Genetics of DNA methylation patterning of arabidopsis.

拟南芥 DNA 甲基化模式的遗传学。

• 批准号: 8402659
• 负责人: STEVEN E JACOBSEN
• 金额: $ 28.77万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8402659
• 关键词: Adopted; Alleles; Animal Model; Arabidopsis; Binding; Biological Assay; Caenorhabditis elegans; Cancer Biology; Cancer Etiology; Chemicals; Chromatin; Chromosomes; Cytosine; DNA; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Sequence; DNA-Binding Proteins; Defect; Developmental Biology; Drosophila genus; Epigenetic Process; Eukaryota; Feedback; Gene Expression Regulation; Gene Silencing; Generations; Genes; Genetic; Genetic Screening; Genetic screening method; Genome; Genomic Imprinting; Genomics; Grant; Health; Histone H3; Histones; Homologous Gene; Hypermethylation; Inverted Repeat Sequences; Lead; Light; Maintenance; Mammals; Methods; Methylation; Methyltransferase; Modification; Molecular Genetics; Mouse-ear Cress; Mutation; Organism; Pattern; Plant Model; Plants; Play; Process; Proteins; Recruitment Activity; Regulator Genes; Research; Role; Series; Site; Small Interfering RNA; Small RNA; Specificity; Study models; System; Tissues; Tumor Suppressor Genes; Work; X Inactivation; Yeasts; abstracting; demethylation; gene function; genetic analysis; genome-wide; histone methyltransferase; human disease; imprint; insight; interest; loss of function; mutant; positional cloning; prevent; research study; tool; tumor

DESCRIPTION (provided by applicant): Project Summary/Abstract Cytosine DNA methylation is an epigenetic mark for gene silencing that is important in many gene regulatory systems including mammalian imprinting, X-chromosome inactivation, and the silencing of transposons and other DNA sequences containing either direct or inverted repeats. Methylation is important in cancer biology, as tumors often show both genome wide demethylation and hypermethylation of specific tumor suppressor genes. We are studying the mechanisms of DNA methylation control in the model plant Arabidopsis, which has methylation systems that have much in common with mammalian systems. Both forward and reverse genetics can be performed, and Arabidopsis can tolerate mutations that virtually eliminate methylation, allowing for detailed analysis. In Arabidopsis, the initial establishment of methylation requires the activity of DRM2, a homolog of mammalian Dnmt3, which is guided to appropriate targets, at least in part, by small interfering RNAs. Once established, DNA methylation is maintained by three different systems for methylation of cytosines in three different sequence contexts, CG, CHG and CHH (asymmetric). CG methylation is maintained by MET1, a homolog of mammalian Dnmt1. CHG methylation is maintained by the CMT3 DNA methyltransferase, which is guided to appropriate targets by the methylation of histone H3. CHH methylation is maintained by the persistent activity of DRM2, which is targeted by small interfering RNAs. In this proposal, we plan to further study these DNA methylation control mechanisms. First, we plan to study the poorly understood process by which DNA methylation is initially established, a process called de novo methylation. We have developed assays that allow us to perform both reverse and forward genetic screens to identify genes required for proper de novo DNA methylation. These mutant screens have already yielded several interesting genes, and a major part of this proposal is to perform a detailed molecular genetic characterization of these genes to understand more about the mechanism by which they contribute to de novo methylation. We also propose to continue additional genetic screens, and to characterize a set of 35 new mutants we have isolated that block de novo methylation. The second major aim of this proposal is to further study the mechanisms by which CG DNA methylation is maintained, by studying a new factor called UHRF1 in mammals and ORTH/VIM in Arabidopsis, which appears to work by binding directly to previously methylated CG sites and recruiting the CG methyltransferase to further maintain methylation. We propose to study the function of six Arabidopsis ORTH/VIM genes by using loss of function genetic alleles, understanding the specificity of chromatin interactions for each gene, and performing molecular and genetic tests of the function of the different domains of each protein. These studies should shed new light on DNA methylation systems in Arabidopsis and have implications for related systems in mammals and other eukaryotes.

描述（由申请人提供）： 项目摘要/摘要胞嘧啶DNA甲基化是基因沉默的表观遗传标记，在许多基因调节系统中都很重要，包括哺乳动物烙印，X染色体失活以及转座子和其他包含直接或反向重复的DNA序列的沉默。甲基化在癌症生物学中很重要，因为肿瘤通常表现出特定肿瘤抑制基因的基因组广泛的脱甲基化和高甲基化。我们正在研究模型植物拟南芥中DNA甲基化控制的机制，该植物具有与哺乳动物系统有很多共同点的甲基化系统。可以进行前进和反向遗传学，拟南芥可以忍受几乎消除甲基化的突变，从而详细分析。在拟南芥中，最初的甲基化建立需要DRM2的活性，DRM2的活性是一种哺乳动物DNMT3的同源物，它至少通过小型干扰RNA被引导到适当的靶标。一旦建立，DNA甲基化将通过三种不同的系统在三种不同的序列上下文中（CG，CHG和CHH（不对称））维持。 CG甲基化由Met1（哺乳动物DNMT1的同源物）维持。 CMT3 DNA甲基转移酶维持CHG甲基化，该甲基转移酶通过组蛋白H3的甲基化引导到适当的靶标。 CHH甲基化由DRM2的持续活性维持，DRM2的持续活性是由小的干扰RNA靶向的。在此提案中，我们计划进一步研究这些DNA甲基化控制机制。首先，我们计划研究最初建立DNA甲基化的知识的过程，这是一种称为从头甲基化的过程。我们开发了使我们能够执行反向和正向遗传筛选的测定方法，以鉴定适当的从头DNA甲基化所需的基因。这些突变筛选已经产生了几个有趣的基因，该建议的主要部分是对这些基因进行详细的分子遗传表征，以更多地了解它们为从头甲基化做出贡献的机制。我们还建议继续其他遗传筛选，并表征一组35个新突变体，我们已经隔离了从头甲基化。该提案的第二个主要目的是进一步研究CG DNA甲基化的机制，通过研究哺乳动物中的一种称为UHRF1的新因素，在拟南芥中的Orth/VIM来维持CG DNA甲基化，该因子似乎通过直接与先前甲基化的CG位点结合并募集CG甲基转移酶来进一步维持甲基化。我们建议通过使用功能遗传等位基因的丧失来研究六个拟南芥Orth/VIM基因的功能，了解每个基因的染色质相互作用的特异性，并对每种蛋白质不同结构域功能的分子和遗传测试进行分子和遗传测试。这些研究应为拟南芥中的DNA甲基化系统提供新的启示，并对哺乳动物和其他真核生物的相关系统产生影响。