Tollefsbol T O, Hutchison C A
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599, USA.
J Mol Biol. 1997 Jun 20;269(4):494-504. doi: 10.1006/jmbi.1997.1064.
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.
甲基化扩展涉及哺乳动物DNA（胞嘧啶-5）-甲基转移酶对预先存在的5-甲基胞嘧啶碱基附近的胞嘧啶-鸟嘌呤二核苷酸（CpG）进行从头甲基化的倾向，这与众多生物学过程的调控有关。我们使用仅甲基化状态不同的合成共聚物和寡核苷酸，在体外评估了小鼠DNA甲基转移酶的甲基化扩展情况。发现双链寡核苷酸总体上比其他相同的单链寡核苷酸经历更高水平的从头甲基化。这种差异反映了双链序列中可从头甲基化的胞嘧啶碱基数量比单链序列更多。所有含有预先存在的5-甲基胞嘧啶的测试寡核苷酸，其从头甲基化速率是未甲基化对照的几倍。除了DNA甲基转移酶外，不需要其他哺乳动物蛋白就能观察到这种从头甲基化的增强。使用预甲基化模式不同的寡核苷酸进行的研究表明，甲基化扩展可由半甲基化或双链甲基化的CpG启动，这表明该酶对5-甲基胞嘧啶的识别足以刺激甲基化扩展。在CpG之间有几个碱基的双链和单链寡核苷酸，每个CpG的从头甲基化程度比含有连续不间断可甲基化位点的序列高得多。在半甲基化寡核苷酸中也观察到了DNA甲基转移酶的间距偏好，这表明这是该酶的一个普遍特性。虽然CpG二核苷酸之外的甲基化扩展相对较少，但与双链DNA相比，单链DNA在CpG以外的位点经历了更高水平的从头甲基化。这表明单链序列中甲基化扩展的特异性较低。最后，双链寡核苷酸中存在完全甲基化的CpG位点时，增强的从头甲基化程度不如相同寡核苷酸中半甲基化CpG的甲基化速率高。这些研究进一步阐明了甲基化扩展过程的机制和调控及其在它所影响的生物学过程中的潜在作用。
