Targeting DNA 5mCpG sites with chimeric endonucleases

Cytosine modification of the dinucleotide CpG in the DNA regulatory region is an important epigenetic marker during early embryo development, cellular differentiation, and cancer progression. In clinical settings, such as anti-cancer drug treatment, it is desirable to develop research tools to characterize DNA sequences affected by epigenetic perturbations. Here, we describe the construction and characterization of two fusion endonucleases consisting of the ⁵mCpG-binding domain of human MeCP2 (hMeCP2) and the cleavage domains of BmrI and FokI restriction endonucleases (REases). The chimeric (CH) endonucleases cleave M.HpaII (C⁵mCGG)—and M.SssI (⁵mCpG)-modified DNA. Unmodified DNA and M.MspI-modified DNA (⁵mCCGG) are poor substrates for the CH-endonucleases. Sequencing cleavage products of modified λ DNA indicates that cleavage takes place outside the ⁵mCpG recognition sequence, predominantly 4-17 bp upstream of the modified base (/N_4-17⁵mCpG, where / indicates the cleavage site). Such ⁵mCpG-specific endonucleases will be useful to study CpG island modification of the regulatory regions of tumor suppressor genes, and for the construction of cell-specific and tumor-specific modified CpG island databases.