Directed Evolution of Improved Zinc Finger Methyltransferases

Abstract

The ability to target DNA methylation toward a single, user-designated CpG site in vivo may have wide applicability for basic biological and biomedical research. A tool for targeting methylation toward single sites could be used to study the effects of individual methylation events on transcription, protein recruitment to DNA, and the dynamics of such epigenetic alterations. Although various tools for directing methylation to promoters exist, none offers the ability to localize methylation solely to a single CpG site. In our ongoing research to create such a tool, we have pursued a strategy employing artificially bifurcated DNA methyltransferases; each methyltransferase fragment is fused to zinc finger proteins with affinity for sequences flanking a targeted CpG site for methylation. We sought to improve the targeting of these enzymes by reducing the methyltransferase activity at non-targeted sites while maintaining high levels of activity at a targeted site. Here we demonstrate an in vitro directed evolution selection strategy to improve methyltransferase specificity and use it to optimize an engineered zinc finger methyltransferase derived from M.SssI. The unusual restriction enzyme McrBC is a key component of this strategy and is used to select against methyltransferases that methylate multiple sites on a plasmid. This strategy allowed us to quickly identify mutants with high levels of methylation at the target site (up to ∼80%) and nearly unobservable levels of methylation at a off-target sites (<1%), as assessed in E. coli. We also demonstrate that replacing the zinc finger domains with new zinc fingers redirects the methylation to a new target CpG site flanked by the corresponding zinc finger binding sequences.

Advances in the directed evolution of proteins

“…Methylation of only a single site in a target DNA was selected for by digesting with a target site-specific restriction endonuclease and a second, unusual restriction enzyme that digests DNA with two distally methylated sites [27]. This method identified methyltransferase variants that showed 80% methylation at the target site and less than 1% methylation at off-target sites.…”

Epigenetic Mechanisms Underlying the Pathogenesis of Neurogenetic Diseases

“…For example, one group of investigators has reported the development of an interesting technology for promoting genomic locus-specific DNA methylation, using 2 different ZFs designed to bind to DNA flanking a target CpG site that are each fused with a component of a bifurcated DNA methyltransferase [115][116][117]. This approach aims to increase the local concentration of both methyltransferase fragments when the ZFs bind to the genome, leading to the assembly of an active methyltransferase enzyme only at the target site.…”

Versatile Thymine DNA Glycosylase (TDG): Attractive Subject of Future Research and Potential Target for Therapeutics

“…The regulation of methylation could be achieved either by 1) site-specific methylation or by 2) sitespecific demethylation of target genes. Engineered zinc fingermethyltransferase domains have been shown promises for site-specific methylation to a hypomethylated region of genome [20,21]. Similarly engineered TDG could offer site-specific demethylation of target genes which are hypermethylated.…”