Post-translational modifications of TRF1 and TRF2 and their roles in telomere maintenance

Mechanisms of Ageing and Development 133 (2012) 421–434

Post-translational modifications of TRF1 and TRF2 and their roles in telomeremaintenance

John R. Walker, Xu-Dong Zhu *

Department of Biology, LSB438, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada

A R T I C L E I N F O

Article history:

Received 12 March 2012

Received in revised form 27 April 2012

Accepted 4 May 2012

Available online 23 May 2012

Keywords:

Post-translational modification

Human

Telomere binding proteins

TRF1

TRF2

A B S T R A C T

Telomeres, heterochromatic structures, found at the ends of linear eukaryotic chromosomes, function to

protect natural chromosome ends from nucleolytic attack. Human telomeric DNA is bound by a

telomere-specific six-subunit protein complex, termed shelterin/telosome. The shelterin subunits TRF1

and TRF2 bind in a sequence-specific manner to double-stranded telomeric DNA, providing a vital

platform for recruitment of additional shelterin proteins as well as non-shelterin factors crucial for the

maintenance of telomere length and structure. Both TRF1 and TRF2 are engaged in multiple roles at

telomeres including telomere protection, telomere replication, sister telomere resolution and telomere

length maintenance. Regulation of TRF1 and TRF2 in these various processes is controlled by post-

translational modifications, at times in a cell-cycle-dependent manner, affecting key functions such as

DNA binding, dimerization, localization, degradation and interactions with other proteins. Here we

review the post-translational modifications of TRF1 and TRF2 and discuss the mechanisms by which

these modifications contribute to the function of these two proteins.

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1. Telomeres and the shelterin complex

Telomeres are specialized heterochromatic structures found atthe ends of linear eukaryotic chromosomes. Human telomeric DNAconsists of double-stranded TTAGGG tandem repeats ranging from 2to 30 kilobase pairs in length (Cheng et al., 1989; de Lange et al.,1990; Moyzis et al., 1988) as well as a G-rich 30 single-strandprotrusion (�150 nt) (Makarov et al., 1997; McElligott and Well-inger, 1997; Wright et al., 1997). Invasion of the 30 G-rich single-strand protrusion into the duplex region of telomeric DNA gives riseto the formation of a higher order structure at telomeres, referred toas the t-loop (Griffith et al., 1999). The t-loop structure has beenfound not only in mammals but also in plants and protozoa,suggesting a conserved role of t-loops in masking chromosome endsfrom being recognized as double-strand breaks (Griffith et al., 1999;Munoz-Jordan et al., 2001; Murti and Prescott, 1999).

Mammalian telomeric DNA is coated with a telomere-specificprotein complex, referred to as shelterin/telosome, which consistsof TRF1, TRF2, TIN2, POT1, TPP1 and hRap1 [reviewed in de Lange2005; Liu et al., 2004a; Palm and de Lange, 2008]. TRF1 interactsdirectly with TIN2 (Kim et al., 1999), which binds to TRF2 and TPP1(Houghtaling et al., 2004; Kim et al., 2004; Liu et al., 2004b; Yeet al., 2004a; Ye et al., 2004b). While POT1 binds to TPP1(Houghtaling et al., 2004; Liu et al., 2004b; Ye et al., 2004b), TRF2

* Corresponding author. Tel.: +1 905 525 9140x27737; fax: +1 905 522 6066.

E-mail address: [email protected] (X.-D. Zhu).

0047-6374/$ – see front matter � 2012 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.mad.2012.05.002

interacts and forms a complex with hRap1 with a �1:1stoichiometry (Li et al., 2000; Zhu et al., 2000). Components ofthe shelterin complex do not exist in an equal abundance in cells(Takai et al., 2010) and, therefore, sub-complexes of shelterinlacking one or more subunits have been observed in cell extracts(Kim et al., 2008b; Liu et al., 2004a; Ye et al., 2004a).

The shelterin complex plays an essential role in maintaining theintegrity of telomere length and structure. Disruption of shelterinproteins at telomeres not only promotes deregulation of telomerelength homeostasis but also induces telomere de-protection,resulting in the formation of telomere abnormalities includingtelomere end-to-end fusions, telomere loss, telomere-containingdouble-minute chromosomes (TDM) and telomere doublets/fragiletelomeres (more than one telomeric signal at a single chromatidend) [reviewed in de Lange, 2005; Palm and de Lange, 2008]. Thesedysfunctional telomeres are recognized as damaged DNA (McKerlieand Zhu, 2011; Mitchell et al., 2009; Sfeir et al., 2009; Takai et al.,2003; Wang et al., 2004) and can contribute to genomic instability, ahallmark associated with tumorigenesis and ageing.

In addition to the shelterin complex, mammalian telomeres arealso associated with a number of accessory factors that areinvolved in DNA metabolism but not unique to telomeres (deLange, 2005; Liu et al., 2004a; Palm and de Lange, 2008). Thesefactors also play an important role in the maintenance of telomereintegrity. Examples include XPF/ERCC1 (Zhu et al., 2003), theMre11 complex (Zhu et al., 2000), Apollo (Lenain et al., 2006; vanOverbeek and de Lange, 2006), Ku70/Ku80 (Hsu et al., 1999; Hsuet al., 2000) and ORC (Atanasiu et al., 2006; Deng et al., 2007).


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J.R. Walker, X.-D. Zhu / Mechanisms of Ageing and Development 133 (2012) 421–434422

2. Double-stranded telomere binding proteins TRF1 and TRF2

Within the shelterin complex, only TRF1, TRF2 and POT1 caninteract with telomeric DNA directly (Palm and de Lange, 2008).POT1 contains multiple OB folds and binds to the G-strandtelomeric DNA (Baumann and Cech, 2001; Kelleher et al., 2005; Leiet al., 2004; Loayza and De Lange, 2003; Loayza et al., 2004). On theother hand, TRF1 and TRF2 are double-stranded telomeric DNAbinding proteins (Bilaud et al., 1997; Broccoli et al., 1997; Chonget al., 1995), and these two proteins will be the focus of this review.

TRF1 and TRF2 are related proteins that share a commonarchitecture (Fig. 1), consisting of an N-terminal domain, a TRFhomology domain (TRFH) which mediates dimerization, a flexiblelinker region and a C-terminal homeodomain which has beenhistorically described as a SANT/Myb-like DNA binding domain(Bilaud et al., 1997; Broccoli et al., 1997; Court et al., 2005). Whilethere is a high degree of sequence similarity for the respectiveTRFH and the Myb-like domains of TRF1 and TRF2, little sequenceand structural similarity is found in their respective flexible linkerregion (Bianchi et al., 1999). Furthermore, the N-terminal domainsof TRF1 and TRF2 differ significantly from one other, with TRF1possessing a unique acidic domain and TRF2 carrying a basicdomain rich in glycine and arginine residues, also referred to as aGAR domain.

Both TRF1 and TRF2 bind to duplex telomeric DNA as a dimer(Bianchi et al., 1997; Bilaud et al., 1997; Broccoli et al., 1997; Shenet al., 1997; van Steensel and de Lange, 1997), but they do notinteract with each other (Broccoli et al., 1997; Zhu et al., 2000).Three-dimensional structural analysis showed that the homo-dimer formation of TRF1 or TRF2 is mediated by their respectiveTRFH domain; however, unique features at their respectivedimerization interface prohibit them from forming a heterodimer(Fairall et al., 2001). The TRFH domains of TRF1 and TRF2 contain asimilar peptide-docking site that is differentially used to interactwith proteins containing the motif FxLxP for docking with TRF1,

Fig. 1. Schematic diagrams of TRF1 (A) and TRF2 (B) along with their respective post-trans

localization signal. Phosphorylation sites are indicated in black, ubiquitylation sites

interpretation of the references to color in this figure legend, the reader is referred to

and YxLxP for docking with TRF2 (Chen et al., 2008; Kim et al.,2009). TRF1 binds to the FxLxP motif of TIN2 whereas TRF2 binds tothe YxLxP motif of several shelterin accessory factors includingApollo, XPF, the Mre11 complex, MCPH1 and PNUTS (Chen et al.,2008; Kim et al., 2009). In addition, TRF1 and TRF2 can also interactwith proteins through regions outside of their TRFH domains. Theacidic domain in the N-terminus of TRF1 contains the RxxADGsequence that is bound by tankyrase 1 and tankyrase 2 (Guettleret al., 2011; Kaminker et al., 2001; Sbodio and Chi, 2002; Sbodioet al., 2002; Smith et al., 1998). Several proteins are found tointeract with either the N-terminus or the C-terminus of TRF2including WRN, ORC and FEN1 (Atanasiu et al., 2006; Muftuogluet al., 2006; Opresko et al., 2002).

