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：
7222723
负责人：
JIAN-KANG ZHU
金额：
$ 27.45万
依托单位：
UNIVERSITY OF CALIFORNIA RIVERSIDE
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 2008-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7222723
关键词：
Affinity Animal Model Arabidopsis Base Excision Repairs Biochemical Biochemical Genetics Chromosomes Cloning Complex DNA DNA Methylation DNA Repair Pathway DNA glycosylase Development Developmental Process Double-Stranded RNA Epigenetic Process Eukaryota Eukaryotic Cell Excision Excision Repair Gene Expression Gene Proteins Gene Silencing Gene Targeting Genes Genetic Genome Genome Stability Genomic Imprinting Histone H3 Homologous Gene Hypermethylation In Vitro Lead Lyase Lysine Maps Mass Screening Methylation Molecular Mouse-ear Cress Mutate Mutation Pathway interactions Phenotype Plants Play Population Production Protein Overexpression Proteins RNA RNA Interference ROS1 gene Role Signal Transduction Site Small RNA Staging Suppressor Mutations System Testing Transgenes Transgenic Organisms Virus Work X Inactivation Yeasts base demethylation in vivo mutant novel prevent promoter response tandem mass spectrometry yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): DNA methylation and transcriptional gene silencing (TGS) play important roles in genome organization and stability, genomic imprinting, X chromosome inactivation and other developmental processes in eukaryotes. Little is known about the initial trigger for DNA methylation that is important for stable TGS, and in particular, the cellular mechanisms for the active suppression of TGS are not understood. We have developed a unique TGS system in the model organism Arabidopsis thaliana. In this system, an active transgene and a homologous endogenous gene become silenced when cellular ROS (repressor or silencing) factors are mutated. We have shown that ROS1 encodes a DNA glycosylase/lyase that prevents the hypermethylation and TGS of the homologous genes by active DNA demethylation via a base excision repair mechanism. We propose here to characterize the putative DNA demethylation activity of ROS1, to clone the ROS2 and ROS3 loci, to identify ROS1-interacting proteins, and to isolate and clone ros1 suppressor mutations. The mechanisms of active DNA demethylation have been very controversial, in part due to a critical lack of genetic evidence for the in vivo function of putative DNA demethylases. Our combined genetic and biochemical analysis of the ROS genes and proteins promises to provide unequivocal evidence that a base excision repair DNA glycosylase is an active demethylase, and will lead to the isolation of novel components of cellular pathways for the suppression of TGS.

描述（由申请人提供）：DNA甲基化和转录基因沉默（TGS）在基因组组织和稳定性，基因组烙印，X染色体灭活和真核生物的其他发育过程中起着重要作用。关于对稳定TG很重要的DNA甲基化的初始触发因素鲜为人知，尤其是不了解用于主动抑制TG的细胞机制。我们已经在模型有机体拟南芥中开发了独特的TGS系统。在该系统中，当细胞ROS（阻遏物或沉默）因子突变时，主动转基因和同源性内源基因会沉默。我们已经表明，ROS1通过通过碱基切除修复机制进行活性DNA脱甲基化来编码DNA糖基化酶/裂解酶/裂解酶，从而防止同源基因的高甲基化和TG。我们在这里提出要表征ROS1的假定DNA去甲基化活性，克隆ROS2和ROS3基因座，以鉴定ROS1相互作用的蛋白质，并分离和克隆ROS1抑制器突变。主动DNA脱甲基化的机制一直非常有争议，部分原因是缺乏假定DNA脱甲基酶体内功能的遗传证据。我们对ROS基因和蛋白质的遗传和生化分析有望提供明确的证据，即碱性切除修复DNA糖基化酶是一种活性去甲基酶，并将导致细胞途径的新成分以抑制TGS。