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Telomeres at a glance

Agnel Sfeir

The Helen L. and Martin S. Kimmel Center for Biologyand Medicine at the Skirball Institute of BiomolecularMedicine, Department of Cell Biology, NYU School ofMedicine, New York, NY 10016, USA

agnel.sfeir@med.nyu.edu

Journal of Cell Science 125, 4173–4178

� 2012. Published by The Company of Biologists Ltd

doi: 10.1242/jcs.106831

The linearity of chromosomes creates two

major problems for eukaryotic cells: the

end-replication problem and the end-

protection problem. The end-replication

problem stems from the inherent inability

of the replication machinery to fully

duplicate linear templates. The end-

protection problem refers to the propensity

of linear chromosome ends to be recognized

as DNA double-strand breaks (DSBs). Both

problems are surmounted by telomeres, the

specific nucleoprotein complexes that adorn

chromosome ends.

In 2009, the Nobel Prize was awarded to

pioneering researchers in the telomere field

in recognition of their seminal findings that

set the stage for, so far, three decades of

vigorous investigation of telomeres and

telomerase. In this article, I review our

current knowledge and recent findings

regarding the function of telomeres,

with a focus on the mammalian system.

Specifically, I delineate the structure and

composition of the telomere and highlight

recent discoveries that have elucidated the

molecular mechanisms underlying the

solutions to both ‘end problems’. Lastly, I

describe the perilous consequences of

telomere dysfunction during tumorigenesis.

The composition of chromosome endsFeatures of telomeric DNA

The telomeric architecture incorporatesthree known entities: tandem repeats of

DNA sequence, a specific set of binding

proteins and a non-coding RNA transcript.In mammals, telomeric DNA consists of

TTAGGG repeats and their complementary

AATCCC sequences and, in humans, its sizeranges between 5 and 15 kb. A key feature

of the telomere end in all organisms is a 39

single-stranded G-rich overhang (Makarovet al., 1997; McElligott and Wellinger,

1997). Although, it should also be noted

that 59 single-stranded C-rich overhangshave been observed in worms (Raices

et al., 2008) and can occur transiently in

some human cancer cells (Oganesian andKarlseder, 2011). Mammalian G-rich

overhangs are 30-500 nucleotides long

(Chai et al., 2005) and are generated by

(See poster insert)

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the removal of the RNA primer from theterminal Okazaki fragment on the lagging

strand, as well as by post-replicativeprocessing events that involve nucleasesperforming 39 resection on both newlysynthesized strands. Recent studies have

started to unravel the molecular details ofsuch processing events by identifying theApollo nuclease that acts on leading end

telomeres (Lam et al., 2010; Wu et al.,2010). G-rich overhangs have a central rolein sustaining a diverse array of telomeric

functions and are thought to invade thepreceding duplex region of the telomere toform a lariat-like structure called the t-loop.This higher-order architecture, which was

revealed by electron microscopy (Griffithet al., 1999; Nikitina and Woodcock,2004; Raices et al., 2008), provides one

mechanism by which chromosome endsmaintain their protective cap.

Telomere-binding proteins

Telomeric DNA is bound by a specialized setof proteins, whose composition, structureand function have diverged across species

(de Lange, 2009). In mammalian cells, thereare six bona fide telomere-specific proteins:TRF1 (telomeric repeat binding factor 1, also

known as TERF1), TRF2 (telomeric repeatbinding factor 2, also known as TERF2),RAP1 (TERF2 interacting protein,

also known as TERF2IP), TIN2 (TRF1interacting nuclear factor 2, also known asTINF2), TPP1 (adrenocortical dysplasia

protein homolog, also known as ACD) andPOT1 (protection of telomeres 1), whichtogether form the shelterin complex (deLange, 2005). The exquisite specificity

with which shelterin binds to the telomericDNA is conferred by its three DNA-bindingmodules. TRF1 and TRF2 bind to the duplex

