The Shelterin Protein TRF2 Inhibits Chk2 Activity at Telomeres in the Absence of DNA Damage

Current Biology 19, 874–879, May 26, 2009 ª2009 Elsevier Ltd All rights reserved

DOI 10.1016/j.cub.2009.03.064

Report The Shelterin Protein TRF2 Inhibits Chk2 Activity at Telomeres in the Absence of DNA Damage Giacomo Buscemi,1 Laura Zannini,1 Enrico Fontanella,1 Daniele Lecis,1 Sofia Lisanti,1 and Domenico Delia1,* 1Department of Experimental Oncology Fondazione IRCCS Istituto Nazionale dei Tumori Milan 20133 Italy

Summary The shelterin complex [1] shapes and protects telomeric DNA from being processed as double strand breaks (DSBs) [2, 3]. Here we show that in human undamaged cells, a fraction of the kinase Chk2, a downstream target of ATM and mediator of checkpoint responses and senescence [4, 5], physically interacts with the shelterin subunit TRF2 and colocalizes with this complex at chromosome ends. This interaction, enhanced by TRF2 binding to telomeric DNA, inhibits the activation and senescence-induced function of Chk2 by a mechanism in which TRF2 binding to the N terminus of Chk2 surrounding Thr68 hinders the phosphorylation of this priming site. In response to radiationinduced DSBs, but not chromatin-remodelling agents, the telomeric Chk2-TRF2 binding dissociates in a Chk2 activity-dependent manner. Moreover, active Chk2 phosphorylates TRF2 and decreases its binding to telomeric DNA repeats, corroborating the evidences on the specific TRF2 relocalization in presence of DSBs [6]. Altogether, the capacity of TRF2 to locally repress Chk2 provides an additional level of control by which shelterin restrains the DNA damage response from an unwanted activation [6, 7] and may explain why TRF2 overexpression acts as a telomerase-independent oncogenic stimulus [8]. Results and Discussion Telomeres are nucleoprotein structures that protect chromosome ends. In mammalian cells, this protection is afforded by the shelterin complex [1], which accumulates on telomeric DNA, hiding chromosome ends from being recognized as sites of DNA damage and avoiding the activation of a DNA damage response (DDR) and inappropriate DNA processing [2]. The shelterin subunit TRF2 plays a key role in suppressing the telomere-associated DDR through its binding to and inhibition of ATM kinase [6, 7]. ATM is the apical kinase that in response to few DSBs activates many subpathways of the DDR [9]. Although it has been proposed that shelterin inhibits the DDR at telomeres and not elsewhere in the genome, this attractive proposal remains elusive because ATM is not detectable at telomeres. The checkpoint kinase Chk2 is phosphorylated by ATM on the S/TQ domain upon DNA damage, thereby promoting Chk2 activation and phosphorylation of molecules involved in transient cell cycle arrest, apoptosis, and senescence [4]. Recent studies have implicated the DDR in senescence, with

*Correspondence: [email protected]

