Mechanisms of Telomere End Protection by TRF2

Author

Audrey Goldfarb

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Agata Smogorzewska

Abstract

Shelterin is a six-subunit protein complex that protects the ends of mammalian chromosomes, called telomeres. The six subunits of shelterin, TRF1, TRF2, TIN2, TPP1, POT1, and Rap1, each have distinct roles in telomere homeostasis. These include telomere length regulation and preventing telomeres from activating various DNA damage response (DDR) signaling and double-strand break repair pathways. The need to suppress these DDR pathways at telomeres is known as the “end protection problem.” The TRF2 subunit of shelterin is responsible for suppressing the ATM DNA damage response kinase, which is activated by exposed DNA ends. TRF2 is also necessary and sufficient for forming t-loops, lariat-like structures that sequester the ends of telomeric DNA. In the absence of TRF2, t-loops are lost, ATM is activated, and telomeres fuse together via cNHEJ. While t-loops have been presented as an elegant solution to the end protection problem, it is yet unproven whether it is the t-loop itself that suppresses the DNA damage response, or whether other activities of TRF2 are equally or more important for end protection. There is evidence for the importance of both. The loss of t-loops coincides with ATM activation, suggesting an essential role for t-loops in ATM suppression. However, some work suggests that TRF2 can suppress ATM and the double-strand break repair pathway cNHEJ via t-loop[1]independent mechanisms. The ultimate proof that t-loops are necessary and sufficient for telomere end protection requires engineering of a TRF2-independent t-loop. This was previously unachievable because thus far, the mechanism of t-loop formation by TRF2 has been unknown. Since the discovery of t-loops 2.5 decades ago, the mechanism of t-loop formation has been a major focus in the field of telomere biology. Here, we propose a mechanism for t-loop formation by TRF2 and generate TRF2-independent t-loops in order to answer these longstanding questions. We first show that TRF2 possesses two dimerization domains and binds telomeric DNA as a tetramer. TRF2 has one dimerization domain in its TRFH that was discovered previously, and we identify a second one formed by regions in its Hinge and Myb domains. Tetramerization is necessary for t-loop formation by TRF2, as a TRF2 mutant lacking part of its second dimerization domain properly localizes to telomeres but is deficient in suppressing ATM and cNHEJ. The ability of this mutant to suppress ATM and cNHEJ was improved by artificial tetramerization. We demonstrate the sufficiency of tetramerization for t-loop formation by showing that TRF1, a dimeric protein that normally does not form t-loops and plays no role in end protection, forms t-loops when induced to tetramerize. Using this system to create t-loops in the absence of TRF2, we show that t-loops themselves are sufficient to block both ATM signaling and cNHEJ. Alt-EJ, however, was not suppressed in this system, indicating that TRF2 suppresses alt-EJ via a mechanism other than tetramerization and t-loop formation. We also investigate TRF2’s other means of protecting chromosome ends and show that the iDDR, a region within TRF2’s Hinge domain, suppresses both ATM signaling and the 53BP1 recruitment downstream of ATM signaling. This likely occurs via two independent mechanisms, one in which the iDDR inhibits ATM through an interaction with MRN, and another in which it recruits additional factors that disrupt the accumulation of 53BP1 downstream of ATM signaling. Additionally, Rap1, a shelterin component brought to the telomere by TRF2, suppresses cNHEJ. This may occur through binding of Rap1’s BRCT domain to Ku, blocking the interaction with Ligase 4 that is required for the completion of cNHEJ. TRF2 employs several mechanisms to suppress ATM and cNHEJ, including t-loop formation, thereby protecting chromosome ends from aberrant DNA damage signaling and repair. We propose that for most of the cell cycle, t-loops serve as the main mechanism of end protection by TRF2. Directly after leading-end DNA replication, the ends of the telomere must be resected to form the 3’ overhang before t-loops can form. During this resection process and delay in t-loop formation, we propose that the iDDR and Rap1 serve to suppress ATM and cNHEJ. Together, our results describe the intricate, elegant, and often redundant mechanisms of end protection by the essential and multifaceted shelterin component, TRF2.

Comments

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

Recommended Citation

Goldfarb, Audrey, "Mechanisms of Telomere End Protection by TRF2" (2024). Student Theses and Dissertations. 806.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/806