosphorylation state of ATM, thereby inhibiting its activation and that of its downstream targets in the NHEJ pathway, such as Nbs1 and p53 (Karlseder et al., 2004). Another target is the kinase Chk2, which was recently reported to localize to undamaged telomeres in a TRF2-dependent manner (Buscemi et al., 2009). A TRF2-Chk2 physical association was found to occur through the Myb domain of TRF2 and the S/TQ phosphorylation motif of Chk2. This interaction prevented Chk2 autophosphorylation and its subsequent activation in a manner akin to TRF2-mediated inhibition of ATM (Buscemi et al., 2009; Karlseder et al., 2004). Notably, telomere-dysfunction-induced activation of ATM and its downstream targets inevitably leads to entry into senescence or apoptosis (Karlseder et al., 1999; Smogorzewska et al., 2002). Therefore, this immediate and strategically targeted binding of TRF2 to these DNA-damage-associated kinases might hamper their activity.

TRF2 is also involved in suppressing illicit homologous recombination events at the telomere. This is important because the D-loop motif within the T-loop resembles a Holliday junction (HJ)-like recombination intermediate, which can be mistakenly processed by the homology-directed repair (HDR) pathway. In support of such a role for TRF2, expression of a mutant allele of TRF2 specifically lacking its B domain (TRF2ΔB) was shown to result in catastrophic T-loop-sized deletions of telomeric DNA and the appearance of extrachromosomal telomeric circles, which provoked a DNA-damage response and the induction of senescence (Wang et al., 2004). These deletion events were dependent on XRCC3 (X-ray repair complementing defective repair in Chinese hamster cells 3), attesting to the involvement of the HDR pathway. Telomere loss also depended on the presence of Nbs1 (Wang et al., 2004) and WRN helicase (Li et al., 2008), although it is not clear whether these proteins act in synergy or are redundant in this T-loop-deletion process. Furthermore, it was recently reported that TRF2ΔB impaired the cleavage activities of several archetypical HJ-resolving enzymes (Poulet et al., 2009), including that of GEN1, a recently identified human resolvase that cleaves HJs specifically and with perfect symmetry (Ip et al., 2008; Poulet et al., 2009). Additionally, it has been demonstrated that TRF2 associates with the nucleotide excision repair (NER) endonuclease complex XPF-ERCC1, which represses the recombination of telomeres with interstitial telomere-related sequences, giving rise to telomeric DNA-containing double minute chromosomes (TDMs) (Zhu et al., 2003). The XPF-ERCC1 complex has also been implicated in promoting overhang removal, resulting in chromosome fusions after TRF2 depletion from telomeres (Zhu et al., 2003). These observations provide important clues as to how TRF2 might deter recombination events at the telomere.

TRF1 negatively regulates telomerase-dependent maintenance of telomere length. This regulation is achieved by a tight collaboration with the TPP1-POT1 complex that oversees the access of telomerase to the G-overhang. It is thought that the extent of TRF1 occupancy on telomeric DNA positively correlates with telomere length, implying that longer telomeres bear more TRF1. This information is relayed to POT1 through TPP1, resulting in enhanced association of POT1 with the G-overhang, rendering it inaccessible to telomerase (Loayza and De Lange, 2003; Xin et al., 2007; Ye et al., 2004b). In support of this model, manipulation of TRF1 or POT1 levels at the telomere (through overexpression, downregulation or dominant-negative approaches) gives rise to telomere shortening and lengthening events, respectively (Ancelin et al., 2002; Loayza and De Lange, 2003; Smogorzewska et al., 2000; van Steensel and de Lange, 1997). This model holds up not only for cell cultures but also for whole organisms, whereby increased expression of TRF1 at a specific cellular compartment results in a corresponding decrease in telomere-length signal specifically within that compartment (Munoz et al., 2009). In the context of the whole organism, the activity of the XPF-ERCC1 nuclease has been implicated in TRF1-dependent telomere shortening (Munoz et al., 2009). Several lines of evidence suggest that the role of TRF1 as a modulator of telomere length is governed by its post-translational modifications that determine its abundance at telomeres; these modifications and their consequent effect on the fate of TRF1 and telomeres are summarized in Table 1.

Table 1.

