Telomere dysfunction may result in chromosomal abnormalities, DNA damage responses, and even cancer. Early studies in lower organisms have helped to establish the crucial role of telomerase and telomeric proteins in maintaining telomere length and protecting telomere ends. In Oxytricha nova, telomere G-overhangs are protected by the TEBP-alpha/beta heterodimer. Human telomeres contain duplex telomeric repeats with 3' single-stranded G-overhangs, and may fold into a t-loop structure that helps to shield them from being recognized as DNA breaks. Additionally, the TEBP-alpha homologue, POT1, which binds telomeric single-stranded DNA (ssDNA), associates with multiple telomeric proteins (for example, TPP1, TIN2, TRF1, TRF2 and RAP1) to form the six-protein telosome/shelterin and other subcomplexes. These telomeric protein complexes in turn interact with diverse pathways to form the telomere interactome for telomere maintenance. However, the mechanisms by which the POT1-containing telosome communicates with telomerase to regulate telomeres remain to be elucidated. Here we demonstrate that TPP1 is a putative mammalian homologue of TEBP-beta and contains a predicted amino-terminal oligonucleotide/oligosaccharide binding (OB) fold. TPP1-POT1 association enhanced POT1 affinity for telomeric ssDNA. In addition, the TPP1 OB fold, as well as POT1-TPP1 binding, seemed critical for POT1-mediated telomere-length control and telomere-end protection in human cells. Disruption of POT1-TPP1 interaction by dominant negative TPP1 expression or RNA interference (RNAi) resulted in telomere-length alteration and DNA damage responses. Furthermore, we offer evidence that TPP1 associates with the telomerase in a TPP1-OB-fold-dependent manner, providing a physical link between telomerase and the telosome/shelterin complex. Our findings highlight the critical role of TPP1 in telomere maintenance, and support a yin-yang model in which TPP1 and POT1 function as a unit to protect human telomeres, by both positively and negatively regulating telomerase access to telomere DNA.

Telomere maintenance has been implicated in cancer and ageing, and requires cooperation between a multitude of telomeric factors, including telomerase, TRF1, TRF2, RAP1, TIN2, Tankyrase, PINX1 and POT1 (refs 1-12). POT1 belongs to a family of oligonucleotide-binding (OB)-fold-containing proteins that include Oxytricha nova TEBP, Cdc13, and spPot1, which specifically recognize telomeric single-stranded DNA (ssDNA). In human cells, the loading of POT1 to telomeric ssDNA controls telomerase-mediated telomere elongation. Surprisingly, a human POT1 mutant lacking an OB fold is still recruited to telomeres. However, the exact mechanism by which this recruitment occurs remains unclear. Here we identify a novel telomere protein, PTOP, which interacts with both POT1 and TIN2. PTOP binds to the carboxyl terminus of POT1 and recruits it to telomeres. Inhibition of PTOP by RNA interference (RNAi) or disruption of the PTOP-POT1 interaction hindered the localization of POT1 to telomeres. Furthermore, expression of the respective interaction domains on PTOP and POT1 alone extended telomere length in human cells. Therefore, PTOP heterodimerizes with POT1 and regulates POT1 telomeric recruitment and telomere length.

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 telomereassociated 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 (1, 2). 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 (3-5). 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 (6 -8). TRF1 may also regulate telomerase activity through its interaction with PINX1 (9). In comparison, TRF2 has an essential role in telomere end protection and t-loop formation (1, 10, 11). Interference of endogenous TRF2 activity by expressing dominant negative forms of TRF2 markedly increased the rate of telomere end-to-end fusions (12). 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 (13-15). 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 (16). 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 (17). In addition, chromosome end-to-end fusion was detected in TRF1 knock-out cells, indicating that telomere end protection was compr...