ATM and Chk2 playing a key role in replicative- and oncogene-induced senescence [10, 11]. These findings have prompted us to investigate the interplay between TRF2 and Chk2 in senescence, specifically whether Chk2, like ATM, was inhibited by TRF2 and whether this occurred at chromosome ends. To explore whether TRF2 and Chk2 physically interacted in vivo, reciprocal coimmunoprecipitation (co-IP) analysis were performed with lysates from undamaged BJ-hTERT immortalized fibroblasts, which revealed (Figure 1A) the existence of an endogenous Chk2/TRF2 complex. Similar findings were seen in LCL normal cells, U2OS and IOSE80 cancer cells (data not shown), thus excluding a cell type specificity for this interaction. This association was also confirmed by pull-down assays with GST-Chk2 and GST-TRF2 that were able to precipitate TRF2 and Chk2 from LCL cell extracts, respectively (Figure S1 available online). To further substantiate the Chk2/TRF2 binding, we assessed other shelterin subunits in undamaged cells and found that RAP1 and TRF1 [1] co-IP with endogenous Chk2 (Figure 1B). The specificity of these interactions was confirmed by the failure of Cdc2 to co-IP RAP1 (Figure S2) and Chk2 to co-IP the nuclear protein RXRa (Figure 1B; Figure S2). These data indicate that endogenous Chk2 forms a complex with TRF2, RAP1, and TRF1. To determine whether TRF2 and Chk2 interacted directly and to verify the role of telomeric DNA, binding assays were performed with recombinant TRF2 and Chk2 wild-type (Chk2WT) or kinase dead (Chk2KD) incubated with or without duplex telomeric oligonucleotide repeats. The TRF2 association with Chk2 was not only direct, but significantly enhanced by telomeric DNA (Figure 1C, compare lanes 2–3 and 4–5). Furthermore, TRF2 interacted more strongly with Chk2KD than with Chk2WT (Figure 1C, compare lane 3 with 5), suggesting a negative role for active Chk2. The telomeric localization of Chk2 was assessed by immunofluorescence (IF) analysis, after validation of antibodies (Figures S3, S4A, and S4B). As telomeric marker, instead of TRF2 we assessed RAP1 because of the bright fluorescence signal it generated in the experimental conditions used here, which required detergent extraction to remove weakly bound or nuclear diffuse Chk2 (Figure S4C for Chk2 IF staining in pre-extraction condition, Figure S4D for RAP1-TRF2 colocalization). In BJ-hTERT cells, the Chk2 fluorescent dots, though faint, mostly colocalized with RAP1 (Figure 1D), consistent with Chk2 being on telomeres. In contrast to the shelterin components, Chk2 was not detected on all telomeres, similar to other telomeric proteins such as Rad50 [12]. Whether this reflects the highly mobile nature of Chk2 in cells [13] is a possibility. These colocalizations with RAP1 were more evident in BJ-hTERT overexpressing Chk2KD than Chk2WT (Figure 1D), suggesting that activity of Chk2 modulates its telomeric localization. Importantly, Chk2 on telomeres were negative for g-H2AX (Figure 1D, bottom), indicating that Chk2 resides on normal and not dysfunctional telomeres, the latter known to elicit a canonical DDR and accumulation of g-H2AX at chromosome ends [10]. These results, besides showing a TRF2/Chk2 interaction in vivo, provide the first direct evidence for a DDR signaling kinase at telomeres, considering that the

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Figure 1. Chk2 Physically Interacts with TRF2 on Telomeres (A) Immunoprecipitations (IPs) were performed on BJ-hTERT extracts with antibodies against Chk2 (left) or TRF2 (right) and immunoblotted (IB) with the indicated antibodies. (B) Western blot analysis of Chk2 IPs from BJ-hTERT cell extracts tested with antibodies against RAP1, TRF1, Chk2, and RXRa. RXRa was used as an additional negative control (see also Figure S2 for similar experiments with additional negative controls). (C) Pull-down analysis performed with sepharose-bound GST-Chk2WT or GST-Chk2KD, and His-TRF2 previously incubated with or without telomeric DNA. After extensive washing, the complexes were analyzed by western blot with TRF2 or Chk2 antibodies. Pre, preclearing; input, 8% of the total cell lysate. (D) Upper panels: double-color IF staining for Chk2 and RAP1 in mock, Chk2WT, or Chk2KD expressing BJ-hTERT cells. Cells were treated with detergent (see Supplemental Experimental Procedures) to extract weakly bound or diffuse Chk2. Nuclei were stained with DAPI. Arrows denote endogenous Chk2/RAP1 colocalizations. Bottom: double-color IF staining for Chk2 and g-H2AX in Chk2KD expressing BJ-hTERT cells. Chk2 IF dots do not colocalize with g-H2AX, as evidenced in merged picture. (E) BJ-hTERT cells treated with siRNA targeting TRF2 or a control sequence (siLuc) were tested for TRF2 depletion by western blot (top) and by IF with antibodies against Chk2 and TRF1. Labeled cells were scored (>100) by fluorescence microscopy and those with more than eight Chk2-TRF1 colocalizing spots were considered positive. Error bars calculated from three independent experiments. In these experiments TRF1, rather than RAP1, was used to label telomeres because depletion of TRF2 results in a diffuse expression of RAP1 (whose localization on telomeres depends on TRF2). (F) The same cells in (E) were tested for Chk2-RAP1 interaction. Pre, preclearing; input, 5% of the total IP input.