3. Roles Of TRF1 and TRF2 at telomeres

TRF1, the first shelterin protein discovered to bind telomericDNA (Chong et al., 1995), is implicated in telomere replication,telomere protection, telomere length maintenance and theresolution of sister telomeres. The gene encoding TRF1 is essentialsince knockout of TRF1 leads to embroynic lethality (Karlsederet al., 2003). Deletion of TRF1 promotes the formation of fragiletelomeres, a type of telomere abnormality that can be caused byreplication-dependent defects (Martinez et al., 2009; Sfeir et al.,2009). It has been suggested that TRF1 is required to supportefficient replication of telomeric DNA (Sfeir et al., 2009). Cells nullor depleted for TRF1 accumulate telomere fusions (Iwano et al.,2004; Martinez et al., 2009; McKerlie and Zhu, 2011; Sfeir et al.,2009), indicative of its role in telomere protection. Overexpressionof TRF1 promotes telomere shortening whereas loss of TRF1 fromtelomeres has been shown to induce telomerase-dependenttelomere lengthening, implying that TRF1 negatively regulatestelomerase-dependent telomere extension, perhaps by restrictingthe access of telomerase to the ends of telomeres (Ancelin et al.,2002; Smogorzewska et al., 2000; van Steensel and de Lange,

lational modification. The major domains of TRF1 and TRF2 are shown. NLS: nuclear

in blue, SUMOylation sites in green, and arginine methylation sites in red (for

the web version of the article).

J.R. Walker, X.-D. Zhu / Mechanisms of Ageing and Development 133 (2012) 421–434 423

1997). Furthermore, TRF1 has been shown to interact with thecohesin protein SA1 (Canudas and Smith, 2009) as well asmicrotuble binding protein EB (Nakamura et al., 2001). TRF1 hasbeen implicated in the regulation of the mitotic spindle as well asthe resolution of sister telomeres (Dynek and Smith, 2004;McKerlie and Zhu, 2011; Nakamura et al., 2002; Shen et al.,1997). Recently it has been shown that overexpression of TRF1lacking the phosphorylation site for cyclin B-dependent kinase 1(Cdk1) leads to the blockage of the resolution of sister telomeres(McKerlie and Zhu, 2011).

TRF2 is best known for its role in telomere protection. Removalof TRF2 from telomeres either through overexpression of adominant-negative allele of TRF2 lacking the basic and the Myb-like domains or TRF2 deletion results in loss of telomere overhangs(Celli and de Lange, 2005; van Steensel et al., 1998), and such loss isthought to be mediated by the XPF/ERCC1 complex (Zhu et al.,2003), an endonuclease known to be involved in nucleotideexcision repair (Sijbers et al., 1996). Degradation of telomere G-rich overhangs promotes the formation of telomere end-to-endfusions (Celli and de Lange, 2005; van Steensel et al., 1998), leadingto the induction of an ATM- and p53-dependent senescence orapoptosis (Karlseder et al., 1999; van Steensel et al., 1998).Overexpression of TRF2 lacking the basic domain promotes DNArecombination at telomeres, resulting in the induction of rapidtelomere deletion and the formation of telomere circles (Wanget al., 2004). Furthermore, amino acid substitutions of arginines tolysines in the basic domain of TRF2 induce the accumulation oftelomere doublets (Mitchell et al., 2009). These findings suggest for

Table 1Summary of post-translational modification of TRF1.

Modification Amino acids ID of sites Modifying

enzymesMS MA Ab

Phosphorylation T122 Yes Yes – CK2

T149 – Yes Yes CDK

S219 – Yes – ATM

T273 – Yes – AKT

S274 Yes Yes – –

S296 – Yes – Aurora A

T344 – Yes – Cdkl

S367 Yes Yes Yes ATM

T371 Yes Yes Yes Cdkl

S435 Yes Yes – Plkl

Ubiquitylation – – – – Fbx4

– – – – RLIM

SUMOylation K337, K338,

K349-K352

– Yes – MMS21

PARsylation – – – – Tankyrase-1

and 2

ID: Identification method; MS: mass spectrometry analysis; MA: mutational analysis;

–: ‘‘Not available’’.

a crucial role of TRF2 in maintaining the integrity of telomerestructure. In addition, TRF2 is also implicated in telomere lengthhomeostasis. While knockdown of TRF2 induces telomere elonga-tion (Takai et al., 2010), overexpression of TRF2 leads to telomereshortening in both telomerase-negative and telomerase-positivecells (Karlseder et al., 2002; Smogorzewska et al., 2000), implyingthat TRF2 acts as a negative mediator of telomere lengthmaintenance independently of the status of telomerase. It hasbeen shown that TRF2-induced telomere shortening is dependentupon XPF/ERCC1 (Munoz et al., 2005; Wu et al., 2008a; Wu et al.,2007b).

It is evident that the abundance of TRF1 and TRF2 at telomeresis crucial for the maintenance of telomere length and structure.Both TRF1 and TRF2 are subject to extensive post-translationalmodification (Fig. 1; Tables 1 and 2), which in turn contribute tothe regulation of their stability, binding activity and localization.Below we discuss recent advances made in the understanding ofthe post-translational control of TRF1 and TRF2, with a focus onphosphorylation. An overview with an emphasis on the role ofubiquitylation and SUMOlyation in telomere maintenance andDNA damage response has recently been published (Peuscher andJacobs, 2012).

4. Phosphorylation

Both TRF1 and TRF2 are phosphorylated in vivo. Recentadvances reveal an important role of phosphorylation in theregulation of TRF1 binding, stability and localization. The

Function References

CK2 inhibition impairs TRF1 binding to

telomeric DNA and its stability.

Kim et al. (2008a)

T149 phosphorylation is required for TRF1

interaction with PIN1.

Lee et al. (2009)

S219 is implicated in DNA damage response. Kishi et al. (2001)

Overexpression of AKT increases TRF1 levels

and promotes telomere shortening.

Chen et al. (2009)

Phosphomimic mutation of S274 impairs TRF1

binding to telomeric DNA.

Wu et al. (2007a)

S296 is implicated in Aurora A-induced

mitotic defects.

Ohishi et al. (2010)

T344 is implicated in priming TRF1

phosphorylation by Plk1.

Wu et al. (2008b)

S367 phosphorylation inhibits TRF1 binding to

telomeric DNA and targets TRF1 to nuclear

proteasome centers.

McKerlie et al. (2012)

T371 phosphorylation keeps TRF1 free of

telomeres and is required for the resolution of

sister telomeres.

McKerlie and Zhu (2011)

S435 phosphorylation is implicated in

stimulating TRF1 binding to telomeric DNA.

Wu et al. (2008b);

Dephoure et al. (2008)

Knockdown of Fbx4, a F-box protein, increases

TRF1 levels, resulting in telomere shortening

and impaired cell growth.

Lee et al. (2006)

Overexpression of RLIM, a RING-H2 zinc-

finger-containing E3 ligase, decreases TRF1

levels, leading to telomere shortening.

Her and Chung (2009)

Depletion of MMS21 promotes telomere

shortening and telomere instability, resulting

in induction of cellular senescence in ALT cells.

Potts and Yu (2007)

Poly(ADP-ribosyl)ation of TRF1 by tankyrase 1

removes TRF1 from telomeres leading to the

increased access of telomerase to the ends of

telomeres.

Cook et al. (2002);

Smith and de Lange (2000)

Ab; site-specific antibody.

Table 2Summary of post-translational modification of TRF2.

Modification Amino acids ID of site Modifying

enzymes

Function References

MS MA Ab

Phosphorylation S20 – Yes – Chk2 Phosphorylation of TRF2 by Chk2 decreases

TRF2 binding to telomeric DNA. S20

phosphorylation is required for TRF2

interaction with G-rich RNA and recruitment

of ORC to OriP.

Buscemi et al. (2009);

Zhou et al. (2010)

T188 – Yes Yes ATM T188 phosphorylation is implicated in DNA

damage response and the fast pathway of DNA

double-strand break repair.

Tanaka et al. (2005);

Huda et al. (2009)

T358 – – – Aurora C Unknown. Spengler (2007)

Ubiquitylation K173 K180 K184 – – – Siah1 Siah1 is a p53-inducible E3 ligase with a

C3H4-type RING finger domain. Knockdown

of Siah1 stabilizes TRF2 and extends the life

span of human primary fibroblasts.

Fujita et al. (2010)

SUMOylation K140, K245 K333 – Yes – MMS21 Simultaneous mutations of all three

SUMOylation sites of TRF2 prevent the

formation of APB in ALT cells.

