Mechanism and Consequences of Telomere Dysfunction

描述（由申请人提供）：端粒完整性对哺乳动物细胞存活和增殖很重要。端粒磨损或暴露的染色体末端可能导致基因组不稳定，DNA损伤反应，并最终导致癌症。端粒酶亚单位或端粒调节子的突变与过早衰老和癌症有关。因此，了解端粒维持的细胞机制可能有助于开发包括癌症在内的疾病的治疗方法。人3'突出端结合蛋白POT 1有助于保护端粒ssDNA并调节端粒酶进入。最近，该实验室发现了一种新的端粒相关蛋白TPP 1/PTOP（也称为PIP 1/TINT 1）。我们发现它直接与POT 1和端粒酶相互作用。此外，TPP 1与POT 1、TRF 1、TRF 2、RAP 1和TIN 2沿着形成人端粒体。我们的研究结果表明，TPP 1通过调节POT 1和端粒酶的端粒募集来控制端粒长度。长期目标是研究端粒组分的失调如何导致端粒功能障碍和癌症发展。本提案的总体目标是了解TPP 1在端粒保护和造血系统癌症中的功能。我们推测TPP 1可能与POT 1异源二聚体化以保护端粒末端，并控制端粒酶的进入和活性。具体目标1。探讨TPP 1介导端粒末端保护和长度调控的分子机制。(1)将研究POT 1-TPP 1复合物在人细胞中的动态组装、蛋白质稳定性和定位。我们将确定蛋白质修饰，如泛素化，和细胞周期调节的POT 1-TPP 1复合物的定位和组织。(2)TPP 1在端粒末端保护、细胞存活和细胞周期检查点激活中的作用将通过敲除、RNAi和显性负表达来确定。目的二：阐明TPP 1在端粒酶活性调控中的作用。TPP 1是如何与端粒酶相互作用并调控端粒酶活性，调节细胞周期依赖的端粒酶进入端粒的，这一点有待进一步研究。具体目标3：研究端粒功能障碍在发育和癌症中的后果。将产生造血系统用表达显性阴性TPP 1和TPP 1 shRNA的细胞重建的嵌合小鼠，并用于确定TPP 1如何影响造血存活和造血癌症的发展。本提案中描述的研究应有助于阐明TPP 1在体内调节端粒维持的信号机制，并提供对TPP 1在发育和端粒保护中，特别是在造血过程中的调节和机制的见解。这项工作可能会提供一个更精确的图片复杂的信号网络，细胞用来控制基因组的稳定性。此外，有价值的目标机制驱动的癌症和衰老治疗的设计可能会通过这个项目确定。

DESCRIPTION (provided by applicant): Telomere integrity is important for mammalian cell survival and proliferation. Telomere attrition or exposed chromosomal ends could result in genome instability, DNA damage response, and ultimately cancer. Mutations in telomerase subunits or telomere regulators have been linked to premature aging and cancer. Understanding the cellular mechanisms of telomere maintenance may therefore help in developing therapies for diseases including cancer. The human 3' overhang binding protein POT1 helps to protect the telomere ssDNA and regulate telomerase access. Recently, the lab identified a new telomere associated protein TPP1/PTOP (also known as PIP1/TINT1). We found it to directly interact with POT1 and the telomerase. Furthermore, TPP1 forms the human telosome, along with POT1, TRF1, TRF2, RAP1, and TIN2. Our findings suggest that TPP1 controls telomere length by regulating telomere recruitment of POT1 and telomerase. The long-term goal is to study how dysregulation of telosome components may lead to telomere dysfunction and cancer development. The overall objective of this proposal is to understand the function of TPP1 in telomere protection and hematopoietic cancer. We hypothesize that TPP1 may heterodimerize with POT1 to protect telomere ends, and control telomerase access and activity. Specific Aim 1. To determine the molecular mechanism of TPP1-mediated telomere end protection and length control. (1) The dynamic assembly, protein stability, and localization of the POT1-TPP1 complex in human cells will be investigated. We will determine protein modifications such as ubiquitination, and the cell cycle regulated localization and organization of the POT1-TPP1 complex. (2) The role of TPP1 in telomere end protection, cell survival, and cell cycle checkpoint activation will be determined through knockout, RNAi, and dominant negative expression. Specific Aim 2: To elucidate the role of TPP1 in controlling telomerase activity. How TPP1 interacts with the telomerase and controls telomerase activity, regulates cell cycle-dependent telomerase access to telomeres will be studied. Specific Aim 3: To investigate the consequences of telomere dysfunction in development and cancer. Chimeric mice whose hematopoietic systems are reconstituted with cells expressing dominant-negative TPP1 and TPP1 shRNA will be generated, and used to determine how TPP1 affects hematopoietic survival and development of hematopoietic cancers. The studies described in this proposal should help to delineate the signaling mechanisms by which TPP1 regulates telomere maintenance in vivo, and provide insight into the regulation and mechanisms of TPP1 in development and telomere protection, especially during hematopoiesis. This work may provide a more refined picture about the complex signaling networks that are employed by the cells to control genome stability. Furthermore, valuable targets for mechanism-driven design of cancer and aging therapeutics may be identified through this project.