Journ
alof
Cell
Scie
nce
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
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)
Cell Science at a Glance 4173
Journ
alof
Cell
Scie
nce
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.
Journal of Cell Science 125 (18)4174
Journ
alof
Cell
Scie
nce
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
Journal of Cell Science 125 (18) 4175
Journ
alof
Cell
Scie
nce
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
Journal of Cell Science 125 (18)4176
Journ
alof
Cell
Scie
nce
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
ReferencesAbreu, E., Aritonovska, E., Reichenbach, P.,
Cristofari, G., Culp, B., Terns, R. M., Lingner, J. andTerns, M. P. (2010). TIN2-tethered TPP1 recruits humantelomerase to telomeres in vivo. Mol. Cell. Biol. 30, 2971-2982.
Artandi, S. E., Chang, S., Lee, S. L., Alson, S., Gottlieb,
G. J., Chin, L. and DePinho, R. A. (2000). Telomeredysfunction promotes non-reciprocal translocations andepithelial cancers in mice. Nature 406, 641-645.
Azzalin, C. M., Reichenbach, P., Khoriauli, L.,
Giulotto, E. and Lingner, J. (2007). Telomeric repeatcontaining RNA and RNA surveillance factors atmammalian chromosome ends. Science 318, 798-801.
Baumann, P. and Cech, T. R. (2001). Pot1, the putativetelomere end-binding protein in fission yeast and humans.Science 292, 1171-1175.
Bianchi, A., Smith, S., Chong, L., Elias, P. and deLange, T. (1997). TRF1 is a dimer and bends telomericDNA. EMBO J. 16, 1785-1794.
Bilaud, T., Brun, C., Ancelin, K., Koering, C. E.,Laroche, T. and Gilson, E. (1997). Telomericlocalization of TRF2, a novel human telobox protein.Nat. Genet. 17, 236-239.
Broccoli, D., Smogorzewska, A., Chong, L. and de
Lange, T. (1997). Human telomeres contain two distinctMyb-related proteins, TRF1 and TRF2. Nat. Genet. 17,231-235.
Bryan, T. M., Englezou, A., Dalla-Pozza, L., Dunham,
M. A. and Reddel, R. R. (1997). Evidence for analternative mechanism for maintaining telomere length inhuman tumors and tumor-derived cell lines. Nat. Med. 3,1271-1274.
Celli, G. B. and de Lange, T. (2005). DNA processing isnot required for ATM-mediated telomere damageresponse after TRF2 deletion. Nat. Cell Biol. 7, 712-718.
Celli, G. B., Denchi, E. L. and de Lange, T. (2006).Ku70 stimulates fusion of dysfunctional telomeres yetprotects chromosome ends from homologousrecombination. Nat. Cell Biol. 8, 855-890.
Chai, W., Ford, L. P., Lenertz, L., Wright, W. E. and
Shay, J. W. (2002). Human Ku70/80 associates physicallywith telomerase through interaction with hTERT. J Biol.
Chem. 277, 47242-47247.
Chai, W., Shay, J. W. and Wright, W. E. (2005). Humantelomeres maintain their overhang length at senescence.Mol. Cell. Biol. 25, 2158-2168.
Chin, K., de Solorzano, C. O., Knowles, D., Jones, A.,Chou, W., Rodriguez, E. G., Kuo, W. L., Ljung, B. M.,
Chew, K., Myambo, K. et al. (2004). In situ analyses ofgenome instability in breast cancer. Nat. Genet. 36, 984-988.
Cohen, S. B., Graham, M. E., Lovrecz, G. O., Bache,
N., Robinson, P. J. and Reddel, R. R. (2007). Proteincomposition of catalytically active human telomerasefrom immortal cells. Science 315, 1850-1853.
Cong, Y. S., Wright, W. E. and Shay, J. W. (2002).Human telomerase and its regulation. Microbiol. Mol.
Biol. Rev. 66, 407-425.
