Mechanism and Consequences of Telomere Dysfunction

描述(申请人提供)：端粒完整性对于哺乳动物细胞的生存和增殖非常重要。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，最终导致癌症。端粒酶亚单位或端粒调节器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。人类3‘悬垂结合蛋白POT1有助于保护端粒单链DNA，调节端粒酶通路。最近，实验室发现了一种新的端粒相关蛋白TPP1/PTOP(也称为PIP1/TINT1)。我们发现它直接与POT1和端粒酶相互作用。此外，TPP1与POT1、TRF1、TRF2、RAP1和TIN2一起形成了人类端粒。我们的发现表明，TPP1通过调节POT1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成分的失调如何导致端粒功能障碍和癌症的发展。这项建议的总体目标是了解TPP1在端粒保护和造血癌中的功能。我们推测，TPP1可能与POT1异源二聚，以保护端粒末端，并控制端粒酶的访问和活性。具体目的1.确定TPP1介导的端粒末端保护和长度控制的分子机制。(1)研究POT1-TPP1复合体在人类细胞中的动态组装、蛋白质稳定性和定位。我们将确定蛋白质的修饰，如泛素化，以及细胞周期调节的POT1-TPP1复合体的定位和组织。(2)TPP1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基因敲除、RNAi和显性负表达来确定。特异性目的2：阐明TPP1在调控端粒酶活性中的作用。TPP1如何与端粒酶相互作用并控制端粒酶活性，调节细胞周期依赖的端粒酶对端粒的访问将被研究。具体目标3：研究端粒功能障碍在发育和癌症中的后果。将产生造血系统与表达显性阴性TPP1和TPP1 shRNA的细胞重组的嵌合小鼠，并用于确定TPP1如何影响血液病的生存和发展。这项研究将有助于阐明TPP1在体内调节端粒维持的信号机制，并有助于深入了解TPP1在发育和端粒保护中的调控和机制，特别是在造血过程中。这项工作可能提供一幅关于细胞用来控制基因组稳定性的复杂信号网络的更精细的图景。此外，通过该项目可以确定癌症和衰老治疗机制驱动设计的有价值的目标。

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.