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：
8607867
负责人：
John Russell Bracht
金额：
$ 5.39万
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/8607867
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 deep sequencing design drug development endonuclease human disease human tissue inhibitor/antagonist innovation insight micronucleus new therapeutic target novel prevent programs promoter public health relevance 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.

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，对哺乳动物发育过程中沉默基因很重要，而癌细胞经常通过异常甲基甲基启动子来使肿瘤抑制基因沉默。当DNA甲基化模式在癌症或发育性疾病中破坏DNA甲基化模式时，基因组稳定性将受到损害。尽管DNA甲基化在基因表达和基因组稳定性中的关键作用，但指导从头DNA甲基化的机制仍然很差。 Trifallax是一种单细胞真核生物，在发育过程中执行戏剧性的基因组重排，该过程将其微核（MIC）基因组转化为分化的大核（MAC）。通过精确消除95％的基因组来完成此过程，并概括了干细胞的关键特性：自我更新（始终保留了MIC的一副副本）和分化（MAC形成）。有趣的是，我在Landweber实验室中的工作表明，这种生物在消除过程中使用了从头DNA甲基化。大多数氧曲基因组是甲基化和消除的事实，使其成为了解甲基化靶向方式的有吸引力的模型系统：异常的甲基化和随后消除5％保留的DNA是致命的。此外，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联系的独特模型，这两者通常与癌症和发育过程有关。目标1：检验以下假设：通过对人工构建的体外甲基化染色体的微分注射，甲基化诱导氧气中的DNA降解，进入了营养细胞的MAC。 AIM 2：将使用甲基-DNA免疫沉淀和高吞吐量测序（MEDIP-SEQ）来定义oxytricha基因组重排期间从头DNA甲基化的内源性结构域。 AIM 3：通过使用化学抑制剂的甲基转移酶的化学抑制剂，然后对治疗细胞的DNA进行深层测序，分析了氧基因组重排中胞嘧啶甲基化的功能相关性。 AIM 4：通过甲基 - 环蛋白染色质IP（ME-CHIP）和质谱分析鉴定新型甲基化途径蛋白。 oxytricha trifallax提供了一个前所未有的机会，可以研究DNA甲基化在发展出精细的基因组重排过程的模型系统中的作用。目前，对人类疾病中从头甲基化的靶向知之甚少，因此来自oxytricha的见解将立即与人类健康和疾病的研究有关。在纤毛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潜在的致癌作用。