Potts and Yu (2007)

PARsylation – – – – PARP1, PARP2 Poly(ADP-ribosyl)ation of TRF2 reduces TRF2

binding to telomeres, which may contribute to

the formation of dysfunctional telomeres

observed in cells deficient in PARP1or PARP2.

Dantzer et al. (2004);

Gomez et al. (2006)

Methylation R13,R17 R18, R21

R25, R27 R28, R30

Yes Yes Yes PRMT1 Lack of TRF2 methylation promotes the

accumulation of telomere doublets, resulting

in the induction of cellular senescence.

Mitchell et al. (2009)

ID: Identification method; MS: mass spectrometry analysis; MA: mutational analysis; Ab; site-specific antibody.

‘‘–’’: Not available.


phosphorylation of TRF2 is less well understood. The fewcharacterized phosphorylation sites of TRF2 are implicated eitherin the DNA damage response or in the regulation of the function ofits basic domain, which is not required for TRF2 interaction withtelomeric DNA.

4.1. TRF1

4.1.1. AKT-mediated phosphorylation of TRF1

TRF1 was found to physically interact with AKT throughcoimmunoprecipitation and GST pulldown assays (Chen et al.,2009). AKT is a serine–threonine kinase that has been implicated invarious cellular processes such as proliferation, apoptosis and cellgrowth (Vivanco and Sawyers, 2002). Previous work showed thatoverexpression of AKT led to an increase in the level of TRF1 and adecrease in telomere length (Chen et al., 2009). AKT was found tophosphorylate threonine 273 of TRF1 in vitro (Chen et al., 2009),however whether this phosphorylation occurs in vivo and whetherit is important for TRF1 stability has not been determined. T273 liesin the flexible linker region of TRF1 known to interact with RLIM(Her and Chung, 2009), an E3 ligase. It would be of interest to knowwhether T273 phosphorylation might interfere with RLIM-mediated ubiquitylation and degradation of TRF1.

4.1.2. ATM-mediated phosphorylation of TRF1

Ataxia telangiectasia mutated (ATM) is a member of thesuperfamily of phosphatidylinositol 3-kinase-related kinases (PI-3). It is a master regulator of the DNA damage response followingionizing irradiation, by transducing the DNA damage signal throughphosphorylation of proteins essential for the activation of the DNAdamage checkpoint, cell cycle arrest, DNA repair or apoptosis (Bhattiet al., 2011; Shiloh, 2003). Mutations in ATM give rise to ataxia-telangiectasia (AT), an autosomal recessive disorder characterizedby immunodeficiency, spontaneous chromosomal instability,hypersensitivity to ionizing irradiation and a predisposition tocancer (Savitsky et al., 1995). Primary fibroblasts derived from AT

patients accumulate telomere abnormalities and show an elevatedrate of telomere shortening as compared to cells from normalindividuals (Hande et al., 2001; Metcalfe et al., 1996; Pandita, 2002;Pandita et al., 1995; Ranganathan et al., 2001; Smilenov et al., 1997),indicative of an important role of ATM at telomeres.

ATM interacts with TRF1 (Kishi et al., 2001) and it has beenshown to phosphorylate TRF1 both in vivo and in vitro (Kishi et al.,2001; Wu et al., 2007a). TRF1 contains three consensus Ser-Gln(SQ) sites for ATM and serines in these sites are S219, S274 andS367. Phosphorylation of S219 by ATM was observed only in cellsin response to ionizing irradiation (Kishi et al., 2001). Mutations atS219 have no effect on the average telomere length (Kishi et al.,2001) or TRF1 interaction with telomeric DNA (Wu et al., 2007a).On the other hand, Lu and co-workers showed that a non-phosphorylatable mutation of S219 increases radiation hypersen-sitivity of AT cells whereas a phosphomimic mutation of S219reduces the hypersensitivity of AT cells to ionizing irradiation(Kishi et al., 2001), indicative of a role of S219 of TRF1 in the cellularresponse to DNA double-strand breaks.

Phosphorylation of TRF1 by ATM has also been observed inundamaged cells (Wu et al., 2007a). It has been suggested that ATMnegatively regulates TRF1 binding to telomeric DNA, which in turnpromotes telomerase-dependent telomere lengthening (Wu et al.,2007a). Phosphomimic mutations at S274 and S367, two of thethree ATM consensus SQ sites in TRF1, have been shown toabrogate TRF1 binding to telomeric DNA in vitro (Wu et al., 2007a),raising the possibility that ATM may phosphorylate S274 and S367in vivo. Mass spectrometry analysis indicated phosphorylation ofS274 of TRF1 in vivo (X.-D. Zhu, unpublished data); however,whether this phosphorylation is mediated by ATM in vivo remainsto be determined.

ATM is found to phosphorylate S367 of TRF1 in undamaged cellsand this phosphorylation remains unchanged in response toionizing irradiation (McKerlie et al., 2012), suggesting thatphosphorylation of S367 by ATM is independent of DNA double-strand breaks. Several lines of evidence suggest that S367


phosphorylation by ATM negatively regulates TRF1 binding totelomeric DNA (McKerlie et al., 2012; Wu et al., 2007a). Firstly, in

vitro gel shift assays showed that a non-phosphorylatablemutation of S367A has little effect on the ability of TRF1 tointeract with telomeric DNA whereas a phosphomimic mutation ofS367D severely impairs its ability to bind telomeric DNA (McKerlieet al., 2012; Wu et al., 2007a). Secondly pre-incubation of wild-type TRF1 with ATM completely abrogates TRF1 binding totelomeric DNA in vitro (McKerlie et al., 2012) whereas preincuba-tion of TRF1 carrying an amino acid substitution of S367A withATM has little effect on TRF1 binding to telomeric DNA in vitro (M.McKerlie and X.-D. Zhu, unpublished data). Thirdly immunofluo-rescence analysis with a phospho-specific anti-pS367 antibodyrevealed that phosphorylated (pS367)TRF1 forms distinct subnu-clear foci that are not associated with telomere chromatin but aredependent upon functional ATM in vivo (McKerlie et al., 2012).

Analysis of cycloheximide chase experiments showed that TRF1carrying a phosphomimic mutation of S367D has a shorter half-lifethan wild-type TRF1 or TRF1 carrying a non-phosphorylatablemutation of S367A (McKerlie et al., 2012), suggesting thatphosphorylation of S367 renders TRF1 susceptible to proteindegradation. In agreement with this notion, analysis of immuno-fluorescence with phospho-specific anti-pS367 antibody showedthat phosphorylated (pS367)TRF1 is associated with nuclearproteasome centers and that such association is completelyabolished when cells are treated with proteasome inhibitor(McKerlie et al., 2012). These findings suggest that phosphoryla-tion of S367 by ATM may target TRF1 for degradation in thenucleus. When not bound to telomeric DNA, TRF1 has been shownto undergo ubiquitylation, which targets it for protein degradation(Chang et al., 2003; Her and Chung, 2009; Lee et al., 2006). WhetherATM-mediated S367 phosphorylation might prime TRF1 forubiquitylation remains to be determined.

4.1.3. Aurora A-mediated phosphorylation of TRF1

Aurora A, a serine-threnonine kinase, plays multiple roles inmitosis including centrosome maturation and separation, bipolarspindle assembly, chromosome alignment and cytokinesis (Mar-umoto et al., 2005; Ohishi et al., 2010). Overexpression of Aurora Ais known to induce mitotic defects such as centrosome amplifica-tion and multinucleation (Meraldi et al., 2002; Ohishi et al., 2010)and these defects can be suppressed by TRF1 depletion (Ohishiet al., 2010), suggesting a functional interaction between TRF1 andAurora A. Recombinant Aurora A was found to interact with in vitro

translated TRF1 (Ohishi et al., 2010). Mutational analysis revealedthat S296 of TRF1 is a substrate of Aurora A in vitro (Ohishi et al.,2010). It has been shown that an amino acid substitution of S296Aprevents Aurora A-induced multinucleation and cytokinetic failure(Ohishi et al., 2010), suggesting that S296 of TRF1 may play a role inAurora A-induced mitotic defects. S296 lies in the region of 20amino acids that are missing in PIN2, a short isoform of TRF1 (Shenet al., 1997). It would be of interest to know whether TRF1 and PIN2may be differentially regulated by Aurora A.