Cristofari, G., Adolf, E., Reichenbach, P., Sikora, K.,
Terns, R. M., Terns, M. P. and Lingner, J. (2007).Human telomerase RNA accumulation in Cajal bodiesfacilitates telomerase recruitment to telomeres andtelomere elongation. Mol. Cell 27, 882-889.
de Lange, T. (2005). Shelterin: the protein complex thatshapes and safeguards human telomeres. Genes Dev. 19,2100-2110.
de Lange, T. (2009). How telomeres solve the end-protection problem. Science 326, 948-952.
Denchi, E. L. and de Lange, T. (2007). Protection oftelomeres through independent control of ATM and ATRby TRF2 and POT1. Nature 448, 1068-1071.
Dimitrova, N. and de Lange, T. (2006). MDC1accelerates nonhomologous end-joining of dysfunctionaltelomeres. Genes Dev. 20, 3238-3243.
Dimitrova, N. and de Lange, T. (2009). Cell cycle-dependent role of MRN at dysfunctional telomeres: ATMsignaling-dependent induction of nonhomologous endjoining (NHEJ) in G1 and resection-mediated inhibitionof NHEJ in G2. Mol. Cell. Biol. 29, 5552-5563.
Ding, Z., Wu, C. J., Jaskelioff, M., Ivanova, E., Kost-
Alimova, M., Protopopov, A., Chu, G. C., Wang, G.,Lu, X., Labrot, E. S. et al. (2012). Telomerasereactivation following telomere dysfunction yieldsmurine prostate tumors with bone metastases. Cell 148,896-907.
Evans, S. K. and Lundblad, V. (1999). Est1 and Cdc13as comediators of telomerase access. Science 286, 117-120.
Flynn, R. L., Centore, R. C., O’Sullivan, R. J., Rai, R.,
Tse, A., Songyang, Z., Chang, S., Karlseder, J. andZou, L. (2011). TERRA and hnRNPA1 orchestrate anRPA-to-POT1 switch on telomeric single-stranded DNA.Nature 471, 532-536.
Garcıa-Cao, M., O’Sullivan, R., Peters, A. H.,
Jenuwein, T. and Blasco, M. A. (2004). Epigeneticregulation of telomere length in mammalian cells by theSuv39h1 and Suv39h2 histone methyltransferases. Nat.
Genet. 36, 94-99.
Gong, Y. and de Lange, T. (2010). A Shld1-controlledPOT1a provides support for repression of ATR signalingat telomeres through RPA exclusion. Mol. Cell 40, 377-387.
Gonzalo, S., Jaco, I., Fraga, M. F., Chen, T., Li, E.,
Esteller, M. and Blasco, M. A. (2006). DNAmethyltransferases control telomere length and telomererecombination in mammalian cells. Nat. Cell Biol. 8, 416-424.
Greider, C. W. and Blackburn, E. H. (1985).Identification of a specific telomere terminal transferaseactivity in Tetrahymena extracts. Cell 43, 405-413.
Griffith, J. D., Comeau, L., Rosenfield, S., Stansel,
R. M., Bianchi, A., Moss, H. and de Lange, T. (1999).Mammalian telomeres end in a large duplex loop. Cell 97,503-514.
Hockemeyer, D., Sfeir, A. J., Shay, J. W., Wright,W. E. and de Lange, T. (2005). POT1 protects telomeresfrom a transient DNA damage response and determineshow human chromosomes end. EMBO J. 24, 2667-2678.
Hockemeyer, D., Daniels, J. P., Takai, H. and deLange, T. (2006). Recent expansion of the telomericcomplex in rodents: Two distinct POT1 proteins protectmouse telomeres. Cell 126, 63-77.
Houghtaling, B. R., Cuttonaro, L., Chang, W. and
Smith, S. (2004). A dynamic molecular link between thetelomere length regulator TRF1 and the chromosome endprotector TRF2. Curr. Biol. 14, 1621-1631.
Hu, J., Hwang, S. S., Liesa, M., Gan, B., Sahin, E.,
Jaskelioff, M., Ding, Z., Ying, H., Boutin, A. T., Zhang,H. et al. (2012). Antitelomerase therapy provokes ALTand mitochondrial adaptive mechanisms in cancer. Cell
148, 651-663.
Kibe, T., Osawa, G. A., Keegan, C. E. and de Lange, T.
(2010). Telomere protection by TPP1 is mediated byPOT1a and POT1b. Mol. Cell. Biol. 30, 1059-1066.
Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley,
C. B., West, M. D., Ho, P. L., Coviello, G. M., Wright,
W. E., Weinrich, S. L. and Shay, J. W. (1994). Specific
association of human telomerase activity with immortal
cells and cancer. Science 266, 2011-2015.
Koo, B. K., Park, C. J., Fernandez, C. F., Chim, N.,
Ding, Y., Chanfreau, G. and Feigon, J. (2011). Structure
of H/ACA RNP protein Nhp2p reveals cis/trans
isomerization of a conserved proline at the RNA and
Nop10 binding interface. J. Mol. Biol. 411, 927-942.
LaBranche, H., Dupuis, S., Ben-David, Y., Bani, M. R.,
Wellinger, R. J. and Chabot, B. (1998). Telomereelongation by hnRNP A1 and a derivative that interacts
with telomeric repeats and telomerase. Nat. Genet. 19,
199-202.
Lam, Y. C., Akhter, S., Gu, P., Ye, J., Poulet, A.,
Giraud-Panis, M. J., Bailey, S. M., Gilson, E., Legerski,
R. J. and Chang, S. (2010). SNMIB/Apollo protects
leading-strand telomeres against NHEJ-mediated repair.
EMBO J. 29, 2230-2241.
Lei, M., Baumann, P. and Cech, T. R. (2002).
Cooperative binding of single-stranded telomeric DNA
by the Pot1 protein of Schizosaccharomyces pombe.
Biochemistry 41, 14560-14568.
Lei, M., Zaug, A. J., Podell, E. R. and Cech, T. R.
(2005). Switching human telomerase on and off with
hPOT1 protein in vitro. J. Biol. Chem. 280, 20449-20456.
Lejnine, S., Makarov, V. L. and Langmore, J. P.
(1995). Conserved nucleoprotein structure at the ends of
vertebrate and invertebrate chromosomes. Proc. Natl.
Acad. Sci. USA 92, 2393-2397.
Li, B. and de Lange, T. (2003). Rap1 affects the length
and heterogeneity of human telomeres. Mol. Biol. Cell 14,
5060-5068.
Lin, T. T., Letsolo, B. T., Jones, R. E., Rowson, J.,
Pratt, G., Hewamana, S., Fegan, C., Pepper, C. and
Baird, D. M. (2010). Telomere dysfunction and fusion
during the progression of chronic lymphocytic leukemia:
evidence for a telomere crisis. Blood 116, 1899-1907.
Liu, L., Bailey, S. M., Okuka, M., Munoz, P., Li, C.,
Zhou, L., Wu, C., Czerwiec, E., Sandler, L., Seyfang, A.
et al. (2007). Telomere lengthening early in development.
Nat. Cell Biol. 9, 1436-1441.
Loayza, D. and de Lange, T. (2003). POT1 as a terminal
transducer of TRF1 telomere length control. Nature 423,
1013-1018.
Maida, Y., Yasukawa, M., Furuuchi, M., Lassmann, T.,
Possemato, R., Okamoto, N., Kasim, V., Hayashizaki,
Y., Hahn, W. C. and Masutomi, K. (2009). An RNA-
dependent RNA polymerase formed by TERT and the
RMRP RNA. Nature 461, 230-235.
Makarov, V. L., Lejnine, S., Bedoyan, J. and
Langmore, J. P. (1993). Nucleosomal organization of
telomere-specific chromatin in rat. Cell 73, 775-787.
Makarov, V. L., Hirose, Y. and Langmore, J. P. (1997).
Long G tails at both ends of human chromosomes suggest
a C strand degradation mechanism for telomere
shortening. Cell 88, 657-666.
Marion, R. M. and Blasco, M. A. (2010). Telomere
rejuvenation during nuclear reprogramming. Curr. Opin.
Genet. Dev. 20, 190-196.
Marion, R. M., Schotta, G., Ortega, S. and Blasco,
M. A. (2011). Suv4-20h abrogation enhances telomere
elongation during reprogramming and confers a higher
tumorigenic potential to iPS cells. PLoS ONE 6, e25680.
