Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells

Abstract

The ability to target methylation to specific genomic sites would further the study of DNA methylation's biological role and potentially offer a tool for silencing gene expression and for treating diseases involving abnormal hypomethylation. The end-to-end fusion of DNA methyltransferases to zinc fingers has been shown to bias methylation to desired regions. However, the strategy is inherently limited because the methyltransferase domain remains active regardless of whether the zinc finger domain is bound at its cognate site and can methylate non-target sites. We demonstrate an alternative strategy in which fragments of a DNA methyltransferase, compromised in their ability to methylate DNA, are fused to two zinc fingers designed to bind 9 bp sites flanking a methylation target site. Using the naturally heterodimeric DNA methyltransferase M.EcoHK31I, which methylates the inner cytosine of 5'-YGGCCR-3', we demonstrate that this strategy can yield a methyltransferase capable of significant levels of methylation at the target site with undetectable levels of methylation at non-target sites in Escherichia coli. However, some non-target methylation could be detected at higher expression levels of the zinc finger methyltransferase indicating that further improvements will be necessary to attain the desired exclusive target specificity.

Figure 1.

The effect of fusion linker length and spacing at the target site on the methyltransferase activity and specificity of M.EcoHK31I-derived constructs with a 42-residue deletion on the β fragment. (A) Schematic of the various constructs tested showing the variability in linker length between zinc finger and methyltransferase fragments and the distance between the zinc finger’s binding site and the target site for methylation. (B) The two plasmids encoding the two methyltransferase fragments and the DNA fragments that result from AfeI and EagI digestion if the EagI site is not methylated (methylation blocks EagI but not AfeI digestion). Methylation creates uniquely sized bands depending on if the target or non-target site is protected. Representative results are shown to demonstrate the observed effects of (C) zinc finger–target site distance, (D) linker lengths between the α fragment and Tyr123 and (E) linker lengths between the β fragment and Tyr456. Linker lengths are designated by 0, 5, 10 or 15 L and the spacing at the target site designated by the number of base pairs between the target site and zinc finger site. Individual plasmids digested with AfeI are also shown for size comparisons (pDIM-N7 and pAR) with (+) and without (−) EagI digestion. The locations of bands resulting from only target methylation, only non-target methylation and methylation at both sites are indicated. The plasmids were prepared from cells grown in LB media.

Figure 2.

The effects of removing zinc finger proteins or zinc finger binding sites on the methylation activity and specificity of M.EcoHK31I-derived constructs with a 67-residue deletion on the β fragment. (A) Schematic of the fusion and control constructs used in these experiments. (B) DNA sequences at the target sites (with and without zinc finger binding sites) and non-target site. All pDIM-N7 plasmids contain a non-target site and either the target site containing zinc finger binding sites (+) or containing a control site for which the zinc fingers lack affinity (–). Gel electrophoresis results for plasmids digested with AfeI and EagI (performed as in Figure 1) are shown for plasmids containing the DNA encoding (C) Tyr123-15L-α and (D) α cotransformed with plasmids encoding βΔ67 or βΔ67-5L-Tyr456. Results for AfeI or AfeI/EagI digestions of unmethylated control plasmids are shown for size comparison. The locations of bands resulting from only target methylation, only non-target methylation and methylation at both sites are indicated. The plasmids were isolated from cells grown in LB media.

Figure 3.

Methylation activity and specificity resulting from a single plasmid encoding Tyr123-15L-α and βΔ67-5L-Tyr456 (A). Schematic of the plasmid and its digestion with BsaAI (not-blocked by methylation) and EagI (blocked by methylation). Gel electrophoresis results for plasmids digested with BsaAI and EagI are shown for plasmids contain the DNA encoding (B) Tyr123-15L-α and (C) α Combinations with DNA encoding either βΔ67 or βΔ67-5L-Tyr456 and with and without zinc finger binding sites flanking the target site were tested. The locations of bands resulting from only target methylation, only non-target methylation and methylation at both sites are indicated. Plasmids were isolated from cells grown in LB media supplemented with 0.2% glucose.

Figure 4.

Tyr123-15L-α/βΔ67-5L-Tyr456 has a high preference for the target site over other M.EcoHK31I methylation sites. (A) The pDIM-N7 plasmid contains five methylation sites for M.EcoHK31I that overlap EaeI restriction enzyme sites (EaeI is blocked by methylation) and one XbaI site used to linearize the plasmid and facilitate analysis. The sizes of the five largest DNA fragments that result from XbaI/EaeI digestion of unmethylated DNA are shown. (B) Agarose gel electrophoresis results of XbaI/EaeI digestions of plasmids expressing Tyr123-15L-α/βΔ67-5L-Tyr456 or Tyr123-15L-α/β-5L-Tyr456 (i.e. control protein with no truncation of the β fragment). Methylation at the target site yields a 2499 bp band if the flanking sites are not methylated. Methylation at the two EaeI sites marked with an asterisk would result in bands at either 3231 bp or 3561 bp if their flanking sites are not methylated. These two EaeI sites are non-target sites that were not tested in previous experiments. Plasmids were isolated from cells grown in LB media supplemented with 0.2% glucose.