TRF2-targeting kinase ATM has not been detected at telomeres [7]. Telomeric Chk2 localization was mainly dependent on TRF2, as shown by the fact that depletion of TRF2 by siRNA markedly reduced the fraction of Chk2-TRF1 colocalizations (Figure 1E), and almost abolished Chk2-RAP1 binding (Figure 1F). To map the Chk2/TRF2 interacting regions, co-IPs were performed in U2OS cells expressing wild-type and deletion forms of FLAG-Chk2 and Myc-TRF2 (Figure S5). Whereas Chk2 lacking the first 17 aa (DN1-17) and 42 aa (DN1-42) or the fork-head domain (DFHA) (a phosphoprotein-binding module) preserved

the interaction with TRF2, Chk2 lacking the S/TQ (DS/TQ) alone or together with the FHA domain (DS/TQ-DFHA) lost the interaction (Figure 2A). Furthermore, the TRF2DBDM lacking both Basic (B) and Myb (M) domains, the former involved in binding to DNA junctions [6], the latter in the interaction with telomeric TTAGGG repeats [14], failed to bind Chk2 (Figure 2B), whereas TRF2DB lacking the Basic domain only (a truncated protein mainly localized on telomeres [14]) weakly interacted with Chk2 (Figure 2B). Thus, the Chk2 S/TQ and the TRF2 Myb domains are crucial for their binding and confirm an interaction preferentially occurring on telomeres. These

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Figure 2. The S/TQ and Myb Domains of Chk2 and TRF2, Respectively, Mediate the Interaction between These Proteins Extracts were prepared from U2OS cells transiently expressing full-length or deleted forms of Myc-TRF2 and FLAG-Chk2 (see Figure S5 for detailed amino acid deletions). For each extract, 5% of the total IPs input was western blotted with an Flag antibody ([A], top). Anti-FLAG immunoprecipitates from these cell extracts were immunoblotted with antibodies against Myc and FLAG ([A], bottom). The corresponding preclearings, immunoblotted on a separate gel, were tested with an Myc antibody (as negative control). The asterisk denotes a nonspecific band. Western blot analysis of lysates from U2OS cells expressing Myc-TRF2 constructs ([B], top). TRF2 immunoprecipitated from these cells with an Myc antibody was immunoblotted with antibodies against Chk2 and Myc ([B], bottom). Inputs, 5% of the total extract; pre, preclearing; IP, immunoprecipitate.

