A critical role for TPP1 and TIN2 interaction in high-order telomeric complex assembly

Abstract

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...

Plk1 Phosphorylation of TRF1 Is Essential for Its Binding to Telomeres

“…Although the phosphorylation site in TRF1 identified here fails to exactly resemble the Plk1 consensus phosphorylation sequence (D/E)X(S/T)⌽ (X, any amino acid; ⌽, a hydrophobic amino acid) (21,35), the sequence context of TRF1-Ser-435 is similar to that identified in other substrates of Plk1 in our laboratory: ESSY 720 in Topors 3 and TASE 195 in CLIP-170. 4 Thus, an aspartic acid or glutamic acid at the ϩ1 or Ϫ1 position appears to be important for phosphorylation of substrates by Plk1.…”

“…We also observed that casein kinase 2 is a TRF1 kinase in vitro, and our mapping experiments indicated that Ser-435 is not the phos-3 X. Yang and X. Liu, unpublished data. 4 H. Li and X. Liu, unpublished data. C-E, Plk1 depletion reduces TRF1 binding to telomeres.…”

“…Although the TRF1 complex contains TRF1, TRF1 interacting partners, TIN2, TPP1, and POT1, the TRF2 complex contains TRF2 and its interacting partner RAP1 (1,4). TRF1 has been shown to negatively regulate telomere length.…”

“…Telomeric DNA together with specific and nonspecific telomere-binding proteins participates in forming highly ordered structures that protect the ends of chromosomes from exonucleolytic attack, end-to-end fusion, and degradation leading to cell death, possible genetic recombination, or survival selection (1,3,4). Most human normal somatic cells show a progressive loss of telomeric DNA during successive rounds of cell division due to incomplete DNA replication at the most terminal lagging-strand synthesis (4,5). Thus, telomere shortening functions as a control mechanism that regulates the replicative capacity of cells and cellular senescence (6).…”

G-quadruplex ligand SYUIQ-5 induces autophagy by telomere damage and TRF2 delocalization in cancer cells

“…A crucial way in which shelterin is thought to affect the structure of telomeric DNA is by forming t-loops. This complex is composed of six proteins including TRF1 (7,8), TRF2 (9), POT1 (10), TIN2 (11), TPP1 (12), and Rap1 (13). TRF2 binds directly to the tandem array of duplex TTAGGG repeats and coats the length of all human telomeres at all stages of the cell cycle (9,14).…”