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Telosome, a Mammalian Telomere-associated Complex Formed by Multiple Telomeric Proteins*

  • Dan Liu
    Correspondence
    To whom correspondence may be addressed. ¶ To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030
    Footnotes
    Affiliations
    Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030
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  • Matthew S. O'Connor
    Footnotes
    Affiliations
    Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030
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  • Jun Qin
    Affiliations
    Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030
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  • Zhou Songyang
    Correspondence
    To whom correspondence may be addressed. ¶ To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030
    Affiliations
    Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030
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  • Author Footnotes
    * This work was supported by grants from the Department of Defense and the National Institute of Health (to Z. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Both authors contributed equally to this work.
Open AccessPublished:September 20, 2004DOI:https://doi.org/10.1074/jbc.M409293200
      In mammalian cells, telomere-binding proteins TRF1 and TRF2 play crucial roles in telomere biology. They interact with several other telomere regulators including TIN2, PTOP, POT1, and RAP1 to ensure proper maintenance of telomeres. TRF1 and TRF2 are believed to exert distinct functions. TRF1 forms a complex with TIN2, PTOP, and POT1 and regulates telomere length, whereas TRF2 mediates t-loop formation and end protection. However, whether cross-talk occurs between the TRF1 and TRF2 complexes and how the signals from these complexes are integrated for telomere maintenance remain to be elucidated. Through gel filtration and co-immunoprecipitation experiments, we found that TRF1 and TRF2 are in fact subunits of a telomere-associated high molecular weight complex (telosome) that also contains POT1, PTOP, RAP1, and TIN2. We demonstrated that the TRF1-interacting protein TIN2 binds TRF2 directly and in vivo, thereby bridging TRF2 to TRF1. Consistent with this multi-protein telosome model, stripping TRF1 off the telomeres by expressing tankyrase reduced telomere recruitment of not only TIN2 but also TRF2. These results help to unify previous observations and suggest that telomere maintenance depends on the multi-subunit telosome.
      The homeostasis of mammalian telomeres is regulated by a number of telomere-associated proteins. Among these proteins, TRF1 and TRF2 directly bind double-stranded telomere DNA and interact with a number of proteins to maintain telomere length and structure (
      • de Lange T.
      ,
      • Kim Sh S.H.
      • Kaminker P.
      • Campisi J.
      ). It has been shown that the amount of telomere-bound TRF1 correlates with telomere length. Overexpression of TRF1 shortened telomeres in human cells, whereas dominant negative TRF1 led to elongated telomeres (
      • van Steensel B.
      • de Lange T.
      ,
      • Smith S.
      • de Lange T.
      ,
      • Smogorzewska A.
      • van Steensel B.
      • Bianchi A.
      • Oelmann S.
      • Schaefer M.R.
      • Schnapp G.
      • de Lange T.
      ). TRF1 may control the length of telomere repeats through multiple mechanisms. For example, TRF1 can control telomerase access through its interaction with TIN2, PTOP/PIP1, and the single-stranded telomere DNA-binding protein POT1 (
      • Loayza D.
      • De Lange T.
      ,
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ,
      • Ye J.Z.
      • Hockemeyer D.
      • Krutchinsky A.N.
      • Loayza D.
      • Hooper S.M.
      • Chait B.T.
      • de Lange T.
      ). TRF1 may also regulate telomerase activity through its interaction with PINX1 (
      • Zhou X.Z.
      • Lu K.P.
      ). In comparison, TRF2 has an essential role in telomere end protection and t-loop formation (
      • de Lange T.
      ,
      • Griffith J.D.
      • Comeau L.
      • Rosenfield S.
      • Stansel R.M.
      • Bianchi A.
      • Moss H.
      • de Lange T.
      ,
      • Wei C.
      • Price M.
      ). Interference of endogenous TRF2 activity by expressing dominant negative forms of TRF2 markedly increased the rate of telomere end-to-end fusions (
      • van Steensel B.