Proteins affiliated with the shelterin complex

Accessory protein Shelterin member Site of interaction Significance of interaction Reference
Ck2 (casein kinase 2)   TRF1   N/D   Phosphorylates TRF1, promotes its binding to telomeric DNA, protects it from degradation   (Kim et al., 2008)  
Fbx4   TRF1   TRFH domain   Targets TRF1 for ubiquitin-mediated degradation   (Lee et al., 2006)  
Pin1   TRF1   TRFH domain   Regulation of TRF1 turnover and stability; targeting of TRF1 for degradation; telomere length regulation   (Lee et al., 2009)  
PinX1   TRF1   TRFH domain   Control of subcellular localization; enhances TRF1 binding to telomeres; inhibits telomerase   (Yoo et al., 2009; Zhou and Lu, 2001)  
RLIM   TRF1   C-terminal aa 265-378   Targets TRF1 for ubiquitin-mediated degradation   (Her and Chung, 2009)  
RNA polymerase II   TRF1   N/D   Might regulate transcription of TERRA   (Schoeftner and Blasco, 2008)  
Tankyrase 1 and 2   TRF1   N-terminal acidic domain   ADP-ribosylation and removal of TRF1 from telomeres; regulation of telomere length   (Cook et al., 2002; Hsiao et al., 2006; Smith et al., 1998)  
ATM   TRF1, TRF2   N/D   Phosphorylates TRF1, impairs its binding to telomeric DNA; autophosphorylation and activation of ATM can be inhibited by TRF2   (Karlseder et al., 2004; Wu et al., 2007)  
Ku   TRF1, TRF2, RAP1   Multiple domains; TRFH domain; N/D   End protection   (Hsu et al., 2000; O'Connor et al., 2004; Song et al., 2000)  
Apollo/human Snm1B   TRF2   TRFH domain   Prevention of a DNA damage response and end-to-end fusions during S phase   (Freibaum and Counter, 2006; Lenain et al., 2006; van Overbeek and de Lange, 2006)  
Chk2   TRF2   N/D   Phosphorylates TRF2, causing reduced binding to telomeres; Chk2 phosphorylation is inhibited by TRF2 in undamaged cells   (Buscemi et al., 2009)  
MCPH1   TRF2   TRFH domain   Involved in end protection; mediates DNA damage response at unprotected telomeres   (Kim et al., 2009)  
MUS81   TRF2   N/D   TRF2 regulates the endonuclease activity of MUS81 in ALT cells   (Zeng et al., 2009)  
PNUTS   TRF2   TRFH domain   Regulation of telomere length   (Kim et al., 2009)  
Topoisomerase IIIα   TRF2   N/D   Sustains TRF2-levels in ALT cells; associates with TRF2/BLM complex   (Temime-Smaali et al., 2008)  
XPF-ERCC1   TRF2   N/D   Removal of the 3′ overhang of in the absence of TRF2; suppression of TDMs (see text)   (Zhu et al., 2003)  
WRN; BLM   TRF2, POT1   N/D   Stimulates helicase activity   (Opresko et al., 2005; Opresko et al., 2002)  
MRN complex (MRE11, RAD50, NBS1)   TRF2, RAP1   N/D; multiple domains   MRN association with TRF2 might be mediated through RAP1; possible role in T-loop assembly and maintenance   (O'Connor et al., 2004; Zhu et al., 2000)  
Accessory protein Shelterin member Site of interaction Significance of interaction Reference
Ck2 (casein kinase 2)   TRF1   N/D   Phosphorylates TRF1, promotes its binding to telomeric DNA, protects it from degradation   (Kim et al., 2008)  
Fbx4   TRF1   TRFH domain   Targets TRF1 for ubiquitin-mediated degradation   (Lee et al., 2006)  
Pin1   TRF1   TRFH domain   Regulation of TRF1 turnover and stability; targeting of TRF1 for degradation; telomere length regulation   (Lee et al., 2009)  
PinX1   TRF1   TRFH domain   Control of subcellular localization; enhances TRF1 binding to telomeres; inhibits telomerase   (Yoo et al., 2009; Zhou and Lu, 2001)  
RLIM   TRF1   C-terminal aa 265-378   Targets TRF1 for ubiquitin-mediated degradation   (Her and Chung, 2009)  
RNA polymerase II   TRF1   N/D   Might regulate transcription of TERRA   (Schoeftner and Blasco, 2008)  
Tankyrase 1 and 2   TRF1   N-terminal acidic domain   ADP-ribosylation and removal of TRF1 from telomeres; regulation of telomere length   (Cook et al., 2002; Hsiao et al., 2006; Smith et al., 1998)  
ATM   TRF1, TRF2   N/D   Phosphorylates TRF1, impairs its binding to telomeric DNA; autophosphorylation and activation of ATM can be inhibited by TRF2   (Karlseder et al., 2004; Wu et al., 2007)  
Ku   TRF1, TRF2, RAP1   Multiple domains; TRFH domain; N/D   End protection   (Hsu et al., 2000; O'Connor et al., 2004; Song et al., 2000)  
Apollo/human Snm1B   TRF2   TRFH domain   Prevention of a DNA damage response and end-to-end fusions during S phase   (Freibaum and Counter, 2006; Lenain et al., 2006; van Overbeek and de Lange, 2006)  
Chk2   TRF2   N/D   Phosphorylates TRF2, causing reduced binding to telomeres; Chk2 phosphorylation is inhibited by TRF2 in undamaged cells   (Buscemi et al., 2009)  
MCPH1   TRF2   TRFH domain   Involved in end protection; mediates DNA damage response at unprotected telomeres   (Kim et al., 2009)  
MUS81   TRF2   N/D   TRF2 regulates the endonuclease activity of MUS81 in ALT cells   (Zeng et al., 2009)  
PNUTS   TRF2   TRFH domain   Regulation of telomere length   (Kim et al., 2009)  
Topoisomerase IIIα   TRF2   N/D   Sustains TRF2-levels in ALT cells; associates with TRF2/BLM complex   (Temime-Smaali et al., 2008)  
XPF-ERCC1   TRF2   N/D   Removal of the 3′ overhang of in the absence of TRF2; suppression of TDMs (see text)   (Zhu et al., 2003)  
WRN; BLM   TRF2, POT1   N/D   Stimulates helicase activity   (Opresko et al., 2005; Opresko et al., 2002)  
MRN complex (MRE11, RAD50, NBS1)   TRF2, RAP1   N/D; multiple domains   MRN association with TRF2 might be mediated through RAP1; possible role in T-loop assembly and maintenance   (O'Connor et al., 2004; Zhu et al., 2000)  

N/D, not determined; aa, amino acid

The conditional deletion of Trf1 in mice revealed a role for this factor in telomere replication. Lack of TRF1, combined with low doses of aphidicolin (a DNA polymerase inhibitor), led to a fragile telomere phenotype and S-phase-dependent signaling of ATR (ataxia telangiectasia and Rad3 related). This suggests that TRF1 plays a major role in replication-fork progression at TTAGGG (human telomeric sequence) repeats, and reveals a novel function for this factor (Sfeir et al., 2009).