4.1.4. Cdk1-mediated phosphorylation of TRF1

Cyclin B-dependent kinase 1 (Cdk1), a key regulator of mitoticentry (Lindqvist et al., 2009; Takizawa and Morgan, 2000), isknown to phosphorylate serine/threonine in the consensus Ser/Thr-Pro (S/TP) sequence. There are four S/TP sites in TRF1 and theyare S11, T149, T344 and T371. While S11 phosphorylation has beenobserved in a global phosphoproteome study (Olsen et al., 2010),its function remains uncharacterized. T149 of TRF1 has beenreported to undergo phosphorylation in mitosis in a Cdk-dependent manner (Lee et al., 2009). T149 lies in the TRFH domainof TRF1; however, mutations at T149 have no effect on the ability ofTRF1 to form a dimer or to interact with TIN2 (Lee et al., 2009), a

TRF1-interacting protein (Kim et al., 1999). On the other hand, ithas been shown that phosphorylation of T149 is required for TRF1interaction with PIN1, a prolyl isomerase known to bind andisomerize-specific phosphorylated S/TP motifs in proteins (Shenet al., 1998; Yaffe et al., 1997). Inhibition of PIN1 not only increasesthe half-life of TRF1 but also enhances TRF1 association withtelomeric DNA, leading to telomere shortening (Lee et al., 2009).These findings led to a model that the phosphorylation of T149 ofTRF1 acts as a bridge for PIN1 to regulate telomere maintenance(Lee et al., 2009).

T149 lies in the dimerization domain of TRF1, in a region criticalfor the mutually exclusive binding of TIN2 and the E3 ligase SCFFbx4

(Zeng et al., 2010), suggesting that phosphorylation of this site andthe subsequent isomerization of the adjacent proline may contributeto its degradation by SCFFbx4. However, structural analysis of theTRFH domain alone or complexed with binding substrates (Chenet al., 2008; Fairall et al., 2001; Zeng et al., 2010) reveals that T149has little surface exposure, implying that this residue may not beeasily accessible. Future studies are needed to investigate how theCdk kinase might gain access to phosphorylate T149.

In addition to T149, Cdk1 has also been shown to phosphorylateboth T344 and T371 of TRF1 in vitro (McKerlie and Zhu, 2011; Wuet al., 2008b); however, only T371 phosphorylation has beenobserved in vivo (McKerlie and Zhu, 2011). Based on biochemicalanalysis from in vitro sequential kinase assays, Liu and co-workersproposed that phosphorylation of T344 and T371 by Cdk1 serves asa docking site for TRF1 phosphorylation by Plk1 to stimulate TRF1binding to telomeric DNA (Wu et al., 2008b). Using multipleapproaches (see below), we have demonstrated that T371phosphorylation by Cdk1 inhibits TRF1 binding to telomericDNA and that it is unlikely that Cdk1-mediated T371 phosphor-ylation could serve as a docking site for TRF1 phosphorylation byPlk1 to promote TRF1 interaction with telomeric DNA (McKerlieand Zhu, 2011).

Firstly, immunofluorescence analysis with a phospho-specificanti-pT371 antibody revealed that TRF1 undergoes robustphosphorylation of T371 by Cdk1 upon mitotic entry and thatmost of the anti-pT371 staining in mitotic cells did not overlapwith DAPI staining (McKerlie and Zhu, 2011). Secondly, analysis ofdifferential salt extraction of chromatin showed that phosphory-lated (pT371)TRF1 in nocodazole-treated cells is predominantlyfound in the chromatin-free fraction (150 mM KCl) (McKerlie andZhu, 2011). Along with the sharp increase in T371 phosphorylationin nocodazole-treated cells, a reduction in the amount of total TRF1in the chromatin-bound fraction (420 mM KCl) and the appearanceof TRF1 in the chromatin-free fraction (150 mM KCl) was observed.Thirdly, chromatin immunoprecipitation showed that nocodazoletreatment results in a 50% decrease in telomeric association of totalTRF1 (McKerlie and Zhu, 2011). Very little association ofphosphorylated (pT371)TRF1 with telomeric DNA was detectedand this association was not affected by nocodazole treatment.Fourthly, in vitro gel shift assays showed that pre-phosphorylationof the recombinant wild-type TRF1 by Cdk1 resulted in a reductionin TRF1 binding to telomeric DNA, whereas pretreatment withCdk1 and ATP had no effect on telomeric DNA binding of TRF1carrying a non-phosphorylatable mutation of T371A (McKerlie andZhu, 2011). Fifthly, no TRF1 phosphorylation by Plk1 was detectedwhen TRF1 was preincubated with Cdk1 and ATP in in vitro

sequential kinase assays despite the fact that Plk1 readilyphosphorylated b-Casein, a Plk1 substrate used as a control(McKerlie and Zhu, 2011). Collectively these findings demonstratethat T371 phosphorylation by Cdk1 does not promote TRF1 bindingto telomeric DNA. Instead T371 phosphorylation negativelyregulates TRF1 binding to telomeric DNA. However, whetherT344 phosphorylation by Cdk1 might serve as a docking site forTRF1 phosphorylation by Plk1 remains to be determined.


Cdk1-mediated T371 phosphorylation only occurs on TRF1 thatis not bound to telomeric DNA since preincubation of TRF1 withtelomeric DNA completely abrogates T371 phosphorylation byCdk1 (McKerlie and Zhu, 2011). Once phosphorylated at T371,TRF1 remains free of chromatin and becomes resistant to proteindegradation (McKerlie and Zhu, 2011). Although it has been shownthat TRF1 is susceptible to proteasome-dependent proteindegradation when not bound to telomeric DNA (Chang et al.,2003; Lee et al., 2006), phosphorylation of TRF1 by Cdk1 has beenreported to slightly inhibit SCFFbx4-mediated TRF1 ubiquitylationin vitro (Zeng et al., 2010). How Cdk1-dependent T371 phosphor-ylation protects TRF1 from proteasome-mediated degradationremains unclear. Mutations at T371 do not affect TRF1 interactionwith TIN2 (M. McKerlie and X.-D. Zhu, unpublished data), whichhas been shown to protect TRF1 from degradation by the E3 ligaseSCFFbx4 (Zeng et al., 2010). Perhaps TIN2 might play a role inprotecting telomere-free phosphorylated (pT371)TRF1 from pro-teasome-mediated degradation.

T371 phosphorylation by Cdk1 in early mitosis is associatedwith loss of telomere heterochromatin and temporal de-protectionof telomeres as evidenced by telomeric accumulation of DNAdamage response factors such as g-H2AX (McKerlie and Zhu,2011). T371 phosphorylation is largely removed when Cdk1activity is down-regulated in telophase, indicative of a tightregulation of T371 phosphorylation. Dephosphorylation of T371allows for the re-association of TRF1 with telomeric DNA and re-establishment of telomere heterochromatin in late mitosis(McKerlie and Zhu, 2011). Mutational analysis revealed that aphosphomimic mutation of T371D prevents TRF1 binding totelomeric DNA, promoting accumulation of telomere loss andtelomere fusions (McKerlie and Zhu, 2011). On the other hand, anon-phosphorylatable mutation of T371A not only leads to anincrease in telomeric association of TRF1 in mitosis but also blocksthe resolution of sister telomeres, resulting in an induction ofmicronuclei formation and impaired cell proliferation (McKerlieand Zhu, 2011). These findings led to a model that upon entry intomitosis, Cdk1-dependent phosphorylation of T371 leads totemporal telomere de-protection, allowing the resolution of sistertelomeres. Once sister chromatids are separated in late mitosis,dephosphorylation of T371 results in the re-association of TRF1with telomeres and the re-establishment of telomere protection(McKerlie and Zhu, 2011).

4.1.5. CK2-mediated phosphorylation of TRF1

Casein kinase 2 (CK2) is a serine/threonine kinase that playsmultiple roles in cell cycle control, DNA repair, cell proliferationand tumorgenesis (Dodson et al., 2010; Duncan and Litchfield,2008; St-Denis and Litchfield, 2009). Using TRF1 as bait in a yeasttwo-hybrid screen, CK2 was identified as a TRF1-interactingprotein (Kim et al., 2008a). The interaction between CK2 and TRF1was further demonstrated through GST pull down and co-immunoprecipitation assays (Kim et al., 2008a). Inhibition ofCK2 has been shown not only to impair TRF1 association withtelomeric DNA but also to shorten the half-life of TRF1 (Kim et al.,2008a), suggesting that CK2 acts as a positive mediator of TRF1binding and stability.

CK2 has been shown to phosphorylate TRF1 at T122 in vitro

(Kim et al., 2008a). In vitro gel shift assays using nuclear extractsprepared from MCF7 cells overexpressing wild type and mutantTRF1 showed that TRF1 carrying a non-phosphorylatable mutationof T122A is defective in binding to telomeric DNA (Kim et al.,2008a). The T122A mutation also affects the ability of TRF1 todimerize with itself (Kim et al., 2008a). These findings suggest thatT122 of TRF1 is important for TRF1 dimerization as well as theability of TRF1 to bind telomeric DNA. However, it remains unclearwhether CK2 phosphorylates T122 of TRF1 in vivo.

