Chromosomal DNA demethylation specified by protein binding

In the present study, we utilize the well-characterized Escherichia coli lac repressor/operator system to demonstrate that protein binding can lead to demethylation at the binding sites in the chromosome. Similar to the findings using the episome, we found that the presence of Lacl in the cells can lead to demethylation of methylated lacO in the chromosome and the Lad inhibitor, isopropyl-beta -D-thiogalactopyranoside (IPTG), can prevent demethylation of the methylated lacO. The lacO sites become progressively more demethylated over time with the presence of Lad, supporting the role of protein occupancy in demethylation targeting. These results validate our earlier conclusions using a stable episomal system, and establish for the first time that protein binding can specify sites of demethylation in the chromosome.