Genetics of DNA methylation patterning of arabidopsis.

描述（由申请人提供）： 胞嘧啶DNA甲基化是基因沉默的表观遗传标记，在许多基因调控系统中很重要，包括哺乳动物印记，X染色体失活，转座子和其他含有直接或反向重复的DNA序列的沉默。甲基化在癌症生物学中是重要的，因为肿瘤通常表现出基因组范围的去甲基化和特定肿瘤抑制基因的超甲基化。我们正在模式植物拟南芥中研究DNA甲基化控制的机制，拟南芥的甲基化系统与哺乳动物系统有很多共同之处。正向和反向遗传学都可以进行，拟南芥可以容忍几乎消除甲基化的突变，从而允许进行详细的分析。在拟南芥中，甲基化的初始建立需要DRM 2的活性，DRM 2是哺乳动物Dnmt 3的同源物，其至少部分地通过小干扰RNA被引导到适当的靶标。一旦建立，DNA甲基化由三种不同序列背景中的胞嘧啶甲基化的三种不同系统维持，CG、CHG和CHH（不对称）。CG甲基化由MET 1维持，MET 1是哺乳动物Dnmt 1的同源物。CHG甲基化由CMT 3 DNA甲基转移酶维持，CMT 3 DNA甲基转移酶通过组蛋白H3的甲基化被引导至适当的靶标。CHH甲基化由DRM 2的持续活性维持，DRM 2被小干扰RNA靶向。在本提案中，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DNA甲基化最初建立的鲜为人知的过程，这一过程称为从头甲基化。我们已经开发了检测方法，使我们能够进行反向和正向遗传筛选，以确定正确的从头DNA甲基化所需的基因。这些突变体筛选已经产生了几个有趣的基因，该提案的主要部分是对这些基因进行详细的分子遗传学表征，以了解更多关于它们促进从头甲基化的机制。我们还建议继续进行额外的遗传筛选，并对我们分离出的一组35个新突变体进行表征，这些突变体可以阻断从头甲基化。该提案的第二个主要目的是进一步研究CG DNA甲基化维持的机制，通过研究哺乳动物中称为UHRF 1的新因子和拟南芥中的ORTH/Vim，其似乎通过直接结合先前甲基化的CG位点并招募CG甲基转移酶来进一步维持甲基化。我们建议研究6个拟南芥ORTH/Vim基因的功能，通过使用功能丧失的遗传等位基因，了解每个基因的染色质相互作用的特异性，并进行分子和遗传测试的功能，每个蛋白质的不同结构域。这些研究将为拟南芥中的DNA甲基化系统提供新的线索，并对哺乳动物和其他真核生物中的相关系统产生影响。公共卫生相关性：该研究旨在了解控制DNA甲基化的基本机制，DNA甲基化是发生在某些基因上的化学修饰，并阻止这些基因在特定组织中发挥作用。当DNA甲基化模式不能正确维持时，这可能导致基因的不适当调节，是癌症的主要原因。因此，对DNA甲基化的进一步理解可能有一天会导致纠正DNA甲基化模式缺陷的方法。

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. PUBLIC HEALTH RELEVANCE: Project Narrative This research aims at understanding the basic mechanisms that control DNA methylation, which is a chemical modification that occurs on certain genes and prevents these genes from functioning in a particular tissue. When DNA methylation patterns are not properly maintained, this can cause inappropriate regulation of genes and is a major cause of cancer. Thus a further understanding of the DNA methylation may someday lead to methods for correcting DNA methylation patterning defects.