Both TRF1 and TRF2 are involved in DNA remodeling. TRF1 can loop, bend and promote synapsis of telomeric DNA in vitro, which are activities mostly attributed to its homodimerization domain (Bianchi et al., 1997; Bianchi et al., 1999; Griffith et al., 1998). TRF2 can induce topological changes in DNA that stimulate strand invasion (Amiard et al., 2007; Griffith et al., 1999; Stansel et al., 2001; Verdun and Karlseder, 2006). The synergistic DNA-remodeling properties of these two shelterin components are thought to give rise to the higher-order architecture of the chromosome end (the T-loop) (Fig. 1). Evidence also suggests that TRF2 can modulate telomeric G-quadruplex assembly (Pedroso et al., 2009). It is conceivable that G-quadruplex formation at the G-overhang could reduce the efficiency of the strand invasion process (Pedroso et al., 2009); alternatively, quadruplex formation at the D-loop might help to stabilize the invasion site (see Fig. 1B). Therefore, TRF2 might be involved in either preventing or promoting G-quadruplex formation, depending on the context. Moreover, TRF2 has been shown to exhibit a strong affinity for four-stranded junctions in vitro, and this preference could assist TRF2 in further stabilizing the overhang invasion site (Fouche et al., 2006).

TIN2

TIN2 was originally identified in a yeast two-hybrid assay (Kim et al., 1999). It was shown to negatively regulate telomerase-dependent telomere elongation, which resembles the function of TRF1 in regulating telomere length. Biochemical analyses revealed that TIN2 stimulates TRF1 homodimerization, thereby allowing for synapsis of telomeric DNA. This indicated that TIN2 imparts its regulation of telomere length by causing conformational changes in TRF1, which in turn shape the structure of telomeric DNA (Kim et al., 2003). TIN2 also directly interacts with TRF2 and this interaction is further stabilized through TRF1-TIN2 binding. Expression of TIN2 mutants with defective binding to either TRF1 or, in particular, to TRF2 yields a DNA-damage response, demonstrated by the presence of TIFs and indicative of telomere de-protection (Kim et al., 2004). Recently, it was proposed that there are two distinct subcomplexes that cooperate and reinforce each other to form the higher-order protective terminal structure at the telomere: TIN2-TRF1 associated with TPP1-POT1; and TIN2-TRF2-hRAP1 associated with TPP1-POT1. The former subcomplex was speculated to enhance the stability and function of the latter (Kim et al., 2008b). A splice variant of TIN2, TIN2L, has now been identified that can be differentiated from its originally described counterpart, TIN2S, by the presence of 97 additional amino acids at the C-terminus. TIN2L maintains a tight association with the nuclear matrix and is thus speculated to tether shelterin-associated telomeres to the matrix (Kaminker et al., 2009). The significance of this finding needs further investigation.

POT1 and TPP1

POT1 and TPP1 are the most evolutionarily conserved components of the shelterin complex in that they represent the human homologs of the α- and β-subunits of the ciliate telomere end-binding proteins (TEBPs), respectively (Baumann and Cech, 2001; Lei et al., 2004; Wang et al., 2007; Xin et al., 2007). POT1 homologs from yeast (Baumann and Cech, 2001), plants (Shakirov et al., 2005) and, more recently, from the ciliate Tetrahymena thermophila (Jacob et al., 2007) and potentially in the nematode C. elegans (Raices et al., 2008) have been identified on the basis of their sequence or structural identity to the oligonucleotide/oligosaccharide-binding (OB) fold within the DNA-binding domain of TEBPs.

As discussed above, the telomeric overhang is the cognate binding substrate of POT1. The DNA-binding motif of hPOT1 resides in its two N-terminal OB folds (OB1 and OB2) (Lei et al., 2004), whereas the C-terminus mediates its interaction with TPP1 (Houghtaling et al., 2004; Liu et al., 2004b; Ye et al., 2004b). The crystal structure of the DNA-binding motif of hPOT1 in association with telomeric ssDNA illustrated that a minimum of ten nucleotides of precise register (5′-TTAGGGTTAG-3′) are required for optimal interaction (Lei et al., 2004). Furthermore, it was evident that the end 3′ nucleotide was buried within the molecular surface of the OB2 domain. This suggests that POT1 alone could be sufficient to confer G-overhang protection (Lei et al., 2004). This prediction was verified with an in vitro biochemical approach that demonstrated that hPOT1 could interact with its decamer recognition sequence in two distinct modes: one in which interaction occurred distally from the 3′ end, leaving a free DNA tail to accommodate for access and elongation by recombinant telomerase; and a second in which access was occluded by binding most proximally to the 3′ end (Lei et al., 2005). Such bimodal G-overhang binding by hPOT1 could explain the observation that artificially perturbed hPOT1 levels exert both positive and negative effects on telomerase-dependent maintenance of telomere length in vivo (Colgin et al., 2003; Loayza and De Lange, 2003).

