Protein binding protects sites on stable episomes and in the chromosome from de novo methylation

We have utilized the Escherichia coli lac repressor-operator system to test whether. protein binding can interfere with de novo DNA methylation in mam malian cells. We find that a DIVA binding protein can protect sites on the episome as well as in the genome from the de novo methylation activity of D nmt3a. Transcriptional machinery moving through the binding sites does not affect the de novo methylation of these sites, anti it does not affect the binding protein protection of these sites from de novo methylation. This st udy and previous studies provide a possible mechanism for the observation t hat an Sp1 site can serve as a cis-acting signal for demethylation anti for preventing de novo methylation of the CpG island upstream of the mouse ade nine phosphoribosyltransferase (Aprt) gene. These findings also support the hypothesis that protein binding may play a crucial role in changes of CpG methylation pattern in mammalian cells.