As with T149, T122 lies in an important region of TRF1 forregulation by TIN2 and SCFFbx4. Conceivably, mutation orphosphorylation of this residue might affect TRF1 interactionwith one or another of these proteins, leading to some of thephenotypes observed by Chung and co-workers.

4.1.6. PLK-mediated phosphorylation of TRF1

The polo-like kinases (Plks) are a conserved superfamily ofserine/threonine protein kinases that are essential for successfulcell division (van de Weerdt and Medema, 2006; van Vugt andMedema, 2005). Plk1, one of four polo-like kinases identified inmammalian cells, is the human homolog of the founding memberPolo in Drosophila (van Vugt and Medema, 2005). Plk1 has beenimplicated in the regulation of many aspects of mitosis, includingmitotic entry, centrosome maturation, spindle formation, chro-mosome segregation and cytokinesis (van de Weerdt and Medema,2006; van Vugt and Medema, 2005). Using Plk1 as bait, TRF1 wasidentified to interact with Plk1 in a yeast two-hybrid screen andtheir interaction was further demonstrated through GST pull downand coimmunoprecipitation assays (Wu et al., 2008b). Baculo-virus-derived GST-tagged Plk1 has been shown to phosphorylaterecombinant TRF1 in vitro (Wu et al., 2008b). However, in aseparate study, no phosphorylation of TRF1 was observed withbaculovirus-derived His-tagged Plk1 although His-Plk1 was able toreadily phosphorylate b-Casein (McKerlie and Zhu, 2011), thelatter arguing against the possibility that the lack of TRF1phosphorylation by His-Plk1 was due to an absence of Plk1activity. Further studies are required to investigate the discrepancybetween these two studies.

Plk1 has been shown to phosphorylate S435 of TRF1 (Wu et al.,2008b). Phosphorylation of S435 has been observed in G1 cells in aglobal phosphoproteome study (Dephoure et al., 2008). Mutationalanalysis showed that a non-phosphorylatable mutation of S435Aimpairs TRF1 binding to telomeric DNA in vitro and in cells arrestedin mitosis (Wu et al., 2008b). These findings led to a model thatphosphorylation of S435 by Plk1 promotes TRF1 interaction withtelomeric DNA (Wu et al., 2008b). This model seems at odds withthe recent finding that Plk1 positively regulates the activity andstability of tankyrase 1 (Ha et al., 2012), a negative mediator ofTRF1 binding to telomeric DNA (Smith et al., 1998). Perhaps onepossible way to reconcile these two studies would be thatphosphorylation of S435 by Plk1 might render TRF1 resistant tothe action of tankyrase 1, which would require further investiga-tion.

4.2. TRF2

4.2.1. ATM- and Chk2-mediated phosphorylation of TRF2

ATM, a master regulator of the DNA damage response, isresponsible for transducing the DNA damage signal throughphosphorylation of a number of downstream target proteinscentral for activation of the DNA damage checkpoint, cell cyclearrest, DNA repair or apoptosis (Bhatti et al., 2011; Shiloh, 2003).Chk2 is one of the downstream targets of ATM and acts as amediator for checkpoint responses and senescence (Ahn et al.,2004; Antoni et al., 2007; Chen et al., 2005; Stracker et al., 2009).Both ATM and Chk2 are found to be associated with humantelomeres in a cell-cycle-dependent manner (Buscemi et al., 2009;Verdun et al., 2005; Verdun and Karlseder, 2006; Zhou et al., 2010).

TRF2, a central player in protecting telomeres from unwarrant-ed activity (Palm and de Lange, 2008), physically interacts withboth ATM and Chk2 (Buscemi et al., 2009; Karlseder et al., 2004).TRF2 is found to interact with a region of ATM that contains S1981(Karlseder et al., 2004), autophosphorylation of which is requiredfor ATM activation in response to DNA double-strand breaks(Bakkenist and Kastan, 2003). Overexpression of TRF2 has been


shown to attenuate the response of ATM to DNA damage, resultingin diminished phosphorylation of a number of downstream targetsincluding Nbs1 and p53 (Bradshaw et al., 2005; Karlseder et al.,2004). It has been suggested that binding of TRF2 to the S1981-containing region of ATM inhibits ATM activation at telomeres(Karlseder et al., 2004). This inhibition is thought to be vital fortelomere integrity since removal of TRF2 from telomeres leads toATM-dependent formation of telomere end-to-end fusions (Celliand de Lange, 2005; Denchi and de Lange, 2007). It has beenreported that ATM phosphorylates T188 of TRF2 in response toDNA damage (Tanaka et al., 2005) and that this phosphorylation isimportant for the fast pathway of DNA double-strand break repair(Huda et al., 2009).

TRF2 binds to the S/TQ domain in the N-terminus of Chk2 andthis interaction mediates Chk2 association with telomeric DNA inundamaged cells (Buscemi et al., 2009). The S/TQ domain of Chk2includes the ATM phosphorylation target T68, which is required forChk2 activation in response to DNA damage (Ahn et al., 2000;Schwarz et al., 2003). It has been suggested that binding of TRF2 tothe S/TQ domain of Chk2 represses Chk2 activity at telomeres(Buscemi et al., 2009). These findings suggest that TRF2 may act atmultiple levels (ATM and Chk2) to restrain the DNA damageresponse at telomeres. Upon radiation-induced DNA double-strandbreaks, Chk2 is dissociated from telomeres in a Chk2 activity-dependent manner (Buscemi et al., 2009). Phosphorylation of TRF2by Chk2 has been shown to decrease TRF2 binding to telomericDNA (Buscemi et al., 2009), implying that Chk2 may inhibittelomeric localization of TRF2 in the presence of DNA damage.

S20 of TRF2 has been shown to be a target of Chk2 (Zhou et al.,2010). An amino acid substitution of S20A abrogates TRF2phosphorylation by Chk2 when in vitro kinase assays wereperformed with recombinant TRF2 containing the first 90 aminoacids (Zhou et al., 2010). However, mutations at S20 have littleeffect on TRF2 phosphorylation when recombinant TRF2 carryingthe first 350 amino acids was used in the Chk2 kinase assays(Buscemi et al., 2009), suggesting that it is likely that Chk2phosphorylates TRF2 at multiple sites. It has been reported thatphosphorylation of S20 by Chk2 is required for TRF2 interactionwith G-rich RNA and the recruitment of ORC needed for OriP DNAreplication (Zhou et al., 2010).

4.2.2. Aurora C-mediated phosphorylation of TRF2

Aurora C is a serine/threonine kinase with sequence homologyto Aurora A and B that have been reported to play a role in mitosis,cell morphology and growth (Carmena and Earnshaw, 2003; Gietet al., 2005; Sasai et al., 2004; Slattery et al., 2009). Using a kinaseinactive N-terminal fragment of Aurora C as bait in a yeast two-hybrid screen, TRF2 was identified to be an Aurora C-interactingprotein (Spengler, 2007). It has been shown that Aurora Cphosphorylates T358 of TRF2 in vitro (Spengler, 2007). However,the physiological function of T358 phosphorylation by Aurora C in

vivo remains to be determined.

5. Ubiquitylation/de-ubiquitylation

Ubiquitylation is a major post-translational process that is usedby cells to target proteins for degradation by the proteosome, aswell as in some cases to serve as a signaling event (Al-Hakim et al.,2010; Kerscher et al., 2006; Mukhopadhyay and Riezman, 2007).Key components of the ubiquitylation pathway include ubiquitin,E1 activating enzymes, E2 conjugating enzymes and E3 ligases(Kerscher et al., 2006; Ye and Rape, 2009). The E1 activatingenzymes use ATP to form a high-energy thioester bond withubiquitin, allowing it to be transferred to E2 conjugating enzymes.E3 ligases are responsible for substrate recognition and, dependingon the type, either catalyze the transfer of ubiquitin from the E2 to

the target protein, or directly transfer the ubiquitin themselves.Several E3 ligases have been reported to bind TRF1 or TRF2 andtarget them for proteasome-mediated degradation.

5.1. TRF1

TRF1 binding to telomeric DNA is very dynamic and there is ahigh exchange rate between bound TRF1 and free TRF1 (Matternet al., 2004). When not bound to telomeric DNA, TRF1 undergoespolyubiquitylation, which was first reported by Smith and co-workers (Chang et al., 2003). Two different E3 ligases SCFFbx4 andRLIM have been shown to mediate polyubiquitylation of TRF1 andtarget TRF1 for proteasome-dependent protein degradation (Herand Chung, 2009; Lee et al., 2006).