      • Smogorzewska A.
      • de Lange T.
      ). Consistent with this role of TRF2, TRF2 forms a complex with RAP1 and associates with several proteins involved in DNA damage and repair responses, notably RAD50/MER11/NBS1, Ku86, and ERCC1/XPF (
      • Zhu X.D.
      • Kuster B.
      • Mann M.
      • Petrini J.H.
      • de Lange T.
      ,
      • Zhu X.D.
      • Niedernhofer L.
      • Kuster B.
      • Mann M.
      • Hoeijmakers J.H.
      • de Lange T.
      ,
      • O'Connor M.S.
      • Safari A.
      • Liu D.
      • Qin J.
      • Songyang Z.
      ). These findings have pointed to distinct biological functions of TRF1 and TRF2. Some recent findings, however, suggest a more complex picture. For instance, overexpression of TRF2 caused telomere shortening in primary cells (
      • Karlseder J.
      • Smogorzewska A.
      • de Lange T.
      ). In mouse embryonic stem cells, the conditional knockout of TRF1 led to significantly reduced levels of TRF2 at the telomeres, suggesting that TRF2 telomere localization may be partially regulated by TRF1 (
      • Iwano T.
      • Tachibana M.
      • Reth M.
      • Shinkai Y.
      ). In addition, chromosome end-to-end fusion was detected in TRF1 knock-out cells, indicating that telomere end protection was compromised. Despite the wealth of information, the functional relationship between TRF1 and TRF2 in telomere maintenance remains unclear. Notably, a recent report demonstrated a direct interaction between TRF2 and the TRF1-interacting protein, TIN2 (
      • Kim S.H.
      • Beausejour C.
      • Davalos A.R.
      • Kaminker P.
      • Heo S.J.
      • Campisi J.
      ). Such findings further suggest that cross-talk probably occurs between the TRF1 and TRF2 complexes. However, whether TIN2 can simultaneously associate with both TRF1 and TRF2 in the same complex remains to be demonstrated.
      In addition to TRF1, several other telomeric proteins have been shown to be regulators of telomere length (
      • de Lange T.
      ,
      • Kim Sh S.H.
      • Kaminker P.
      • Campisi J.
      ,
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ,
      • Ye J.Z.
      • Hockemeyer D.
      • Krutchinsky A.N.
      • Loayza D.
      • Hooper S.M.
      • Chait B.T.
      • de Lange T.
      ,
      • Kim S.H.
      • Kaminker P.
      • Campisi J.
      ,
      • Li B.
      • Oestreich S.
      • de Lange T.
      ,
      • Baumann P.
      • Cech T.R.
      ,
      • Colgin L.M.
      • Baran K.
      • Baumann P.
      • Cech T.R.
      • Reddel R.R.
      ). Both inhibition of endogenous RAP1, TIN2, POT1, or PTOP expression through RNA interference (RNAi)
      The abbreviations used are: RNAi, RNA interference; TANK, tankyrase; GST, glutathione S-transferase; PD, poly(ADP-ribose) polymerase-dead.
      1The abbreviations used are: RNAi, RNA interference; TANK, tankyrase; GST, glutathione S-transferase; PD, poly(ADP-ribose) polymerase-dead.
      and expression of dominant negative forms of these four proteins resulted in elongated telomeres in cultured cells (
      • van Steensel B.
      • de Lange T.
      ,
      • Loayza D.
      • De Lange T.
      ,
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ,
      • Ye J.Z.
      • Hockemeyer D.
      • Krutchinsky A.N.
      • Loayza D.
      • Hooper S.M.
      • Chait B.T.
      • de Lange T.
      ,
      • O'Connor M.S.
      • Safari A.
      • Liu D.
      • Qin J.
      • Songyang Z.
      ,
      • Ye J.Z.
      • de Lange T.