The phenotypic consequences of POT1 loss in vivo are somewhat inconsistent across species. Ablation of the pot1 gene in fission yeast results in complete loss of telomeric DNA and circularization of its chromosomes, and allows for survival in the absence of chromosome-end protection (Baumann and Cech, 2001). By contrast, in human cells, where only partial depletion of POT1 is feasible, the resulting phenotype is far more modest: fusions are not always evident, the G-overhang length is somewhat altered, a transient DNA-damage response ensues, and in some cases there are chromosomal abnormalities accompanied by cell-cycle arrest or apoptosis (Hockemeyer et al., 2005; Veldman et al., 2004). In rodents, the situation is different again. In most vertebrates POT1 is encoded by a single gene, but in rodents a recent gene duplication event gave rise to two POT1 orthologs, POT1a and POT1b, each exhibiting a distinct role in telomere protection (Hockemeyer et al., 2006; Wu et al., 2006). Conditional deletion of Pot1a alone or together with Pot1b sparks a severe ATR-driven DNA-damage response (Denchi and de Lange, 2007; Hockemeyer et al., 2006; Wu et al., 2006), whereas deletion of Pot1b alone induces progressive telomere shortening and excessive resection of the 5′ strand, resulting in long G-overhangs (Hockemeyer et al., 2006; Hockemeyer et al., 2008). Both POT1a and POT1b have been shown to play a role in controlling telomeric recombination, as assessed by telomere sister chromatid exchange (t-SCE) events (Hockemeyer et al., 2005) or detection of extrachromosomal telomeric circles (Wu et al., 2006). Domain-swapping experiments showed that hPOT1 contains features of its two mouse counterparts (Palm et al., 2009) in that it has both a strong effect on the ATR-governed DNA-damage response and ensures overhang protection from unwanted processing activities or telomerase. Therefore, it appears that POT1 in mice and humans mediates protection specifically from the ATR arm of the DNA-damage response (Denchi and de Lange, 2007). This also holds true for chicken POT1 (Churikov and Price, 2008), establishing some parallels for the role of vertebrate POT1 in end protection.

In C. elegans, as mentioned earlier, there are two telomere-specific ssDNA-binding proteins, CeOB1 and CeOB2; these share structural homology with the OB2 and OB1 folds of hPOT1, respectively. Interestingly, deletion of CeOB1 results in overhang and telomere lengthening, whereas loss of CeOB2 causes telomere-length heterogeneity (Raices et al., 2008). This indicates that, on one hand, CeOB1 might regulate telomerase access to the overhang; on the other hand, CeOB2 could prevent uncontrolled losses or gains of telomeric DNA through dysregulated recombination.

The involvement of POT1 in the regulation of telomere-length dynamics is mostly linked with its ties to TPP1. It has been proposed that TPP1 plays an instrumental role in coordinating the assembly of the whole shelterin complex (O'Connor et al., 2006), and it is thought to stabilize the TIN2-TRF2 interaction and the connectivity of TRF1 and TRF2 subcomplexes. Interference with TIN2-dependent TPP1 localization to telomeres disrupts proper shelterin assembly, leading to excessive telomere elongation in cells that express telomerase (Houghtaling et al., 2004; O'Connor et al., 2006). Disturbing the POT1-TPP1 link gives rise to a similar outcome, accompanied by a DNA-damage response (Xin et al., 2007). For a long time it was not understood how the shelterin complex communicates with telomerase and controls its access to the G-overhang. It was then realized that the OB domain of TPP1 could physically interact with the catalytic subunit of human telomerase, hTERT, and promote its recruitment to the chromosome end (Xin et al., 2007). Moreover, the POT1-TPP1 heterodimer was reported to enhance the activity and processivity of recombinant human telomerase in vitro (Wang et al., 2007). These findings provide some insight into how the POT1-TPP1 duo might modulate changes in telomere length. Recently, Miyoshi and co-workers identified tpz1 as the TPP1 homolog in fission yeast (Miyoshi et al., 2008). A pot1-tpz1 complex is also involved in regulation of telomere length and end protection in fission yeast, although with contribution from two novel interacting partners, poz1 and ccq1, which dictate its function in length regulation in a negative and positive manner, respectively (Miyoshi et al., 2008).

Overall, POT1 appears to serve two key roles at the telomeric overhang: TPP1-assisted (in most species) control of telomere length fluctuations, as mediated by telomerase or recombination events, and protection of the overhang from insults by the DNA-damage machinery.

Fig. 3.

Mechanisms of telomerase-driven (A) and homologous-recombination-driven (B) synthesis of telomeric DNA. (AI) DNA synthesis is initiated with the alignment of the 3′ telomeric G-overhang (in blue) with the RNA template (in red). (AII) Telomerase catalyzes nucleotide addition to the overhang until the 5′ end of the template is reached. (AIII) The enzyme translocates and realigns with the newly synthesised 3′ end of the overhang (primer). (AIV) A second round of nucleotide addition ensues. (B) Both inter- and intratelomeric recombination events have been hypothesised to foster telomere maintenance in ALT. In both types of recombination, the 3′ overhang of the telomere initiates invasion and uses its host DNA as a template for DNA copying. (BI) Intermolecular recombination might involve telomeres from two adjacent chromosomes or chromatids. Alternatively, telomere alignment and invasion into circular or linear (not shown) ECTR could also promote DNA synthesis. (BII) The telomere could also use itself as a template and synthesize DNA via rolling circle replication.

