Use of restriction enzymes to study eukaryotic DNA methylation: II. The symmetry of methylated sites supports semi-conservative copying of the methylation pattern

Abstract

Two experiments with Xenopus laevis ribosomal DNA have demonstrated that in the base-paired sequence ▪ either both cytosines are methylated or neither is methylated. Half-methylated sites are not found. The first experiment involved reassociation of a labelled methylated ribosomal DNA restriction fragment (4.65-RI) with an excess of the unmethylated fragment in order to expose any half-methylated sites to CpG-enzyme digestion. Subsequent treatment with HpaII and HhaI showed that methylated/unmethylated rDNA hybrids are no more sensitive to digestion than the native rDNA. The result indicates that fewer than 2% of methylated sites are half-methylated. In the second experiment erythrocyte rDNA was denatured and allowed to self-reassociate. This procedure rendered the 4.65 kilobase EcoRI fragment immune to digestion with HpaII, AvaI and HhaI at all sites except the HhaI hotspot. Since the native fragment was partially digested at many sites by HpaII, AvaI and HhaI prior to annealing, its subsequent immunity indicates that most paired CpGs are symmetrically methylated. The result also verifies the presence of a specific undermethylated recognition sequence for HhaI. Finally it has been shown for X. laevis cultured cells that following DNA replication new methyl groups are added to the progeny strand. The parental strand was not detectably labelled with [ methyl- 3H]methionine. Taken together these results indicate that any pattern of methylated and unmethylated paired CpGs in the genome of a cell will be inherited by descendants of that cell.