region of the DNA (Bianchi et al., 1997;Broccoli et al., 1997; Bilaud et al., 1997),whereas POT1 coats the overhang with

its oligonucleotide/oligosaccharide binding(OB) folds (Baumann and Cech, 2001; Leiet al., 2002; Loayza and de Lange, 2003).Rodents express two POT1 paralogs (POT1a

and POT1b) that are structurally similar, yetfunctionally divergent (Hockemeyer et al.,2006; Wu et al., 2006). TIN2 has a bridging

function, as it interacts with both TRF1 andTRF2 while simultaneously recruiting TPP1(Ye et al., 2004a; Ye et al., 2004b; O’Connor

et al., 2006; Takai et al., 2011; Houghtalinget al., 2004). In vivo data has suggested thatPOT1 accumulation at telomeres relies on its

interaction with TPP1, as its OB folds areinsufficient to tether it to DNA (Kibe et al.,2010). RAP1 is the sixth and most

evolutionary conserved member of shelterin(Li and de Lange, 2003). Furthermore, it is

the only subunit known to have non-telomeric functions: it also operates as atranscriptional regulator (Martinez et al.,

2010) and impinges on nuclear factorkappa B (NF-kB) signaling (Teo et al.,2010).

Biochemical studies have uncovered anincreasing number of telomere-associatedproteins that are primarily recruited by

shelterin and act as accessory factors for thecomplex (Palm and de Lange, 2008). Theyinclude DNA damage factors, nucleases,helicases and DNA replication proteins.

Recently, a trimeric complex termed CST,which contains DNA polymerase a, theprimase accessory factors CTC1 (for CTS

telomere maintenance complex component1) and STN1 (for suppressor of cdc thirteen1) in addition to TEN1 (for telomeric

pathways with STN1), was found toassociate with ,20% of telomeres (Miyakeet al., 2009; Surovtseva et al., 2009). Little isknown about the function of CST, but studies

on human cells have suggested that STN1interacts with TPP1 and limits overhanglength (Wan et al., 2009).

A long non-coding telomeric RNA

An RNA component, TERRA (fortelomeric-repeat-containing RNA), hasbeen identified as the third entity of the

telomere nucleoprotein complex (Azzalinet al., 2007). TERRA transcription ismediated by RNA polymerase II and isinitiated from the sub-telomeric regions

that are found near chromosome ends(Porro et al., 2010). TERRA levels areregulated during the cell cycle, and its

localization at telomeres is modulated bythe nonsense-mediated decay machinery(Azzalin et al., 2007). It has been

postulated that this non-coding RNA isimportant for telomere maintenance andfunction (Redon et al., 2010; Flynn et al.,

2011), yet the molecular mechanismsunderlying this proposed function remainto be uncovered.

Shelterin – the elegant, but not sosimple solution to the end-protection problemThe ability of cells to distinguish nativechromosome ends from broken DNA –

which is unstable and fusogenic – was firstdocumented in the 1940s by McClintockand Muller (Muller, 1938; McClintock,

1941). Linear ends of plasmids areunstable when introduced into eukaryoticcells and often recombine with the genome

(Orr-Weaver et al., 1981). Furthermore,

when DSBs accumulate in cells, a

signaling cascade that leads to cell cycle

arrest is initiated (Weinert and Hartwell,

1988). By contrast, natural chromosome

ends are inherently stable: they are not

targeted by DNA repair pathways, namely

non-homologous end-joining (NHEJ) and

homology-directed repair (HDR), and do

not activate the major DNA damage-

induced kinases ataxia telangiectasia

mutated (ATM) and ATM and Rad3

related (ATR). It was later recognized

that telomeres accomplish chromosome

end-protection by means of their protein

elements (de Lange, 2005). In recent years,

major strides have been made in

understanding how shelterin is employed

to disguise chromosome ends from DNA

damage-sensing and repair pathways.