Fig. 3.

Mechanisms of telomerase-driven (A) and homologous-recombination-driven (B) synthesis of telomeric DNA. (AI) DNA synthesis is initiated with the alignment of the 3′ telomeric G-overhang (in blue) with the RNA template (in red). (AII) Telomerase catalyzes nucleotide addition to the overhang until the 5′ end of the template is reached. (AIII) The enzyme translocates and realigns with the newly synthesised 3′ end of the overhang (primer). (AIV) A second round of nucleotide addition ensues. (B) Both inter- and intratelomeric recombination events have been hypothesised to foster telomere maintenance in ALT. In both types of recombination, the 3′ overhang of the telomere initiates invasion and uses its host DNA as a template for DNA copying. (BI) Intermolecular recombination might involve telomeres from two adjacent chromosomes or chromatids. Alternatively, telomere alignment and invasion into circular or linear (not shown) ECTR could also promote DNA synthesis. (BII) The telomere could also use itself as a template and synthesize DNA via rolling circle replication.

RAP1

RAP1 is perhaps the least well-understood member of the human shelterin complex. It was first identified as a TRF2-interacting protein that exhibited significant sequence identity with the Rap1p protein from Saccharomyces cerevisiae (Li et al., 2000), known to play a key role in regulation of telomere length (Marcand et al., 1997). Exogenous expression of full-length or mutant alleles of human RAP1 resulted in telomere elongation, consistent with the idea that the exogenous protein either displaced its endogenous counterpart from telomeres or titrated out other factors involved in negative regulation of telomere length; in both scenarios, fluctuations in telomere length were dependent on telomerase expression (Li et al., 2000; O'Connor et al., 2004). Deletion analyses revealed that the central linker domain of human RAP1 (hRAP1) was responsible for its negative regulatory role (O'Connor et al., 2004), whereas the N-terminal BRCT domain was implicated in influencing the heterogeneity of telomere length (Li et al., 2000). Apart from its involvement in telomere-length homeostasis, hRAP1 has also been shown to assist TRF2 in inhibiting end-to-end fusion events at telomeric substrates in vitro, suggesting that TRF2 alone is not sufficient for halting NHEJ-mediated chromosome-end fusions, and that hRAP1 is integral to end protection (Bae and Baumann, 2007).

Accessory proteins

TRF1 and TRF2, the focal point of protein-protein interactions at telomeres, share a common TRF homology (TRFH) domain that appears to be necessary for homodimerization of each individual protein; heterodimerization of the two proteins has not been observed. Curiously, the two proteins exploit the TRFH motif to recruit different interacting partners. TRF1 uses the TRFH molecular surface to recognize TIN2 and an accessory protein, PinX1 (Zhou and Lu, 2001), whereas TRF2 uses this surface to bind to Apollo (Freibaum and Counter, 2006; Lenain et al., 2006; van Overbeek and de Lange, 2006), another accessory affiliate. TRF2-TIN2 interaction occurs outside of the TRFH domain. These findings have led to the proposal that TRFH is a docking site that recognizes proteins based on a consensus sequence [F/Y]xLx(P) (Chen et al., 2008). Based on this sequence motif, two novel TRF2-interacting partners were identified, phosphatase nuclear-targeting subunit (PNUTS) and microcephalin 1 (MCPH1) (Kim et al., 2009). For a comprehensive list of proteins affiliated with the human shelterin complex, see Table 1.

Telomere maintenance pathways

DNA is lost each time a cell divides in the absence of telomerase (Harley et al., 1990; Lundblad and Szostak, 1989). This loss is mainly attributed to the `end replication problem' (see below) (Olovnikov, 1973; Watson, 1972) and processing events that follow semi-conservative DNA replication. Although most cells in the human soma are fated to undergo telomere attrition, which eventually ushers them into senescence, cells of the germline and the majority (>85%) of those that have undergone malignant transformation express telomerase (Shay and Bacchetti, 1997; Wright et al., 1996), the enzyme that corrects telomere loss (Box 1, Fig. 3A). Telomere maintenance in the absence of telomerase relies on mechanisms that involve inter- or intratelomeric recombination, collectively termed ALT (Box 1, Fig. 3B).

Telomere replication and processing

Replication of telomeres and their subsequent processing steps are poorly understood, particularly in the context of activated telomere maintenance pathways. Telomere replication is probably unidirectional because it is thought to originate at the subtelomere, with the replication fork moving towards the chromosome end. Given the conserved orientation of telomeric repeats, the G- and C-rich strands are inevitably synthesized by leading- and lagging-strand polymerases, respectively (Gilson and Geli, 2007) (Fig. 4A). The conventional DNA replication machinery only works in the 5′-3′ direction and relies on the presence of an RNA primer to initiate synthesis as short pieces of DNA (Okazaki fragments) that are eventually ligated to form a continuous DNA strand. The template sequence between the extreme end of the DNA strand and the most distal Okazaki fragment cannot be replicated after removal of the RNA primer, resulting in loss of DNA at the 5′ end of the newly synthesized C-strand; this is known as the `end replication problem'. Although lagging-strand synthesis yields a short 3′ overhang, the product of leading-strand synthesis is blunt-ended. This presents a problem for telomerase because it requires a G-rich overhang as its substrate. Generation of the 3′ telomeric overhang presumably entails re-section of the C-rich strand; however, the nuclease(s) responsible for this DNA-processing event has yet to be identified. Telomerase elongation of the G-rich overhang is accompanied by semi-conservative replication of the C-rich strand, which is accomplished by conventional polymerases (Verdun and Karlseder, 2007) (Fig. 4A). Further processing of the 3′ DNA ends probably follows the cooperative syntheses of the G- and C-rich strands to facilitate formation of the T-loop. It appears that recognition of replicated telomeres as DNA damage is a necessary step that triggers the molecular machinery involving the aforementioned HR and telomere proteins to promote generation of overhang and the assembly of this protective structure (Verdun et al., 2005; Verdun and Karlseder, 2006).

