NSF Award
1505793
1505793/1510652 <br/>Ostermeier/Novina<br/><br/>Every cell in the body contains the same DNA sequence. However, the different types of cells in the body (brain, liver, blood, skin, etc.) perform different functions. For example, certain genes that are 'off' in one cell type might be 'on' in another, which enables each cell's unique function. Whether a gene is off or on in a particular cell is due in part to differences in a modification of DNA called CpG methylation. Additionally, certain diseases are caused by abnormal CpG methylation. In this research, a protein will be developed that can alter CpG methylation in a very precise and controlled fashion. This protein will provide other researchers with a tool to study CpG methylation and its effects, to alter cell behavior, and to develop therapies based on the selective altering of a single gene function. A pre-doctoral student and a postdoctoral fellow will be trained in research while performing this research. <br/><br/>Modular, targeted cytosine DNA methyltransferases (MTases) will be developed that are capable of methylating a target CpG site (i.e. 5'-CG-3') at >95% efficiency while leaving non-target CpG sites unmethylated (< 1%). High specificity will be achieved by requiring that the MTase assembles into a functional form at the desired target site in a sequence-dependent manner. These MTases will be optimized using directed evolution. An E. coli reverse selection system will be developed for the rapid assessment of methylation specificity. To adapt the targeted MTases for the repression of mammalian promoters, a user-friendly platform for rapidly identifying CpGs whose methylation leads to repression of eukaryotic promoters will be developed. This work will result in a pipeline of assays and reagents for identifying repressive promoter CpG methylation mediated silencing of endogenous promoters in eukaryotes. The pipeline will be optimized for maximal on-target and minimal off-target gene repression and will be scalable for genome-wide applications. Our research activities will empower researchers to: (1) study methylation's effect on transcription, (2) investigate the mechanisms of spreading of methylation patterns and epigenetic regulation, (3) use targeted MTases as a tool for silencing genes of interest or controlled differentiation of stem cells, and (4) develop targeted MTases as therapeutic agents for the selective silencing of genes (e.g. cancers or viral infections).<br/><br/>This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Cellular Dynamics and Function Program of the Division of Molecular and Cellular Biology.
