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

项目总结/摘要 端粒保护线性染色体的自然末端免受DNA损伤的识别 反应机制。毫不奇怪，极短或不适当的“帽”端粒是主要来源， 基因组不稳定，并与过早衰老、血液恶性肿瘤和实体瘤有关 阵因此，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，对我们理解细胞转化至关重要。本提案的目的 是进一步确定调节端粒DNA损伤反应的机制， 在这个过程中促进了基因组的不稳定性，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认识，并将为我们的癌症研究奠定基础。 疾病诊断和/或治疗进展的基础。具体目标概述如下。 具体目标1将使用生物化学和细胞生物学的组合，以了解人类如何 POT 1蛋白通过两种不同的功能保护端粒。在小鼠中，POT 1分化成两个基因， 编码mPOT 1a和mPOT 1b蛋白，每种蛋白在端粒末端保护中具有独特的功能。在 为了达到这个目的，我将使用小鼠mPOT 1a和mPOT 1b来确定这两种蛋白质在它们的能力上是如何不同的， 特异性抑制ATR以及人类中的单个POT 1蛋白如何实现这一功能。梳理出 人类POT 1的功能域将使我们能够更好地了解POT 1在端粒中的功能， POT 1突变如何损害端粒末端保护并促进肿瘤发生。 具体目标2将使用生物化学和细胞生物学的组合，以确定如何TRF 2 在端粒抑制ATM。这一目标的目的是了解TRF 2的功能机制， 抑制ATM活化并最终保持基因组稳定性。使用新的体内和体外试验，我将 测试TRF 2通过拮抗DNA损伤传感器的结合来抑制ATM激活的假设， 端粒DNA TRF 2是端粒长度维持和信号传导的关键因子，因此，剖析TRF 2在端粒长度维持和信号传导中的作用。 TRF 2在ATM抑制中的作用不仅将推进我们目前对正常端粒如何维持的认识， 以及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被诱发并随后在癌症中被绕过的。 具体目标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DNA损伤反应。TERRA对于维持基因组稳定性至关重要， 在人类癌症的一个子集中下调，表明TERRA的缺陷有助于端粒的形成。 功能障碍并最终导致细胞转化这一目标的目的是确定造成这一现象的因素。 调节TERRA的转录、降解和/或定位，并剖析TERRA在 维持基因组的稳定性。 尽管我最近接受了生物化学方面的培训，但我还需要1-2年的培训来确立自己的地位 尤其是作为一个端粒生物化学家。这是一个在端粒生物学领域代表性不足的利基， 我相信，通过进一步的培训，我可以为这个领域做出重大贡献。作为一个独立 调查员，我将调整我的研究从全球DNA损伤和基因组维护，端粒 稳态和基因组维持。此外，我将继续从事癌症领域的研究 生物学，并将开始解决这个建议中概述的问题。进一步界定机制 调节端粒稳定性将不可避免地导致更好地理解细胞转化， 最终提供急需的治疗见解。我渴望剖析端粒的调控机制 稳态，并将非常感谢有机会进行这项研究的支持下，K99 奖获得这个奖项不仅使我能够扩大我的研究计划，而且还建立了自己的地位。 作为癌症生物学领域的主要研究者。

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.