This has been predominantly based on

dissecting the phenotypes that are

associated with the functional impairment

of individual shelterin subunits using

conditional knockout mice.

TRF2, the master repressor of ATM

signaling and NHEJ

Deleting TRF2 results in the activation of

the kinase ATM (Celli and de Lange,

2005) and triggers the accumulation of

DNA damage factors, including H2AX

(H2A histone family, member X), and

53BP1 (p53 binding protein 1) at telomeres

(Dimitrova and de Lange, 2006; Denchi

and de Lange, 2007; Dimitrova and de

Lange, 2009). ATM activates p53, which

induces cell cycle arrest. Furthermore, in

the absence of TRF2, ligase 4- and Ku-

mediated NHEJ repair is activated at

telomeres, which results in chromosome

end-to-end fusions (Celli and de Lange,

2005). The mechanism by which TRF2

normally represses ATM and NHEJ is

currently under investigation. One model

has been proposed, whereby TRF2 is able

to sequester the telomere terminus in the t-

loop structure. Purified TRF2 binds at the

junction of the t-loop (Stansel et al., 2001)

and can promote the formation and

stabilization of structures resembling t-

loops in vitro (Poulet et al., 2009). A t-loop

configuration would prevent detection of

the telomere end by the MRN (for MRE11,

RAD50 and NBS1) complex, which is the

DNA damage sensor of the ATM pathway.

Similarly, the t-loop structure would

exclude the binding of the Ku70–Ku80

heterodimer, thereby blocking NHEJ.

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Silencing the kinase ATR by the POT1proteins

The task of suppressing the second majorcheckpoint activator, the kinase ATR, isascribed to POT1 in humans and to POT1ain mice, whereas the main role of mouse

POT1b is to regulate the length of the single-stranded overhang (Hockemeyer et al.,2005; Hockemeyer et al., 2006; Wu et al.,

2006). The mechanism by which POT1 andPOT1a block ATR activation pertains totheir ability to prevent replication protein A

(RPA), the sensor of the ATR pathway, frombinding to single-stranded telomeric DNA.Indeed, RPA foci have been detected attelomeric DNA that has been depleted of

POT1 (Gong and de Lange, 2010). DeletingTPP1 or TIN2, which leads to loss of POT1from telomeres, prompts similar ATR

activation (Kibe et al., 2010; Takai et al.,2011).

Cooperative inhibition of HDR by RAP1,POT1 and the Ku70–Ku80 complex

HDR is typically repressed at telomeres, butoccurs in a small subset of tumors that do notuse telomerase for telomere maintenance

(Bryan et al., 1997) as well as during theearly cleavage cycles during embryonicdevelopment (Liu et al., 2007). Recent

work has indicated that recombination isrepressed at telomeres in a highly redundantmanner. First, it is repressed by the Ku70–

Ku80 complex, which inhibits general HDRin the nucleus. Additionally, is it repressedin a telomere-specific manner by RAP1 and

POT1 (Palm et al., 2009; Sfeir et al., 2010).The mechanism by which Rap1, POT1 andKu cooperate to block recombination is notunderstood.

A distinct protective function for TRF1

The G-rich and highly repetitive nature oftelomeric DNA poses major challenges to the

DNA replication machinery. The shelterinsubunit TRF1 assists the semi-conservativereplication machinery during the duplication

of bulk telomeric DNA, thereby protectingtelomeres from breakage and from acquiringfeatures that are reminiscent of commonfragile sites (Martınez et al., 2009; Sfeir et al.,

2009). The function of TRF1 is mainlyachieved by recruiting helicases, includingregulator of telomere length 1 (RTEL1)

and Bloom syndrome, RecQ helicase-like(BLM), which unwind spurious secondarystructures that can hinder fork progression.

How do shelterin-free telomeres behave?

