(72h) Targeted DNA Methylation Using a Bisected M.Hhal Fused to Zinc Fingers

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 engineered a targeted methyltransferase by splitting a prokaryotic, monomeric methyltransferases into a heterodimeric methyltransferase and fusing each heterodimeric fragment to a sequence specific DNA binding protein. The recognition sequences for the DNA binding protein flank a desired CpG site. When bound, the DNA binding proteins increase the local concentration of the methyltransferase fragments over the CpG site, forcing functional reassembly of the enzyme only over the target site. Thus, the enzyme activity at a desired site will be contingent upon DNA binding recognition of the adjacent sites. This strategy works because the fragments are engineered to have reduced affinity for another, and do not efficiently assemble away from the target DNA sequence. Using this strategy we created a target methyltransferase that methylates ~60% of a desired site with undetectable methylation at non-targeted sites. In vivo experimentation and in silico modeling of these methyltransferases has elucidated some of the design parameters and current limitations of these targeted methyltransferases.