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

Abstract

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. 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 (1de Lange T. Oncogene. 2002; 21: 532-540Crossref PubMed Google Scholar, 2Kim Sh S.H. Kaminker P. Campisi J. Oncogene. 2002; 21: 503-511Crossref PubMed Google Scholar). 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 (3van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1055) Google Scholar, 4Smith S. de Lange T. Curr. Biol. 2000; 10: 1299-1302Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 5Smogorzewska A. van Steensel B. Bianchi A. Oelmann S. Schaefer M.R. Schnapp G. de Lange T. Mol. Cell. Biol. 2000; 20: 1659-1668Crossref PubMed Scopus (630) Google Scholar). 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 (6Loayza D. De Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar, 7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar, 8Ye J.Z. Hockemeyer D. Krutchinsky A.N. Loayza D. Hooper S.M. Chait B.T. de Lange T. Genes Dev. 2004; 18: 1649-1654Crossref PubMed Scopus (351) Google Scholar). TRF1 may also regulate telomerase activity through its interaction with PINX1 (9Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). In comparison, TRF2 has an essential role in telomere end protection and t-loop formation (1de Lange T. Oncogene. 2002; 21: 532-540Crossref PubMed Google Scholar, 10Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. de Lange T. Cell. 1999; 97: 503-514Abstract Full Text Full Text PDF PubMed Scopus (1946) Google Scholar, 11Wei C. Price M. Cell Mol. Life Sci. 2003; 60: 2283-2294Crossref PubMed Scopus (44) Google Scholar). Interference of endogenous TRF2 activity by expressing dominant negative forms of TRF2 markedly increased the rate of telomere end-to-end fusions (12van Steensel B. Smogorzewska A. de Lange T. Cell. 1998; 92: 401-413Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar). 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 (13Zhu X.D. Kuster B. Mann M. Petrini J.H. de Lange T. Nat. Genet. 2000; 25: 347-352Crossref PubMed Scopus (512) Google Scholar, 14Zhu X.D. Niedernhofer L. Kuster B. Mann M. Hoeijmakers J.H. de Lange T. Mol. Cell. 2003; 12: 1489-1498Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 15O'Connor M.S. Safari A. Liu D. Qin J. Songyang Z. J. Biol. Chem. 2004; 279: 28585-28591Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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 (16Karlseder J. Smogorzewska A. de Lange T. Science. 2002; 295: 2446-2449Crossref PubMed Scopus (668) Google Scholar). 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 (17Iwano T. Tachibana M. Reth M. Shinkai Y. J. Biol. Chem. 2003; 279: 1442-1448Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). 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 (18Kim S.H. Beausejour C. Davalos A.R. Kaminker P. Heo S.J. Campisi J. J. Biol. Chem. 2004; 279: 43799-43804Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). 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 (1de Lange T. Oncogene. 2002; 21: 532-540Crossref PubMed Google Scholar, 2Kim Sh S.H. Kaminker P. Campisi J. Oncogene. 2002; 21: 503-511Crossref PubMed Google Scholar, 7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar, 8Ye J.Z. Hockemeyer D. Krutchinsky A.N. Loayza D. Hooper S.M. Chait B.T. de Lange T. Genes Dev. 2004; 18: 1649-1654Crossref PubMed Scopus (351) Google Scholar, 19Kim S.H. Kaminker P. Campisi J. Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (429) Google Scholar, 20Li B. Oestreich S. de Lange T. Cell. 2000; 101: 471-483Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 21Baumann P. Cech T.R. Science. 2001; 292: 1171-1175Crossref PubMed Scopus (810) Google Scholar, 22Colgin L.M. Baran K. Baumann P. Cech T.R. Reddel R.R. Curr. Biol. 2003; 13: 942-946Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Both inhibition of endogenous RAP1, TIN2, POT1, or PTOP expression through RNA interference (RNAi) 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 (3van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1055) Google Scholar, 6Loayza D. De Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar, 7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar, 8Ye J.Z. Hockemeyer D. Krutchinsky A.N. Loayza D. Hooper S.M. Chait B.T. de Lange T. Genes Dev. 2004; 18: 1649-1654Crossref PubMed Scopus (351) Google Scholar, 15O'Connor M.S. Safari A. Liu D. Qin J. Songyang Z. J. Biol. Chem. 2004; 279: 28585-28591Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 23Ye J.Z. de Lange T. Nat. Genet. 