Control of methylation spreading in synthetic DNA sequences by the murine DNA methyltransferase

Methylation spreading, which involves a propensity for the mammalian DNA-(cytosine-5)-methyltransferase to de novo methylate cytosine-guanine dinucleotides (CpGs) near pre-existing 5-methylcytosine bases, has been implicated in the control of numerous biological processes. We have assessed methylation spreading by the murine DNA methyltransferase in vitro using synthetic copolymers and oligonucleotides which differ only in their methylation state. Double-stranded oligonucleotides were found to undergo higher levels of de novo methylation overall than otherwise identical single-stranded oligonucleotides. This difference reflects the greater number of de novo methylatable cytosine bases in double-stranded than single-stranded sequences. All tested oligonucleotides containing pre-existing 5-methyl-cytosine(s) were de novo methylated at several fold the rates of non-methylated controls. No mammalian proteins besides the DNA methyltransferase were required for this observed enhancement of de novo methylation. Studies using oligonucleotides differing in patterns of pre-methylation showed that methylation spreading can be initiated by hemimethylated or duplex methylated CpGs indicating that recognition of 5-methylcytosine by the enzyme is sufficient to stimulate methylation spreading. Double and single-stranded oligonucleotides with several bases between CpGs underwent considerably more de novo methylation per CpG than sequences containing sequential uninterrupted methylatable sites. Spacing preferences by the DNA methyltransferase were also observed in hemimethylated oligonucleotides, suggesting that this is a general property of the enzyme. Although methylation spreading outside of CpG dinucleotides was relatively rare, single-stranded DNA incurred higher levels of de novo methylation at sites other than CpG as compared to double-stranded DNA. This indicates less specificity of methylation spreading in single-stranded sequences. Finally, enhanced de novo methylation in the presence of fully methylated CpG sites in double-stranded oligonucleotides was not as high as the rates of methylation of hemimethylated CpGs in otherwise identical oligonucleotides. These studies provide further elucidation of the mechanisms and regulation of the methylation spreading process and its potential role in the biological processes it influences.