Suppression of genomic instability by tuning the DNA damage response at telomeres

项目摘要/摘要 端粒保护线性染色体的自然末端不被dna损伤识别。 应对机制。不足为奇的是，端粒过短或不适当的“封顶”是 基因组不稳定，并与早衰、血液系统恶性肿瘤和实体瘤有关 队形。因此，研究端粒是如何维持的以及异常的端粒是如何向DNA发出信号的 损伤反应，对于我们理解细胞转化是必不可少的。这项提议的目的是 是进一步定义调节端粒DNA损伤反应的机制，并定义缺陷如何 在这个过程中，会促进基因组的不稳定，并最终导致癌症的进展。这里描述的研究将 毫无疑问，这将进一步加深我们对癌症进展机制的了解，并将为 疾病诊断和/或治疗进步基金会。具体目标概述如下。 特殊目标1将结合生物化学和细胞生物学来了解人类是如何 POT1蛋白通过两种不同的功能保护端粒。在小鼠中，POT1分化为两个基因 编码mPOT1a和mPOT1b蛋白，每个蛋白在端粒末端保护中都具有独特的功能。在……里面 为此，我将使用小鼠mPOT1a和mPOT1b来确定这两种蛋白质在能力上的不同 具体地说，抑制ATR以及人类中单一的POT1蛋白如何完成这一功能。梳理出 人类POT1的功能域将使我们更好地了解POT1如何在端粒和 POT1的突变如何损害端粒末端保护并促进肿瘤的发生。 特定目标2将使用生物化学和细胞生物学的组合来确定TRF2如何 抑制ATM的端粒。这个目标的目的是从机械上理解TRF2是如何发挥作用的 抑制ATM的激活，最终保持基因组的稳定性。使用新的体内和体外测试，我将 验证TRF2通过拮抗DNA损伤感应器的结合而抑制ATM激活的假设 端粒DNA。TRF2是端粒长度维持和信号转导的关键因子，因此，剖析了TRF2在端粒长度维持和信号转导中的作用 ATM抑制中的TRF2不仅将促进我们目前对正常端粒如何维持的了解， 而且还有功能失调的端粒如何引发DNA损伤反应。我在这里提出的研究可能 阐明了端粒检查点是如何在癌症中被激发并随后被绕过的。 特定目标3将使用细胞生物学来研究非编码RNA Terra在调控中的作用 端粒的DNA损伤反应。Terra对维持基因组稳定至关重要，而且 人类癌症亚群中的下调表明Terra缺陷与端粒有关 功能障碍，最终导致细胞转化。这一目标的目的是找出导致 调节Terra的转录、降解和/或定位，并剖析Terra在 保持基因组的稳定。 尽管我最近接受了生物化学方面的培训，但我还需要1-2年的额外培训才能站稳脚跟 特别是作为一名端粒生物化学家。这是一个在端粒生物学领域被低估的利基市场， 有了额外的培训，我觉得我可以为这个领域做出实质性的贡献。作为一个独立的 调查员，我将把我的研究从全球DNA损伤和基因组维护调整到端粒 动态平衡和基因组维护。此外，我将继续从事癌症领域的研究 生物学，并将开始解决这项提案中概述的问题。进一步明确机制(S) 调节端粒稳定性将不可避免地导致对细胞转化的更好理解，并可能 最终提供亟需的治疗洞察力。我渴望剖析调节端粒的机制。 并将非常感谢有机会在K99的支持下进行这项研究 获奖。获得这一奖项不仅可以让我扩大我的研究计划，而且还可以确立我的地位 作为癌症生物学领域的主要研究员。

Project Summary/Abstract Telomeres protect the natural ends of linear chromosomes from recognition by the DNA damage response machinery. Not surprisingly, critically short or improperly 'capped' telomeres are major sources of genomic instability and have been linked to premature aging, hematological malignancies, and solid tumor formation. Thus, investigating how telomeres are maintained and how aberrant telomeres signal a DNA damage response, is essential to our understanding of cellular transformation. The objective of this proposal is to further define the mechanisms regulating the DNA damage response at telomeres and define how defects in this process promote genomic instability and ultimately, cancer progression. The studies described here will undoubtedly further our knowledge of the mechanisms involved in cancer progression and will lay the foundation for advances in disease diagnosis and/or treatment. The specific aims are outlined below. Specific Aim 1 will use a combination of biochemistry and cell biology to understand how the human POT1 protein protects telomere through two distinct functions. In mice, POT1 diverged into two genes encoding mPOT1a and mPOT1b proteins each possessing a unique function in telomere end protection. In this aim, I will use mouse mPOT1a and mPOT1b to determine how these two proteins differ in their ability to specifically repress ATR and how the single POT1 protein in humans accomplishes this function. Teasing out the functional domains in human POT1 will allow us to better understand how POT1 functions at telomeres and how mutations in POT1 may impair telomere end protection and contribute to tumorigenesis. Specific Aim 2 will use a combination of biochemistry and cell biology to determine how TRF2 represses ATM at telomeres. The goal of this aim is to understand mechanistically how TRF2 functions to inhibit ATM activation and ultimately, preserve genomic stability. Using new in vivo and in vitro assays, I will test the hypothesis TRF2 inhibits ATM activation by antagonizing binding of the DNA damage sensors at telomeric DNA. TRF2 is a key factor in telomere length maintenance and signaling, thus, dissecting the role of TRF2 in ATM inhibition will not only advance our current knowledge of how normal telomeres are maintained, but also how dysfunctional telomeres evoke a DNA damage response. The studies I have proposed here may shed light on how the telomere checkpoint is evoked and subsequently bypassed in cancers. Specific Aim 3 will use cell biology to investigate the role of the non-coding RNA TERRA in regulation of the DNA damage response at telomeres. TERRA is critical for maintaining genomic stability and is downregulated in a subset of human cancers suggesting that defects in TERRA contribute to telomere dysfunction and eventually, cellular transformation. The goal of this aim is to identify factors responsible for regulating the transcription, degradation, and/or localization of TERRA and to dissect the function of TERRA in maintaining genome stability. Despite my recent training in biochemistry, I will need 1-2 additional years of training to establish myself specifically as a telomere biochemist. This is a niche that is underrepresented in the field of telomere biology, and with additional training I feel I can make substantial contributions to the field. As an independent investigator, I will adapt my research from global DNA damage and genome maintenance, to telomere homeostasis and genome maintenance. In addition, I will continue to pursue research in the field of cancer biology and will begin to address the questions outlined in this proposal. Further defining the mechanism(s) regulating telomere stability will inevitably lead to a better understanding of cellular transformation and may ultimately provide much needed therapeutic insight. I am eager to dissect the mechanisms regulating telomere homeostasis and would greatly appreciate the opportunity to conduct this research with the support of a K99 award. Receipt of this award would not only allow me to expand my research plan, but also establish myself as a primary investigator in the field of cancer biology.