Genetic Analysis of RNA-directed DNA Methylation

描述(申请人提供)：单个基因和每个细胞内DNA的其他非编码区的表达必须受到精确的调控，才能正常生长和发育，这从无数与基因表达异常相关的疾病中可见一斑。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甲基化对于沉默基因组中的高重复元件，包括转座子和逆转录病毒，以及一些表观遗传调控过程，如双亲印迹和X染色体失活，是重要的。尽管这些生物过程很重要，尽管发现DNA甲基化的错误调控会导致与癌症相关的高甲基化和低甲基化状态，但建立和维持DNA甲基化的机制仍然知之甚少。这一建议旨在进一步阐明拟南芥中RNA诱导的DNA甲基化(RdDM)的机制，该机制是由哺乳动物Dnmts de nevo甲基转移酶的同源物结构域重排的METHYLASE 2(DRM2)催化的。研究将从这一途径中一种新发现的蛋白质组蛋白甲基转移酶的活性开始，它是DRM2 DNA甲基化(MOD)的中介。然后，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MOD在RdDM期间何时和在哪里起作用。此外，还将进行基因筛查，以确定RdDM所需的其他蛋白质。更透彻地了解DRM2介导的甲基化和RdDM将是回答重要生物学问题的关键，这些问题包括甲基化最初是如何靶向的，这种甲基化是如何维持的，以及这一过程是如何被破坏的，从而导致基因表达的变化。相关：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，当这些系统出现故障时，蛋白质可能会错误表达，并导致癌症等疾病。在分子水平上阐明DNA甲基化这一系统的机制，对于理解该系统的中断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.