Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships

Abstract

DNA methylation is now seen as a primary signal in the cell for mediating transcriptional repression through chromatin formation. The construction and evaluation of enzymes capable of influencing this process in vivo is therefore of significant interest. We have fused the C5-cytosine DNA methyltransferases, M.HhaI and M.HpaII, which both methylate 4 bp sequences containing a CpG dinucleotide, to a three zinc finger protein recognising a 9 bp DNA sequence. DNA methylation analyses demonstrate specific DNA methylation by both enzymes at target sites comprising adjacent methyltransferase and zinc finger subsites, targeted M.HpaII being the most specific. Binding analysis of the targeted M.HpaII enzyme reveals an 8-fold preference for binding to its target site, compared to binding to a zinc finger site alone, and an 18-fold preference over binding to a methyltransferase site alone, thereby demonstrating enhanced binding by the fusion protein, compared to its component proteins. Both DNA binding and methylation are specific for the target site up to separations of approximately 40 bp between the zinc finger and methyltransferase subsites. Ex vivo plasmid methylation experiments are also described that demonstrate targeted methylation. These targeted enzymes, however, are shown to be not fully mono-functional, retaining a significant non-targeted activity most evident at elevated protein concentrations.

Targeted cytosine methylation for in vivo detection of protein–DNA interactions

“…We conclude that M.CviPI is efficiently and directly targeted (see Fig. 1) to C residues of GC sites 13 and 26a on the lower strand of the PHO5 promoter, which agrees well with the optimal distance range of 10-40 bp observed for targeting DNA MTases to oligodeoxynucleotide substrates in vitro (16,22). It is likely that the MTase can reach sites within this distance range when the targeting factor (i.e., Pho4) is specifically bound to its UAS.…”

“…5me C has been selectively targeted to oligonucleotides in vitro by fusing C5 MTases to heterologous DNA-binding factors (16,22). To date, however, attempts to reproduce this capability in vivo have been unsuccessful (22).…”

“…5me C has been selectively targeted to oligonucleotides in vitro by fusing C5 MTases to heterologous DNA-binding factors (16,22). To date, however, attempts to reproduce this capability in vivo have been unsuccessful (22). As a first step toward targeting C5 DNA methylation in vivo, we tested whether a native yeast protein could specifically target a C5 MTase and hence increase 5me C levels at promoters in the tractable eukaryote, Saccharomyces cerevisiae ( Fig.…”

“…In Fig. 3, the marked methylation of site 93 compared with other sites on the upper strand of the PHO5 promoter suggests that M.CviPI is targeted at distances (93 bp from UASp2 and 202 bp from UASp1), well beyond the optimal targeting distance of 10-40 bp observed in vitro (16,22). To investigate this possibility further, we determined methylation levels at GC sites farther upstream in the PHO5 promoter (Fig.…”

Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

“…This enzyme predominately methylates cytosines in CpG sites to form 5-methylcytosine but can also modify general CpN sites less efficiently. Methylation was chosen deliberately as a marker activity because it is practically absent from mtDNA (17), it is easy to assay (18), and it had been previously used in chimeric ZFP enzymes (19)(20)(21).…”