Genetic Analysis of RNA-directed DNA Methylation

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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.