SCFFbx4 was first discovered as a TRF1/Pin2 interacting proteinin a yeast two-hybrid screen (Lee et al., 2006). SCFFbx4 is a memberof the F-box protein subfamily that does not contain either WD-40repeat or leucine-rich repeat domains (Jin et al., 2004). The F-boxproteins typically act as substrate-specific adaptor subunits of theSCF (Skp1-Cul1/Rbs1-F-box protein) ubiquitin E3 ligases (Petroskiand Deshaies, 2005). SCFFbx4 interacts with the TRFH domain ofTRF1 (Lee et al., 2006), which is also known to interact with TIN2(Chen et al., 2008; Kim et al., 1999). SCFFbx4 ubiquitylates TRF1both in vivo and in vitro (Lee et al., 2006; Li and Hao, 2010; Zenget al., 2010). Overexpression of SCFFbx4 reduces TRF1 levels andleads to telomere elongation, whereas depletion of SCFFbx4

increases TRF1 levels and promotes telomere shortening, resultingin impaired cell growth (Lee et al., 2006). Structural analysisreveals that the binding site of SCFFbx4 on TRF1 overlaps with thatof TIN2 on TRF1 (Zeng et al., 2010). Biochemical analysis showedthat TIN2 inhibits SCFFbx4-mediated polyubiquitylation of TRF1 in

vitro (Zeng et al., 2010). Overexpression of TIN2 has been shown tostabilize the level of TRF1 whereas depletion of TIN2 leads to areduction in the level of TRF1 protein in an SCFFbx4-dependentmanner (Canudas et al., 2007; Zeng et al., 2010). Based on thesefindings, it has been suggested that TIN2 protects TRF1 fromprotein degradation by sequestering SCFFbx4 from binding to TRF1(Zeng et al., 2010).

Bhanot and Smith showed that Siah2, an E3 ligase, ubiquitylatesTIN2 and targets TIN2 for proteasome-mediated protein degrada-tion (Bhanot and Smith, 2012). Overexpression of Siah2 results in areduction in the level of TIN2, which is not associated with adecrease in the level of TRF1 (Bhanot and Smith, 2012). It has beensuggested that TIN2 might inhibit SCFFbx4-mediated TRF1 ubiqui-tylation in the cytoplasm whereas Siah2 might act on nuclear TIN2,the loss of which might open up binding sites on TRF1 but notnecessarily lead to TRF1 degradation (Bhanot and Smith, 2012).

In addition to TIN2, guanine nucleotide binding protein-like 3(GNL3L), a GTP-binding protein most similar to nucleostemin, hasalso been reported to bind to the TRFH domain of TRF1, preventingSCFFbx4 from binding and ubiquitylating TRF1 (Zhu et al., 2009). Ithas been suggested that GNL3L stabilizes TRF1 in mitosis topromote the metaphase-to-anaphase transition (Zhu et al., 2009).

RLIM (also known as RNF12), a RING-H2 zinc-finger protein, isthe second E3 ligase that has been shown to ubiquitylate TRF1 bothin vitro and in vivo (Her and Chung, 2009). RLIM acts as a negativeco-regulator for LIM homeodomain transcription factors (Bachet al., 1999). It is able to target CLIM cofactors for proteasome-mediated degradation, and thereby inhibit the function of LIMhomeodomain transcription factors (Ostendorff et al., 2002).

RLIM was isolated as a TRF1-interacting protein in a yeast two-hybrid screen (Her and Chung, 2009). The N-terminal region ofRLIM binds to the flexible linker region of TRF1 spanning aminoacids 265–378 (Her and Chung, 2009). RLIM ubiquitylates TRF1both in vivo and in vitro (Her and Chung, 2009). Overexpression ofRLIM reduces the TRF1 protein level whereas depletion of RLIM not


only increases the level of TRF1 but also results in telomereshortening (Her and Chung, 2009). Overexpressed TRF1 is found tocolocalize with overexpressed RLIM in distinct subnucleardomains (Her and Chung, 2009). Recently we have reported thatATM phosphorylates S367 of TRF1, which targets TRF1 to nuclearproteasome centers for degradation (McKerlie et al., 2012). Itwould be of interest to know whether phosphorylation of S367 byATM might prime TRF1 for RLIM-mediated protein ubiquitylation.

Pluripotency factors such as Nanog, Oct4 and Sox2 have beenshown to down-regulate RLIM expression in embryonic stem cells(Navarro et al., 2011). Interestingly, high levels of TRF1 have beenreported in murine pluripotent stem cells, coincident with highlevels of Sox2 and Oct3/4 (Varela et al., 2011). These findings raisethe possibility that RLIM-mediated ubiquitylation of TRF1 mightbe disabled in embryonic stem cells.

In addition to ubiquitylation, TRF1 also undergoes USP22-mediated de-ubiquitylation. USP22, a deubiquitylating enzyme, isa member of the SAGA complex, a large protein assemblycomposed of more than 20 protein subunits including the nuclearhistone acetyl transferase GCN5. The SAGA complex, best knownfor its role in transcriptional activation, contains two enzymaticdomains that mediate the acetylation and de-ubiquitylation ofhistones as well as non-histone proteins (Koutelou et al., 2010).Depletion of USP22 decreases TRF1 protein levels whereasoverexpression of wild-type USP22 increases TRF1 levels in amanner dependent upon its enzymatic activity (Atanassov et al.,2009). Moreover, knockdown of GCN5 and another SAGA compo-nent ATXN7L3 also leads to an increase in the level of ubiquitylatedTRF1. A catalytically dead mutation of GCN5 has no effect on thesteady state levels of TRF1 (Atanassov et al., 2011). It has beensuggested that USP22 and the deubiquitylating module of SAGAare required for TRF1 stability, deregulation of which can lead touncapped telomeres as evidenced by the observed accumulation oftelomere fusions in GCN5 null mice embryos (Atanassov et al.,2009; Smith, 2009).

The discovery of de-ubiquitylation of TRF1, along with the factthat there exist two separate but potentially redundant pathwaysto ubiquitylate TRF1 for degradation, demonstrate the complexityof regulating the level of TRF1 in cells. These pathways permit thefine tuning of this regulation, as the capability of two E3 ligases andthe de-ubiquitylating enzyme to ubiquitylate and de-ubiquitylateTRF1 may be subject to control by various mechanisms. RLIMcontains a putative nuclear localization signal and is directed to thenucleus (Bach et al., 1999) whereas SCFFbx4 is reported topredominantly reside in the cytoplasm (Lin et al., 2006). Accessto a substrate by virtue of compartmentalization of each ligase ineither the cytoplasm or the nucleus could be one of thesemechanisms. In addition, phosphorylation of TRF1 could affect itsbinding with these ligases or de-ubiquitylase since severalreported phosphorylation sites of TRF1 are associated with theregulation of TRF1 stability (McKerlie and Zhu, 2011; McKerlieet al., 2012; Zhou et al., 2000). Furthermore, the activity of the de-ubiquitylating enzyme USP22 is regulated by its interaction withother subunits of the SAGA complex (Lang et al., 2011), whichcould add another dimension to the control of TRF1 stability.

5.2. TRF2

TRF2 is found to be ubiquitylated and subject to proteasome-mediated degradation in vivo (Fujita et al., 2010). Mass spectrom-etry analysis revealed K173 of TRF2 to be the major ubiquitylationsite, with K180 and K184 being additional sites for ubiquitylation(Fujita et al., 2010). Siah1, an E3 ubiquitin ligase with a C3H4-typeRING finger domain (Horikawa et al., 2011), has been reported tophysically interact with and ubiquitylate TRF2 (Fujita et al., 2010).Knockdown of Siah1 stabilizes TRF2 and delays the onset of cellular

senescence in human primary fibroblasts (Fujita et al., 2010). TheSiah1 gene is transcriptionally induced by p53 (Matsuzawa andReed, 2001), which is up-regulated in senescent cells. Over-expression of p53 up-regulates Siah1, which is associated with adecrease in the level of TRF2 protein (Fujita et al., 2010). Inhibitionof p53 down-regulates Siah1, which coincides with an increase inthe level of TRF2 (Fujita et al., 2010). These findings led to a modelthat p53-induced ubiquitylation of TRF2 by Siah1 targets TRF2 fordegradation (Fujita et al., 2010). TRF2 is a central player intelomere protection. Loss of TRF2 from telomeres induces telomereuncapping and the formation of telomere end-to-end fusions,resulting in the induction of cellular senescence or apoptosis in anATM- and p53-dependent manner (Karlseder et al., 1999; Takaiet al., 2003; van Steensel et al., 1998). It has been suggested thatp53-induced degradation of TRF2 may help maintain/reinforce theuncapped state of telomeres, preventing DNA-damage-carryingcells from escaping cellular senescence or apoptosis to contributeto tumor formation (Fujita et al., 2010; Horikawa et al., 2011).

