Epigenetic regulation of programmed genome instability in O. trifallax

三法螟编程基因组不稳定性的表观遗传调控

基本信息

批准号：
8397434
负责人：
John Russell Bracht
金额：
$ 5.22万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8397434
关键词：
Aberrant DNA Methylation Antibodies Artificial Chromosomes Binding Proteins Biological Assay Biological Models Cancer Model Candidate Disease Gene Cells Chemicals Chromatin Chromosomes Cytosine DNA DNA Methylation DNA Sequence DNA Sequence Rearrangement Data Development Developmental Process Disease Epigenetic Process Eukaryota Exonuclease Fellowship Gene Expression Gene Silencing Genome Genome Stability Genomic Instability Goals Health Human Immunofluorescence Immunologic Immunoprecipitation In Vitro Injection of therapeutic agent Laboratories Left Link Macronucleus Malignant Neoplasms Methylation Methyltransferase Microinjections Minor Modeling Mus Oncogenic Organism Oxytricha Pathway interactions Pattern Process Property Proteins Protocols documentation Publications RNA Interference Regulation Reproducibility Research Role Spectrophotometry Stem cells Study models System Techniques Testing Time Tumor Suppressor Genes Variant Work cancer cell cancer initiation chromatin immunoprecipitation crosslink design drug development endonuclease human disease human tissue inhibitor/antagonist innovation insight micronucleus new therapeutic target novel prevent programs promoter research study self-renewal tissue culture

项目摘要

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.

描述（由申请人提供）：DNA 的胞嘧啶甲基化是一种抑制性染色质标记，对于哺乳动物发育过程中的基因沉默非常重要，并且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。当癌症或发育性疾病中 DNA 甲基化模式被破坏时，基因组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发挥着关键作用，但引导 DNA 从头甲基化的机制仍不清楚。 Oxytricha trifallax 是一种单细胞真核生物，在将其微核 (MIC) 基因组转变为分化的大核 (MAC) 的发育过程中进行剧烈的基因组重排。这一过程是通过精确消除 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 MIC 的一个副本）和分化（MAC 形成）。有趣的是，我在 Landweber 实验室的工作表明，这种生物体在消除过程中使用从头 DNA 甲基化。大多数尖毛虫基因组都被甲基化并被消除，这一事实使其成为了解甲基化如何靶向的有吸引力的模型系统：异常甲基化和随后 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 甲基化和基因组稳定性之间联系的独特模型，这两者通常与癌症和发育过程相关。目标 1：通过将人工构建的体外甲基化染色体显微注射到营养细胞的 MAC 中，检验甲基化诱导尖毛虫 DNA 降解的假设。目标 2：甲基 DNA 免疫沉淀和高通量测序 (meDIP-Seq) 将用于定义尖毛虫基因组重排过程中 DNA 从头甲基化的内源域。目标 3：将通过使用甲基转移酶的化学抑制剂来分析尖毛虫基因组重排中胞嘧啶甲基化的功能相关性，然后对处理细胞的 DNA 进行深度测序。目标 4：通过甲基胞嘧啶染色质 IP (me-ChIP) 和质谱分析鉴定新型甲基化途径蛋白。三毛尖毛虫为研究 DNA 甲基化在模型系统中的作用提供了前所未有的机会，该模型系统已经进化出了复杂的基因组重排过程。目前，人类疾病中从头甲基化的靶向作用尚不清楚，因此来自 Oxytricha 的见解将立即与人类健康和疾病的研究相关。纤毛虫模型中鉴定出的任何保守候选蛋白或基序都可以在人体组织培养物或小鼠癌症模型中测试其潜在的致癌作用。公共卫生相关性：异常 DNA 甲基化会导致多种人类发育疾病，并与基因组不稳定、癌症发生和致癌进展相关。然而，DNA 甲基化靶向正确序列的机制仍然不清楚。拟议研究的目标是利用纤毛虫的独特特性来发现治疗药物开发的新靶点。最终，纠正 DNA 甲基化错误的能力将使我们能够预防或治疗人类疾病。