Epigenetic regulation of programmed genome instability in O. trifallax

描述（由申请人提供）：DNA的胞嘧啶甲基化是一种抑制染色质标记，对哺乳动物发育过程中的基因沉默很重要，癌细胞经常通过异常甲基化其启动子来沉默肿瘤抑制基因。当DNA甲基化模式在癌症或发育性疾病中被破坏时，基因组的稳定性就会受到损害。尽管DNA甲基化在基因表达和基因组稳定性中起着关键作用，但指导从头DNA甲基化的机制仍然不明确。三叶草氧tricha trifallax是一种单细胞真核生物，在其微核（MIC）基因组向分化大核（MAC）转变的发育过程中进行了戏剧性的基因组重排。这个过程是通过精确地消除95%的基因组来完成的，并概括了干细胞的关键特性：自我更新（始终保留一个MIC拷贝）和分化（MAC形成）。有趣的是，我在兰德韦伯实验室的工作表明，这种生物体在消除过程中使用了从头DNA甲基化。大多数Oxytricha基因组被甲基化和消除的事实使其成为一个有吸引力的模型系统，以了解甲基化是如何靶向的：异常甲基化和随后的5%保留DNA的消除将是致命的。此外，这种纤毛虫基因组重排的可重复性使其成为研究DNA甲基化与基因组稳定性之间关系的独特模型，这两者通常与癌症和发育过程有关。目的1：通过将人工构建的体外甲基化染色体微注射到营养细胞的MAC中，验证甲基化诱导氧化毛霉DNA降解的假设。目的2：甲基DNA免疫沉淀和高通量测序（meDIP-Seq）将用于确定Oxytricha基因组重排过程中从头DNA甲基化的内源性结构域。目的3：利用甲基转移酶的化学抑制剂分析胞嘧啶甲基化在氧化毛霉基因组重排中的功能相关性，然后对处理细胞的DNA进行深度测序。目的4：通过甲基胞嘧啶染色质IP （me-ChIP）和质谱分析鉴定新的甲基化途径蛋白。三叶草提供了一个前所未有的机会来研究DNA甲基化在一个模型系统中的作用，这个模型系统已经进化出了一个复杂的基因组重排过程。目前，人们对人类疾病中新生甲基化的靶向性知之甚少，因此来自Oxytricha的见解将立即与人类健康和疾病的研究相关。在纤毛虫模型中发现的任何保守的候选蛋白或基序都可以在人类组织培养或小鼠癌症模型中测试潜在的致癌作用。

DESCRIPTION (provided by applicant): Cytosine methylation of DNA is a repressive chromatin mark important for silencing genes during mammalian development, and cancer cells frequently silence tumor suppressor genes by aberrantly methylating their promoters. Genome stability is compromised when DNA methylation patterns are disrupted in cancer or in developmental disease. Despite the critical role of DNA methylation in gene expression and genome stability, the mechanisms guiding de novo DNA methylation remain poorly defined. Oxytricha trifallax is a unicellular eukaryote that performs dramatic genome rearrangements in a developmental process that transforms its micronucleus (MIC) genome into a differentiated macronucleus (MAC). This process is accomplished by the precise elimination of 95% of the genome, and recapitulates the key property of stem cells: self-renewal (one copy of the MIC is retained at all times) and differentiation (MAC formation). Interestingly, my work in the Landweber lab has shown that this organism uses de novo DNA methylation in the elimination process. The fact that most of the Oxytricha genome is methylated and eliminated makes it an attractive model system to understand how methylation is targeted: aberrant methylation and subsequent elimination of the 5% retained DNA would be fatal. In addition, the reproducibility of genome rearrangements in this ciliate makes it a unique model for studying the connection between DNA methylation and genome stability, both of which are relevant to cancer and developmental processes generally. AIM 1: Test the hypothesis that methylation induces DNA degradation in Oxytricha through microinjection of artificially constructed, in vitro methylated chromosomes into the MAC of vegetative cells. AIM 2: Methyl-DNA immunoprecipitation and high throughput sequencing (meDIP-Seq) will be used to define the endogenous domains of de novo DNA methylation during genome rearrangements in Oxytricha. AIM 3: The functional relevance of cytosine methylation in Oxytricha genome rearrangements will be analyzed by use of chemical inhibitors of methyltransferases, followed by deep sequencing the DNA from treated cells. AIM 4: Identification of novel methylation pathway proteins by methyl-cytosine chromatin IP (me-ChIP) and mass-spec analysis. Oxytricha trifallax provides an unprecedented opportunity to study the role of DNA methylation in a model system that has evolved an elaborate genome rearrangement process. Currently, the targeting of de novo methylation in human disease is poorly understood, so insights from Oxytricha will be immediately relevant to studies of human health and disease. Any conserved candidate proteins or motifs identified in the ciliate model can be tested in human tissue culture or mouse cancer models for potential oncogenic roles. PUBLIC HEALTH RELEVANCE: Aberrant DNA methylation causes several human developmental diseases and is correlated with genome instability, cancer initiation, and oncogenic progression. However the mechanism by which DNA methylation is targeted to the correct sequences remains obscure. The goal of the proposed research is to use the unique properties of ciliates to uncover new targets for therapeutic drug development. Ultimately, the ability to correct errors of DNA methylation will allow us to prevent or treat human disease.