2004; 36: 618-623Crossref PubMed Scopus (155) Google Scholar). 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 (7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar, 8Ye J.Z. Hockemeyer D. Krutchinsky A.N. Loayza D. Hooper S.M. Chait B.T. de Lange T. Genes Dev. 2004; 18: 1649-1654Crossref PubMed Scopus (351) Google Scholar, 20Li B. Oestreich S. de Lange T. Cell. 2000; 101: 471-483Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar), 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. 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 (23Ye J.Z. de Lange T. Nat. Genet. 2004; 36: 618-623Crossref PubMed Scopus (155) Google Scholar). 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 (24Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). 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 (7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar) 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 of the were performed as described previously (7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar). cell nuclear extracts were on gel filtration The resulting were resolved by SDS-PAGE and with of and Cell of and and were in the as previously described M.S. Safari A. Liu D. Qin J. Songyang Z. J. Biol. Chem. 2004; 279: 28585-28591Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The and were a from de Lange (23Ye J.Z. de Lange T. Nat. Genet. 2004; 36: 618-623Crossref PubMed Scopus (155) Google Scholar). The were used to cells to for the of or HT1080 These cells were with 2 μg/ml for 3 to cells expressing and its or and and nuclear were by SDS-PAGE and to The primary antibodies anti-FLAG M2 (Sigma) and anti-TRF2 The was human POT1 protein anti-TIN2C, and anti-PTOP 466 antibodies were previously described (7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar). These antibodies were by the was a from the de Lange (3van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1055) Google Scholar). The antibodies and In POT1, RAP1, and TIN2 were using beads 1 μg of fusion proteins on beads was used for each In and of human TRF1 and TRF2 were using the In The were washed times with NETN, eluted with 2× SDS buffer, resolved by and to followed by using a localization of telomere-associated proteins were visualized through as previously described B. Oestreich S. de Lange T. Cell. 2000; 101: 471-483Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). were grown on in the in phosphate-buffered with in phosphate-buffered and in the containing mm The cells were for 1 h at °C in with primary and antibodies for 1 h each at and then visualized a The primary antibodies used and anti-TRF2 antibodies were and of a proteins can be for and of proteins by immunoprecipitation and mass We a to the molecular that regulate human telomeres. In of the RAP1 protein we identified several proteins that are to interact with RAP1, including and TRF2 (13Zhu X.D. Kuster B. Mann M. Petrini J.H. de Lange T. Nat. Genet. 2000; 25: 347-352Crossref PubMed Scopus (512) Google Scholar, 15O'Connor M.S. Safari A. Liu D. Qin J. Songyang Z. J. Biol. Chem. 2004; 279: 28585-28591Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). the of the TIN2 as a of the RAP1 complex as The same RAP1 complex was to form in the of suggesting that the between the were not through DNA not The of TIN2 in the complex was TIN2 is a TRF1-interacting protein S.H. Kaminker P. Campisi J. Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (429) Google Scholar). and of proteins further the of a protein as we found TRF2 and RAP1 to with TRF1, TIN2, PTOP, and POT1 (7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar). mass spectrometry the six proteins to be the of the complex (7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar). TIN2, PTOP, and POT1 have been shown to complex with TRF1 (6Loayza D. De Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar, 7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar, 8Ye J.Z. Hockemeyer D. Krutchinsky A.N. Loayza D. Hooper S.M. Chait B.T. de Lange T. Genes Dev. 2004; 18: 1649-1654Crossref PubMed Scopus (351) Google Scholar), that TRF2 and RAP1 may interact with the TRF1 resulting in the formation of a complex at the telomeres. the interaction between the six telomeric proteins, we co-immunoprecipitation using nuclear extracts from cells and antibodies endogenous POT1, PTOP, TIN2, RAP1, or TRF2. Consistent with previous observations (7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar), endogenous POT1 and PTOP with TIN2, whereas immunoprecipitation with anti-PTOP and antibodies POT1 Notably, TRF2 was also to with POT1, PTOP, and TIN2. In the antibodies TRF2 or RAP1 POT1 and TIN2 as These 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 using nuclear shown in endogenous TRF1, TRF2, TIN2, RAP1, PTOP, and POT1 in a molecular complex indicating that the proteins indeed form a complex that contains the telomeric proteins identified to in mammalian on the we this containing the six telomeric proteins, the telosome. It be that of the telomeric proteins RAP1 and were also at molecular weight may be other telomere complexes in addition to the telosome. TIN2 TRF2 Both in and in and TRF2 to the TRF1 how the was to the TRF1 in the telosome. RAP1 contains an a and a RAP1 B. Oestreich S. de Lange T. Cell. 2000; 101: 471-483Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). We to of the of RAP1 were for its with TIN2. shown in anti-FLAG immunoprecipitation of RAP1 endogenous TRF2 and TIN2. An of a of RAP1 in the M.S. Safari A. Liu D. Qin J. Songyang Z. J. Biol. Chem. 2004; 279: 28585-28591Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) that the and were in RAP1 interaction with TRF2 and TIN2 However, the RAP1 to with not only endogenous TRF2 but also TIN2, indicating that RAP1 may associate with TIN2 through TRF2. these suggest a direct interaction between TRF2 and TIN2 other of the TRF1 this TRF2 was in and incubated with fusion telomeric proteins. shown in in TRF2 bound a to but not but not in TRF1 both TRF1 and TRF2 can directly interact with TIN2. between TRF2 and TIN2 were detected in cells not Consistent with a recent report on TIN2 interaction with TRF2 (18Kim S.H. Beausejour C. Davalos A.R. Kaminker P. Heo S.J. Campisi J. J. Biol. Chem. 2004; 279: 43799-43804Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), results that TIN2 the between the TRF1 and TRF2 complexes. of by further the functional of telosome as as the interaction between TRF1 and TRF2, we whether telomere localization of telosome in TRF2, was regulated by TRF1 using HT1080 cells expressing or is a poly(ADP-ribose) S. A. de Lange T. Science. 1998; PubMed Scopus Google Scholar). can TRF1, resulting in TRF1 and by the stripping TRF1 off the telomeres S. A. de Lange T. Science. 1998; PubMed Scopus Google Scholar, S. Genes Dev. 2003; PubMed Scopus Google Scholar). In these cells, and were at whereas TIN2 and TRF2 levels were not reduced not We then the levels of TRF1, TRF2, and TIN2 in these cells using TRF1, TRF2, and TIN2 of telomeric proteins previously (17Iwano T. Tachibana M. Reth M. Shinkai Y. J. Biol. Chem. 2003; 279: 1442-1448Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), telomere-bound TRF1 was reduced in cells expressing with cells the number and of TIN2 significantly in cells The direct interaction between TIN2 and TRF2 that TRF2 telomere localization may be in cells as in cells in TRF1 levels were reduced of anti-TRF2 also Consistent with this the of TIN2 and TRF2 in cells were also as by These results are not only with the that TRF2 telomere localization was in TRF1 knock-out cells (17Iwano T. Tachibana M. Reth M. Shinkai Y. J. Biol. Chem. 2003; 279: 1442-1448Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) but also further for the telosome In this TRF1 telosome thereby telomere localization of TIN2 and TRF2. telomere