Telomeres and telomerase dance to the rhythm of the cell cycle

Telomeres and telomerase dance tothe rhythm of the cell cycleJ. Arturo Londono-Vallejo1,2 and Raymund J. Wellinger3

1 Laboratoire Te lome res et Cancer, UMR3244, Institut Curie, 26 rue d’Ulm, 75248 Paris, France2 UPMC Universite Paris 06, F-75005 Paris, France3 Department of Microbiology and Infectiology, Groupe ARN/RNA Group, Faculty of Medicine, Universite de Sherbrooke,

3201 rue Jean Mignault, Sherbrooke, Quebec, J1E 4K8, Canada

Review

The stability of the ends of linear eukaryotic chromo-somes is ensured by functional telomeres, which arecomposed of short, species-specific direct repeatsequences. The maintenance of telomeres depends ona specialized ribonucleoprotein (RNP) called telomerase.Both telomeres and telomerase are dynamic entitieswith different physical behaviors and, given their sub-strate–enzyme relation, they must establish a productiveinteraction. Regulatory mechanisms controlling this in-teraction are key missing elements in our understandingof telomere functions. Here, we review the dynamicproperties of telomeres and the maturing telomeraseRNPs, and summarize how tracking the timing of theirdance during the cell cycle will yield insights into chro-mosome stability mechanisms. Cancer cells often dis-play loss of genome integrity; therefore, these issues areof particular interest for our understanding of cancerinitiation or progression.

A dance in the nucleusEven though our genomes are broken up into chromo-somes, at the molecular level single chromosomes areformidably large entities. They require continuous main-tenance, large parts of them are transcribed into RNA, andthey need to be duplicated in each S-phase and thenproperly partitioned into the two dividing cells. All of thisrequires transactions with the underlying DNA, which areof utmost importance for continued cell viability. In addi-tion, sophisticated regulatory circuitries must be able torapidly and precisely reach chromosomes within the con-fines of the densely packed nucleus. Chromosomes are notrandomly mixed up in the nucleus but rather occupyrelatively distinct and nonoverlapping territories [1]. How-ever, neither the specific local organization of DNA intoparticular chromatin nor the localization of chromosomalloci in the nucleus is static. The constraints imposed on thechromosomes by the cell division cycle and fluctuatingchromosomal metabolism require frequent changes tothe chromosomes. These include reorganization or modifi-cation of chromatin and sometimes large-scale movementsof whole chromosomes or portions of chromosomes. There-fore, some of the greatest challenges in the field are todecipher how the dynamic behavior of chromosomes in the

Corresponding author: Wellinger, R.J. ([email protected]).Keywords: telomere dynamics; nuclear organization; telomerase; RNP maturation;telomerase trafficking.

0968-0004/$ – see front matter � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tib

nucleus is kept nonrandom and how this behavior servesthe complex control mechanisms that need to find them.Furthermore, it is still unclear how key enzymes or otherfactors required for genome stability find their cognatesubstrate region on the chromosomes in a timely manner.Advances in live cell microscopy have allowed an approachto those questions, and here we review these results asthey apply to a specific part of chromosomes, namelytelomeres. This proposition appears particularly timely,because real-time in vivo visualization of telomeres andtheir interactions with the enzyme telomerase has recentlybeen achieved.

Telomeres are the terminal areas of chromosomes thatfulfill at least two essential functions: they must be recog-nized as functional domains, thus distinguishing themfrom random chromosomal breaks that will induce DNAdamage checkpoint signaling, and they must prevent theseDNA ends from fusing with other DNA ends (Figure 1; forrecent reviews, see [2–4]). To fulfill these functions, anadequate amount of telomeric repeat sequences must bemaintained at each telomere, for which the RNP enzymetelomerase is required (Box 1) [5]. Thus, telomerase isessential for maintaining genome stability because it mustmaintain adequate telomeric repeats at chromosome ends.Because the telomerase RNP is composed of RNA andseveral protein subunits, a relatively complex maturationpathway is required to generate the active RNP that mustsubsequently find its substrate. Slight variations of thesefundamental concepts have to be dealt with by all eukary-otic cells that have linear chromosomes, and problemsalong this road could have dire consequences, such as celldeath or genome instability, which is a common prelude tocancer in humans.

How this dance of telomerase with its substrate, thetelomere, is initiated and controlled has predominantlybeen elucidated from studies in budding yeast (Saccharo-myces cerevisiae) and mammalian cells. Therefore, wefocus our description on those systems.

Budding yeast: a proposal between two very differentpartnersChromosomes, in budding yeast nuclei, occupy relativelyconstrained spaces in a configuration that, in an approxi-mate manner was first described by C. Rabl for chromosomesin salamander cell nuclei [6]. This Rabl configuration refersto a stable arrangement of chromosomes in a relatively fixed

s.2012.05.004 Trends in Biochemical Sciences, September 2012, Vol. 37, No. 9 391

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Glossary

3C analysis: chromosome conformation capture. This technique links together

the DNA of two distinct loci in the genome that happen to be physically close in

the living cell. Thus, although the technique does not identify the location of

chromosome areas with respect to nuclear landmarks, it documents them

relative of any other individual chromosomal locus.

