TIN2-Tethered TPP1 Recruits Human Telomerase to Telomeres In Vivo

Abstract

Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase; however, the molecular basis for recruitment is not known. Here, we have directly investigated the process of telomerase recruitment via fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (ChIP). We find that depletion of two components of the shelterin complex that is found at telomeres-TPP1 and the protein that tethers TPP1 to the complex, TIN2-results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Deletion of the oligonucleotide/oligosaccharide binding fold (OB-fold) of TPP1 disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere.

Replication of Telomeres and the Regulation of Telomerase

“…At the telomere, Tpp1 and Tin2 are important for telomerase recruitment or its retention, as depletion of either of these factors reduces (but does not abolish) telomerase association with telomeres (Abreu et al 2010). Depletion and rescue studies further identified the aminoterminal OB-fold of Tpp1 as important for telomerase recruitment (Abreu et al 2010), a domain that was found to also interact with telomerase in extracts (Xin et al 2007).…”

“…Depletion and rescue studies further identified the aminoterminal OB-fold of Tpp1 as important for telomerase recruitment (Abreu et al 2010), a domain that was found to also interact with telomerase in extracts (Xin et al 2007). Whether telomerase recruitment is regulated in mammals by Atm or Atr, as in fission and budding yeast, is unknown, nor is it clear if in addition to Tpp1 other factors contribute to recruitment.…”

A quantitative telomeric chromatin isolation protocol identifies different telomeric states

“…For DNA extraction, the QIAquick PCR Purification kit (Qiagen) was used. Telomeric and Alu-repeat DNA were detected successively 37 . Telomeric DNA was detected using a probe 4 that was generated as follows: a template mixture of 1-5 kb long telomeric DNA fragments was synthesized by ligating double-stranded telomeric DNA oligonucleotides ((TTAGGG) 5 , (CCCTAA) 5 ) that were amplified by PCR.…”

Phosphorylation of TPP1 regulates cell cycle-dependent telomerase recruitment

“…It has been reported that the TPP1-POT1 complex can recruit and stimulate telomerase activity through direct interaction between TPP1 and telomerase. This interaction depends on the OB fold of TPP1 (15)(16)(17)(18)20). Recent studies have identified the residues that are required for the interface of TPP1-telomerase interaction (20)(21)(22).…”

“…Of the six proteins, TPP1 contains an N-terminal oligonucleotide-and oligosaccharide-binding (OB) fold and is tethered to telomeres through interactions with telomere DNA binding proteins TRF1, TRF2, and POT1 (13,14). Work from us and others has shown that the TPP1 OB fold interacts with the telomerase and is required for telomere recruitment of telomerase and high telomerase processivity (15)(16)(17)(18)(19). Recently, key residues that define the interface for human TPP1 OB foldtelomerase interaction have been identified (20)(21)(22).…”