      ). These observations suggest that RAP1, TIN2, POT1, and PTOP may function in the same pathway. All four proteins, RAP1, TIN2, POT1, and PTOP, directly or indirectly associate with TRF1 or TRF2 (
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ,
      • Ye J.Z.
      • Hockemeyer D.
      • Krutchinsky A.N.
      • Loayza D.
      • Hooper S.M.
      • Chait B.T.
      • de Lange T.
      ,
      • Li B.
      • Oestreich S.
      • de Lange T.
      ), pointing to a possible functional connection among these six telomeric proteins. In this report, we present evidence demonstrating that the TRF1 and TRF2 complexes do indeed interact with each other, as TRF1, TRF2, RAP1, TIN2, POT1, and PTOP can form a protein complex in vivo to regulate telomeres.

      MATERIALS AND METHODS

      Preparation of Nuclear Extracts—HeLa S3 cells grown in suspension to 1 × 106 cells/ml were collected and washed in cold phosphate-buffered saline and hypotonic buffer (10 mm Tris, pH 7.3, 10 mm KCl, 1.5 mm MgCl2, 0.2 mm phenylmethylsulfonyl fluoride, and 10 mm 2-mercaptoethanol). The cells were then allowed to swell for 15 min in hypotonic buffer, homogenized until cell membrane lysis was ∼80%. The lysates were resuspended in low salt buffer (20 mm KCl, 20 mm Tris, pH 7.3, 25% glycerol, 1.5 mm MgCl2, and 0.2 mm EDTA) and homogenized briefly to break the nuclear membrane. An equal volume of high salt buffer (1.2 m KCl, 20 mm Tris, pH 7.3, 25% glycerol, 1.5 mm MgCl2, and 0.2 mm EDTA) was added followed by agitation for 30 min at 4 °C and centrifuged at 20,000 × g for 30 min. The supernatant was dialyzed in BC0 buffer (20 mm Tris, pH 7.3, 20% glycerol, 0.2 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, and 10 mm 2-mercaptoethanol) for 3 h and centrifuged again. The cleared supernatant was then aliquoted and stored at -80 °C.
      Salt extraction and fractionation of HT1080 cells were performed as previously described previously (
      • Ye J.Z.
      • de Lange T.
      ). The cells were extracted in a low salt buffer (20 mm Hepes pH 7.9, 5 mm MgCl2, 1 mm DTT, 25% glycerol, protease inhibitors, 0.2% Nonidet P-40, and 150 mm KCl). The resulting supernatant was the 150 mm fraction. The pellet was further extracted with a similar buffer but containing 420 mm KCl. Chromatin-bound proteins were in the 420 mm KCl fraction.
      Immunoprecipitation and Mass Spectrometry—For large-scale affinity purification, ∼70 mg of nuclear protein extracts were incubated with 100 μl of anti-FLAG M2-agarose beads (Sigma) for 3 h at 4 °C. The beads were then washed 4 times with NETN (20 mm Tris, pH 8.0, 100 mm NaCl, 0.5% Nonidet P-40, and 1 mm EDTA), and the bound protein was eluted twice with 100 μlof200 μg/ml FLAG peptide-(DYKDDDDK) (Sigma) in NETN. The eluent was resolved on a 8–12% SDS-PAGE gradient gel (Bio-Rad) and visualized by Coomassie Blue staining. Specific bands were then excised, digested with trypsin, and subjected to ion-trap mass spectrometry as previously described (
      • Ogryzko V.V.
      • Kotani T.
      • Zhang X.
      • Schiltz R.L.
      • Howard T.
      • Yang X.J.
      • Howard B.H.
      • Qin J.
      • Nakatani Y.
      ). Peptides were identified using PROWL (prowl.rockefeller.edu/).