53BP1: p53-binding protein 1, a component of the DNA damage pathway in

mammalian cells. Its phosphorylated form is recruited to telomeres upon

uncapping.

ALT: alternative lengthening of telomeres, a general term to describe all

telomerase-independent telomere maintenance mechanisms. Most, if not all,

of them are based on recombination.

APB: ALT-associated PML body, a hallmark of ALT, is a PML body that is

modified by the presence of telomeric material (DNA and proteins) and the

association of DNA repair proteins.

ATM: ataxia telangectasia mutated, a kinase that plays a major role in the

initiation of the DNA damage signaling pathway.

Cajal bodies: nuclear bodies whose main constituent is the protein coilin. They

are thought to be implicated in RNA-related metabolic processes. Furthermore,

they associate with the telomerase RNP and deliver them to telomeres.

CST complex: a complex of three proteins that are essential for telomere

capping and telomerase recruitment in budding yeast (Cdc13, Stn1, Ten1).

These proteins interact genetically and biochemically, and might exist as a

complete three member complex and/or as subcomplex of the Stn1/Ten1

proteins. Proteins with striking functional similarities to the yeast members

have also been found in plants and mammals (Ctc1, Stn1, and Ten1).

Dyskerin: a protein that is implicated in several aspects of rRNA processing and

modification. It is essential for normal accumulation of mature TERC.

FISH: fluorescence in situ hybridization, a technique using sequence specific

probes to allow the visualization of specific chromosomal loci/structures or

RNA in fixed cells.

GFP tagging: the green fluorescent protein naturally emits fluorescent light. It

is often fused to another protein to allow tracking of that protein via

microscopy; it usually does not perturb the intrinsic properties of the fusion

partner.

ICF syndrome: immunodeficiency, centromeric instability, and facial anomalies

syndrome; a rare autosomal recessive disorder that is caused by mutations in

the gene coding for the de novo DNA methyltransferase DNMT3B, which leads

to deficiencies in the methylation of satellite and pericentromeric DNA.

LacO/LacI system: the LacI protein, a repressor of the expression of the

bacterial lacZ gene, binds strongly and stably to the lacO operator, a DNA

element that is found immediately downstream of the lacZ promoter. Each

lacO operator can bind a LacI protein; therefore, insertion of tandem lacO

repeats into a eukaryote genome can be combined with expression of a

fluorescently tagged LacI protein to detect a particular DNA sequence by

microscopy.

MS2 coat protein: a bacteriophage MS2-encoded protein that binds a short

RNA stem–loop in a sequence-specific manner with high affinity. The presence

of this protein is very well tolerated in eukaryotic cells. It can be fused to a

fluorescent indicator protein, such as GFP, in combination with insertion of

cognate short stem–loops into targeted RNAs, to allow in vivo visualization of

single RNA molecules.

PML bodies: promyelocytic leukemia bodies, or ND10 (nuclear domain 10), are

punctuated subnuclear structures that are found in many mammalian cells.

Many DNA–protein and protein–protein interactions are thought to occur

specifically within these bodies, and they are also thought to be important for

specific protein trafficking, although the molecular mechanisms involved

remain mysterious.

Pontin/Reptin: related members of the AAA+ (ATPases associated with diverse

cellular activities) superfamily. Pontin and reptin are involved with many

functions, including transcription regulation, DNA damage repair, and

telomerase RNP biogenesis.

RNP (ribonucleoprotein) complex: a particle that contains both RNA and

protein.

Shelterin: a six member protein complex that specifically associates with

telomeres and is essential for telomere capping.

SM7 complex: a highly conserved seven member complex that is required for

the biogenesis and stability of the majority of the U snRNAs and the

telomerase RNA (Tlc1) in yeast. The ring-shaped structure binds to a

consensus motif, usually near the 30-end of its target RNA, and is present in

the final active complexes (splicing or telomerase RNPs). Although it might

function in splicing, it is not clear whether it is involved in telomerase activity.

snoRNA: small nucleolar RNA are small noncoding RNAs that participate in the

metabolism (maturation, modification, and editing) of other RNAs (such as

rRNA).

snRNA: small nuclear RNA are small noncoding RNAs that participate in the

metabolism of other RNAs, including mRNA splicing.

SUMO: small ubiquitin-like modifier is a small protein that can be covalently

attached to other proteins to stimulate or inhibit their activity, provoke their

redistribution, or induce their degradation.

TCAB1: telomerase Cajal body protein 1 (also known as WRAP53) is a major

factor in the retention of telomerase RNP in Cajal bodies through its interaction

with a specific RNA motif in the 30-end of TERC.

TERC, TERT: telomerase RNA component (TERC) and the telomerase reverse

transcriptase (TERT). Together, they form the minimal components of an active

telomerase RNP in mammalian cells.

TERRA: telomere repeat-containing RNA denominates transcripts that are

produced from subtelomeric promoters near the end of chromosomes.

