THE ROLE OF TELOMERASE REGULATORS IN TELOMERE MAINTENANCE AND GENOMIC INSTABILITY

项目摘要 哺乳动物染色体末端或端粒受到端粒酶的严格调控， 端粒延伸和端粒结合蛋白，其加帽并保护端粒末端。端粒DNA 通常采用封闭构象，由大量端粒结合蛋白质加帽和保护， 防止DNA损伤和基因组不稳定。端粒在DNA复制过程中变得开放和线性， 使端粒酶能够接近端粒延长。暴露的和短的端粒，由于 端粒酶和端粒调节因子的突变，也变得开放，容易受到损伤和基因组 不稳定，最终导致癌症。端粒结合蛋白和TERT启动子的突变 在许多癌症中被发现。大多数癌细胞具有上调的端粒酶表达， 活性，并且癌细胞似乎对端粒酶活性和端粒的扰动高度敏感 加帽，使得端粒酶对于治疗靶向具有吸引力。端粒酶的综合研究 因此，调节剂应该大大促进我们对癌症中端粒酶失调的理解， 发现新的药物靶点。我们建立了一个阵列式全基因组蛋白质相互作用网络 基于双分子荧光蛋白互补（BiFC）测定的筛选策略。一个试点 TERTBiFC筛选鉴定了几种蛋白质作为端粒酶复合物的关键组分，包括 我们将其命名为TARP 1，以前从未被鉴定过。我们建议在基因组范围内进行筛选 端粒酶的细胞周期依赖性调节剂，并检查其机制和功能， TARP 1-端粒酶复合物。我们也将使用由CRISPR/Cas9产生的诱导型TARP 1敲除细胞， 作为这些研究的小鼠异种移植模型。我们的工作将对制定有效的 用于由端粒功能障碍引起的癌症的治疗策略诱导基因组不稳定性。

Project Summary Mammalian chromosome ends or telomeres are tightly regulated by the telomerase that mediates telomere elongation and telomere-binding proteins that cap and protect telomere ends. Telomere DNA normally adopts a closed conformation, capped and protected by a multitude of telomere-binding proteins, to prevent DNA damage and genome instability. Telomeres become open and linear during DNA replication to enable telomerase access for telomere elongation. Exposed and critically short telomeres, as a result of mutations in telomerase and telomere regulators, also become open and susceptible to damage and genome instability, ultimately leading to cancer. Mutations in telomere-binding proteins and the TERT promoter have been identified in a number of cancers. Most cancer cells have up-regulated telomerase expression and activities, and cancer cells appear highly sensitive to perturbations in telomerase activities and telomere capping, making the telomerase attractive for therapeutic targeting. A comprehensive study of telomerase regulators therefore should greatly facilitate our understanding of telomerase dysregulation in cancer and the discovery of new drug targets. We have developed an arrayed whole-genome protein interaction network screening strategy based on the Bi-molecular Fluorescence Protein Complementation (BiFC) assay. A pilot TERT BiFC screen identified several proteins as key components of the telomerase complex, including a protein we named TARP1 that has never been characterized before. We propose here to screen genome wide for cell cycle-dependent regulators of the telomerase, and to examine the mechanisms and function of the TARP1-telomerase complex. We will use inducible TARP1 knockout cells generated by CRISPR/Cas9 as well as mouse xenograft models for these studies. Our work will have important implications in devising effective treatment strategies for cancers that result from telomere dysfunction induced genome instability.