DNA methylation plays important roles in development, differentiation, X inactivation, genomic imprinting, and silencing of transposable elements through the regulation of transcription [1
]. This usually occurs in mammalian cells at cytosine residues in the context of cytosine-phosphate-guanine dinucleotide (CpG), and approximately 60%–90% of cytosines at these sites are methylated [4
]. Methyl-CpG serves as the physiological ligand for a family of proteins containing a highly conserved methyl-CpG binding domain (MBD) [5
]. These proteins recruit various chromatin-modifying complexes to methyl-CpG sites to cause further chromatin structural changes that result in transcriptional silencing.
Alterations of the patterns of normal DNA methylation result in many human diseases, including cancer [6
]. Aberrant patterns of DNA methylation in cancers are associated with tumor type, stage, prognosis, and response to chemotherapy. Human tumors undergo global DNA demethylation, including of Line-1 repetitive elements, and then DNA hypermethylation of certain gene promoters, including those of tumor suppressor genes [7
]. Aberrant DNA methylation in cancers is not restricted to protein-coding genes; it is also observed in microRNA (miRNA) genes. MicroRNAs are small noncoding regulatory RNAs of 20–24 nucleotides that reduce the stability and/or translation of fully or partially sequence-complementary target mRNAs. MicroRNAs can act as oncogenes or tumor suppressors, and can contribute to cancer initiation and progression [8
Genome-wide analysis of DNA methylation is an important issue in epigenetic research. The oldest technology for genome-wide analysis of DNA methylation, Restriction Landmark Genomic Scanning, which utilizes two-dimensional gel electrophoresis of genomic DNA, was developed in 1991 [9
]. This technology has been used in the cloning of many imprinted genes [10
] and tumor suppressors [11
]. In this century, development of genome-wide analysis technology such as microarrays and next-generation sequencers brought about several new methods for analyzing DNA methylation [13
]. Among these methods, capture and next-generation sequencing of methyl-CpG binding domains of MBD1 protein is especially useful for analysis because the binding activity of this protein is stronger than that of antibodies [14
Although epigenetic silencing of miRNAs has been reported in many cancers, systematic studies of DNA methylation and miRNA transcription have not yet been reported. In addition, differentially methylated regions were found only in the CpG islands just upstream of miRNAs in most reports on the epigenetic silencing of miRNAs because such cases are easily discovered. Therefore, it has not been clarified whether the transcriptional start sites far upstream of miRNAs are influenced by DNA methylation or not. It has also not been clarified whether the CpG-poor promoters of miRNA are influenced by DNA methylation. Furthermore, miRNAs are not only transcribed by RNA polymerase II, which is responsible for most protein-coding genes, but also transcribed by RNA polymerase III. Although the relation between DNA methylation and silencing has been extensively studied in genes transcribed by RNA polymerase II, there are few reports for genes transcribed by RNA polymerase III. Therefore, systematic analyses of the methylation and expression of miRNAs are required. To clarify the relation between DNA methylation and transcription of miRNAs, here we systematically analyzed the methylation and expression of human genes encoding miRNAs, proteins, other non-coding RNAs (ncRNAs), and pseudogenes using next-generation sequencing and microarray analysis.