Fig. 4.

Telomere replication and processing in cells that utilize the telomerase (A) and ALT (B) pathways of telomere maintenance. (A) Telomeric replication is initiated at subtelomeric origins. The T-loop structure at the chromosome end must be resolved for successful replication; the factors responsible for T-loop disassembly, if required, are unknown. The product of lagging-strand synthesis bears a short G-rich overhang, whereas that of leading-strand synthesis is blunt-ended and thus needs to be processed to yield the correct substrate for telomerase. It is unclear whether further processing also needs to occur at the lagging-strand synthesis product. Telomerase elongates the newly formed overhangs and conventional DNA polymerases fill in the opposite strand. At this point, fully replicated telomeres are envisaged to provoke DNA damage, which is thought to be the trigger for end processing and T-loop assembly to reinstate a protected telomere end. (B) Post-replication telomeres at sister chromatids are of equivalent length. In cells that use the ALT pathway, asymmetric alignment could promote unbalanced telomere elongation of one telomere at the expense of the other, resulting in very long and short telomeres. Such alignments could also initiate uneven t-SCE events (not shown) in the absence of DNA synthesis. All replicated telomeres of sufficient length can then take up the T-loop structure, whereby further self-templated telomere synthesis could ensue via rolling circle replication. Alternatively, T-loops could be inappropriately resolved, resulting in release of free telomere circles and severely shortened telomeres with high recombinogenic potential.

Fig. 4.

Telomere replication and processing in cells that utilize the telomerase (A) and ALT (B) pathways of telomere maintenance. (A) Telomeric replication is initiated at subtelomeric origins. The T-loop structure at the chromosome end must be resolved for successful replication; the factors responsible for T-loop disassembly, if required, are unknown. The product of lagging-strand synthesis bears a short G-rich overhang, whereas that of leading-strand synthesis is blunt-ended and thus needs to be processed to yield the correct substrate for telomerase. It is unclear whether further processing also needs to occur at the lagging-strand synthesis product. Telomerase elongates the newly formed overhangs and conventional DNA polymerases fill in the opposite strand. At this point, fully replicated telomeres are envisaged to provoke DNA damage, which is thought to be the trigger for end processing and T-loop assembly to reinstate a protected telomere end. (B) Post-replication telomeres at sister chromatids are of equivalent length. In cells that use the ALT pathway, asymmetric alignment could promote unbalanced telomere elongation of one telomere at the expense of the other, resulting in very long and short telomeres. Such alignments could also initiate uneven t-SCE events (not shown) in the absence of DNA synthesis. All replicated telomeres of sufficient length can then take up the T-loop structure, whereby further self-templated telomere synthesis could ensue via rolling circle replication. Alternatively, T-loops could be inappropriately resolved, resulting in release of free telomere circles and severely shortened telomeres with high recombinogenic potential.

In cells that rely on the ALT pathway of telomere maintenance, loss of telomeric DNA due to replication and processing is mainly counteracted post-replicatively via inter- and intramolecular recombination events (Fig. 3B and Fig. 4B). It is thought that alignment of sister chromatids (or chromatids on neighboring chromosomes; Fig. 3B) allows for invasion of the 3′ overhang of one chromatid into the duplex region of its sister, which could then serve as the template for telomere copying. If the alignment is asymmetric, unequal telomere elongation of one chromatid would occur at the expense of the other, giving rise to extremely long and short telomeres, which are typical of cells in which ALT is active. Asymmetric chromatid alignment could also lead to unequal t-SCE events in the absence of DNA synthesis; herein, a chromatid might sacrifice the entirety of its telomeric DNA to the sister. The daughter cell that receives a telomere-less chromosome will undergo senescence and be lost from the population, whereas the one with longer telomeres will have a proliferative advantage (Muntoni et al., 2009; Muntoni and Reddel, 2005). The Reddel group has now presented evidence for T-loop-mediated intrachromatid copying (Muntoni et al., 2009). This scenario is based on self-templated telomere synthesis and could thus occur pre- or post-replication. Chromosomes with telomere-signal-free ends are often a prominent feature of cells in which the ALT pathway is active. This phenomenon might be attributed to T-loop junction-resolution events that result in the formation of free T-circles that contribute to the pool of extrachromosomal telomeric repeats (ECTRs) and severely truncated telomeres that are likely to be highly recombinogenic (Cesare and Reddel, 2008) (Fig. 4B).

Consequently, although telomere replication and processing machineries are probably shared between cells that express telomerase and those that engage in ALT, the latter appear to be in a far more precarious state in terms of telomere protection. T-loops have been observed in both cell types (Cesare and Griffith, 2004; Griffith et al., 1999); however, it seems that telomeric chromatin within this structure in cells that use ALT is far more loosely organized. Furthermore, a large proportion of telomeres might not bear a T-loop due to insufficient length. It is conceivable that shelterin function, particularly that of TRF2, is somehow compromised, although specific defects have not been reported for any shelterin member in cells with activated ALT.

