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

描述（由申请人提供）：端粒保护线性染色体的天然末端不被DNA损伤反应机制识别。毫不奇怪，非常短或不适当的“加帽”端粒是基因组不稳定性的主要来源，并与过早衰老，血液恶性肿瘤和实体瘤形成有关。因此，研究端粒如何维持以及异常端粒如何发出DNA损伤反应的信号，对我们理解细胞转化至关重要。该提案的目的是进一步确定调节端粒DNA损伤反应的机制，并确定该过程中的缺陷如何促进基因组不稳定性并最终导致癌症进展。本文所述的研究无疑将进一步加深我们对癌症进展机制的认识，并为疾病诊断和/或治疗的进展奠定基础。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来了解人类POT 1蛋白如何通过两种不同的功能保护端粒。在小鼠中，POT 1分化为两个编码mPOT 1a和mPOT 1b蛋白的基因，每个蛋白在端粒末端保护中具有独特的功能。在这个目标中，我将使用小鼠mPOT 1a和mPOT 1b来确定这两种蛋白质在特异性抑制ATR的能力方面有何不同，以及人类中的单一POT 1蛋白质如何实现这一功能。梳理出人类POT 1的功能结构域将使我们能够更好地了解POT 1如何在端粒中发挥作用，以及POT 1的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来确定TRF 2如何在端粒抑制ATM。该目标的目的是从机制上了解TRF 2如何发挥作用以抑制ATM激活并最终保持基因组稳定性。使用新的体内和体外试验，我将测试的假设TRF 2抑制ATM激活拮抗结合的DNA损伤传感器在端粒DNA。TRF 2是端粒长度维持和信号传导的关键因素，因此，剖析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知识，而且还将推进功能失调的端粒如何引起DNA损伤反应的知识。我在这里提出的研究可能会阐明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将使用细胞生物学研究非编码RNA TERRA在调节端粒DNA损伤反应中的作用。TERRA对于维持基因组稳定性至关重要，并且在人类癌症的一个子集中下调，这表明TERRA中的缺陷有助于端粒功能障碍并最终导致细胞转化。该目标的目的是确定负责调节TERRA的转录、降解和/或定位的因子，并剖析TERRA在维持基因组稳定性中的功能。 尽管我最近接受了生物化学方面的培训，但我还需要1-2年的培训才能使自己成为一名端粒生物化学家。这是一个在端粒生物学领域代表性不足的利基，通过额外的培训，我觉得我可以为这个领域做出实质性的贡献。作为一个独立的研究者，我将调整我的研究从全球DNA损伤和基因组的维护，端粒稳态和基因组的维护。此外，我将继续从事癌症生物学领域的研究，并将开始解决本提案中概述的问题。进一步定义调节端粒稳定性的机制将不可避免地导致更好地理解细胞转化，并最终提供急需的治疗见解。我渴望剖析调节端粒稳态的机制，并将非常感谢有机会在K99奖的支持下进行这项研究。获得这个奖项不仅使我能够扩大我的研究计划，而且还确立了自己在癌症生物学领域的主要研究者地位。 公共卫生相关性：端粒保护线性染色体的自然末端不被识别为DNA损伤位点。鉴于DNA损伤是基因组不稳定性的主要来源，本提案的目的是进一步剖析调节基因组不稳定性的机制。 端粒的维持以及这一过程中的缺陷如何促进与癌症发展相关的基因组不稳定性。

DESCRIPTION (provided by applicant): 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 ma 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. PUBLIC HEALTH RELEVANCE: Telomeres protect the natural ends of linear chromosomes from being recognized as sites of DNA damage. Given that DNA damage is a major source of genomic instability, the objective of this proposal is to further dissect the mechanisms regulating telomere maintenance and how defects in this process promote the genomic instability associated with cancer development.