TLC1, Est2: telomerase component 1 (TLC1) is the yeast homolog of

mammalian TERC, and the ever shorter telomere 2 protein (Est2) is the yeast

homolog of mammalian TERT. Together, they form the minimal components

for an active telomerase RNP in budding yeast.

T-Rec: telomere recruitment clusters. T-Recs form in cells that express an

exogenous MS2–GFP fusion protein and a telomerase RNA that has several

engineered MS2-specific stem–loops. The resulting very bright fluorescent foci

are observed in living yeast cells during S-phase. These foci are composed of

several telomerase RNA molecules, are associated with telomeres, and reflect

actively elongating telomere–telomerase interactions.

TRF1, TRF2: telomere repeat-binding factors 1 and 2 are the two components of

shelterin that directly bind double-stranded telomere repeats, thus providing

the basis for shelterin build-up.

yKu: the yeast homolog of the mammalian KU complex. It is composed of two

proteins (Yku70 and Yku80), which form a heterodimer that nonspecifically

binds double-stranded DNA ends. This binding is required for efficient

nonhomologous end joining (NHEJ)-mediated DNA repair. However, the

complex also has important functions in the conservation of telomeric DNA

structure. Furthermore, the KU complex can associate with telomerase RNA.

Abolishing this latter interaction in budding yeast leads to telomerase

mislocalization.

Review Trends in Biochemical Sciences September 2012, Vol. 37, No. 9

392

configuration in the nuclear volume. Recent chromosomeconformation capture (3C) methods (see Glossary), fluores-cent in situ hybridization (FISH) experiments, and live-cellmicroscopy techniques have further revealed that buddingyeast centromeric regions cluster at the spindle pole body onone side of the cell, and the nucleolus is located at theopposite side of the nucleus, creating an axis that can bedrawn through the middle of the nucleus (Figure 2; [7–11]).The localization of telomeres, one of the partners in thedance, was first established by immunofluorescence usingantibodies against telomeric proteins and, later, by FISH oftelomere-associated sequences. The images appeared toshow clusters of telomeres at the nuclear periphery[12,13]. More recent whole genome analyses indeed placetelomeres between the nucleolus and the spindle pole body[10,14,15] (see [11,16] for reviews). However, telomeres donot cluster randomly: as expected for a Rabl configuration,the two telomeres of a chromosome that has equal armlengths preferentially colocalize together [7,17], and perhapswith the telomeres of other chromosomes that have similararm lengths [14]. This prominent localization pattern is alsoknown as telomere anchoring or telomere tethering. Geneticexperiments have revealed that at least two pathways con-tribute to telomere anchoring: one that is mediated by thetelomere associated Sir4 protein, and one that is orchestrat-ed by the yeast Ku complex (yKu) and telomerase [16].However, not all telomeres localize to the nuclear periphery;for those that do, they do not remain there indefinitely, asthey become displaced during S-phase [18–20]. Therefore, itremains difficult to unambiguously assign a functional rolefor telomere anchoring. Indeed, comparing telomere func-tions in strains that have marked differences in perinucleartelomere localization has not revealed a clear correlationbetween anchoring defects and telomere function. One prev-alent idea is that perinuclear tethering is important forprotection against unwarranted telomere recombination,and perhaps even inhibition of elongation by telomerase,because shifting telomeres to the vicinity of a nuclear pore

Box 1. Components of the telomerase RNP

In all organisms where telomerase has been examined, it exists as a

ribonucleoprotein (RNP) particle. It contains at least one RNA

molecule (TLC1 in yeast, TERC in mammals) and several protein

components. In yeast, the telomerase RNA displays similarities to

small nuclear RNAs (snRNAs), as it has a 50-trimethyl cap structure

and the SM7 complex is associated near its nonpolyadenylated 30-

end (Figure 1). The mammalian telomerase RNA also has a 50-

trimethyl cap, but its 30-end bears similarities to small nucleolar

RNAs (snoRNAs) and is bound by Dyskerin and the TCAB1 protein.

Telomerase RNAs from different organisms vary enormously in size

and sequence, but nevertheless harbor certain similar core structur-

al features. The most important of those are a templating area and a

pseudoknot structure, which are thought to form the catalytic

center. This center is bound by the catalytic subunit protein (Est2 in

yeast, TERT in mammals). The RNA, together with this protein

subunit, can reconstitute a minimal telomerase activity in vitro.

Consistent with the fact that telomerase adds DNA telomeric repeats

via an RNA-templated mechanism, Est2/TERT proteins show clear

similarities to reverse transcriptases. Finally, there are additional

protein subunits of telomerase that are essential for in vivo activity

(Est1, Est3, and Sm7 in yeast; Pontin/Reptin in mammals) and there

is evidence that telomerase RNP assembly is aided by chaperone

proteins. Finally, the telomerase RNP might vary in composition as

cells progress through the cell cycle or in cells of different origins,

but there is currently very little evidence of such changes.


complex allows recombinational repair and telomere elon-gation [21,22].