Martınez, P., Thanasoula, M., Munoz, P., Liao, C.,
Tejera, A., McNees, C., Flores, J. M., Fernandez-
Capetillo, O., Tarsounas, M. and Blasco, M. A. (2009).
Increased telomere fragility and fusions resulting from
TRF1 deficiency lead to degenerative pathologies and
increased cancer in mice. Genes Dev. 23, 2060-2075.
Martinez, P., Thanasoula, M., Carlos, A. R., Gomez-
Lopez, G., Tejera, A. M., Schoeftner, S., Dominguez,
O., Pisano, D. G., Tarsounas, M. and Blasco, M. A.
(2010). Mammalian Rap1 controls telomere function and
gene expression through binding to telomeric and
extratelomeric sites. Nat. Cell Biol. 12, 768-780.
McClintock, B. (1941). The stability of broken ends of
chromosomes in Zea mays. Genetics 26, 234-282.
McElligott, R. and Wellinger, R. J. (1997). The terminal
DNA structure of mammalian chromosomes. EMBO J. 16,
3705-3714.
Journal of Cell Science 125 (18) 4177
Journ
alof
Cell
Scie
nce
Meeker, A. K., Sommerfeld, H. J. and Coffey, D. S.(1996). Telomerase is activated in the prostate and seminalvesicles of the castrated rat. Endocrinology 137, 5743-5746.Miyake, Y., Nakamura, M., Nabetani, A., Shimamura,
S., Tamura, M., Yonehara, S., Saito, M. and Ishikawa,F. (2009). RPA-like mammalian Ctc1-Stn1-Ten1 complexbinds to single-stranded DNA and protects telomeresindependently of the Pot1 pathway. Mol. Cell 36, 193-206.Muller, H. J. (1938). The remaking of chromosomes. The
Collecting Net. Woods Hole 8, 182-195.Nikitina, T. and Woodcock, C. L. (2004). Closedchromatin loops at the ends of chromosomes. J. Cell
Biol. 166, 161-165.O’Connor, M. S., Safari, A., Xin, H., Liu, D. andSongyang, Z. (2006). A critical role for TPP1 and TIN2interaction in high-order telomeric complex assembly.Proc. Natl. Acad. Sci. USA 103, 11874-11879.Oganesian, L. and Karlseder, J. (2011). Mammalian 59
C-rich telomeric overhangs are a mark of recombination-dependent telomere maintenance. Mol. Cell 42, 224-236.Orr-Weaver, T. L., Szostak, J. W. and Rothstein, R. J.
(1981). Yeast transformation: a model system for thestudy of recombination. Proc. Natl. Acad. Sci. USA 78,6354-6358.Palm, W. and de Lange, T. (2008). How shelterinprotects mammalian telomeres. Annu. Rev. Genet. 42,301-334.Palm, W., Hockemeyer, D., Kibe, T. and de Lange, T.
(2009). Functional dissection of human and mouse POT1proteins. Mol. Cell. Biol. 29, 471-482.Park, J. I., Venteicher, A. S., Hong, J. Y., Choi, J., Jun,S., Shkreli, M., Chang, W., Meng, Z., Cheung, P., Ji, H.
et al. (2009). Telomerase modulates Wnt signalling byassociation with target gene chromatin. Nature 460, 66-72.Peterson, S. E., Stellwagen, A. E., Diede, S. J., Singer,
M. S., Haimberger, Z. W., Johnson, C. O., Tzoneva, M.and Gottschling, D. E. (2001). The function of a stem-loop in telomerase RNA is linked to the DNA repairprotein Ku. Nat. Genet. 27, 64-67.Porro, A., Feuerhahn, S., Reichenbach, P. and Lingner,
J. (2010). Molecular dissection of telomeric repeat-containing RNA biogenesis unveils the presence ofdistinct and multiple regulatory pathways. Mol. Cell.
