Model systems for the investigation of DNA methylation and drug repurposing

描述（由申请人提供）：在本提案中，我们将使用两种生物模型来研究DNA甲基化，其在肿瘤发生中起关键作用，但在许多模型系统中不存在。我们利用的一个系统是纤毛类Oxytricha trifallax，它最近才被发现在一个显着的基因组重排过程中使用胞嘧啶甲基化。目的1从尖孢霉核裂解液中分离纯化一种新型甲基转移酶复合物，并对其作用机理进行研究。一个复杂的成员，DNA-PKcs同源物，在检测异常染色体和指导DNA甲基化的作用是一个特别的焦点。目的2是联合小鼠细胞系（10 T1/2）和Oxytricha筛选FDA批准的临床化合物，以鉴定具有甲基化调节特性的化合物。Oxytricha和小鼠10 T1/2细胞都提供了强大的DNA甲基化驱动的表型，允许快速筛选新的活性化合物。鉴于蠕虫、苍蝇和酵母中缺乏DNA甲基化，这些模型系统为快速科学进步以及药物筛选和再利用工作提供了理想的平台。近期和长期职业目标我的最终职业目标是成为一名创新的终身教职员工，为表观遗传学这一令人兴奋的领域的基础和临床研究做出重要贡献。这一目标的核心是从纯粹的基础研究转向以Oxytricha和其他模式系统的独特生物学为中心的基础和应用研究相结合。这项拟议的研究阐述了Oxytricha在临床和基础研究轨迹中的优势，并重新点燃了一种较老的小鼠细胞系10 T1/2，用于甲基化药物筛选，这是一种新的应用。这项工作将满足模型系统研究DNA甲基化与临床应用的深刻需求。为了过渡到一个独立的教师职位，我将立即进行具体目标1中所描述的基础研究，最终出版一份描述Oxytricha中新型甲基化机制的手稿。第二年将致力于进行甲基化抑制剂的化学筛选（目标2），并使用所得数据编写R 01资助申请。我希望 在第二年结束前完成筛选，第三年进行甲基化增强子筛选，并对有趣的命中，验证研究和手稿写作的作用机制进行后续研究。这个职业发展奖将允许必要的保护时间来建立出版物，并给予必要的支持，快速开始运行我自己的实验室。

DESCRIPTION (provided by applicant): In this proposal, we will use two biological models to investigate DNA methylation, which plays key roles in oncogenesis yet is absent in many model systems. One system we utilize is the ciliate Oxytricha trifallax, which was only recently discovered to use cytosine methylation in a remarkable genome rearrangement process. Aim 1 is a mechanistic investigation of a novel methyltransferase complex biochemically purified from Oxytricha nuclear lysate. The role of one complex member, a DNA-PKcs homolog, in detecting aberrant chromosomes and guiding DNA methylation is a particular focus. Aim 2 is a combined mouse cell line (10T1/2) and Oxytricha screen of FDA-approved clinical compounds to identify those with methylation-modulating properties. Both Oxytricha and mouse 10T1/2 cells provide robust DNA methylation-driven phenotypes, allowing rapid screening for novel active compounds. Given the lack of DNA methylation in worms, flies, and yeast, these model systems provide ideal platforms for rapid scientific advance and for drug screening and repurposing efforts. Immediate and Long-term Career Goals My ultimate career goal is to be an innovative tenure-track faculty member who makes important contributions to basic and clinical research in the exciting field of epigenetics. Central to this goal is a shift from purely basic research to a combination of basic and applied research centered on the unique biology of Oxytricha and other model systems. The proposed research elaborates the advantages of Oxytricha into both clinical and basic research trajectories, and also rekindles an older mouse cell line, 10T1/2, for methylation drug screening, a novel application. This work will meet a deep need for model systems to study DNA methylation with clinical applications. In order to transition into an independent faculty position, I will perform the basic research described in Specific Aim 1 immediately, culminating in the publication of a manuscript describing the novel methylation machinery in Oxytricha. Year 2 will be dedicated to performing the chemical screen for methylation inhibitors (Aim 2) and writing an R01 grant application with the resultant data. I hope to have the screen finished by the end of Year 2, leaving Year 3 for a methylation enhancer screen and follow-up on mechanisms of action of the interesting hits, validation studies, and manuscript writing. This Career Development Award will allow the protected time necessary to establish publication and grant support necessary for a fast start in running my own laboratory.