Mechanisms of Telomere Cohesion

项目摘要/摘要 端粒是位于染色体末端的特殊结构，由TTAGGG重复序列、端粒重复序列组成。 含有RNA(Terra)和谢尔特林蛋白复合体。姐妹染色单体从一开始就保持在一起 在S期复制，直到有丝分裂时被粘附素环分离。环介导的凝聚力是 在有丝分裂中确保染色体准确分配到子细胞所必需的。姐妹之间的凝聚力 对重组和修复也很重要，特别是在像端粒这样的重复序列中，它保持在 他们对齐了。为此，需要更多的亲密接触(除了粘附环)，因此，有 是端粒特有的凝聚力要求。端粒凝聚力的建立需要保护 亚基和相关蛋白质。端粒间凝聚力的解析需要PARP，tankyrase。 在S/G2细胞中，Tankyrase通过与TRF1亚基中的TTAGGG-Repeat结合屏蔽而定位于端粒 来解决凝聚力问题。在Tankyrase缺失的细胞中，姐妹端粒在有丝分裂中保持粘连，尽管正常 武器和着丝粒的分辨率。这种持久的端粒凝聚力不仅仅是一种由 Tankyrase耗尽，它自然发生在某些类型的人类细胞中，这些细胞缺乏端粒酶，并具有严重的 端粒短小：正常衰老细胞和癌症ALT细胞。出乎意料的是，(我们的实验室在过去的几个月中显示了这一点 这种持久的凝聚力对细胞是有益的；它起到了保护作用，防止过早衰老 衰老的细胞和ALT癌细胞的生长停滞。我们未来五年的研究目标是阐明 建立和分解凝聚力所需的蛋白质和机制。我们将在之前的基础上继续发展 我们在工作中确定了亚单位和相关因素中的庇护，这是凝聚力所必需的，我们将 关注我们最新的发现，表明RNA(Terra和Terra R-Loop)在端粒中的作用 凝聚力。我们将确定端粒组件如何有助于建立、维持和 正常人类细胞、衰老和癌症中端粒凝聚力的解析。为了建立凝聚力，我们将 确定所需蛋白质在端粒上的沉积方式和时间，以及进入染色体的程度 它们延伸开来。对于凝聚力解析，我们将确定如何招募tankyrase来解析凝聚力。 我们将对tankyrase本身招募到端粒的蛋白质进行表征，并研究tankyrase如何使用 功能划分，以协调解决过程。我们将确定Terra RNA和 Terra R环有助于正常细胞的端粒凝聚力，并有助于 衰老和ALT癌症的病理条件。最后，我们将阐明凝聚体的完整蛋白质组 端粒状态。最终，对端粒凝聚力的完整理解将阐明基本的 正常人类细胞中染色体稳定性和基因组完整性的机制，并为 防止衰老细胞的过早衰老和促进ALT癌细胞的过早死亡。

Project Summary/Abstract Telomeres, the specialized structures at chromosome ends, are comprised of TTAGGG repeats, telomere repeat containing RNA (TERRA), and the shelterin protein complex. Sister chromatids are held together from the time of their replication in S phase until their separation in mitosis by cohesin rings. Ring-mediated cohesion is essential to ensure accurate distribution of chromosomes to daughter cells in mitosis. Cohesion between sisters is also important for recombination and repair, particularly at repetitive sequences like telomeres, where it keeps them aligned. For this, more intimate contacts (in addition to the cohesin ring) are needed and as such, there are telomere-specific requirements for cohesion. Establishment of cohesion at telomeres requires shelterin subunits and associated proteins. Resolution of cohesion between telomeres requires the PARP, tankyrase. Tankyrase localizes to telomeres by binding to the TTAGGG-repeat binding shelterin subunit TRF1, in late S/G2 to resolve cohesion. In tankyrase-depleted cells sister telomeres remain cohered in mitosis despite normal resolution of arms and centromeres. This persistent telomere cohesion is not just an aberrant state induced by depletion of tankyrase, it occurs naturally in certain human cell types that lack telomerase and have critically short telomeres: normal aged cells and cancer ALT cells. Unexpectedly, (and shown by our lab in the last few years) this persistent cohesion is beneficial to cells; it serves a protective role to prevent premature senescence in aged cells and growth arrest in ALT cancer cells. The goal of our research for the next five years is to elucidate the proteins and mechanisms required for establishment and resolution of cohesion. We will build on our previous work where we identified shelterin subunits and associated factors that are required for cohesion and we will focus on our most recent discovery indicating a role for RNA (TERRA and TERRA R-Loops) in telomere cohesion. We will determine how the telomeric components contribute to the establishment, maintenance, and resolution of telomere cohesion in normal human cells, aging, and cancer. For cohesion establishment, we will determine how and when the required proteins are deposited on telomeres and how far into the chromosome they extend. For cohesion resolution, we will determine how recruitment of tankyrase serves to resolve cohesion. We will characterize the proteins that tankyrase itself recruits to telomeres and investigate how tankyrase uses functional compartmentalization to orchestrate the resolution process. We will determine how TERRA RNA and TERRA R-loops contribute to telomere cohesion in normal cells and to persistent telomere cohesion in pathological conditions of aging and ALT cancer. And finally, we will elucidate the full proteome of the cohered telomeric state. Ultimately, a complete understanding of telomere cohesion will elucidate fundamental mechanisms of chromosome stability and genome integrity in normal human cells and provide insights into prevention of premature senescence in aging cells and into promotion of premature death in ALT cancer cells.