Telomere length homeostasis

Telomerase access and levels

Even in malignant cells that express telomerase at high levels, telomere length is kept in check. An extensive body of evidence indicates that the access of telomerase to its substrate is tightly regulated, which offers one possible explanation for this balance in length; another plausible explanation is that telomerase levels are limiting. Indeed, augmented expression of hTERT and telomerase RNA (hTR) levels in cells that already express telomerase results in massive telomere elongation, concomitant with enhanced loading of telomeric proteins (Cristofari and Lingner, 2006). This suggests that, despite the increased load of TRF1 and TRF2, long telomeres are not permanently locked into an inaccessible state and, more importantly, that telomere homeostasis requires that telomerase levels are limiting (Cristofari and Lingner, 2006). One study reported that, in the context of telomerase excess due to hTR overexpression in telomerase-positive cells, telomeres did lengthen several-fold beyond their physiological norm. However, this length increase was not exponential; rather it appeared to plateau after extensive culture. This stabilization in telomere length was accompanied by heterogeneity of telomere length, the appearance of circular and single-stranded extrachromosomal telomeric DNA, and formation of ALT-associated PML body (APB)-like nuclear foci; these are all features characteristic of cells that use the ALT pathway (Pickett et al., 2009). On the basis of these observations, the authors proposed that cells possess an intrinsic mechanism that allows for trimming of telomeres once they reach a certain threshold, although the determinants of this threshold are not known. Importantly, the over-elongated telomeres did not trigger a DNA-damage response, suggesting that they were sufficiently protected (Pickett et al., 2009).

Apart from its canonical role in counteracting loss of telomeric DNA, telomerase has also been suggested to play a more direct role in telomere protection; this is proposed to entail `capping' of the chromosome end through sustained interaction with the G-overhang (Chan and Blackburn, 2002). Although such a role could only be applicable to transformed or malignant cells that express telomerase at high levels, there is some evidence that there is modest S-phase-specific expression of this enzyme in primary cells (Masutomi et al., 2003).

Recently, it came to light that telomere-length homeostasis might also be achieved by a distinct, hitherto uncovered mechanism – the production of telomeric transcripts.

TERRA

For a long time, telomeres and the adjacent subtelomeric region were considered to be transcriptionally silent. This perception was largely based on observations of enriched DNA methylation at the subtelomere and of overrepresentation of heterochromatic marks (namely trimethylation of histones H3 and H4 at lysines 9 and 20, respectively) and binding of heterochromatin protein 1 within both telomeric and subtelomeric regions (Blasco, 2007). However, two groups recently reported that telomeres are, in fact, transcribed in several species and result in UUAGGG-containing RNA repeats referred to as telomeric repeat-containing RNA (TERRA) (Azzalin et al., 2007) or TelRNA (Schoeftner and Blasco, 2008). Transcription is strand-specific and is mediated by RNA polymerase II (Luke et al., 2008; Schoeftner and Blasco, 2008). The transcripts are subject to negative regulation by the 5′-3′ exonuclease activities of Rat1p in S. cerevisiae (Luke et al., 2008) and by effectors of nonsense-mediated messenger RNA decay, known as SMG proteins in yeast (Azzalin et al., 2007). TERRA localize to telomeres (Azzalin et al., 2007; Schoeftner and Blasco, 2008) and have been speculated to negatively influence telomerase activity in one of three proposed ways: by hybridization of TERRA to the complementary template region within the RNA moiety of telomerase (Schoeftner and Blasco, 2008); by forming RNA-DNA duplexes (Luke et al., 2008); or by forming G-quadruplexes at telomeres (Xu et al., 2008). In accordance with these propositions, TERRA levels are strongly reduced in immortalized telomerase-positive cell lines compared with primary cells or cells engaged in ALT (Ng et al., 2009). In addition, TERRA levels are highly upregulated in patients with immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome, whose cells exhibit abnormally short telomeres (Yehezkel et al., 2008), further substantiating the potential involvement of TERRA in telomere-length homeostasis.

Telomerase-independent homeostasis

Telomere-length homeostasis in a telomerase-deficient setting might be mediated through recombination initiated by the shortest telomeres. This idea is supported by recent findings from the Greider laboratory, indicating that some elements of the ALT mechanism, such as t-SCE and subtelomeric recombination events, might be used by non-transformed cells to maintain stable telomeres without a net gain in length (Morrish and Greider, 2009).

Telomeric chromatin

Mammalian telomeres contain nucleosomes (Lejnine et al., 1995; Tommerup et al., 1994) and mouse telomeres bear marks of constitutive heterochromatin (Blasco, 2007) (Box 3). Recent findings in rodents have unraveled ties between the epigenetic state of telomeric chromatin and maintenance of telomere length. For example, shortened telomeres in telomerase-null mice exhibited loss of trimethylated H3K9 and H4K20 heterochromatic motifs, diminished HP1 binding to telomeric chromatin, and loss of subtelomeric DNA methylation. Together, these findings suggest that shorter telomeres are associated with a more `open' chromatin state that is amenable to homologous recombination, marked by a rise in the frequency of t-SCE events and APB foci (Benetti et al., 2007). In support of this idea, methylation of subtelomeric DNA repeats has been negatively correlated with telomere length and recombination in a large panel of human cancer cell lines (Vera et al., 2008). In addition, cells genetically deficient for histone or DNA methyltransferases, or null for Retinoblastoma (Rb) family members, were found to have dramatically elongated telomeres and were more prone to recombination (Garcia-Cao et al., 2002; Garcia-Cao et al., 2004; Gonzalo and Blasco, 2005; Gonzalo et al., 2006), further highlighting the impact of chromatin state on telomere length. Therefore, telomeric heterochromatin in its compact configuration is thought to be a negative regulator of telomere length.

