Chromosome end protection in stem cells and development

端粒已经进化为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。对于“末端保护”至关重要的是庇护素亚基，TRF 2，其抑制DDR并促进T环形成以隔离端粒末端，从而将其隐藏于混杂的DNA修复。“末端保护”的重要性在去除TRF 2后被揭示，TRF 2触发DDR和由末端连接介导的快速端粒-端粒融合。虽然在许多体细胞类型中已经看到了对TRF 2的“末端保护”的依赖性，但我们最近发现TRF 2在干细胞和早期发育中具有“末端保护”作用。Trf 2-/-干细胞正常增殖并保持不受端粒融合的影响，但迅速转变为依赖TRF 2，以防止分化后的端对端融合和多能性丧失。这些发现挑战了当前的教条，并揭示了多能细胞和体细胞状态之间“末端保护”的根本差异。在这个ERC提案中，我们将利用遗传，蛋白质组学和超分辨率成像方法来解决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？为了进一步了解端粒的维持机制，我们还将利用我们最近在生物化学和单分子（SM）生物物理学方面的进展，在体外重建端粒。使用荧光标记的Shelterin，端粒酶和端粒相关因子，我们将询问1）T-环组装/拆卸，2）端粒酶募集到端粒，和3）在端粒的Shelterin-染色质相互作用的机制。我们的多学科方法将建立多能细胞中端粒末端保护的机制基础，并将提供实时和SM水平的端粒维持机制的前所未有的见解。

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.