      For small-scale immunoprecipitation experiments, 1 mg of nuclear extracts was incubated for 2 h at 4 °C with 5 μg of anti-FLAG M2 (Sigma), anti-hRap1 (Bethyl Laboratories), anti-TRF2 (Oncogene), anti-POT1N, anti-TIN2C, or anti-PTOP 466 antibodies (
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ) and 15 μl of protein A or protein G-agarose beads (Santa Cruz Biotechnology). The beads were then washed four times with 0.5 ml of NETN, boiled in 2× SDS loading buffer, and resolved on 8 or 10% SDS-PAGE gels.
      Fractionation of the Telomere-associated Complex, Telosome—Chromatographic experiments were performed as described previously (
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ). HeLa cell nuclear extracts were fractionated on ΔKTA Superose 6 HR 10/30 gel filtration columns (Amersham Biosciences). The resulting fractions were resolved by SDS-PAGE and probed with various antibodies.
      Generation of Constructs and Cell Lines of hRap1 and Its Deletion Mutants—FLAG-tagged full-length hRap1 and various hRap1 mutants were cloned in the pBabe-puro retroviral vector as previously described (
      • O'Connor M.S.
      • Safari A.
      • Liu D.
      • Qin J.
      • Songyang Z.
      ). The retroviral wild type and mutant FLAG-tankyrase (TANK) vectors were a generous gift from Dr. Titia de Lange (
      • Ye J.Z.
      • de Lange T.
      ). The retroviral vectors were used to transfect BOSC23 cells to produce retroviruses for the subsequent infection of HeLa or HT1080 cells. These cells were selected with 2 μg/ml puromycin for 3 days after infection to obtain cells stably expressing hRap1 and its mutants or TANK.
      Antibodies and Western Blotting Analysis—For Western analysis, immunoprecipitates and nuclear extract controls were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The primary antibodies included anti-FLAG M2 (Sigma) and anti-TRF2 (Oncogene). The rabbit polyclonal anti-POT1N antibody was generated against GST-tagged human POT1 protein (amino acids 1–253). Anti-RAP1, anti-TIN2C, and anti-PTOP 466 antibodies were previously described (
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ). These antibodies were generated by the Bethyl Laboratories. Anti-TRF1 antibody was a generous gift from the de Lange laboratory (
      • van Steensel B.
      • de Lange T.
      ). The secondary antibodies included anti-mouse horseradish peroxidase and anti-rabbit horseradish peroxidase (Bio-Rad).
      In Vitro Binding Assays—Bacterially expressed GST full-length POT1, RAP1, and TIN2 were purified using glutathione-agarose beads (Molecular Probes). Approximately 1 μg of GST fusion proteins on beads was used for each binding reaction. In vitro translation and [35S]Met labeling of human TRF1 and TRF2 were carried out using the In Vitro TnT kit (Promega). The mixtures were washed three times with NETN, eluted with 2× SDS buffer, resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membranes followed by analysis using a PhosphorImager (Amersham Biosciences).
      Indirect Immunofluorescence—The localization of telomere-associated proteins were visualized through indirect immunofluorescence as previously described (
      • Li B.
      • Oestreich S.
      • de Lange T.
      ). Cells were grown overnight on poly-d-lysine-coated coverslips, permeabilized in the Triton X-100 solution (0.5% Triton X-100 in phosphate-buffered saline), fixed with 3.7% paraformaldehyde in phosphate-buffered saline, and permeabilized again in the Triton X-100 solution containing 300 mm sucrose. The cells were subsequently blocked for 1 h at 37 °C in 5% goat serum, stained with various primary and fluorescence-conjugated secondary antibodies for 1 h each at 37 °C, and then visualized under a Nikon TE200 fluorescence microscope. The primary antibodies used were: polyclonal anti-TRF1; anti-TIN2C antibody; and monoclonal anti-TRF2 antibody (Oncogene). Secondary antibodies were AlexaFluor 488-conjugated goat anti-mouse antibody (Molecular Probes) and Texas Red goat anti-rabbit antibody (Molecular Probes).