Mammalian telomeric proteins function through dynamic interactions with each other and telomere DNA. We previously reported the formation of a high-molecular-mass telomeric complex (the mammalian telosome) that contains the six core proteins TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 (formerly named PTOP͞PIP1͞ TINT1) and mediates telomere end-capping and length control. In this report, we sought to elucidate the mechanism of six-protein complex (or shelterin) formation and the function of this complex. Through reconstitution experiments, we demonstrate here that TIN2 and TPP1 are key components in mediating the six-protein complex assembly. We demonstrate that not only TIN2 but also TPP1 are required to bridge the TRF1 and TRF2 subcomplexes. Specifically, TPP1 helps to stabilize the TRF1-TIN2-TRF2 interaction and promote six-protein complex formation. Consistent with this model, overexpression of TPP1 enhanced TIN2-TRF2 association. Conversely, knocking down TPP1 reduced the ability of endogenous TRF1 to associate with the TRF2 complex. Our results suggest that coordinated interactions among TPP1, TIN2, TRF1, and TRF2 may ensure robust assembly of the telosome, telomere targeting of its subunits, and, ultimately, regulated telomere maintenance.protein complex ͉ telomere ͉ telosome M ammalian telomeres are regulated by the telomerase and telomeric proteins (1-6). Among the telomere-associated proteins important for mammalian telomere homeostasis, POT1 is likely the major regulator of telomere length control (7-9). POT1 binds the 3Ј G-rich telomere overhangs through its oligonucleotide-binding folds (7, 10, 11) and may regulate telomerase access (12)(13)(14). The telomere recruitment of POT1 thus constitutes an important step in telomere end-capping and length control. Recently, a new telomeric protein TPP1 (previously PTOP͞PIP1͞TINT1) was identified as a regulator of POT1 (9,15,16). The telomeric targeting of POT1 depends on its interaction with TPP1 (9). It remains to be determined how TPP1 interacts with other telomeric proteins and whether TPP1 has any function other than targeting POT1.In contrast to POT1, TRF1 and TRF2 directly bind doublestranded telomere DNA and interact with a number of proteins to maintain telomere structure and length (1-5). It has been shown that TRF1 counts and controls the length of telomere repeats, probably through its interaction with TIN2, Tankyrase, PINX1, TPP1, and POT1 (7,9,12,15,(17)(18)(19)(20)(21)(22)(23)(24). In comparison, TRF2 has an essential role in end protection and the telomeric recruitment of several proteins, including the BRCA1 Cterminal domain-containing protein RAP1, the nucleotide excision repair protein ERCC1͞XPF, BLM, and the DNA repair MRN complex (24-31). Because of their abilities to interact with multiple proteins, TRF1 and TRF2 are by definition hubs of protein-protein interaction at the telomeres (32). Recent studies have established multiple pairwise interactions among the six telomeric proteins (TRF1, TRF2, RAP1, TIN2, POT1, and TPP1), including the asso...

Proper maintenance of telomere length and structure is necessary for normal proliferation of mammalian cells. Mammalian telomere length is regulated by a number of proteins including human repressor activator protein (hRap1), a known association factor of TRF2. To further delineate hRap1 function and its associated proteins, we affinity-purified and identified the hRap1 protein complex through mass spectrometry analysis. In addition to TRF2, we found DNA repair proteins Rad50, Mre11, PARP1 (poly(ADP-ribose) polymerase), and Ku86/Ku70 to be in this telomeric complex. We demonstrated by deletional analysis that Rad-50/Mre-11 and Ku86 were recruited to hRap1 independent of TRF2. PARP1, however, most likely interacted with hRap1 through TRF2. Interestingly, knockdown of endogenous hRap1 expression by small hairpin interference RNA resulted in longer telomeres. In addition, overexpression of full-length and mutant hRap1 that lacked the BRCA1 C-terminal domain functioned as dominant negatives and extended telomeres. Deletion of a novel linker domain of hRap1 (residues 199 -223), however, abolished the dominant negative effect of hRap1 overexpression. These results indicate that hRap1 negatively regulates telomere length in vivo and suggest that the linker region of hRap1 may modulate the recruitment of negative regulators of telomere length.Telomere-binding proteins TRF1 and TRF2 play pivotal roles in telomere protection and maintenance in mammalian cells (1-5). Several proteins have been shown to associate with TRF1 and TRF2 (3,5,6). Recently, a novel telomere regulator human repressor activator protein (hRap1) 1 was identified as a protein that specifically interacts with TRF2 (7). hRap1 is the human homologue of yeast RAP1. In yeast, RAP1 is a negative regulator of telomere length as well as a regulator of transcription (for review, see Refs. 8 -13). The RAP1 mutants in Saccharomyces cerevisiae (scRAP1) and Schizosaccharomyces pombe (spRAP1) are defective in telomere length control and telomere position effects (14 -16). In human cells overexpression of hRap1 extends telomeres (7,17). It has yet to be fully determined, however, whether endogenous hRap1 is a negative or positive regulator of telomere length. scRAP1 contains two Myb domains and binds telomeric DNA (18,19). scRAP1 has been shown to recruit Rif1, Rif2, and Sir proteins to regulate telomere length, structure, and transcriptional silencing (11,20). Whether similar proteins, as found with yeast RAP1, are complexed with hRap1 remains unknown. In contrast, spRAP1 has only one Myb domain and is recruited to telomeres through Taz1, a yeast homologue of TRF2 (15). Similar to spRAP1, hRap1 also contains a single Myb domain without detectable DNA binding activity (7,21). In addition to the Myb domain, hRap1 has three putative protein-protein interaction domains; they are a BRCT domain, a coiled-coil domain, and a TRF2 interacting RCT domain (7). The function of the hRap1 BRCT and coiled-coil domains has yet to be described.TRF2 has been shown to associate with t...