Chromosome end protection in stem cells and development

端粒的进化是为了保护线性染色体末端，防止它们降解并激活DNA损伤反应(DDR)途径。“末端保护”的关键是掩蔽素亚基TRF2，它抑制DDR并促进T环的形成，以隔离端粒末端，从而阻止DNA的混杂修复。TRF2的去除揭示了“末端保护”的重要性，它触发了DDR，并通过末端连接介导了快速的端粒-端粒融合。虽然在许多体细胞类型中都可以看到TRF2对“末端保护”的依赖性，但我们最近发现，在干细胞和早期发育中，TRF2对于“末端保护”是必不可少的。TRF2-/-干细胞正常增殖并保持不受端粒融合的影响，但在分化和丧失多能性后，迅速转换为依赖TRF2以防止端到端融合。这些发现挑战了当前的教条，并揭示了多能和体细胞状态之间在“末端保护”方面的根本差异。在这个ERC提案中，我们将利用遗传学、蛋白质组学和超分辨率成像方法来解决在多能细胞中如何实现末端保护，这与体细胞有何不同，以及为什么不同的细胞状态进化出不同的末端保护机制？为了加深我们对端粒维持机制的理解，我们还将利用我们在生物化学和单分子(SM)生物物理学方面的最新进展来重建体外端粒。利用荧光标记的保护素、端粒酶和端粒相关因子，我们将探讨1)T环组装/拆解，2)端粒酶重新聚集到端粒，以及3)保护素-染色质在端粒相互作用的机制。我们的多学科方法将在多能细胞中建立端粒末端保护的机制基础，并将提供前所未有的实时和SM水平的端粒维持机制。

Telomeres have evolved to protect linear chromosome ends, preventing them from degradation and activating DNA damage response (DDR) pathways. Essential for "end protection" is the Shelterin subunit, TRF2, which inhibits the DDR and facilitates T-loop formation to sequester the telomere end, thereby hiding it from promiscuous DNA repair. The essentiality of "end protection" is revealed upon removal of TRF2, which triggers the DDR and rapid telomere-telomere fusions mediated by end joining. While dependency of "end protection" on TRF2 has been seen in numerous somatic cell types, we recently discovered that TRF2 is dispensable for "end protection" in stem cells and early development. Trf2-/- stem cells proliferate normally and remain free from telomere fusions, but rapidly switch to become reliant on TRF2 to prevent end-to-end fusions upon differentiation and loss of pluripotency. These findings challenge current dogma and reveal fundamental differences in "end protection" between pluripotent and somatic cell states. In this ERC proposal, we will exploit genetic, proteomic and super-resolution imaging methods to address how end protection is achieved in pluripotent cells, how this differs from somatic cells, and why different cell states have evolved distinct end protection mechanisms? To further our understanding of telomere maintenance mechanisms, we will also exploit our recent advances in biochemistry and single molecule (SM) biophysics to reconstitute telomeres in vitro. Using fluorescently labelled Shelterin, telomerase and telomere-associated factors, we will interrogate the mechanism of 1) T-loop assembly/disassembly, 2) telomerase recruitment to telomeres, and 3) Shelterin-chromatin interactions at telomeres. Our multi-disciplinary approach will establish the mechanistic basis of telomere end protection in pluripotent cells and will provide unprecedented insight into telomere maintenance mechanisms in real-time and at a SM level.