The studies described above exemplify howthe deletion of individual shelterin subunits

activates specific DNA damage responsepathway(s) without greatly destabilizing the

remaining components of the complex.To uncover possible redundancies in thefunction of shelterin components, we haveinvestigated the consequences of completely

depleting telomeres of all shelterincomponents (Sfeir and de Lange, 2012),and we have identified two additional

pathways that can threaten telomereintegrity. The first is the alternative NHEJDNA repair pathway that is mediated by

ligase 3 and poly(ADP-ribose) polymerase 1(PARP1). The second is nucleolyticdegradation, which is a marked outcome oftelomere deprotection in yeast and has not

been previously observed in mammaliancells. Interestingly, both pathways arerepressed in a highly redundant manner by

shelterin components, as well as by DNAdamage factors, whereby alternative NHEJ isinhibited by Ku70–Ku80 and nucleolytic

degradation is blocked by 53BP1.

After nearly a decade of investigation,we now have a clear definition of the end-

protection problem and are able to betterunderstand the nature of its solution. Insummary, with their six-member shelterincomplex, mammalian telomeres are

properly armed to face the six differentchallenges (namely ATM, ATR, NHEJ,HDR, alternative NHEJ and resection)

that are imposed by the linearity ofchromosome ends.

Telomerase – the enzymatic solutionto the end-replication problemIn almost all eukaryotes, the task ofsolving the end-replication problem and

counteracting telomere erosion is assignedto the telomerase enzyme complex(Greider and Blackburn, 1985). The active

telomerase holoenzyme in mammalian cellsexists as a dimer and consists of thetelomerase reverse transcriptase (TERT),

the telomerase RNA component (TERC)and dyskerin (Cohen et al., 2007). Inhumans, telomerase is expressed during theearly stages of embryogenesis, and its

expression is subsequently repressed inmost somatic cells, except the male germline, activated lymphocytes and stem cells

found in certain regenerative tissues (Wrightet al., 1996). Furthermore, the vast majorityof human cancer cells reactivate telomerase,

and are thus capable of proliferatingindefinitely (Kim et al., 1994). Regulationof telomerase is primarily exerted at the

level of TERT transcription. Extensiveanalysis of the promoter region hasuncovered many transcriptional binding

sites and regulatory elements, including GC-boxes and E-boxes. In addition, TERT

transcription is regulated by oncogenes andtumor suppressor genes, and is influenced bythe surrounding chromatin. Post-transcriptionaland translational processes are likely to control

telomerase activity. Alternative splice variantsof human TERT have been reported insome cancer cells. Telomerase is also

subject to post-translational modificationsincluding sumoylation, phosphorylationand ubiquitylation, all of which can

impact on its activity (Cong et al., 2002).

In vivo, the maturation to a fully activetelomerase depends on its assembly into aribonucleoprotein (RNP) complex and is

influenced by several factors, includingpontin, reptin and others proteins that bindto TERT and TERC (Venteicher et al.,

2008 and see poster). Another importantstep for telomerase maturation is itstrafficking through Cajal bodies, the

dynamic subnuclear sites that areinvolved in RNP biogenesis (Zhu et al.,2004; Tomlinson et al., 2006; Cristofariet al., 2007). Telomerase transit through

Cajal bodies is mediated by telomeraseCajal body protein 1 (TCAB1) (Venteicheret al., 2008; Venteicher et al., 2009; Koo

et al., 2011).