These dynamic changes of telomeric areas occur in a timeframe of several minutes, and some are dictated by cell cycleprogression. Therefore, such gross telomere and chromo-some movements are usually monitored by time lapseexperiments. Real-time recordings of telomeres over muchshorter time spans have also been achieved and essentiallyshow the expected behavior of a very constrained particle[7,23]. This means that continuous tracking of telomeresduring intervals of seconds to minutes reveals a randomwalk-type movement within very little space; that is,telomeres do not move very far. In fact, such analysesrevealed that telomeric and centromeric loci are much moreconstrained than the areas between them [7,11,23].

By contrast, tracking the localization of telomeraseRNP, the other partner of the dance, is a challenge inyeast. This is because there are only approximately 25telomerase RNA molecules per cell, and the RNP has tofirst be assembled from several components [24,25].Nevertheless, RNA FISH experiments and imaging ofGFP-tagged telomerase protein components have shownthat these components accumulate in the nucleus and thatthe nucleolus might play a role in telomerase maturation[25,26] (Figure 2). Moreover, these tools have determinedthat telomerase import and/or retention in the nucleusdepends on the yKu complex and some of the telomerasecomponents [25]. However, the available genetic and bio-chemical tools have not allowed unambiguous determina-tion of when and how the two very different partners, thetelomerase RNP and the telomeres, come together suchthat the enzyme can act on its substrate, or when and howthe dance begins and ends.

Now watch out for them to put on the moves!Recently, direct single cell in vivo studies of telomeraselocalization have been achieved through tagging the yeast

telomerase RNA moiety with short stem–loop motifs thatare recognized by a coexpressed viral MS2–GFP fusion coatprotein [27]. These experiments enabled real-time moni-toring of telomerase RNP for up to a minute, and alsoexamined the regulation of the observed behavior.Strikingly, during G1 and G2/M phases of the cell cycle,telomerase was found to behave like a diffusive particle,within the constraints of the nucleus (Figure 2a). Thismeans that it roams at relatively high speeds and isunhindered in the nucleus because it only slows downfor periods of less than 3 s (Figure 2a, left). However, inlate S-phase the behavior of some of these particleschanges, because they slow down in a dramatic mannerand assume a dynamic behavior that is indistinguishablefrom that of a telomere (Figure 2a, left). In addition, thereis an accumulation of several telomerase RNPs at a veryslow moving spot (Figure 2a, left bottom). Further geneticand colocalization data indicate that these clusters of atleast one telomere and several telomerase RNPs corre-spond to actively replicating telomeres [27]. A surprisingfinding from this study is that the yeast Rif proteins, whichhave been known to limit telomerase-mediated telomereextension, do so by restricting access of the telomeraseRNP to the telomeres to late S-phase [27]. The cluster ofactive telomerase on telomeres was named T-Rec, fortelomerase recruitment cluster. This study reported, forthe first time, the telomere–telomerase dance in singleliving cells during different phases of the cell cycle(Figure 2a). Gratifyingly, the results obtained with thistechnology are consistent with previous findings that pre-dicted there are only a few telomere elongation events on aper cell and per S-phase basis [28]. Furthermore, elaborategenetic setups suggest that the observed T-Recs preferen-tially occur on telomeres with short repeat tracts; that is,those that require elongation to prevent dysfunction [27].Such telomere length-biased association has been deducedfrom a large body of recent work [29], and direct in vivorecording of this aspect now becomes a possibility.

Thus, recent advances in live cell microscopy combinedwith fluorescent tagging of the yeast telomerase RNA haveallowed real-time observations of the whereabouts of thisessential RNP. The experiments showed that the telome-rase–telomere interaction is tightly regulated in a spatialand temporal manner. Surprisingly, the data also showedthat the Rif proteins are important for the cell cyclerestricted access of telomerase to telomeres. Refinementof the above methodology should soon allow the uncoveringof other details of this enzyme–substrate interplay, andalso tell us how and where the telomerase RNP is assem-bled and matured.

The dancing partners in mammalian cellsTelomeres in higher eukaryotes play the same essentialroles as in yeast. Initial pioneering work indicated that,contrary to yeast, mammalian telomeres appear to berandomly distributed throughout the nucleus and theyseem to not show particular aggregative behavior orpreferential subnuclear localization [30]. Telomeres werefound to be tightly associated with the nuclear ‘matrix’, alose term that defines the insoluble remnant after saltextraction and nuclease treatment of nuclei. These initial

393

Budding yeast

Rap1 CST

Rif1/2Sir2/3/4

yKu?

?Est2

Est3Est1

TLC1

Sm7

Telomerase

yKu

3′

Telomere

Mammals

TIN2 TPP1POT1

Shelterin

TRF1 TRF2RAP1

TERT

TERC

Dyskerin

Telomerase

TCAB1

TiBS

Figure 1. Telomeres and telomerase in budding yeast and in mammals. Telomeric DNA is composed of short, direct repeats: (TTAGGG)n in humans, (TG)0–6TGGGTG1–2 in

budding yeast (blue and red parallel lines indicate the telomeric G-rich and C-rich strands). In both systems, there is a single-stranded overhang of the 30-end (blue strand).

