Student Theses and Dissertations

Jill Donigian

Date of Award

2008

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

de Lange Laboratory

Abstract

Tankyrase1 is a multifunctional poly(ADP-ribose) polymerase that can localize to telomeres through its interaction with the shelterin component TRF1. Tankyrase1 poly(ADP-ribosyl)ates TRF1 in vitro, and its nuclear overexpression leads to loss of TRF1 and telomere elongation, suggesting that tankyrase1 is a positive regulator of telomere length. In agreement with this proposal, we showed that tankyrase1 RNA interference results in telomere shortening proportional to the level of knockdown, while a tankyrase1-resistant form of TRF1 enforced normal telomere length control. Thus, in human cells, tankyrase1 appears to act upstream of TRF1, promoting telomere elongation through the removal of TRF1. This pathway appears absent from mouse cells. We demonstrated that murine TRF1, which lacks the tankyrase1-binding motif, is not a substrate for tankyrase1 poly(ADP-ribosyl)sylation in vitro. Furthermore, overexpression of tankyrase1 in mouse nuclei did not remove TRF1 from telomeres and had no detectable effect on other components of mouse shelterin. We propose that the tankyrase1-controlled telomere extension is a human-specific elaboration that allows additional control over telomere length in telomerase positive cells. TIN2 interacts with the double-stranded telomeric DNA-binding proteins TRF1 and TRF2 independently or simultaneously, acting as a bridge linking TRF1 and TRF2 to TPP1 and POT1, the single-stranded telomeric DNA-binding protein. To gain further insight into the function of the TRF2-TIN2 complex, we created a TRF2 mutant that no longer associates with TIN2. Employing protein overlay assays, we established that TIN2 binds TRF2 within its hinge domain from residues 352 to 367. Deletion of this region led to the production of a TRF2 TIN2-binding mutant, TRF2ΔT, which abrogated TRF2-TIN2 binding in protein overlay assays and in immunoprecipitation analysis. Expression of TRF2ΔT in MEFs that contain a conditionally null allele of TRF2 resulted in substantial loss of TIN2 from telomeres, the formation of telomere dysfunction induced foci (TIFs), and the appearance of multiple telomeric signals and telomere loss at chromatid ends. We show that the ATM signaling pathway is activated in response to the telomere dysfunction induced by loss of TIN2 from the TRF2 complex, suggesting that TIN2 assists TRF2 in suppressing ATM activation at telomeres.

Comments

A Thesis Presented to the Faculty of The Rockefeller University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

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