A key step with regards to telomeraseaction is its recruitment to chromosome

ends in a timely manner. The most coherentpicture of telomerase recruitment hasemerged from studies in Saccharomyces

cerevisiae that have shown that this processis mediated by an interaction between EST1(ever-shorter telomeres 1), a component ofthe telomerase complex, and Cdc13, a

protein that binds to the single-strandedoverhang (Evans and Lundblad, 1999; Qiand Zakian, 2000). Another recruitment

pathway involves the Ku complex, whichbinds to the telomeric DNA, as well astelomerase RNA (Peterson et al., 2001;

Stellwagen et al., 2003). Our currentunderstanding of telomerase recruitment inmammalian cells is much more limited. TheKu70–Ku80 complex has been shown to

associate with telomerase by interactingwith both human TERT and TERC (Chaiet al., 2002; Ting et al., 2005). However,

Ku70-deficient mice do not show a defect intelomere maintenance (Celli et al., 2006),which possibly argues against a recruitment

role for Ku in mammals. In vitro studieshave exposed an interaction between TPP1and TERT (Wang et al., 2007; Xin et al.,

2007) that enhances the processivity oftelomerase. More recent experiments havesuggested that TPP1 might also be involved

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in recruiting telomerase to the telomere end(Abreu et al., 2010). This has been difficult

to prove, because the TERT interactiondomain within TPP1 is required to repressthe DNA damage response. Therefore, it isdifficult to establish a specific function for

TPP1 in the context of telomeraserecruitment without the confoundingactivation of the DNA damage response.

Another factor that has been suggested tobridge telomerase to the DNA is theheterogenous nuclear RNP protein A1

(hnRNPA1) (LaBranche et al., 1998).Again, this observation has been difficultto confirm, because hnRNPA1 is involvedin the biogenesis of a wide spectrum of

RNA transcripts. Given the natural lowabundance of the telomerase enzyme, moresophisticated methods of telomerase

detection are needed to resolve suchconfounding results and fully characterizethe telomerase pathway.

Telomere length regulation

Telomerase research has moved into extradimensions in recent years, as the enzyme has

been found to modulate the Wnt signalingpathways (Park et al., 2009), display anRNA-dependent RNA polymerase activity

(Maida et al., 2009) and has been spotted inthe mitochondria (Sharma et al., 2012).Whereas the function and impact of

telomerase in these pathways is a subjectof debate, its indisputable chief role inmammalian cells remains to combat

telomere erosion and maintain telomerelength. In fact, appropriate telomere lengthis crucial for cell survival. This leads to thefollowing question: what determines

telomere length and how is this processregulated?

The first genes to influence telomere

length regulation were identified in S.

cerevisiae and, so far, the mostcomprehensive understanding of telomere

length regulation comes from studies infungi (Shore and Bianchi, 2009). Themechanism underlying telomere lengthregulation in mammalian cells, whose

telomeres are orders of magnitude longerthan those of budding yeast, is not fullyunderstood. Nevertheless, some factors are

known to influence telomere length; inparticular, certain shelterin subunits areknown to establish a negative-feedback

loop that controls the addition of repeatsby telomerase. In effect, overexpressingTRF1 in cancer-derived human cell lines

leads to telomere shortening, whereasdepleting telomeric DNA of TRF1 resultsin telomere elongation (van Steensel and de

Lange, 1997). Reducing the levels of TIN2

or TRF2 (Ye and de Lange, 2004; Takaiet al., 2010) at telomeres leads to a similarextension phenotype. The message from the

duplex region of the telomere is relayed tothe terminus via POT1, which has beenshown in vivo, to act as a negative regulatorof telomerase activity (Loayza and de

Lange, 2003; Lei et al., 2005). However,as mentioned previously, mechanisticdetails regarding the regulation of

telomerase accessibility and activity at thetelomere terminus are still lacking.

Another layer of complexity is added bytelomeric chromatin. In contrast with yeast

telomeres, which lack histones (Wrightet al., 1992), mammalian telomeres arenucleosomal (Makarov et al., 1993; Lejnine

et al., 1995) and their chromatin is enrichedwith repressive histone marks, includingH3K9me3 and H4K20me3 (me3 represents

trimethylation), and the heterochromatin-specific factors chromobox homolog(CBX) 1, 3 and 5 (Garcıa-Cao et al., 2004;

Gonzalo et al., 2006). Depletion of theseheterochromatic marks correlates withremarkable elongation of telomeres(Marion and Blasco, 2010; Marion et al.,

2011; Varela et al., 2011).

