Presence of 5-methylcytosine in CpNpG trinucleotides in the human genome

While the methylation machinery of mammalian cells has been shown to be capable of both maintenance and de novo methylation at CpNpG sites, CpNpG methylation in the human genome has not been demonstrated. Here, we report the first observation of 5-methylcytosines in CpNpG triplets in the human genome. We identify the existence of CpNpG methylation in a number of genes which contain trinucleotide repeat regions, including the androgen receptor (AR). We further analyzed DNA extracted from primary tissue samples and found the same pattern of CpNpG methylation. To confirm our results, we performed Southern blot analysis by analyzing the cleavage sites of restriction enzymes within exon 1 of the AR gene and found direct evidence of the presence of 5mCs in CpNpG triplets in the human genome. Our results also suggest that this methylation pattern may be due to the human DNA methyltransferases DNMT1 and DNMT3A. Although the functional significance needs to be tested further, the discovery of inheritable CpNpG methylation in the human genome may have important implications in our understanding of gene regulation and of the development of various diseases, including cancer.