Human telomeres can comprise 5–15 kb of such repeats, whereas yeast telomeric repeats are typically approximately 300 bp. Mammalian telomeric DNA is thought to be

organized into a looped structure (top panel), at least for part of the cell cycle. In this so-called t-loop structure, the terminal single-stranded DNA invades into the duplex

DNA internally. However, at least during or after replication, the t-loop is resolved and the telomeric DNA is in a linear arrangement. Telomere-associated proteins are

indicated; importantly, human telomeric repeats also contain occasional nucleosomes (not shown). Shelterin refers to a complex of up to six members (TRF1, TRF2, RAP1,

TIN2, TPP1, and POT1) that is essential for telomere capping. Although there is evidence for a mammalian CST complex (not shown), it is not yet clear whether it is always

associated with telomeres. Human telomerase is outlined with its main in vivo components: TERC (telomerase RNA, black curvilinear form), TERT (catalytic subunit, light

red), Dyskerin (peach oval), and TCAB1 (telomerase Cajal body protein 1, dark red oval). Yeast telomeres (lower panel) are directly bound by Rap1 (green) on the double-

stranded portion, and by members of the Cdc13 complex (or CST, yellow) on the 30-extension. Sir2/3/4 (black) and Rif1/2 (blue and white) proteins associate with telomeres

via their interactions with Rap1. The yeast homolog of the mammalian Ku complex (yKu) (light and dark red ring) also localizes to telomeres, although its binding mode is

not clear (indicated by ‘?’). The yeast telomerase is shown with its main in vivo components: TLC1 (telomerase RNA, black curvilinear form), the Est1/2/3 proteins (dark,

medium, and light red ovals) and the Sm7 complex (red star). Abbreviation: CST complex, a complex of three proteins that are essential for telomere capping and

telomerase recruitment in budding yeast (Cdc13, Stn1, Ten1).


biochemical descriptions thus yielded a somewhat staticportrayal of mammalian telomere behavior. However,these concepts have been recently challenged by observa-tions that specific chromosome ends have preferentialassociations with nuclear domains or territories. For in-stance, because certain very short chromosome arms carrytandem ribosomal DNA repeats, telomeres at these endsare typically found close to nucleoli [31]. Similarly, becauseall chromosomes of inbred mice have one very short armand one long arm [32], the short arm telomeres are local-ized near the chromocenters that are formed by the aggre-gation of all heterochromatic areas, including thecentromeres, of the chromosomes [33]. However, eventhough it has been recognized for some time that specificchromosome ends, such as 4qter and 10qter, are oftenassociated with the nuclear periphery in human cells[34], it came as a surprise to find that other chromosomeextremities (2p, 3p, 6q, 12q) display a similar trend [35].

394

Unfortunately these studies used fixed cells; therefore, thekinetics of association of native telomeres with the nuclearperiphery, chromosome arm-specific or not, remain to bedefined.

The dynamics of telomere movements have been studiedin live human cells using GFP-tagged components of shel-terin, a protein assembly that caps mammalian telomeres(Figure 1) [36]. The association of shelterin proteins withtelomeres has also been shown to be very dynamic, with theshelterin proteins TRF1 and TRF2 undergoing rapidexchanges between the free and chromosome-associatedforms. The half-life of residency for these proteins attelomeres is only approximately 8 s, which is much shorterthan, for example, the half-life of occupancy by histones(over 3 min). Such behavior might reflect a constant moni-toring of telomere states, as suggested by the presence ofprotein pools that bind with different dynamics [37]; thisbehavior might also result from protein turnover that is


controlled by modifications that affect DNA binding affini-ties, as has been shown for TRF1 [38,39]. Nevertheless,although they are highly mobile, there are always TRF1and TRF2 proteins at telomeres, and these proteins cantherefore be used as markers to monitor telomere move-ment. Specifically, TRF1– and TRF2–GFP fusion proteinshave been expressed at low levels in cancer cells and theobservable fluorescent spots have been monitored for 200 s[40]. In this setting, telomeres moved independently ofeach other; some telomeres were immobile, whereas otherswere extremely mobile (Figure 2b). The mobility was non-directional, occurred throughout the nucleus including theperiphery, and the extent of mobility of each telomere didnot vary during the time of observation. Nevertheless, theevidence suggests a correlation between mobility and telo-mere length: the majority of highly mobile telomeres arevery short telomeres. However, in a more recent study thatincluded longer follow-up times (up to 100 min) this corre-lation was not observed, suggesting that telomere mobilityvaries on different time scales (200 s vs 100 min) [41,42].For reasons of convenience, these studies used tumor cellsof different origins; therefore, some heterogeneity in telo-mere behavior can be expected, depending on the differ-ences between cell types, in telomere metabolism.Telomere mobility has also been evaluated and comparedin normal (primary) dermal fibroblasts and fibroblastscarrying mutations in the lamin A gene, whose productis a major component of the nuclear lamina [43]. Telomeremobility was reduced in the presence of progerin accumu-lation (as a consequence of a missense mutation in LMNA)and significantly increased in the absence of lamin Aexpression (due to a homozygous nonsense mutation inLMNA) [43]. Further experiments supported the sugges-tion that both abnormal lamin A processing and a decreasein its expression led to increased telomere motility [43],thus connecting, for the first time, nuclear integrity andmechanics to telomere motion in human cells.