findings raise the possibility that TRF2 binding to the S/TQ might preclude phosphorylation of this domain or hinder Chk2 dimerization and subsequent autophosphorylation, essential steps for Chk2 activation [15]. These repression mechanisms might be similar to those described for ATM [7], suggesting the presence of a conserved activity in the TRF2dependent inhibition of DDR. Upon DNA damage, ATM phosphorylates Chk2 on threonine 68 (T68) located in the S/TQ domain [15], leading to its activation by auto/transphosphorylation at several additional sites including threonine 387 (T387) [15, 16]. Of note, overexpressed Chk2, while undergoing similar modifications, is constitutively active in the absence of DNA damage [15]. TRF2 might antagonize Chk2 by blocking its activation process, so we monitored the phosphorylation of Chk2 on T68 and T387 in BJ-hTERT-overexpressing Chk2. Quite interestingly, the fraction of phosphorylated Chk2 relative to the total amount was considerably reduced in cells stably expressing TRF2 (Figure S6) at 6 and 32 days after Chk2 transfection (Figure 3A), indicating that TRF2 interferes with Chk2 phosphorylation. Neither KU-55933 nor caffeine, inhibitors of ATM and ATM/ATR, respectively [9, 17], affected the phosphorylation of ectopic Chk2-T68 (Figure 3B), thus excluding the inhibitory effect of TRF2 on ATM as the mechanism responsible for reduced Chk2 phosphorylation. Likewise, GW843682 [18], an inhibitor of PLKs that targets Chk2, had no effect on T68 phosphorylation (Figure 3B). To verify the role of DNA-PK, we tested the effects of wortmannin and SU11752 inhibitors. The former works by forming a covalent adduct with the catalytic domain of PI-3 kinases and the dose used here is specific for DNA-PK [19]; the latter selectively inhibits DNA-PK by ATP competition [20]. Both agents markedly attenuated Chk2-T68 phosphorylation (Figure 3B). Ectopic Chk2 did not trigger the activation of DNA-PKcs, ATM, or ATR (Figure S7), so these results would suggest that the phosphorylation of Chk2 is mediated by the basal activity of DNA-PKcs, which would agree with Chk2 being an effective substrate for DNA-PKcs [21, 22] and with a detectable basal DNA-PK activity in unstressed cells [23]. Intriguingly, DNA-PKcs is involved in telomere homeostasis, in signaling short telomeres as DNA damage, and in functional interaction with TRF1 and TRF2 [24]. If DNA-PKcs was actually implicated in Chk2 activation, then TRF2 might have been expected to block Chk2 by inhibiting DNA-PKcs. However,

the autophosphorylation of DNA-PKcs-S2056 in TRF2-overexpressing BJ-hTERT cells was not inhibited either before or after IR, whereas conversely the activity of ATM, assessed by the phosphorylation of its substrate SMC1-S996, was strongly repressed (Figure S8). Therefore, in overexpression conditions, the TRF2-mediated inhibition of Chk2 phosphorylation is not consequent to the suppression of ATM or DNA-PKcs. To determine whether endogenous TRF2 inhibited Chk2 locally on telomeres, we evaluated by IF the phosphorylation of Chk2KD on T68. Despite being constitutively phosphorylated on T68 [16], the fraction of telomeric Chk2KD molecules were unphosphorylated (Figure 3C), confirming the ability of TRF2 to locally repress Chk2. The major biological effect of ectopic Chk2WT in BJ-hTERT cells was, as reported [5], the induction of senescence and senescence-associated marker b-galactosidase in >50% and 27% of cells at 6 and 32 days, respectively (Figure 3D). Similar findings were seen in U2OS cells (data not shown), which use ALT to maintain telomeres, indicating that Chk2 induces senescence irrespective of telomere length and elongation mechanism. TRF2 expression in BJ-hTERT cells markedly antagonized Chk2-induced senescence up to 32 days after transfection (Figure 3D). KU-55933 did not abrogate senescence (Figure S9), demonstrating that the TRF2-mediated inhibition of Chk2 was not ensuing from ATM repression. These data well fit with the aforementioned TRF2-dependent and ATM-independent repression of Chk2 (Figure 3B). Oxidative stress, a major contributor to DNA damage and senescence, was not required for Chk2-induced senescence, according to the comparable b-galactosidase positivity of cells grown in hypoxic and normoxic conditions (Figure S9). Chk2 overexpression resulted in a growth inhibition that was attenuated by TRF2 expression (Figure S9; Chk2 alone, S+G2/M = 13%; Chk2 plus TRF2, S+G2/M = 27%), but in contrast to its apoptotic activity in some cancer cells [5], it did not have such effect in BJ-hTERT (Figure S9). The endogenous activation of Chk2 by ATM involves in trans- and autophosphorylation steps occurring within 3 hr [15]. To determine whether TRF2 affected these events, co-IP experiments were performed at different times after IR. The TRF2/Chk2 binding seen in undamaged cells was lost at 3 hr, but not 30 min after IR (Figure 4A), suggesting that

