DNA Demethylation and Transcriptional Gene Silencing

DNA 去甲基化和转录基因沉默

• 批准号: 7466563
• 负责人: JIAN-KANG ZHU
• 金额: $ 30.12万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7466563
• 关键词: Adenosine; Animal Model; Base Excision Repairs; Biochemical; Chromatin Structure; Complex; DNA; DNA Methylation; DNA glycosylase; Data; Development; Disease; Environment; Enzymes; Epigenetic Process; Functional RNA; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Genome Stability; Genomic Imprinting; Genomics; Goals; Histones; Homologous Gene; Hypermethylation; Investigation; Lyase; Malignant Neoplasms; Methylation; Methyltransferase; Modification; Mouse-ear Cress; Mutate; Nucleic Acids; Numbers; Pathway interactions; Pattern; Phosphotransferases; Physiological; Play; Property; Proteins; RNA; RNA Binding; RNA-Binding Proteins; ROS1 gene; Reaction; Regulation; Research; Role; Small Interfering RNA; Small RNA; Sulfur; Sulfur Metabolism Pathway; System; Testing; Transgenes; Variant; demethylation; in vivo; mutant; prevent; promoter

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 regulation, 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, small RNAs and histone modification patterns have been studied extensively. In contrast, the mechanism of active DNA demethylation and its relation with small RNAs and histone modifications are poorly understood. Our long-term goal is to understand the mechanism of active DNA demethylation and its role in epigenetic regulation. We have developed a unique system in the model organism Arabidopsis thaliana for dissecting DNA demethylation and other anti-silencing mechanisms. In this system, an active transgene and a homologous endogenous gene become silenced when cellular ROS (repressor of silencing) factors are mutated. ROS1 encodes a DNA glycosylase/lyase that prevents the hypermethylation of specific loci by active DNA demethylation through a base excision repair pathway. ROS2 encodes an adenosine phosphosulfate (APS) kinase, an enzyme involved in sulfur metabolism, which together with other data implicates sulfur modification of nucleic acids or proteins in preventing DNA hypermethylation. ROS3 encodes a 24-nt small RNA-binding protein that also functions in preventing DNA hypermethylation. We hypothesize that ROS1 functions in a DNA demethylase complex that includes ROS3 and other regulatory components, and small RNAs bound to ROS3 may guide the demethylase complex for locus-specific demethylation. To test this hypothesis, we propose the following studies: 1) Continue to characterize the biochemical properties of ROS1, investigate the physiological function of ROS1 and other related demethylases in environment-triggered DNA methylation changes, determine the mechanism of regulation of ROS1 transcription by genome methylation status, and test the function of ROS1-interacting proteins; 2) Investigate the mechanism of ROS2 and ROS3 function in preventing DNA hypermethylation; and 3) Characterize the ros4 and ros5 mutants and isolate the ROS4 and ROS5 genes, which may encode additional components of the DNA demethylation pathway.

描述（由申请人提供）：由DNA甲基化，组蛋白修饰和组蛋白变体引起的表观遗传变化在调节染色质结构和基因组稳定性，发育特定的特定创伤调节，基因组烙印，转录和转录沉默的转录基因和其他异物DNA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。通过广泛研究了通过甲基转移酶和DNA甲基化，小RNA和组蛋白修饰模式之间的相互作用的DNA甲基化。相反，对活性DNA脱甲基化的机制及其与小的RNA和组蛋白修饰的关系知之甚少。我们的长期目标是了解主动DNA脱甲基化的机制及其在表观遗传调节中的作用。我们已经在模型有机体拟南芥中开发了一个独特的系统，用于剖析DNA脱甲基化和其他抗沉降机制。在该系统中，当细胞ROS（沉默的阻遏物）因子突变时，主动转基因和同源性内源基因会沉默。 ROS1编码DNA糖基化酶/裂解酶，该糖基化酶/裂解酶通过通过碱基切除修复途径通过活性DNA脱甲基来防止特异性基因座的过度甲基化。 ROS2编码参与硫代谢的酶的腺苷磷酸硫酸盐（APS）激酶，它与其他数据一起暗示核酸或蛋白质的硫修饰在防止DNA高甲基化方面。 ROS3编码一个24-NT小的RNA结合蛋白，该蛋白还起作用，可预防DNA高甲基化。我们假设ROS1在包括ROS3和其他调节成分在内的DNA脱甲基酶配合物中起作用，与ROS3结合的小RNA可能引导脱甲基酶复合物以特异性脱甲基化。为了检验这一假设，我们提出了以下研究：1）继续表征ROS1的生化特性，研究ROS1和其他相关脱甲基酶在环境触发的DNA甲基化变化中的生理功能，确定ROS1转录的调节机制，并通过基因组甲基化状态，并测试Ros1 in-Ros1-Inintract蛋白质蛋白质的功能； 2）研究ROS2和ROS3在预防DNA高甲基化方面的机制； 3）表征ROS4和ROS5突变体，并分离ROS4和ROS5基因，该基因可能编码DNA去甲基化途径的其他成分。