There is also a relation between the capped state of atelomere and its mobility. For instance, the incorporationof mutated telomeric repeats at chromosome ends inducedan increase in telomere mobility. However, such an effectwas not observed upon induction of random double-strandbreaks in the DNA by radiomimetic drugs, which suggeststhat DNA breaks are sensed differently by the cell than adysfunctional telomere generated by noncanonical termi-nal repeats [40]. That damaged telomeres gain motilitywas confirmed in another study using mouse cells, in whichtelomere deprotection was induced by deleting TRF2 [44].Strikingly, the increase in mobility was dependent on ATMand 53BP1 but not on a functional DNA repair pathway,suggesting that local modifications at the chromatin levelcould be involved in this change in behavior. The nuclearlamina might provide a potential molecular link betweenthe telomere damage response and telomere movement,because there are demonstrated links between ATMsignaling and lamin B1 expression and between lamin Aand 53BP1 stability [45,46].

Strikingly, transcription on telomeres also impactstelomere mobility, as demonstrated in a recent study inwhich increasing the production of telomeric repeat-containing RNA (TERRA) enhanced telomere mobility

[47]. This higher mobility was not associated with telomereshortening, uncapping or modification of shelterin compo-sition, and was also indifferent to the presence of telome-rase. An intriguing possibility that remains to be tested isthat physiological modulations of TERRA levels helpdetermine locus- or cell cycle-specific telomere mobility.

It is conceivable that telomere mobility might changeduring the cell cycle. Indeed, studies carried out on fixedcells suggest that the spatial distribution of telomereschanges as cells enter G2, when they adopt a more compactdisk-like shape [48]. Although the significance of thisdistribution remains to be determined, it is possible thatthis positioning prepares the chromosome to move to theequatorial disk. During mitosis, sister chromatid cohesionmust be loosened while centromeres and chromatid armsstart migrating towards opposite poles. In this choreogra-phy, it is possible that telomeres are last in initiatingmigration, as suggested by the fact that sister telomereseparation requires a specific mechanism that involves theenzymatic activity of tankyrase, an ADP-ribosyl transfer-ase that can modify TRF1 [49]. Nothing is known abouttelomere motion right after chromosome segregation andnuclear envelope reformation in telophase. During thisperiod the most dramatic chromosome movements in so-matic cells occur; therefore, telomere movements must becoordinated and their position in the nucleus is directlycorrelated with the creation of the new chromosometerritories.

Taken together, telomeres in mammalian cells, as inyeast cells, are dynamic entities. This applies to theiractual chromatin organization, their intranuclear position-ing, and their movements.

What makes telomeres dance together?Static 3D studies in cells expressing mutated lamins haverevealed a trend for telomeres to aggregate, a behavior thathas also been observed in senescent mesenchymal stemcells [50], certain transformed post-germinal lymphoidcancer cells [51], and telomerase positive cancer cells thatexpress c-Myc [52]. Currently, it is not known whetherthese structures are mobile or whether they representtransient states. In addition, because particular mamma-lian cell types are normally devoid of lamin A expression(at least at specific stages of development [53,54]), theabove observations open the possibility that telomeremobility and aggregation patterns display cell-type speci-ficities. We also do not know whether these aggregateshave particular relations with subnuclear structures,constitute preferential targets for elongation or repair,or if the kinetics of their formation is regulated by the cellcycle. Nevertheless, these studies suggest that telomeresmay spontaneously aggregate in particular settings.

A considerable amount of attention has been focused ontelomere aggregation in cells where telomerase is absentand thus telomere maintenance mechanisms depend onrecombination through alternative lengthening of telo-meres (ALT). ALT cells are characterized, among otherthings, by the presence of nuclear bodies called APBs (ALT-associated PML bodies). APBs are modified PML bodiesthat contain telomeric DNA, shelterin proteins, and areenriched in recombination factors. Telomeric DNA in APBs

395

Cytoplasm

Nucleolus

Nucle us

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N

?

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T-Rec

(i)

(ii )

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MMG

TMGSm7

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Tel ome raseSm7

?? CB

Cytoplasm

Nucle us

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NucleolusPontin/Reptin

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(b) Mammal ian Ce lls

TERC

(i)

(ii)

(iii)

(iv)

(v)

(vi)

TERT

TCAB1

TERT

Tel ome rase

TiBS

Figure 2. Going to the telomere–telomerase dance. A schematic drawing of telomere and telomerase movements leading up to a productive interaction in (a) yeast and (b)

mammalian cells. (a) Positioning of the centromeres (Cen) to one side, and the nucleolus on the other, reflects their opposing positions in the cell. (i) The yeast Tlc1

telomerase RNA is generated by RNA polymerase II as a nonpolyadenylated and monomethylguanosine-capped (MMG, peach rectangle) RNA. (ii) Tlc1 is transported to the

nucleolus where it is modified by Tgs1 (not shown) to obtain a trimethyl cap (TMG, green rectangle) and presumably is bound by the Sm7 protein complex (red star). (iii)

The modified RNA is then released from the nucleolus and (iv) exported to the cytoplasm in a Crm1-dependent manner. (v) Tlc1 acquires telomerase protein components

(Est1, Est2, Est3; red ovals) in the cytoplasm. The yeast homolog of the mammalian Ku complex (yKu) (light and dark red ring) might also associate with the complex there.