Biol. 30, 4808-4817.Poulet, A., Buisson, R., Faivre-Moskalenko, C.,
Koelblen, M., Amiard, S., Montel, F., Cuesta-Lopez,S., Bornet, O., Guerlesquin, F., Godet, T. et al. (2009).TRF2 promotes, remodels and protects telomeric Hollidayjunctions. EMBO J. 28, 641-651.Qi, H. and Zakian, V. A. (2000). The Saccharomycestelomere-binding protein Cdc13p interacts with both thecatalytic subunit of DNA polymerase alpha and thetelomerase-associated est1 protein. Genes Dev. 14, 1777-1788.Raices, M., Verdun, R. E., Compton, S. A., Haggblom,C. I., Griffith, J. D., Dillin, A. and Karlseder, J. (2008).C. elegans telomeres contain G-strand and C-strandoverhangs that are bound by distinct proteins. Cell 132,745-757.Redon, S., Reichenbach, P. and Lingner, J. (2010). Thenon-coding RNA TERRA is a natural ligand and direct
inhibitor of human telomerase. Nucleic Acids Res. 38,5797-5806.
Rudolph, K. L., Millard, M., Bosenberg, M. W. and
DePinho, R. A. (2001). Telomere dysfunction andevolution of intestinal carcinoma in mice and humans.Nat. Genet. 28, 155-159.
Sfeir, A. and de Lange, T. (2012). Removal of shelterinreveals the telomere end-protection problem. Science 336,593-597.
Sfeir, A., Kosiyatrakul, S. T., Hockemeyer, D.,
MacRae, S. L., Karlseder, J., Schildkraut, C. L. and
de Lange, T. (2009). Mammalian telomeres resemblefragile sites and require TRF1 for efficient replication.Cell 138, 90-103.
Sfeir, A., Kabir, S., van Overbeek, M., Celli, G. B. and
de Lange, T. (2010). Loss of Rap1 induces telomererecombination in the absence of NHEJ or a DNA damagesignal. Science 327, 1657-1661.
Sharma, N. K., Reyes, A., Green, P., Caron, M. J.,
Bonini, M. G., Gordon, D. M., Holt, I. J. and Santos, J.
H. (2012). Human telomerase acts as a hTR-independentreverse transcriptase in mitochondria. Nucleic Acids Res.
40, 712-725.
Shore, D. and Bianchi, A. (2009). Telomere lengthregulation: coupling DNA end processing to feedbackregulation of telomerase. EMBO J. 28, 2309-2322.
Stansel, R. M., de Lange, T. and Griffith, J. D. (2001).T-loop assembly in vitro involves binding of TRF2 nearthe 39 telomeric overhang. EMBO J. 20, 5532-5540.
Stellwagen, A. E., Haimberger, Z. W., Veatch, J. R.
and Gottschling, D. E. (2003). Ku interacts withtelomerase RNA to promote telomere addition at nativeand broken chromosome ends. Genes Dev. 17, 2384-2395.
Surovtseva, Y. V., Churikov, D., Boltz, K. A., Song, X.,
Lamb, J. C., Warrington, R., Leehy, K., Heacock, M.,
Price, C. M. and Shippen, D. E. (2009). Conservedtelomere maintenance component 1 interacts with STN1and maintains chromosome ends in higher eukaryotes.Mol. Cell 36, 207-218.
Takai, K. K., Hooper, S., Blackwood, S., Gandhi, R.
and de Lange, T. (2010). In vivo stoichiometry ofshelterin components. J. Biol. Chem. 285, 1457-1467.
Takai, K. K., Kibe, T., Donigian, J. R., Frescas, D. and
de Lange, T. (2011). Telomere protection by TPP1/POT1requires tethering to TIN2. Mol. Cell 44, 647-659.
Teo, H., Ghosh, S., Luesch, H., Ghosh, A., Wong, E. T.,
Malik, N., Orth, A., de Jesus, P., Perry, A. S., Oliver,
J. D. et al. (2010). Telomere-independent Rap1 is an IKKadaptor and regulates NF-kappaB-dependent geneexpression. Nat. Cell Biol. 12, 758-767.
Ting, N. S., Yu, Y., Pohorelic, B., Lees-Miller, S. P. and
Beattie, T. L. (2005). Human Ku70/80 interacts directlywith hTR, the RNA component of human telomerase.Nucleic Acids Res. 33, 2090-2098.