localization of TRF2 may the formation of telosome. All six proteins, TRF1, TRF2, TIN2, RAP1, POT1, and PTOP, have been shown to to the telomeres in mammalian interference with of the six proteins by or dominant negative expression has been to telomere length or end (3van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1055) Google Scholar, 6Loayza D. De Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar, 7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar, 8Ye J.Z. Hockemeyer D. Krutchinsky A.N. Loayza D. Hooper S.M. Chait B.T. de Lange T. Genes Dev. 2004; 18: 1649-1654Crossref PubMed Scopus (351) Google Scholar, Steensel B. Smogorzewska A. de Lange T. Cell. 1998; 92: 401-413Abstract Full Text Full Text PDF PubMed Scopus (1456) Google Scholar, 15O'Connor M.S. Safari A. Liu D. Qin J. Songyang Z. J. Biol. Chem. 2004; 279: 28585-28591Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 23Ye J.Z. de Lange T. Nat. Genet. 2004; 36: 618-623Crossref PubMed Scopus (155) Google Scholar). these proteins probably are the of telomere In a recent by (18Kim S.H. Beausejour C. Davalos A.R. Kaminker P. Heo S.J. Campisi J. J. Biol. Chem. 2004; 279: 43799-43804Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), the a direct interaction between TIN2 and TRF2. In this report, we demonstrated that interaction only of the six telomeric proteins are to a high molecular weight the telosome. this was in J.Z. M. Loayza D. Y. Krutchinsky A.N. Chait B.T. De Lange T. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) also the of the telomeric is with The telosome to similar telomere in human cells were RAP1, POT1, PTOP, or TIN2 was through or dominant negative expression (3van Steensel B. de Lange T. Nature. 1997; 385: 740-743Crossref PubMed Scopus (1055) Google Scholar, 6Loayza D. De Lange T. Nature. 2003; 424: 1013-1018Crossref PubMed Scopus (544) Google Scholar, 7Liu D. Safari A. O'Connor M.S. Chan D.W. Laegeler A. Qin J. Songyang Z. Nat. Cell Biol. 2004; 6: 673-680Crossref PubMed Scopus (335) Google Scholar, 8Ye J.Z. Hockemeyer D. Krutchinsky A.N. Loayza D. Hooper S.M. Chait B.T. de Lange T. Genes Dev. 2004; 18: 1649-1654Crossref PubMed Scopus (351) Google Scholar, 15O'Connor M.S. Safari A. Liu D. Qin J. Songyang Z. J. Biol. Chem. 2004; 279: 28585-28591Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 23Ye J.Z. de Lange T. Nat. Genet. 2004; 36: 618-623Crossref PubMed Scopus (155) Google Scholar). of the dominant negative forms of a the telosome may its and of of the six proteins by probably telosome the of telosome subunits may be crucial to its proper For example, the of TIN2 through led to reduced TRF1 localization at the telomeres (23Ye J.Z. de Lange T. Nat. Genet. 2004; 36: 618-623Crossref PubMed Scopus (155) Google Scholar). In further of a role of the telosome in telomere of TIN2 or TRF1 in resulted in embryonic X.D. Niedernhofer L. Kuster B. Mann M. Hoeijmakers J.H. de Lange T. Mol. Cell. 2003; 12: 1489-1498Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, S. L. Campisi J. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). In the TRF1 knock-out telomeric localization of TRF2 and TIN2 was also (17Iwano T. Tachibana M. Reth M. Shinkai Y. J. Biol. Chem. 2003; 279: 1442-1448Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Both TRF1 and TRF2 can bind telomeric double-stranded The functional between these proteins is probably to to complexes. TRF1 a primary role in telomere length control and cell whereas TRF2 telomere from as DNA It was whether between the TRF1 and TRF2 complexes. results that TRF1 and TRF2 interact with each other through TIN2 and the functional connection between TRF1 and TRF2. The of the telosome essential in telomere telomere length, and end protection and and functional cross-talk between its similar to the telosome J.H. Genes Dev. 6: PubMed Scopus Google Scholar, K. S. S.M. Curr. Cell Biol. 2001; 13: PubMed Scopus Google Scholar), the mammalian telosome may the telomere-associated complex telomere maintenance in mammalian of the six telomeric proteins may interact with other proteins to form for the and of signals from We Chan and Safari for