(vi) The import and retention of the complete telomerase ribonucleoprotein (RNP) is dependent on Est1, Est2, and the yKu complex. (vii) During G1 and G2/M phases of the

cell cycle, the telomerase RNP is highly mobile, displaying dynamic properties consistent with diffusive movements (broken line). (viii) Only during late S-phase do several

telomerase RNPs accumulate on at least one telomere, forming a structure called T-Rec (telomerase recruitment cluster). T-Rec formation is dependent on positive

telomerase regulators such as Cdc13 and Tel1 (not shown), occurs preferentially on short telomeres, and its dynamic behavior matches that of a telomere. (ix) Although

telomerase may occasionally cross paths with a telomere outside S-phase, the establishment of a productive interaction in the form of a T-Rec is inhibited by Rif1/2 proteins

(not shown). Monomer telomerase RNPs are drawn for convenience; there is evidence that it may be acting as a dimer in vivo. (b) In mammalian cells: (i) telomerase RNA is

transcribed by RNA polymerase II as a 50 monomethylguanosine capped (peach rectangle) and 30-extended precursor. (ii) Maturation and stabilization of the transcript

(green rectangle), perhaps during a transit step through the nucleolus, requires its association with Dyskerin (peach oval) and its cofactors GAR1, NOP10, and NHP2 (not

shown). (iii) The ATPases Pontin/Reptin (purple circles) appears to be required both for the accumulation of a TERC (telomerase RNA component)–Dyskerin complex, as

well as for the assembly of a precursor telomerase RNP complex [77]. (iv) TERC and its associated proteins are directed to the Cajal bodies (CBs, pink circles) where further

methylation of the 50 guanosine and assembly of a fully active RNP takes place. Retention in the CBs is mediated by the interaction of 30-end of TERC with the TCAB1 protein


396


can be in the form of extrachromosomal material [55] ornative chromosome ends [56]. Clustering of telomeres isthought to favor intertelomere contacts and to promoterecombination. APBs are constitutively present, exceptwhen they disaggregate in M-phase to allow proper chro-mosome segregation [56]. APBs are highly enriched fortelomeric DNA, which greatly facilitates their visualiza-tion in live cells; this advantage has been amply exploitedto study the kinetics of APB formation. In cells using ALT,telomeres have been shown to associate with and dissoci-ate from each other, with a few telomeres displayingunconstrained directional movements [57]. These dynamicinteractions included the PML bodies, which telomeresassociated with and dissociated from in relatively shortperiods of time (less than 10 min) clearly indicating thatAPBs are not static structures [57]. Although PML bodiesare normally relatively immobile structures, they are rap-idly redistributed upon various stress treatments [58]. InALT cells, after recovery from genotoxic stress, PML bodiesare frequently reformed in association with telomeric ma-terial [59]. This association requires SUMOylation of PML[59], further supporting the notion that SUMOylation,which also occurs on the shelterin proteins TRF1 andTRF2, is an essential step in the formation of APBs [60].

The kinetics of APB appearance has been modeled bystudying the formation of PML bodies around newly cre-ated telomeres. In these experiments, telomeres weremarked with a lacO array in the subtelomeric position,which was visualized by specific binding of either fluores-cently tagged LacI or a LacI derivative that could recruitGFP-tagged proteins [61,62]. These studies found thatAPBs formed both by association of pre-existing PMLbodies with the lacO carrying telomere and by associationof free PML protein with telomeres. Further experimentsshowed that tethering either a nonconjugable SUMO1, theMMS21 SUMO E3 ligase, TRF1 or TRF2 to the subtelo-meric lacO arrays by expressing them as a fusion proteinwith LacI readily promoted APB formation [62]. Althoughthese newly formed APBs had a protein composition thatwas similar to native APBs and were able to supporttelomere replication, most of the time they failed to recruitother telomeres. One possible explanation is suggested bythe particular behavior of PML bodies in cells frompatients with ICF (immunodeficiency, centromeric insta-bility, and facial dysmorphy) syndrome, who are deficientin DNA methyltransferase activity. As a consequence,large genomic heterochromatic regions are hypomethy-lated in these cells, which promotes the formation of largeDNA-containing PML bodies with a protein compositionsimilar to APBs [63]. In contrast to native APBs, in whichchromosome ends appear to be associated with the surfaceof the PML body [56], DNA in both ICF cells and in newlyformed lacO-promoted APBs appears to be surrounded bythe proteinacious structure of the PML body. This diver-gence perhaps reflects a fundamental difference in the

(telomerase Cajal body protein 1, dark red oval). (v) Telomerase RNP-containing CBs tr

substrate during S-phase. (vi) Telomeres are also mobile structures and particularly shor

of collision with a CB. Telomerase RNP may also be prepositioned on its substrate, perha

by the fact that telomerase can extend, at least in human cancer cells, all telomeres in a s

telomeres remain to be identified.

mechanism for telomere recruitment in artificial versusnatural APBs in that the chromatin structure of the formermay be closer to that of an ICF pericentromeric region,which contains highly repetitive and hypomethylated DNAelements, than to a bona fide subtelomeric region, in whichoverall DNA methylation levels are highly heterogeneous[64,65].

