Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins

Cytosine-5 DNA methylation is a critical signal defining heritable epigenetic states of transcription. As aberrant methylation patterns often accompany disease states, the ability to target cytosine methylation to preselected regions could prove valuable in re-establishing proper gene regulation. We employ the strategy of targeted gene methylation in yeast, which has a naturally unmethylated genome, selectively directing de novo DNA methylation via the fusion of C5 DNA methyltransferases to heterologous DNA-binding proteins. The zinc-finger proteins Zif268 and Zip53 can target DNA methylation by M.CviPI or M.SssI 5-52 nt from single zinc-factor binding sites. Modification at specific GC (M.CviPI) or CG (M.SssI) sites is enhanced as much as 20-fold compared with strains expressing either the free enzyme or a fusion protein with the zinc-finger protein moiety unable to bind to DNA. Interestingly, methylation is also selectively targeted as far as 353 nt from the zinc-finger protein binding sites, possibly indicative of looping, nucleosomes or higher-order chromatin structure. These data demonstrate that methylation can be targeted in vivo to a potentially broad range of sequences using specifically engineered zinc-finger proteins. Further more, the selective targeting of methylation by zinc-finger proteins demonstrates that binding of distinct classes of factors can be monitored in living cells.