      RESULTS

      Identification of a Telomere-associated Protein Complex, Telosome—Individual proteins can be epitope-tagged, which allows for easy isolation and identification of their associated proteins by immunoprecipitation and mass spectrometry. We undertook such a proteomic approach to understand the molecular mechanisms that regulate human telomeres. In our analysis of the purified RAP1 protein complex, we identified several proteins that are known to interact with RAP1, including RAD50, Mre11, Ku86/70, and TRF2 (Fig. 1A) (
      • Zhu X.D.
      • Kuster B.
      • Mann M.
      • Petrini J.H.
      • de Lange T.
      ,
      • O'Connor M.S.
      • Safari A.
      • Liu D.
      • Qin J.
      • Songyang Z.
      ). Intriguingly, the sequencing of the 40-kDa band revealed TIN2 as a component of the RAP1 complex as well. The same RAP1 complex was able to form even in the presence of ethidium bromide, suggesting that the interactions between the various components were not mediated through DNA (data not shown). The presence of TIN2 in the RAP1·TRF2 complex was surprising, because TIN2 is a TRF1-interacting protein (
      • Kim S.H.
      • Kaminker P.
      • Campisi J.
      ).
      Figure thumbnail gr1
      Fig. 1Association of TRF2·RAP1 with components of the TRF1 complex. A, nuclear extracts from HeLa cells expressing FLAG-RAP1 were immunoprecipitated with anti-FLAG antibodies and resolved by SDS-PAGE. Specific bands were excised and sequenced by mass spectrometry. MW, molecular weight; Ab, antibody heavy chain. B, HeLa nuclear extracts were immunoprecipitated with anti-TIN2, anti-POT1, anti-PTOP, anti-TRF2, or anti-RAP1 antibodies. The immunoprecipitates were resolved by SDS-PAGE and Western blotted with the indicated antibodies. IP, immunoprecipitation. (Note: we could not Western blot PTOP or TRF1 in these co-immunoprecipitation experiments, because PTOP and TRF1 migrate to almost exactly the same location as antibodies.)
      Our purification and characterization of TIN2-associated proteins further confirmed the existence of a RAP1·TIN2 protein complex, as we found TRF2 and RAP1 to co-purify with TRF1, TIN2, PTOP, and POT1 (
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ). Furthermore, mass spectrometry sequencing revealed the six proteins to be the major components of the isolated complex (
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ). Because TIN2, PTOP, and POT1 have been shown to complex with TRF1 (
      • Loayza D.
      • De Lange T.
      ,
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ,
      • Ye J.Z.
      • Hockemeyer D.
      • Krutchinsky A.N.
      • Loayza D.
      • Hooper S.M.
      • Chait B.T.
      • de Lange T.
      ), it suggests that TRF2 and RAP1 may interact with the TRF1 complex, resulting in the formation of a six-protein complex at the telomeres.
      To determine the interaction between the six telomeric proteins, we carried out co-immunoprecipitation experiments using nuclear extracts from HeLa cells and antibodies against endogenous POT1, PTOP, TIN2, RAP1, or TRF2. Consistent with our previous observations (
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ), endogenous POT1 and PTOP co-immunoprecipitated with TIN2, whereas immunoprecipitation with anti-PTOP and anti-TIN2 antibodies brought down POT1 (Fig. 1B). Notably, TRF2 was also able to co-immunoprecipitate with POT1, PTOP, and TIN2. In the reciprocal experiment, antibodies against TRF2 or RAP1 brought down POT1 and TIN2 as well. These data strongly support our findings that TRF2 and RAP1 associate with the TRF1 complex and suggest a cross-talk between the TRF1 and TRF2 complexes.
