(205a) Evolving a User-Defined, Targeted DNA Methyltransferase

CpG methylation of promoters is an important form of epigenetic control in eukaryotic cells, causing transcriptional repression. It is therefore involved in many important cellular processes, such as cellular differentiation, X-chromosomal inactivation, and chromatin remodeling. As such, aberrant methylation patterns have been implicated in many disease states such as cancer, psychological disorders and a range of diseases caused by the deregulation of epigenetic markers. The ability to target methylation toward a specific site within the genome would have broad applicability as a tool to study the effect of single CpG methylation events on transcription and as a potential therapeutic to target diseases resulting from the hypomethylation of particular promoters. 

We have designed an in vitro directed evolution strategy to create methyltransferases that recognize non-palindromic, user-defined sequences. The strategy is both simple and rapid; it has enabled us to quickly optimize an engineered, heterodimeric methyltransferase previously described in our lab. This methyltransferase consists of two heterodimeric methyltransferase fragments fused to separate zinc fingers, whose recognition sequences flanks a targeted CpG site. Fragments are engineered to have reduced affinity for one another, and do not efficiently assemble away from the target DNA sequence. Our newly optimized enzyme methylates ~80% of a desired site with minimal methylation at non-targeted sites. Early initial experiments in eukaryotic cells will be discussed.