Genetic Analysis of RNA-directed DNA Methylation

描述（由申请人提供）：每个细胞内单个基因和其他DNA非编码区的表达必须被精确调节以正常生长和发育，这一点通过与异常基因表达相关的无数疾病而得到证实。在真核生物中，控制基因表达的一种机制是通过DNA中胞嘧啶残基的甲基化。DNA甲基化对于基因组中的高度重复元件（包括转座子和逆转录病毒）的沉默以及对于几种表观遗传调节过程（如亲本印记和X染色体失活）是重要的。尽管这些生物学过程的重要性，尽管发现DNA甲基化的错误调节可导致与癌症相关的高甲基化和低甲基化状态，但对DNA甲基化的建立和维持的机制知之甚少。本研究旨在进一步阐明拟南芥中RNA介导的DNA甲基化（RdDM）的机制，该机制是由哺乳动物DnmtS从头甲基转移酶的同源物--结构域重排甲基化酶2（DRM 2）催化的。研究将开始，首先表征该途径中新鉴定的蛋白质--DRM 2 DNA甲基化介质（MOD）的组蛋白甲基转移酶活性。MOD在RdDM过程中何时何地起作用将通过评估mdd突变对免疫荧光实验中其他RdDM蛋白定位的影响来确定。此外，还将进行遗传筛查，以确定RdDM所需的其他蛋白质。更深入地了解DRM 2介导的甲基化和RdDM将是回答重要生物学问题的关键，例如特定位点最初如何靶向甲基化，这种甲基化如何维持，以及这个过程如何被破坏，从而导致基因表达的变化。相关性：在真核生物中，已经开发了许多系统来确保遗传信息的正确表达，当这些系统发生故障时，蛋白质可能会错误表达并导致疾病，如癌症。在分子水平上阐明这样一个系统的机制，DNA甲基化，对于理解该系统的破坏如何导致与癌症相关的甲基化状态的增加和减少将是重要的。

DESCRIPTION (provided by applicant): The expression of individual genes and other non-coding regions of DNA within each cell must be precisely regulated for normal growth and development as is evident by the innumerable diseases associated with aberrant gene expression. In eukaryotes, one mechanism by which gene expression is controlled is through the methylation of cytosine residues in the DNA. DNA methylation is important for silencing of highly repetitive elements in the genome including transposons and retroviruses and for several epigenetic regulatory processes such as parental imprinting and X-chromosome inactivation. Despite the importance of these biological processes, and despite findings that mis-regulation of DNA methylation can lead to hyper and hypo methylated states associated with cancer, the mechanisms for the establishment and maintenance of DNA methylation are very poorly understood. This proposal is aimed at further elucidating the mechanism of RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana, which is catalyzed by the DOMAINS REARRANGED METHYLASE2 (DRM2), a homolog of the mammalian DnmtS de novo methyltransferase. Investigation will begin with the characterization of the histone methyltransferase activity of a newly identified protein in this pathway, MEDIATOR OF DRM2 DNA METHYLATION (MOD). When and where MOD acts during RdDM will then be determined by assessing the effects of an mdd mutation on the localization of other RdDM proteins in immunofluorescence experiments. In addition, a genetic screen will be conducted to identify other proteins required for RdDM. A more thorough understanding of DRM2 mediated methylation and RdDM will be key for answering important biological questions such as how specific loci are initially targeted for methylation, how this methylation is maintained, and how this process is disrupted, leading to changes in gene expression. Relevance: In eukaryotes many system have developed to ensure the proper expression of genetic information and when these systems malfunction proteins can become mis-expressed and cause diseases such as cancer. Elucidating the mechanism of one such system, DNA methylation, on the molecular level will be important for understanding how disruptions in this system can lead to the increased and decreased methylation states associated with cancer.