Tomlinson, R. L., Ziegler, T. D., Supakorndej, T.,
Terns, R. M. and Terns, M. P. (2006). Cell cycle-regulated trafficking of human telomerase to telomeres.Mol. Biol. Cell 17, 955-965.
van Steensel, B. and de Lange, T. (1997). Control oftelomere length by the human telomeric protein TRF1.Nature 385, 740-743.
Varela, E., Schneider, R. P., Ortega, S. and Blasco,
M. A. (2011). Different telomere-length dynamics at theinner cell mass versus established embryonic stem (ES)cells. Proc. Natl. Acad. Sci. USA 108, 15207-15212.
Venteicher, A. S., Meng, Z., Mason, P. J., Veenstra,
T. D. and Artandi, S. E. (2008). Identification ofATPases pontin and reptin as telomerase componentsessential for holoenzyme assembly. Cell 132, 945-957.
Venteicher, A. S., Abreu, E. B., Meng, Z., McCann,
K. E., Terns, R. M., Veenstra, T. D., Terns, M. P. and
Artandi, S. E. (2009). A human telomerase holoenzymeprotein required for Cajal body localization and telomeresynthesis. Science 323, 644-648.
Wan, M., Qin, J., Songyang, Z. and Liu, D. (2009). OBfold-containing protein 1 (OBFC1), a human homolog ofyeast Stn1, associates with TPP1 and is implicated intelomere length regulation. J. Biol. Chem. 284, 26725-26731.
Wang, F., Podell, E. R., Zaug, A. J., Yang, Y., Baciu, P.,
Cech, T. R. and Lei, M. (2007). The POT1-TPP1telomere complex is a telomerase processivity factor.Nature 445, 506-510.
Weinert, T. A. and Hartwell, L. H. (1988). The RAD9gene controls the cell cycle response to DNA damage inSaccharomyces cerevisiae. Science 241, 317-322.
Wright, J. H., Gottschling, D. E. and Zakian, V. A.
(1992). Saccharomyces telomeres assume a non-nucleosomal chromatin structure. Genes Dev. 6, 197-210.
Wright, W. E., Piatyszek, M. A., Rainey, W. E., Byrd,
W. and Shay, J. W. (1996). Telomerase activity in humangermline and embryonic tissues and cells. Dev. Genet. 18,173-179.
Wu, L., Multani, A. S., He, H., Cosme-Blanco, W.,
Deng, Y., Deng, J. M., Bachilo, O., Pathak, S., Tahara,
H., Bailey, S. M. et al. (2006). Pot1 deficiency initiatesDNA damage checkpoint activation and aberranthomologous recombination at telomeres. Cell 126, 49-62.
Wu, P., van Overbeek, M., Rooney, S. and de Lange, T.
(2010). Apollo contributes to G overhang maintenanceand protects leading-end telomeres. Mol. Cell 39, 606-617.
Xin, H., Liu, D., Wan, M., Safari, A., Kim, H., Sun, W.,
O’Connor, M. S. and Songyang, Z. (2007). TPP1 is ahomologue of ciliate TEBP-beta and interacts with POT1to recruit telomerase. Nature 445, 559-562.
Ye, J. Z. and de Lange, T. (2004). TIN2 is a tankyrase 1PARP modulator in the TRF1 telomere length controlcomplex. Nat. Genet. 36, 618-623.
Ye, J. Z., Donigian, J. R., van Overbeek, M., Loayza,
D., Luo, Y., Krutchinsky, A. N., Chait, B. T. and de
Lange, T. (2004a). TIN2 binds TRF1 and TRF2simultaneously and stabilizes the TRF2 complex ontelomeres. J. Biol. Chem. 279, 47264-47271.
Ye, J. Z., Hockemeyer, D., Krutchinsky, A. N., Loayza,
D., Hooper, S. M., Chait, B. T. and de Lange, T.
(2004b). POT1-interacting protein PIP1: a telomere lengthregulator that recruits POT1 to the TIN2/TRF1 complex.Genes Dev. 18, 1649-1654.
Zhu, Y., Tomlinson, R. L., Lukowiak, A. A., Terns,
R. M. and Terns, M. P. (2004). Telomerase RNAaccumulates in Cajal bodies in human cancer cells. Mol.
Biol. Cell 15, 81-90.
Journal of Cell Science 125 (18)4178