Telomere dysfunction andtumorigenesisBecause most somatic cells lack telomeraseactivity, they are prone to telomereshrinkage. When telomeres become too

short and lose the binding of the protectiveshelterin complex, the cells face twoopposing fates. In cells with intact

checkpoints, telomere erosion leads tosenescence. By contrast, in the absence ofp53, telomere dysfunction promotesgenomic instability and fuels tumor

progression.

Early work on telomerase-knockoutmice has provided in vivo evidence in

support of telomere dysfunction as an earlydriver in the evolution of cancer genomes.The short and dysfunctional telomeres inthe late-generation telomerase-deficient

mice, which usually result in tissuedegeneration, are tolerated when p53 isabrogated. The outcome is an increase in

the occurrence of epithelial tumors thatdisplay hallmarks of genomic instability(Artandi et al., 2000). The progression of

these tumors is constrained by the lack oftelomerase, which would restore telomerefunction and allow further growth and

more aggressive behavior of tumors. Theparadigm has been re-established byrecent work that expressed an engineered

inducible version of telomerase in mousemodels of prostate cancer and T-cell

lymphoma (Ding et al., 2012; Hu et al.,2012). Indeed, when telomerase isreactivated in mice that have witnessed astage of telomere dysfunction, especially

aggressive cancers, which possess anincreasing propensity of undergoingmetastasis, develop.

Telomere dysfunction also drives theprogression of human cancers. Many solidtumors, including those of the breast (Chin

et al., 2004), prostate (Meeker et al., 1996)and colon (Rudolph et al., 2001), haveshorter telomeres when compared with thoseof the respective normal tissue. Anaphase

bridges have been documented at thehyperplasia-to-carcinoma transition, whichcoincides with telomerase re-activation

(Chin et al., 2004). The most compellingevidence supporting a role for telomeredysfunction in tumor progression has

emerged from the examination of telomeredynamics in hematological malignancies,particularly in chronic lymphocytic

leukemia (CLL). This type of tumor offersa unique opportunity to perform high-resolution telomere length measurementsin B cells from patients at different stages

of the disease. The results demonstrate thattelomere shortening strongly correlates withdisease progression, and telomere fusions

were noted in the earlier stages of thedisease, perhaps setting the stage for thegross genomic rearrangements that drive

progression of CLL (Lin et al., 2010).

What lies ahead?After three decades of research, it is now

well established that telomeres, whichaccount for less than 1% of the humangenome, have a pivotal role in normal

cellular function and that cellulardysfunction ensues if their integrity iscompromised. However, despite substantial

progress in our understanding of howtelomeres protect the integrity of thegenetic material, there are manyunanswered questions. We have yet to

unravel the intricacies by which all theDNA damage pathways are evaded andexperimentally validate the function for the

t-loop. The interplay between telomere-associated proteins and telomerase, whichsets the appropriate telomere length in

different cellular stages and cell types, is anarea that also requires rigorous investigation.Answering these and other questions will

bring us closer to fully understanding theimpact of telomere biology on cellularfunction and might lead to profound

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improvements in cancer therapy as well as inregenerative medicine.

Acknowledgements

I gratefully acknowledge Titia de Lange

for her support during my post-doctoral

training. I am thankful to Peng Wu, Eros

Lazzerini-Denchi, Nadya Dimitrova and

members of the de Lange laboratory for

commenting on the manuscript. I apologize

to the authors whose publications have not

been cited owing to space limitations.

Funding

This research received no specific grant

from any funding agency in the public,

commercial or not-for-profit sectors.

A high-resolution version of the poster is available for

downloading in the online version of this article at

jcs.biologists.org. Individual poster panels are available

as JPEG files at http://jcs.biologists.org/lookup/suppl/

doi:10.1242/jcs.106831/-/DC1

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