6. Sumoylation

SUMOylation is similar to ubiquitination, as it requires a similarenzyme cascade, with an SUMO-activating enzyme, a SUMOconjugating enzyme and a SUMO protein ligase to assist thetransfer of SUMO from the conjugating enzyme to its target(Kerscher et al., 2006; Praefcke et al., 2012). Generally SUMOylatedproteins are not targeted for degradation, but instead SUMOylationcontrols their cellular location, protein interactions or otherregulatory functions. SUMOylation has been shown to playimportant roles in the DNA damage response and DNA repair(Bekker-Jensen and Mailand, 2011; Dou et al., 2011).

Components of the shelterin complex, including TRF1, TRF2,TIN2 and hRap1, are SUMOylated by MMS21 (Potts and Yu, 2007), aSUMO ligase that is a member of the multi-subunit SMC5/6complex implicated in the DNA damage response, regulation ofrDNA stability and telomere maintenance [reviewed in De Piccoliet al., 2009]. It has been shown that SUMOylation of TRF1 and TRF2is required for telomere maintenance in a subset of cancer cells(Potts and Yu, 2007) that lack telomerase but maintain theirtelomere length through homologous recombination, referred toas alternative lengthening of telomeres (ALT) (Bryan et al., 1995;Dunham et al., 2000; Henson et al., 2002). Telomeres in most ALTcells are recruited to promyelocytic leukemia (PML) bodies, termedALT-associated PML bodies (APBs), where telomere replication andrecombination are thought to take place (Draskovic et al., 2009;Wu et al., 2003; Yeager et al., 1999). Mutating SUMOylation sites inTRF1 or TRF2 blocks the formation of APB bodies (Potts and Yu,2007). Depletion of MMS21 or SMC5 complex promotes telomereshortening and telomere instability, leading to the induction ofcellular senescence in ALT cells (Potts and Yu, 2007). These findingsled to a model that MMS21-mediated SUMOylation of TRF1 andTRF2 facilitates homologous recombination at telomeres bypromoting the formation of APB bodies in ALT cells (Potts andYu, 2007).

7. Other modifications

7.1. Arginine methylation

Protein arginine methyltransferases (PRMTs) represent a familyof enzymes that catalyze the direct transfer of the methyl groupfrom a methyl donor S-adenosyl methionine to one or two of theguanidine nitrogen atoms of arginine (Bedford and Richard, 2005;McBride and Silver, 2001). In mammalian cells, 11 protein PRMTshave been identified and can be grouped as either type I or type IIenzymes depending on their respective ability to catalyze arginine


methylation asymmetrically or symmetrically (Bedford andRichard, 2005; McBride and Silver, 2001). PRMT1, the predominantmammalian type I enzyme (Tang et al., 2000), asymmetricallymethylates proteins involved in a diverse range of cellularprocesses (Bedford, 2007; Bedford and Richard, 2005).

PRMT1 is known to methylate proteins that contain acharacteristic motif rich in glycines and arginines, referred to asthe GAR motif (Boisvert et al., 2003; Najbauer et al., 1993). The N-terminal basic domain of TRF2 is rich in glycines and arginines,similar to the GAR motif. Coimmunoprecipitation studies showedthat endogenous TRF2 interacts with PRMT1 (Mitchell et al., 2009).PRMT1 methylates TRF2 both in vivo and in vitro (Mitchell et al.,2009). Biochemical analysis suggested that PRMT1 is capable ofmethylating essentially all arginines in the basic domain of TRF2(Mitchell et al., 2009). Mass spectrometric analysis of TRF2immunoprecipitated from HeLa cells revealed that TRF2 containsdimethylated arginine at position 17 and monomethylatedarginine at position 18 (Mitchell et al., 2009). In vivo methylationof TRF2 at arginine 17 was further confirmed with an antibodyraised specifically against a TRF2 peptide containing dimethylatedarginine 17 (Mitchell et al., 2009). Overexpression of TRF2 carryingamino acid substitutions of arginines to lysines in the basic/GARdomain promotes the formation of telomere doublets, resulting intelomeric accumulation of DNA damage response factors such as53BP1 and induction of cellular senescence (Mitchell et al., 2009).Depletion of PRMT1 also induces the formation of telomeredoublets and cellular senescence in telomerase-expressing normalhuman cells (Mitchell et al., 2009). These findings suggest thatmethylation of TRF2 by PRMT1 plays an important role in themaintenance of telomere structure essential for cellular prolifera-tion. The basic/GAR domain of TRF2 binds to WRN and ORC as wellas Holiday junctions and telomeric repeat-containing RNA (TERRA)(Atanasiu et al., 2006; Deng et al., 2009; Opresko et al., 2002; Pouletet al., 2009). Whether PRMT1-mediated TRF2 methylation mightmodulate the ability of TRF2 to interact with WRN, ORC, Holidayjunctions and TERRA remains to be determined.

Knockdown of PRMT1 in transformed cells increases theamount of telomere-bound TRF2 (Mitchell et al., 2009), implyingthat arginine methylation may negatively regulate TRF2 binding totelomeric DNA. In agreement with this notion, immunofluores-cence with antibody specifically against methylated TRF2 showedthat methylated TRF2 is associated with the nuclear matrix insteadof telomeres (T.R.H. Mitchell and X.-D. Zhu, unpublished data). Thephysiological importance of this association remains to bedetermined.

7.2. Poly (ADP-ribosy)lation (PARsylation)

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymesthat use NAD+ as a substrate to generate ADP-ribose polymers ontoglutamic acid residues of protein acceptors. Such post-translationalmodification can drastically change the properties of proteinacceptors (Ame et al., 2004; Gagne et al., 2006; Hsiao and Smith,2008; Schreiber et al., 2006). Tankyrases are PARPs that have inaddition to their PARP catalytic domain a series of ankyrin repeatsthat are used in substrate recognition (Hsiao and Smith, 2008;Sbodio and Chi, 2002). Using TRF1 as bait in a yeast two-hybridscreen, tankyrase 1 and its closely related homolog tankyrase 2 wereidentified as TRF1-interacting proteins (Kaminker et al., 2001; Smithet al., 1998). Both tankyrases bind to a short RxxADG sequencelocated in the N-terminal acidic domain of TRF1 (Guettler et al.,2011; Kaminker et al., 2001; Sbodio and Chi, 2002; Sbodio et al.,2002; Smith et al., 1998). Poly(ADP-ribosy)lation of TRF1 bytankyrase 1 and tankyrase 2 occurs both in vivo and in vitro (Cooket al., 2002; Kaminker et al., 2001; Smith et al., 1998). Suchmodification has been shown to cause the release of TRF1 from

telomeres (Cook et al., 2002; Seimiya et al., 2004; Smith and deLange, 2000; Smith et al., 1998), rendering TRF1 susceptible toproteasome-dependent degradation (Chang et al., 2003; Lee et al.,2006). Overexpression of tankyrase 1 promotes telomerase-depen-dent telomere elongation (Cook et al., 2002; Smith and de Lange,2000) whereas knockdown of tankyrase 1 leads to telomereshortening (Donigian and de Lange, 2007). It has been suggestedthat poly(ADP-ribosy)lation of TRF1 by tankyrase 1 may allow theaccess of telomerase to the ends of telomeres (Smith and de Lange,2000). Tankyrase 1-mediated release of TRF1 from telomeres hasalso been implicated in the resolution of sister telomeres (Canudaset al., 2007; Dynek and Smith, 2004).

Poly(ADP-ribosy)lation has also been observed for TRF2(Dantzer et al., 2004; Gomez et al., 2006). The Myb-like domainof TRF2 is found to bind to the N-termini of PARP1 and PARP2(Dantzer et al., 2004; Gomez et al., 2006), the enzymatic activity ofwhich is stimulated by DNA breaks (Ame et al., 2004). Both PARP1and PARP2 have been reported to be required for the maintenanceof telomere integrity (Dantzer et al., 2004; Gomez et al., 2006).PARP1 deficiency induces the formation of telomere end-to-endfusions and telomere loss, the latter of which has also beenobserved in cells deficient for PARP2 (Dantzer et al., 2004; Gomezet al., 2006). In addition, PARP1 and PARP2 are capable of thepoly(ADP-ribosyl)ation of TRF2, and thereby reduce TRF2 bindingto telomeric DNA (Dantzer et al., 2004; Gomez et al., 2006).