      We performed gel filtration experiments next using HeLa nuclear extracts. As shown in Fig. 2, endogenous TRF1, TRF2, TIN2, RAP1, PTOP, and POT1 co-eluted in a large molecular complex (∼1 MDa), indicating that the six-telomeric proteins could indeed form a physical complex that contains the major telomeric proteins identified to date in mammalian cells. Based on the above data, we named this large complex, containing the six telomeric proteins, the telosome. It should be noted that some of the telomeric proteins (e.g. RAP1 and TRF2) were also co-eluted at lower molecular weight fractions. Therefore, there may be other telomere complexes in addition to the telosome.
      Figure thumbnail gr2
      Fig. 2The six-telomeric proteins form a high molecular weight protein complex, the telosome. HeLa S3 cell nuclear extracts were fractionated on a Superose 6 gel filtration column. Individual fractions (numbered) were collected, resolved by SDS-PAGE, and Western blotted with the indicated antibodies. Gel filtration molecular standards are indicated by arrows.
      TIN2 Directly Binds TRF2 Both in Vitro and in Vivo and Tethers TRF2 to the TRF1 Complex—We next examined how the TRF2·RAP1 subcomplex was connected to the TRF1 subcomplex in the telosome. RAP1 contains an N-terminal BRCA1 C-terminal domain, a Myb domain, and a C-terminal TRF2-binding RAP1 C-terminal domain (RCT) (
      • Li B.
      • Oestreich S.
      • de Lange T.
      ). We first set out to determine which of the domains of RAP1 were necessary for its association with TIN2. As shown in Fig. 1A, anti-FLAG immunoprecipitation of full-length RAP1 brought down endogenous TRF2 and TIN2. An analysis of a series of RAP1 deletion mutants available in the laboratory (
      • O'Connor M.S.
      • Safari A.
      • Liu D.
      • Qin J.
      • Songyang Z.
      ) revealed that the BRCA1 C-terminal and Myb domains were dispensable in mediating RAP1 interaction with TRF2 and TIN2 (Fig. 3A). However, the RAP1 C-terminal domain deletion mutant (ΔRCT) failed to co-immunoprecipitate with not only endogenous TRF2 but also TIN2, indicating that RAP1 may associate with TIN2 through TRF2. Furthermore, these data suggest a direct interaction between TRF2 and TIN2 (or other components of the TRF1 complex). To test this hypothesis, TRF2 was in vitro translated and incubated with GST fusion telomeric proteins. As shown in Fig. 3B, in vitro translated TRF2 specifically bound GST-TIN2 (at a level comparable to GST-RAP1) but not GST-POT1. GST-TIN2 but not GST-RAP1 specifically pulled down in vitro translated TRF1 (Fig. 3B, right panel). Therefore, both TRF1 and TRF2 can directly interact with TIN2. Moreover, specific interactions between V5-tagged TRF2 and FLAG-tagged TIN2 were detected in 293T cells (data not shown). Consistent with a recent report on TIN2 interaction with TRF2 (
      • Kim S.H.
      • Beausejour C.
      • Davalos A.R.
      • Kaminker P.
      • Heo S.J.
      • Campisi J.
      ), our results indicate that TIN2 provides the link between the TRF1 and TRF2 complexes.
      Figure thumbnail gr3
      Fig. 3TIN2 directly interacts with TRF2. A, extracts from cells expressing FLAG-tagged full-length RAP1, RAP1 ΔBRCT, RAP1 ΔMyb, and RAP1 ΔRCT were immunoprecipitated with anti-FLAG antibodies. The immunoprecipitates were resolved on SDS-PAGE and Western blotted using anti-TRF2 and anti-TIN2 antibodies. B, cDNAs encoding full-length TRF2 or TRF1 were cloned into the pcDNA3 vector and in vitro translated in the presence of [35S]Met. GST-POT1, GST-TIN2, and GST-RAP1 fusion proteins were used in pull-down reactions.
      Telomeric Localization of Telosome Subunits Is Regulated by TRF1—To further address the functional relevance of telosome as well as the interaction between TRF1 and TRF2, we investigated whether telomere localization of telosome subunits, in particular TRF2, was regulated by TRF1 using HT1080 cells expressing FLAG-tagged wild type TANK or catalytically inactive TANK (TANK-PD). TANK is a TRF1-associated poly(ADP-ribose) polymerase (
      • Smith S.