Box 3. Introduction to chromatin

Eukaryotic chromatin constitutes DNA packaged with histones. The basic building block of chromatin is the nucleosome particle (Kornberg and Lorch, 1999), which contains approximately 147 base pairs of DNA wrapped around the surface of an octamer of histone proteins. The histone octamer consists of a central (H3-H4)2 tetramer that is flanked on either side by two H2A-H2B dimers. Further compaction of chromatin is achieved by the linker histone H1. Telomeric chromatin in mammals, similarly to that in yeast, has been shown to exert a silencing effect on the expression of nearby genes (Baur et al., 2001), a phenomenon termed telomere-position effect (TPE).

Chromatin exists in two states: euchromatin and heterochromatin. Telomeres and adjacent sub-telomeric sequences are considered as gene-less regions and are representative of heterochromatin. A signature DNA modification in heterochromatin is the addition of a methyl group at the fifth position of cytosine within CG dinucleotides. This reaction is catalyzed by three DNA methyltransferases – DNMT1, DNMT3a and DNMT3b. Mammalian telomeric DNA is devoid of CG dinucleotides, but these are in abundance within the sub-telomere, which is thus subject to heavy methylation. The flexible N-terminal tails of histones are amenable to a variety of post-translational modifications, such as trimethylation at H3K9 or H4K20, that further define telomeric heterochromatin. These histone modifications are mediated by the histone methyltransferases SUV39H1 and H2 (for H3K9), and SUV420H1 and H2 (for H4K20). Heterochromatin assembly at telomeric and sub-telomeric regions also entails the binding of chromobox proteins CBX1, CBX3 and CBX5 (homologs of Drosophila melanogaster heterochromatin protein 1, HP1) to those regions (Blasco, 2007).

Loss of heterochromatic marks at mouse telomeres does not affect the telomeric occupancy of TRF1 and TRF2 proteins (Benetti et al., 2007); nonetheless, both of these proteins have been implicated in the epigenetic regulation of telomeres. TRF1 overexpression in human cultured cells was found to transiently alleviate telomeric repression of an artificially inserted subtelomeric reporter gene, suggesting a release from telomere-position effect (TPE); this de-repression event correlated with displacement of HP1 isoforms from the telomere (Koering et al., 2002). TRF2 overexpression in mouse primary keratinocytes resulted in a telomere-specific loss of heterochromatin features, decreased abundance of core histones, and increased nucleosomal spacing, which together are the signature of a more relaxed telomere chromatin state. This suggests a potential involvement of TRF2 in chromatin assembly (Benetti et al., 2008), providing a link between telomere chromatin organization and end protection. Notably, de-protected telomeres that are susceptible to either NHEJ or HDR repair pathways do not experience loss of nucleosome occupancy, as is characteristic for other parts of the genome undergoing DNA repair (Wu and de Lange, 2008). This indicates that the nucleosomal organization at telomeres might be uniquely accessible to repair activities (Wu and de Lange, 2008). Recently the de Lange group reported that de-protected telomeres devoid of TRF2 were more mobile and sampled larger territories within the nucleus, and that this increase in the dynamics of telomeric chromatin facilitated 53BP1-mediated NHEJ (Dimitrova et al., 2008).

It has been suggested that nucleosomes provide an added layer of protection to telomeres, as the binding of telomeric proteins such as TRF1 does not result in nucleosome release from telomeric chromatin. Rather, this binding introduces changes to nucleosome structure that might be conducive to formation of the protective telomere cap (Galati et al., 2006).

In the context of cancer, telomerase-positive tumor cell lines exhibit densely methylated subtelomeres, whereas tumors originating from cells that use ALT are associated with heterogeneous methylation patterns; the former is correlated with severely reduced levels of telomeric transcript. This observation allows for the speculation that epigenetic differences between telomerase- and ALT-positive tumors might underpin the mechanism of telomere-length maintenance in human tumorigenesis (Ng et al., 2009).

Perspectives

Although we have made significant progress in uncovering the intricate layers of chromosome-end protection, many aspects of this process remain poorly understood. We have yet to identify the enzyme(s) involved in overhang generation, which is a prerequisite for T-loop formation. The prevalence of the T-loop in vivo is unknown, meaning that other modes of protection are possible. Furthermore, the ambiguous interplay between shelterin and the DNA-damage sensor and repair machinery has yet to be dissected. Recent findings regarding telomeric chromatin and its involvement in homeostasis of telomere length have introduced a new facet of telomere biology, the understanding of which is still in its infancy. Finally, the importance of potential differences between mouse and human telomere biology has yet to be explored in detail. Therefore, despite much progress, many challenges in improving our understanding of telomere protection still lie ahead.

J.K. acknowledges support by the NIH (ROI GM06525 and ROI AG 025837). Deposited in PMC for release after 12 months.

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