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.

We thank Susan Smith for kindly providing us with the TIN2 antibodies and Peter Baumann and Titia de Lange for POT1 antibodies. We are grateful to David Hall (University of Georgia) for overseeing statistical analysis.

This work was supported by a grant from the National Cancer Institute (RO1 CA104676) to M.P.T. and R.M.T. and by a Swiss National Science Foundation grant, the European Community's Seventh Framework Programme FP7/2007-2011 (grant agreement number 200950), and a European Research Council advanced investigator grant (grant agreement number 232812) to J.L. Eladio Abreu was supported by an NIH (NRSA) Predoctoral Fellowship Award To Promote Diversity in Health-Related Research (F31GM087949).