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Figure 3. TRF2 Represses Chk2 Phosphorylation and Chk2-Induced Senescence (A) Chk2 phosphorylation on T68 and T387 was evaluated by western blot in mock, Chk2, TRF2, or Chk2/TRF2 expressing BJ-hTERT cells. Lysates were prepared from these cells at 6 and 32 days after being transduced with Chk2. (B) The phosphorylation of T68 was evaluated by western blot in BJ-hTERT cells transiently expressing Chk2 and treated for 48 hr with the indicated inhibitors (Ve, vehicle; KU, KU-55933; Caff, caffeine; Wort, wortmannin; SU, SU11752; GW, GW843682). Error bars calculated from three independent experiments. (C) IF analysis of Chk2 in combination with RAP1 and phospho-T68 in Chk2KD expressing BJ-hTERT. Cells were treated with detergent to extract free Chk2. Chk2 small dots do not colocalize with phospho-T68 as evidenced by arrows that mark representative Chk2-RAP1 colocalizing spots, demonstrating that telomeric Chk2 is unphosphorylated. Nuclei were stained with DAPI. (D) BJ-hTERT cells stably expressing TRF2 (or a mock control) were transduced with a plasmid expressing Chk2 and assessed for b-galactosidase positivity 6, 20, and 32 days later. The data in the histogram were obtained from three independent experiments 6 SD. Representative images of cells at day 6 are shown.

upon DNA damage endogenous Chk2 is displaced from telomeres. To verify the role played by enzymatic activity of Chk2, IF analyses were performed in BJ-hTERT expressing Chk2WT or Chk2KD. Before damage, both Chk2WT and Chk2KD were found at telomeres (Figure 4B), though the former showed a faint signal, as reported above (Figure 1D), but after IR the telomeric dots dropped in the case of Chk2KD and became undetectable in the case of Chk2WT (Figure 4B). Therefore, DNA damage and catalytic activity both enhance the dissociation of Chk2 from telomeres, in accordance with the in vitro binding assays (Figure 1C). Chromatin remodelling did not contribute to telomeric dissociation of Chk2, because in Chk2KD-expressing cells, neither hypotonic stress nor trichostatin A, both inducing chromatin remodelling and ATM activation, affected Chk2 (Figure S10). The stronger binding of TRF2 to Chk2KD than to Chk2WT prompted us to verify whether Chk2 catalyzes the phosphorylation of TRF2. In vitro, TRF2 was phosphorylated by recombinant Chk2 (Figure 4C) on residues located in the first 350 aa (Figure S11). Because this fragment contains on Ser20 a consensus motif (RxxS) for Chk2, we generated a TRF2 substrate carrying a Ser to Ala substitution at aa 20 (S20A), but its phosphorylation was not abrogated or attenuated (Figure S11, right) suggesting the involvement of other residue(s). It should be noted that not all in vivo targets of Chk2 are phosphorylated on residues matching the RxxS motif (e.g., Brca1, p53, Rb, Xrcc1, TTK). Importantly, in vivo a fraction of the TRF2 molecules appeared phosphorylated on serine in a Chk2-dependent manner, as evident from the phosphoserine signal detected on TRF2 immunoprecipitated from control (siLuc) and Chk2-depleted (siChk2) irradiated cells (Figure 4D).

