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Targeted DNA Methylation Using an Artificially Bisected M.HhaI Fused to Zinc Fingers

Figure 2

A schematic of the restriction enzyme protection assay for targeted methylation.

(A) A single plasmid, pDIMN8, encodes genes for both methyltransferase fragment-zinc finger fusion proteins, as well as two sites for assessing the degree of targeted methyltransferase activity. Expression of both protein fragments was induced in ER2267 cells and plasmid DNA was isolated. (B) Plasmid DNA was linearized by NcoI-HF digestion and incubated with FspI, an endonuclease whose activity is blocked by methylation. In the absence of methylation, the plasmid is digested twice by FspI and once by NcoI-HF as shown. (C) Methylation at one or both of the FspI containing sites creates unique digestion patterns as assessed by agarose gel electrophoresis. Unique bands are diagnostic of no methylation (∼4600 bp), methylation at site 1 (∼5210 bp), methylation at site 2 (∼5830 bp), or methylation at both sites (∼6580 bp). (D) A schematic of the functional methyltransferase at a target site. Zinc finger/DNA recognition mediates methyltransferase assembly. (E) This assembly is designed not to occur at the non-target control site, which lacks zinc finger binding sites.

Figure 2