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

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Abstract
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.