In all, although mammalian telomeres appear to behaveas individual and independent entities, they do interactwith each other within particular nuclear territories inways that remain to be fully explored. Clearly, their chro-matin status is a key variable of such behavior and an issuethat needs to be carefully addressed in future live-imagingstudies.

Telomere meets telomerase: make your move already!As in yeast, the mammalian telomerase RNP (Box 1)provides the enzymatic activity and physiological mecha-nism for telomere elongation. Constitutive expression oftelomerase RNP is the most common mechanism used byhuman tumor cells to achieve unrestricted replicationcapacity [66]. The assembly of the mammalian holoenzymeis not fully understood, but it is expected to occur in thenucleus. In humans, the RNA subunit (TERC) does notappear to be exported to the cytoplasm during biogenesis,as suggested by RNA–FISH localization studies using ahuman/mouse heterokaryon assay [67], but rather accu-mulates in the Cajal body (CB) [68,69], where it probablymeets the catalytic subunit (TERT). The accumulation ofTERC in CBs requires TCAB1 (telomerase Cajal bodyprotein 1), a Dyskerin-associated protein that associateswith all small Cajal body specific RNAs (scaRNAs), whichare a subset of H/ACA small nucleolar RNAs (snoRNAs)that also carry a CAB box sequence [70]. Strikingly, TERC-containing CBs transiently associate with telomeres in acell cycle regulated manner [71,72]. Time-lapse microscopyshowed that CBs rapidly translocate throughout the nu-cleoplasm and can associate with telomeres for up to40 min during S-phase [71], supporting the idea thatCBs actively deliver telomerase to the telomeres [73].Although TERC does not accumulate in CBs in the mouse,it is also found at foci associated with a subset of telomeresduring replication [74].

Remarkably, recent studies using a biochemical ap-proach revealed that, at least in human cancer cells, everysingle telomere is elongated by telomerase within each S-phase, in a processive reaction that is mediated by a singletelomerase complex that can add up to 60 nucleotides[75,76]. However, complementary experiments performedunder nonequilibrium conditions suggested that short tel-omeres undergo a distributive mode of elongation; that is,multiple telomerase molecules are able to work on thesame chromosome end [75]. How unique or multipletelomerase molecules are targeted to every chromosomeend (under conditions where the number of telomerase

avel around the nucleus to meet telomeres and to allow telomerase to work on its

t telomeres may be able to travel longer distances which may increase the chances

ps independently of CBs via an unknown mechanism (marked by ‘?’), as suggested

ingle S-phase [75]. The actual mechanisms of CB or telomerase RNP recruitment to

397


molecules per cell is limiting) is unclear. In particular,because CBs are found to associate with only five to seventelomeres during the entire S-phase in the same cell type[71], it is possible that telomerase is delivered by alterna-tive means within this context. This alternate preposition-ing could occur prior to S-phase on most telomeres, thusenabling an elongation activity to engage the moment thetelomere adopts an accessible conformation for elongation,most probably during or right after the passage of thereplication fork [75]. How and when such prepositioningoccurs, and whether or not the higher mobility of shorttelomeres increases the chance of an interaction withtelomerase, CBs, or other telomerase-containing nuclearbodies, remain key questions to be addressed in the future.The development of more powerful techniques allowing thevisualization of single telomerase complexes in live humancells will be crucial for further investigations of the dy-namics of telomerase–telomere transactions.

Concluding remarksEver increasing technological advances in microscopy al-low for a better understanding of the dynamic behaviors oftelomeres and telomerase during the cell cycle. The estab-lishment of a productive enzyme–substrate interactionthus depends on several conditions; some are dictated byvery local parameters (telomere length and factors regu-lating end-accessibility), whereas others ensue from moreglobal circumstances in the nucleus (telomere positioningand movements, telomerase maturation and trafficking).Nevertheless, how a telomere elongation reaction is actu-ally initiated and how this event is integrated in a harmo-nious manner into the complex events of the nuclear cyclewill require even more detailed investigations in livingcells. Such next steps, however, are required to understandtelomerase biology more completely and perhaps evenexploit that knowledge for medical applications.

AcknowledgmentsWe apologize to researchers whose work could not be mentioned owing tospace constraints. Thanks to N. Laterreur for her expert support ingraphics. Research in the authors’ laboratories is funded by theAssociation pour la Recherche contre le Cancer (ARC) (J.A.L-V.), theLigue contre le Cancer (‘equipe labellisee’) (J.A.L-V.), the InstitutNational du Cancer (INCa) (J.A.L-V.) and the Canadian Institutes ofHealth Research (R.J.W.). R.J.W. holds a Canada Research Chair inTelomere Biology.

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Telomeres and telomerase dance to the rhythm of the cell cycle