8. Post-translational modifications of TRF1 and TRF2 orthologsin other species

Our understanding of the role of post-translation modificationin regulating the function of TRF1 and TRF2 orthologs in otherspecies is very limited. To our knowledge, Xenopus TRF1 (xTRF1)has so far been the only TRF1 ortholog that has been reported to besubject to regulation by post-translational modification. It hasbeen shown that Xenopus TRF1 (xTRF1) binds to telomerechromatin specifically in mitotic Xenopus egg extracts and thatthis binding diminishes upon mitotic exit (Nishiyama et al., 2006).Phosphorylation of xTRF1 was observed when recombinant xTRF1was incubated with Xenopus polo-like kinase (Plx1) but not withAurora B immunoprecipitated from mitotic extracts (Nishiyamaet al., 2006). In addition, immunodepletion of Plx1 has been shownto reduce xTRF1 association with telomere chromatin (Nishiyamaet al., 2006). These findings led the authors to suggest that Plx1phosphorylates and promotes xTRF1 binding to telomere chroma-tin in mitosis (Nishiyama et al., 2006). Although we have not beenable to detect TRF1 phosphorylation by Plk1 (McKerlie and Zhu,2011), it has been reported that human TRF1 undergoes Plk1-dependent phosphorylation at S435 and that this phosphorylationpromotes TRF1 binding to telomeric DNA in mitosis (Wu et al.,2008b). While a residue corresponding to S435 of human TRF1 ispresent in a wide range of species including Xenopus, it has beensuggested that Plx1 phosphorylates the linker region of xTRF1upstream of this location (Nishiyama et al., 2006). Further studiesare required to investigate whether Plk1-mediated TRF1 associa-tion with telomere chromatin in mitosis may be a conservedmechanism.

Sequence comparison suggests that the majority of phosphor-ylation sites reported for human TRF1 and TRF2 are also present intheir mammalian orthologs although the consensus sequence for aparticular kinase may not necessarily be conserved. In some cases,the conservation of phosphorylation sites can be extended to non-mammalian orthologs including T149 and S435 of TRF1 and T358of TRF2, indicative of a possible conserved role of these residues inregulating TRF1 and TRF2 function.

Key amino acids in TRF1 important for its interaction with theE3 ligase SCFFbx4 as well as those involved in its interaction with


TIN2 (Chen et al., 2008; Zeng et al., 2010) are highly conserved inmammals, suggesting that the interplay between SCFFbx4-mediat-ed protein degradation and TIN2-mediated protein protection maybe a conserved regulatory feature of TRF1 among mammals.SUMOylation sites identified in human TRF1 are, for the most part,conserved in human, chimpanzee and cow orthologs whereasSUMOylation sites identified in human TRF2 (Fujita et al., 2010;Potts and Yu, 2007) are conserved in mammals, implying that it islikely that SUMOylation may be a conserved mechanism regulat-ing TRF1 and TRF2 orthologs in a subset of mammals.

The glycine- and arginine-rich motif present in the N-terminaldomain of human TRF2 is also found in mammalian TRF2orthologs, suggesting that arginine methylation of TRF2 byPRMT1 may be conserved among mammals. On the other hand,the tankrayse-interacting sequence (RxxADG) identified in the N-terminal acidic domain of human TRF1 is found in its orthologsfrom chimpanzee and cow but absent in its orthologs frommouse and rats (Muramatsu et al., 2007; Sbodio and Chi, 2002). Ithas been reported that mouse TRF1 does not undergo poly(ADP-ribosyl)ation by tankyrase 1 (Donigian and de Lange, 2007;Muramatsu et al., 2007), suggesting that tankyrase-dependentregulation of TRF1 may not be a conserved mechanism inmammals. Perhaps it is not unexpected that there existvariations in some of the post-translational modifications ofTRF1 and TRF2 orthologs given the observed variation amongspecies in telomere maintenance and protection including thecomposition of the shelterin complex (Hockemeyer et al., 2006;Wu et al., 2006).

9. Concluding remarks

TRF1 and TRF2 bind sequence specifically to double-strandedtelomeric DNA and they both play crucial roles in the maintenanceof telomere length and structure, which is intimately associatedwith cancer and ageing. The abundance of TRF1 and TRF2 and theirbinding to telomeric DNA is subject to extensive post-translationalcontrol. For TRF1, phosphorylation at nine different sites (T122,T149, T273, S274, S296, T344, S367, T371 and S435) by six differentkinases has been reported to modulate its binding to telomericDNA and its stability. These kinases include AKT, ATM, Aurora A,CDK1, CK2 and Plk1. The majority of the well-characterized TRF1phosphorylation sites are located in the flexible linker region ofTRF1 between the dimerization and DNA binding domains. Two ofthem, S367 and T371, are very close to each other, only four aminoacids apart, and yet modification of these residues has differenteffects. Phosphorylation at both sites has been shown to inhibitTRF1 binding to telomeric DNA, suggesting that phosphorylation inthis portion of the linker region may interfere with TRF1 binding totelomeric DNA, perhaps due to the creation of unfavorableelectrostatic interactions. On the other hand, their phosphoryla-tion has an opposing effect on TRF1 stability. While S367phosphorylation by ATM targets TRF1 to proteasome-dependentnuclear foci (McKerlie et al., 2012), Cdk1-mediated T371 phos-phorylation has been shown to increase the half-life of TRF1(McKerlie and Zhu, 2011). How phosphorylation of two closelylinked amino acids (S367 and T371) differentially regulates TRF1stability remains unknown. Nevertheless, these results highlightthe tight regulation of TRF1 function and the important role ofphosphorylation in this control.

The discovery of specific phosphorylated forms of TRF1 with anon-telomeric localization is aided by the use of phospho-specificantibodies that can recognize a subset of TRF1, which otherwiseescapes detection with anti-TRF1 antibody, possibly due to theirlow abundance compared to the total TRF1 in cells (Takai et al.,2010). Interestingly, the observed localization of two of theseforms is cell-cycle regulated. For example, phosphorylated

(pS367)TRF1 is found to colocalize with nuclear proteasomecenters mostly in S and G2 phases of cells. On the other hand,phosphorylated (pT371)TRF1 is predominantly found in earlymitosis with enriched localization at centrosomes (M. McKerlieand X.-D. Zhu, unpublished data). It would be of interest to knowwhether phosphorylation of TRF1 at additional reported sitesregulates cellular localization of TRF1, which would require thedevelopment of a phospho-specific antibody against each of thesesites.

In contrast to TRF1, control of TRF2 by phosphorylation is lesswell understood. So far, only two phosphorylation sites of TRF2have been characterized and neither of them has been documentedto modulate TRF2 binding to telomeric DNA or TRF2 stability.Phosphorylation of T188 by ATM is implicated in the DNA damageresponse whereas phosphorylation of S20 of TRF2 is involved in itsbinding to G-rich RNA and Holiday junctions.

Aside from phosphorylation, TRF1 is also subject to tankyrase-dependent poly(ADP-ribosy)lation. Poly(ADP-ribosy)lation of TRF1removes TRF1 from telomeric DNA, rendering it susceptible toproteasome-mediated degradation. Indeed, two independentpathways responsible for ubiquitylation of TRF1 have beenidentified, one being mediated by SCFFbx4 and the other by RLIM.TRF1 also undergoes de-ubiquitylation, adding another level ofcontrol to TRF1 stability. Furthermore, SUMOylation has beenreported to facilitate TRF1 association with telomeric DNA neededfor telomere recombination in ALT cells.

Like TRF1, TRF2 is also ubiquitylated and SUMOylated in vivo.Siah1-mediated ubiquitylation targets TRF2 for proteasome-mediated degradation, which is thought to help reinforce thestate of uncapped telomeres and to prevent senescent or apoptoticcells from escaping the DNA damage checkpoint (Fujita et al.,2010). SUMOylation of TRF2 is also required for efficient telomererecombination in ALT cells. In addition, PRMT1-mediated argininemethylation has been suggested to negatively regulate TRF2interaction with telomeric DNA.

It is apparent that the network of post-translational control ofTRF1 and TRF2 is very complex. Elucidating how these pathwayscommunicate with one another to control TRF1 and TRF2 couldlead to a better understanding of the mechanism underlying themaintenance of telomere integrity, disruption of which canpromote tumorigenesis and ageing.

Acknowledgements

We thank Megan McKerlie, Taylor Mitchell and KajaparanJeyanathan for their comments. This work was supported byOntario Early Researcher Award (ER07-04-157), Canadian Insti-tutes of Health Research New Investigator Award (MSH-76599)and Canadian Institutes of Health Research (MOP-86620) to X.-D.Z.

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Post-translational modifications of TRF1 and TRF2 and their roles in telomere maintenance