DNA Demethylation and Transcriptional Gene Silencing

DNA 去甲基化和转录基因沉默

基本信息

批准号：
8371550
负责人：
JIAN-KANG ZHU
金额：
$ 33.96万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-05-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8371550
关键词：
Aberrant DNA Methylation Affinity Chromatography Animal Model Arabidopsis Base Excision Repairs Biochemical Chromatin Structure DNA DNA Ligases DNA Markers DNA Methylation DNA Methylation Regulation DNA Repair DNA glycosylase DNA-Directed DNA Polymerase Deoxyribonucleases Development Disease Enhancers Environment Enzymes Epigenetic Process Equilibrium Functional RNA Funding Gene Expression Gene Silencing Genes Genetic Screening Genome Genome Stability Genomic Imprinting Goals Histones Homologous Gene Hypermethylation Knowledge Lyase Malignant Neoplasms Methylation Methyltransferase Modeling Molecular Mouse-ear Cress Mutate Pathway interactions Pattern Phosphoric Monoester Hydrolases Plant Extracts Play Proteins RNA RNA-Binding Proteins ROS1 gene Reaction Regulation Regulatory Pathway Repair Complex Reporter Research Role Ros protein Small Interfering RNA Small RNA System Testing Transgenes United States National Institutes of Health Variant Work active control base demethylation histone modification mutant novel prevent protein complex repaired response tool

项目摘要

DESCRIPTION (provided by applicant): Epigenetic changes caused by DNA methylation, histone modifications, and histone variants play crucial roles in the regulation of chromatin structure and genome stability, developmental specific gene expression, genomic imprinting, and transcriptional silencing of transgenes and other foreign DNA. Cancers and many other diseases are associated with epigenetic alterations. The status of DNA methylation is determined by both methylation and demethylation reactions. DNA methylation by methyltransferase enzymes and the interplay between DNA methylation, non-coding RNAs, and histone modification patterns have been studied extensively. In contrast, the mechanism of active DNA demethylation and its relation with non-coding RNAs and histone modifications are less well understood. Our long-term goal is to understand the dynamic regulation of DNA methylation and its role in development and in responses to the environment. We have developed a unique system in the model organism Arabidopsis thaliana for dissecting active DNA demethylation and RNA-directed DNA methylation (RdDM). In this system, an active transgene (RD29A-LUC) and a homologous endogenous gene become silenced when cellular ROS (repressor of silencing) factors are mutated. ROS1 encodes a 5-methylcytosine-specific DNA glycosylase/lyase that prevents the hypermethylation and silencing of specific loci by initiating a base excision repair pathway for active DNA demethylation. ROS3 encodes a small non-coding RNA-binding protein that regulates active DNA demethylation. We have also discovered several new components of the RdDM pathway using ros1 suppressor mutants, and we have developed a new genetic screen to identify DNA hypermethylation mutants. Our working model is that the RD29A-LUC transgene and some endogenous loci are dynamically regulated by RdDM and active DNA demethylation pathways, and that these marker loci can serve as powerful tools for discovering new components of the DNA methylation and demethylation pathways, i.e., these markers can be used to screen Arabidopsis mutants for altered DNA methylation and expression. This renewal will vigorously test our working model by using the mutant screens and biochemical approaches to discover additional enzymes in the active DNA demethylation pathway and regulatory factors of the pathway. The proposed work will advance new concepts and fill major gaps in our understanding of the dynamic control of DNA methylation, and thus will contribute significantly to elucidating the molecular basis of epigenetic regulation. PUBLIC HEALTH RELEVANCE: Cancers and many other diseases are associated with aberrant DNA methylation and histone modification patterns, but the mechanistic basis of these epigenetic changes is poorly understood. Supported by NIH funding, we have broken new grounds in understanding the control of DNA methylation by discovering a DNA glycosylase-based mechanism of active DNA demethylation and its regulation by non-coding RNAs. The research proposed here will continue to generate exciting new knowledge on the mechanisms and function of active DNA demethylation.

描述（由申请人提供）：由DNA甲基化，组蛋白修饰和组蛋白变异引起的表观遗传变化在调节染色质结构和基因组稳定性，发育特异性基因表达，基因组烙印以及转录基因和其他异物DNA的转录沉默中起着至关重要的作用。癌症和许多其他疾病与表观遗传改变有关。 DNA甲基化的状态是通过甲基化和脱甲基反应确定的。已经广泛研究了通过甲基转移酶和DNA甲基化，非编码RNA和组蛋白修饰模式之间的相互作用的DNA甲基化。相反，活性DNA脱甲基化的机制及其与非编码RNA和组蛋白修饰的关系不太了解。我们的长期目标是了解DNA甲基化的动态调节及其在发育和对环境的反应中的作用。我们已经在模型生物拟南芥中开发了一个独特的系统，用于剖析活性DNA脱甲基化和RNA指导的DNA甲基化（RDDM）。在该系统中，当细胞ROS（沉默的阻遏物）因子突变时，活性转基因（RD29A-LUC）和同源性内源基因会沉默。 ROS1编码5-甲基环胞嘧啶特异性的DNA糖基酶/裂解酶，该酶/裂解酶通过启动碱基切除修复途径以进行主动DNA脱甲基化，从而防止特定基因座的高甲基化和沉默。 ROS3编码一个小的非编码RNA结合蛋白，该蛋白调节活性DNA脱甲基化。我们还使用ROS1抑制剂突变体发现了RDDM途径的几个新组件，并且我们开发了一种新的遗传筛选来鉴定DNA高甲基化突变体。我们的工作模型是，RD29a-luc转基因和某些内源基因座受RDDM和主动DNA脱甲基化途径的动态调节，并且这些标记基因座可以用作发现DNA甲基化的新组件的强大工具，以发现这些标记和脱甲基化途径，即这些标记可用于筛选拟南西的替代品和甲基化的DNA甲基化DNA甲基化的DNA甲基化DNA。这种更新将通过使用突变筛选和生化方法来大力测试我们的工作模型，以发现活性DNA脱甲基途径和途径的调节因子中的其他酶。提出的工作将在我们对DNA甲基化的动态控制方面提高新概念并填补主要空白，因此将显着促进表观遗传调节的分子基础。公共卫生相关性：癌症和许多其他疾病与异常的DNA甲基化和组蛋白修饰模式有关，但是这些表观遗传变化的机械基础知之甚少。在NIH资助的支持下，我们通过发现基于DNA糖基化酶的活性DNA脱甲基化机制及其通过非编码RNA的调节来理解对DNA甲基化的控制。此处提出的研究将继续为主动DNA脱甲基化的机制和功能产生令人兴奋的新知识。