Similar results were seen in extracts from irradiated cells cultured with or without the Chk2 inhibitor VRX0466617 [25] (Figure S12). To evaluate whether Chk2 activity affected the binding of TRF2 for telomeric TTAGGG repeats in duplex DNA, electrophoretic mobility shift assays (EMSA) were performed in the presence of ATP to allow phosphorylation (Figure S13 shows the specificity of TRF2 binding to telomeric DNA in the assay conditions) and found that in contrast to Chk2KD, Chk2WT decreased the binding of TRF2 to DNA (Figure 4E). This was not attributable to the formation of a Chk2/TRF2 complex (data not shown). Besides, TRF2 interacted more strongly with Chk2KD than with Chk2WT (Figure 1C). Of note, in vivo the telomeric binding of TRF2 and of its direct interactor RAP1 was not affected by overexpressed Chk2WT, according to immunoblots of cell lysates fractionated in extraction buffers containing 50–450 mM KCl (Figure 4F). Hence, although ATM plays an essential role in TRF2 delocalization [6], we cannot as yet ascribe to Chk2 a similar function in vivo. Altogether, these findings draw an analogy between Chk2 and ATM in the interplay with TRF2, with TRF2 being an inhibitor of ATM [6, 7] and of Chk2 and a substrate of ATM after DNA damage [26] and Chk2. These data underline the level of redundancy and complexity in the ATM-Chk2 pathway, because ATM and Chk2 share not only activation but also repression mechanisms. TRF2 represents an additional example of target substrate common to both ATM and Chk2, like HdmX, E2F1, BRCA1, and Che-1. Our work supports the view that shelterin avoids the activation of the canonical DDR in two ways, by concealing chromosome ends probably by maintaining telomeric t-loops [1] and by repressing the ATM-Chk2 signaling pathway at normal telomeres.

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Figure 4. DSBs Disrupt TRF2/Chk2 Interaction, whereas Chk2 Phosphorylates TRF2 and Reduces TRF2 Binding to Telomeric DNA (A) Chk2 immunoprecipitates from LCL cells harvested before, 30 min after, and 3 hr after 6Gy were tested for TRF2 by western blotting. (B) BJ-hTERT cells expressing Chk2WT or Chk2KD were treated with 20Gy IR and 75 min later fixed and labeled by double-color IF with Chk2 (green) and RAP1 (red) antibodies. (C) Autoradiographs of in vitro Chk2 kinase assays performed in the presence of active GST-Chk2 and full-length His-TRF2. Cdc25C served as a positive control substrate. Chk2 autophosphorylation is also detectable. The Coomassie-stained gels evidence the loaded proteins per sample. (D) TRF2 immunoprecipitated from U2OS cells transfected for 48 hr with siLuc or siChk2, treated with 20Gy IR 2 hr, and collected 2 hr later, was western blotted with antibodies against phospho-Serine and Myc (for normalization). Input, 5% of the total cell lysate; pre, preclearing; IP, immunoprecipitate. (E) EMSA performed in presence of TRF2, Chk2WT, or Chk2KD and telomeric DNA (see Figure S13 and Supplemental Experimental Procedures). The graph shows the quantification of bound and free DNA from three independent experiments 6 SD. (F) Mock and Chk2-transfected U2OS cells were lysed with an extraction buffer containing 50 mM up to 450 mM KCl. 450p indicates the pellet obtained from 450 mM KCl lysis.

TRF2 might repress ATM and Chk2 by a common mechanism involving the binding to the kinases’ critical segments (the S/TQ region of Chk2 spanning T68, and the ATM region spanning S1981) thereby constraining key steps in their activation. Finally, the suppression by TRF2 of the ATM/Chk2 pathway, whose integrity is essential for DNA damage surveillance and genome stability [27], further supports the view that TRF2 acts as a potently oncogenic stimulus in vivo, independently of telomerase activity [8], and explains the TRF2 overexpression in certain cancers [28].

Acknowledgments We thank Titia de Lange for kindly providing TRF2-expressing vectors and Patrizia Casalini for help with triple IF staining. This work was financially supported by the Italian Association for Cancer Research (AIRC), Fondazione Cariplo, Fondazione Telethon, and Italian Ministry of Health (Progetto Ordinario and Progetto Malattie Rare). Received: October 14, 2008 Revised: March 24, 2009 Accepted: March 26, 2009 Published online: April 16, 2009

Supplemental Data References Supplemental Data include Supplemental Experimental Procedures and 13 figures and can be found with this article online at http://www.cell. com/current-biology/supplemental/S0960-9822(09)00923-3.

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