      • Giriat I.
      • Schmitt A.
      • de Lange T.
      ). TANK can ADP-ribosylate TRF1, resulting in TRF1 ubiquitination and degradation by the proteasome pathway, effectively stripping TRF1 off the telomeres (
      • Smith S.
      • Giriat I.
      • Schmitt A.
      • de Lange T.
      ,
      • Chang W.
      • Dynek J.N.
      • Smith S.
      ). In these cells, FLAG-TANK and TANK-PD were expressed at comparable levels, whereas total TIN2 and TRF2 levels were not reduced (data not shown). We then compared the levels of telomere-localized TRF1, TRF2, and TIN2 in these cells using indirect immunofluorescence. Endogenous TRF1, TRF2, and TIN2 exhibited punctate staining patterns, characteristic of telomeric proteins (Fig. 4). As previously reported (
      • Iwano T.
      • Tachibana M.
      • Reth M.
      • Shinkai Y.
      ), telomere-bound TRF1 was greatly reduced in cells expressing wild type TANK compared with TANK-PD-expressing cells (Fig. 4A). Similarly, the number and intensity of TIN2 foci decreased significantly in wild type TANK-expressing cells (Fig. 4B). The direct interaction between TIN2 and TRF2 suggests that TRF2 telomere localization may be affected in TANK-expressing cells as well. Indeed, in cells in which TRF1 levels were reduced because of TANK expression, anti-TRF2 staining also decreased (Fig. 4A). Consistent with this observation, the amounts of chromatin-bound TIN2 and TRF2 in TANK-expressing cells were also reduced, as analyzed by Western blotting (Fig. 4C). These results are not only consistent with the finding that TRF2 telomere localization was altered in TRF1 knock-out cells (
      • Iwano T.
      • Tachibana M.
      • Reth M.
      • Shinkai Y.
      ) but also provide further support for the six-protein core telosome model. In this case, eliminating TRF1 could prevent telosome formation, thereby preventing telomere localization of TIN2 and TRF2. Therefore, telomere localization of TRF2 may depend upon the formation of telosome.
      Figure thumbnail gr4
      Fig. 4Telomeric localization of telosome subunits is regulated by TRF1. HT1080 cells expressing wild type TANK (two left panels) or its catalytically inactive mutant (TANK-PD, two right panels) were twice permeabilized and stained with antibodies against TRF1, TRF2 (A), or TIN2 (B). C, HT1080 cells expressing TANK wild type or TANK-PD were extracted sequentially with buffer containing 150 and 420 mm KCl (chromatin-bound fraction) (
      • Ye J.Z.
      • de Lange T.
      ). The cell extracts were then Western blotted with anti-TIN2, anti-TRF2, and anti-Ku86 (loading control) antibodies.

      DISCUSSION

      All six proteins, TRF1, TRF2, TIN2, RAP1, POT1, and PTOP, have been shown to specifically localize to the telomeres in mammalian cells. Functional interference with any of the six proteins by RNAi or dominant negative expression has been known to affect telomere length or end capping (
      • van Steensel B.
      • de Lange T.
      ,
      • Loayza D.
      • De Lange T.
      ,
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ,
      • Ye J.Z.
      • Hockemeyer D.
      • Krutchinsky A.N.
      • Loayza D.
      • Hooper S.M.
      • Chait B.T.
      • de Lange T.
      ,
      • van Steensel B.
      • Smogorzewska A.
      • de Lange T.
      ,
      • O'Connor M.S.
      • Safari A.
      • Liu D.
      • Qin J.
      • Songyang Z.
      ,
      • Ye J.Z.
      • de Lange T.
