Design and Application of 6mA-Specific Zinc-Finger Proteins for theReadout of DNA Methylation.

Abstract

Designed zinc-finger (ZnF) proteins can recognize AT base pairs by H-bonds in the major groove, which are disrupted, if the adenine base is methylated at the N6 position. Based on this principle, we have recently designed a ZnF protein, which does not bind to DNA, if its recognition site is methylated. In this review, we summarize the principles of the recognition of methylated DNA by proteins and describe the design steps starting with the initial bacterial two-hybrid screening of three-domain ZnF proteins that do not bind to CcrM methylated target sites, followed by their di- and tetramerization to improve binding affinity and specificity. One of the 6mA-specific ZnF proteins was used as repressor to generate a methylation-sensitive promoter/repressor system. This artificial promoter/repressor system was employed to regulate the expression of a CcrM DNA methyltransferase gene, thereby generating an epigenetic system with positive feedback, which can exist in two stable states, an off-state with unmethylated promoter, bound ZnF and repressed gene expression, and an on-state with methylated promoter and active gene expression. This system can memorize transient signals approaching bacterial cells and store the input in the form of DNA methylation patterns. More generally, the ability to bind to DNA in a methylation-dependent manner gives ZnF and TAL proteins an advantage over CRISPR/Cas as DNA-targeting device by allowing methylation-dependent genome or epigenome editing.