title: Identification of restriction endonucleases sensitive to 5-cytosine methylation at non-CpG sites, including expanded (CAG)n/(CTG)n repeats.
abstract: Most epigenetic studies assess methylation of 5'-CpG-3' sites but recent evidence indicates that non-CpG cytosine methylation occurs at high levels in humans and other species. This is most prevalent at 5'-CHG-3', where H = A, C or T, and it preferentially occurs at 5'-CpA-3' and 5'-CpT-3' sites. With the goal of facilitating the detection of non-CpG methylation, the restriction endonucleases ApeKI, BbvI, EcoP15I, Fnu 4HI, MwoI and TseI were assessed for their sensitivity to 5-methylcytosine at GpCpA, GpCpT, GpCpC or GpCpG sites, where methylation is catalyzed by the DNA 5-cytosine 5'-GpC-3' methyltransferase M.CviPI. We tested a variety of sequences including various plasmid-based sites, a cloned disease-associated (CAG)83•(CTG)83 repeat and in vitro synthesized tracts of only (CAG)500•(CTG)500 or (CAG)800•(CTG)800. The repeat tracts are enriched for the preferred CpA and CpT motifs. We found that none of the tested enzymes can cleave their recognition sequences when they are 5'-GpC-3' methylated. A genomic site known to convert its non-CpG methylation levels upon C2C12 differentiation was confirmed through the use of these enzymes. These enzymes can be useful in rapidly and easily determining the most common non-CpG methylation status in various sequence contexts, as well as at expansions of (CAG)n•(CTG)n repeat tracts associated with diseases like myotonic dystrophy and Huntington disease.
authors.affiliation: Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: Arturo
authors.initials: A
authors.lastname: López Castel
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nakamori
authors.forename: Charles A
authors.initials: CA
authors.lastname: Thornton
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Pearson
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics; D019680:Trinucleotide Repeat Expansion
chemical_list: D044503:5-Methylcytosine; D004247:DNA; D004262:DNA Restriction Enzymes