Modulation of DNA binding protein affinity directly affects target site demethylation

It has recently been shown that in Xenopus, DNA demethylation at promoter r egions may involve protein-DNA interactions, based on the specificity of th e demethylated sites. Utilizing a stable episomal system in human cells, we recently mapped the sites and dissected the steps of demethylation at oriP sites bound by EBNA1 protein. Although it is clear that protein binding is required for demethylation of the oriP sites, it is uncertain whether this is a unique feature of the replication origin or whether it is a general p henomenon for all DNA sequences to which sequence-specific proteins are bou nd. In the present study, we utilize the well-defined Escherichia coli lac repressor/operator system in human cells to determine whether protein bindi ng to methylated DNA, in a region that is neither a replication origin nor a promoter, can also lead to demethylation of the binding sites. We found t hat demethylation specified by protein binding is not unique to the replica tion origin or to the promoter. We also found that transcriptional activity does not influence demethylation of the lac operator. Isopropyl-beta-D-thi ogalactopyranoside (IPTG), an inhibitor of the lac repressor, can prevent d emethylation of the lac operator DNA sites and can modulate demethylation o f the lac operator by affecting the binding affinity of the lac repressor. Using this system, a titration of protein binding can be done. This titrati on permits one to infer that protein binding site occupancy is the determin ant of demethylation at DNA sites and permits a determination of how this p rocess progresses over time.