Mechanism of the telomeric proliferation limit

Rockefeller University, New York NY

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Abstract

This long-term project is focused on replicative senescence, the process whereby telomere shortening limits the proliferation of human cells. In the past, our work on this project showed how telomeres are protected by shelterin and how loss of shelterin-mediated protection can induce senescence. Recently, we have focused more narrowly on aspects of telomere biology specifically relevant to replicative senescence of primary human cells. Our preliminary data has revealed that replicative senescence is induced by the activation of the ATM (not ATR) kinase at critically-short telomeres that have become deprotected because they lack sufficient TRF2. Furthermore, we have addressed the question of why cells grown at 3% (physiological) oxygen levels show an extended replicative life span compared to cultures in normoxia. In both conditions, ATM inhibition (ATMi) or overexpression of TRF2 resulted in life-span extension. Life-span extension at 3% oxygen was not due to a difference in telomere shortening rates or altered expression of TRF2 or ATM pathway factors. Instead, we found that the ATM kinase is less responsive to DSBs and damaged telomeres at 3% oxygen, leading to a greater tolerance for short telomeres and an extension of replicative life span. These observations form the basis for AIM 1, which is focused on further defining the mechanisms of proliferative senescence, understanding how ATM is regulated by oxygen levels, and determining what aspect of TRF2-mediated protection is lacking at critically-short telomeres. As part of our long-term interest in understanding how TRF2 protects telomeres, we have obtained preliminary data that formation of the t-loop, which represses ATM signaling, involves tetramerization of TRF2. We found that TRF2 binds to telomeric DNA as a tetramer whereas its paralog TRF1, which does not protect telomeres, binds as a dimer. A dimeric form of TRF2 is defective in telomere protection but could be partially restored by enforced tetramerization. Furthermore, an engineered TRF1 tetramer formed t-loops and protected telomeres in vivo. In AIM 2, we will further test the model that t-loop formation requires TRF2 tetramers. We recently reported that the highly conserved iDDR domain of TRF2, which binds to the Rad50 subunit of the Mre11/Rad50/Nbs1 (MRN) complex, prevents MRN from associating with CtIP and thus blocks it from becoming an active endonuclease. Interestingly, the iDDR is functionally (but not structurally) similar to MRN inhibitory modules in yeast telomeric proteins, pointing to convergent evolution. Our preliminary data suggest that the iDDR also interferes with the ability of MRN to active the ATM kinase. In AIM 3, we will determine the mechanism of ATM inhibition and explore the physiological role of the iDDR, seeking to explain its evolutionary conservation. Together, these experiments will provide insights into the ATM kinase, its regulation, and its repression by TRF2, revealing mechanistic insights into how shortening telomeres limit the proliferation of human cells.