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Methylation of cytosine to 5-methylcytosine (mC) is the most important epigenetic DNA alteration in eukaryotes. Cytosine methylation is involved in establishing a silenced chromatin stage through interaction with DNA-binding proteins and recruitment of histone deacetylases and other histone-modifying enzymes leading to chromatin remodeling. In this fashion, DNA methylation is connected with processes of normal and pathological gene regulation, DNA replication, virus latency, parental imprinting, embryonic development, carcinogenesis, and genetic diseases in higher organisms (1–4). Analogous to the terms transcriptome and proteome, the neologism methylome has been proposed to describe the complete set of DNA methylation in a cell, which carries information in addition to the naked DNA sequence. The methylome changes over time and, depending on its alterations, is linked to aging, cancer, and polymorphic variation in populations (5). Until now, most attention has been focused on the appearance and functions of mC in the context of CpG sequences. However, for years it has been known that in genomic DNA of animal cells (e.g., human spleen DNA), methylated cytosine appears as often in CpT or CpA as in CpG sequences (6). When considering more complex sequences as the sites of cytosine methylation, the presence of mC in the CpNpG context, especially in the internal cytosine of the CC(A/T)GG sequence, is particularly important both in plant (7) and mammalian cells (8–11). In its sequence specificity, this methylation corresponds to the prokaryotic Dcm methylation, which has been known for many years (12). In mammalian cells, CpNpG methylation might be catalyzed by the methylases Dnmt3A and/or Dnmt2 (for a review see Reference 13). It has been shown that the occurrence of CmC(A/T)GG in the DNA of mammalian cells is able to repress promoter activity and to replace transcription factors with new protein complexes (11). Thus, not only CpG methylation but also CC(A/T)GG methylation is involved in epigenetic regulation. Until now, in contrast to CpG, neither the prevalence nor the genomic distribution of CmC(A/T)GG have been systematically addressed. One of the most important and easiest methods to identify the presence and location of sequence-specific methylation in DNA, and to estimate the proportion of methylated versus nonmethylated sites in bulk DNA, is the use of methylation-sensitive restriction enzymes (MSREs). In combination with either PCR or Southern blotting analysis, MSRE can be used for sensitive analysis of site-specific methylation in large and complex genomes. CC(A/T)GG methylation patterns can be determined by comparative DNA cleavage with the methylation-sensitive restriction endonuclease EcoRII and a restriction endonuclease not affected by methylation of the internal cytosine, such as BstNI or MvaI. However, EcoRII is a member of the type IIE restriction endonucleases, which require the interaction with two copies of the respective recognition sequence for their activity (14). When the DNA molecule contains only a single CC(A/T)GG site, or the distance between two copies of the site in the DNA molecule is too large to allow functional interaction with the same dimeric EcoRII molecule, the sites remain uncleaved even when nonmethylated. This intrinsic cleavage resistance could be erroneously interpreted as proof of CC(A/T)GG methylation, and it cannot be ruled out that publications describing the occurrence of CmC(A/T)GG on the basis of comparative MSRE analysis by EcoRII include bias from this source. Unfortunately, the opportunity to create

Reliable detection of DNA cytosine methylation at CpNpG sites using the engineered restriction enzyme EcoRII-C