      ). Therefore, these proteins probably are the major players of telomere maintenance. In a recent study by Kim et al. (
      • Kim S.H.
      • Beausejour C.
      • Davalos A.R.
      • Kaminker P.
      • Heo S.J.
      • Campisi J.
      ), the authors showed a direct interaction between TIN2 and TRF2. In this report, we demonstrated that TIN2/TRF2 interaction provides only one piece of the puzzle, because all six telomeric proteins are able to assemble into a high molecular weight complex, the telosome. While this paper was in review, Ye et al. (
      • Ye J.Z.
      • Donigian J.R.
      • Van Overbeek M.
      • Loayza D.
      • Luo Y.
      • Krutchinsky A.N.
      • Chait B.T.
      • De Lange T.
      ) also reported the discovery of the six-protein telomeric complex, which is consistent with our findings. The telosome model helps to explain why similar telomere extension phenotypes in human cells were obtained when RAP1, POT1, PTOP, or TIN2 was inhibited through RNAi or dominant negative expression (
      • van Steensel B.
      • de Lange T.
      ,
      • Loayza D.
      • De Lange T.
      ,
      • Liu D.
      • Safari A.
      • O'Connor M.S.
      • Chan D.W.
      • Laegeler A.
      • Qin J.
      • Songyang Z.
      ,
      • Ye J.Z.
      • Hockemeyer D.
      • Krutchinsky A.N.
      • Loayza D.
      • Hooper S.M.
      • Chait B.T.
      • de Lange T.
      ,
      • O'Connor M.S.
      • Safari A.
      • Liu D.
      • Qin J.
      • Songyang Z.
      ,
      • Ye J.Z.
      • de Lange T.
      ). Incorporation of the dominant negative forms of a subunit into the telosome may prevent its normal function, and knock-down of one of the six proteins by RNAi will probably hinder telosome formation. Additionally, the stoichiometry of telosome subunits may be crucial to its proper assembly. For example, the knock-down of TIN2 through RNAi led to reduced TRF1 localization at the telomeres (
      • Ye J.Z.
      • de Lange T.
      ). In further support of a critical role of the telosome in maintaining telomere integrity, inactivation of TIN2 or TRF1 in mice resulted in embryonic lethality (
      • Zhu X.D.
      • Niedernhofer L.
      • Kuster B.
      • Mann M.
      • Hoeijmakers J.H.
      • de Lange T.
      ,
      • Chiang Y.J.
      • Kim S.
      • Tessarollo L.
      • Campisi J.
      • Hodes R.J.
      ). In the TRF1 knock-out mouse, telomeric localization of TRF2 and TIN2 was also disrupted (
      • Iwano T.
      • Tachibana M.
      • Reth M.
      • Shinkai Y.
      ).
      Both TRF1 and TRF2 can bind telomeric double-stranded DNA. The functional difference between these two proteins is probably due to their abilities to recruit different signaling complexes. TRF1 plays a primary role in telomere length control and cell cycle, whereas TRF2 protects telomere ends from being recognized as DNA breaks. It was unclear whether communication could occur between the TRF1 and TRF2 complexes. Our results suggested that TRF1 and TRF2 interact with each other through TIN2 and highlight the functional connection between TRF1 and TRF2. The identification of the telosome unites two essential pathways in telomere maintenance, telomere length, and end protection and suggests coordinated action and functional cross-talk between its subcomplexes. Functionally similar to the budding yeast telosome (
      • Wright J.H.
      • Gottschling D.E.
      • Zakian V.A.
      ,
      • Dubrana K.
      • Perrod S.
      • Gasser S.M.
      ), the mammalian telosome may represent the core telomere-associated complex mediating telomere maintenance in mammalian cells. Each of the six telomeric proteins may interact with many other different proteins to form unique subcomplexes, allowing for the dynamic integration and processing of signals from diverse pathways.

      Acknowledgments

      We thank Doug Chan and Amin Safari for technical assistance.

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