Model systems for the investigation of DNA methylation and drug repurposing

描述(申请人提供)：在这项提案中，我们将使用两个生物学模型来研究DNA甲基化，DNA甲基化在肿瘤发生中发挥关键作用，但在许多模型系统中缺失。我们使用的一个系统是纤毛虫Oxytricha trifalax，它最近才被发现在一个引人注目的基因组重排过程中使用胞嘧啶甲基化。目的1是对一种新的甲基转移酶复合体的机理进行研究，该复合体是从Oxytricha核裂解物中生化纯化的。一个复合体成员，DNA-PKcs同源物，在检测异常染色体和指导DNA甲基化中的作用是一个特别的焦点。AIM 2是一种组合的小鼠细胞系(10T1/2)和Oxytricha筛选FDA批准的临床化合物，以鉴定那些具有甲基化调节特性的化合物。Oxytricha和小鼠10T1/2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努力提供了理想的平台。近期和长期的职业目标我的最终职业目标是成为一名创新的终身教职教员，为令人兴奋的表观遗传学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单纯的基础研究转向以Oxytricha和其他模型系统的独特生物学为中心的基础研究和应用研究的结合。这项拟议的研究阐述了Oxytricha在临床和基础研究中的优势，并重新点燃了一种较老的小鼠细胞系10T1/2，用于甲基化药物筛选，这是一种新的应用。这项工作将满足对模型系统的深入需求，以研究具有临床应用的DNA甲基化。为了过渡到一个独立的教员职位，我将立即进行特定目标1中描述的基础研究，最终出版一份描述Oxytricha中新的甲基化机制的手稿。第二年将致力于进行甲基化抑制剂的化学筛选(目标2)，并用结果数据编写R01拨款申请。我希望 在第二年年底前完成筛选，将第三年留给甲基化增强剂筛选和对有趣的命中、验证研究和手稿写作的作用机制的后续研究。这一职业发展奖将允许有必要的保护时间来建立出版物，并为快速开始运营我自己的实验室提供必要的支持。

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.