C(m)C(a/t)GG methylation: a new epigenetic mark in mammalian DNA?

T years ago, based on the knowledge of cytosine methylation in higher organisms and the newly discovered bacterial adenine methyltransferase, Riggs (1) and Holliday and Pugh (2) independently proposed that the covalent modification of DNA by methylation might serve as a means to propagate heritable expression states in eukaryotes. In the years since, the association between cytosine methylation and transcriptional silencing in mammalian cells has become well established (3), and a number of proteins that catalyze the transfer of a methyl group to the 5-carbon of the cytosine pyrimidine ring have been cloned and characterized. These DNA methyltransferases (m5C-MTases) are encoded by a diverse family of genes found in prokaryotes as well as all four groups of eukaryotes (4). In mammals, cytosines are methylated predominantly in the context of the 59-CpG-39(CG) dinucleotide, and the majority of these sites are methylated. Only the short CG-rich regions known as CpG islands are methylation free in normal tissues. However, non-CG cytosine methylation has also been reported, primarily in viral or stably integrated plasmid DNA sequences. Genomic sequencing of an integrated adenovirus vector, for example, revealed methylation of cytosines in the context of 59-CpW-39 (CW, where W 5 A or T) dinucleotides (5). Subsequently, bisulfite sequencing analysis of endogenous LINE-1 retroelements (6) and stably integrated plasmid DNA (7) confirmed the presence of CW methylation in mammalian cells. In the latter study, such cytosine methylation was found predominantly in the sequence CWG. Interestingly, the methylation state of plasmid DNA premethylated at such CWG sites was faithfully maintained in vivo (7), suggesting that mammalian cells are capable of establishment and maintenance of cytosine methylation in CWG as well as CG sites. Taken together, these results suggested that CWG methylation might be used to stably ‘‘tag’’ DNA in eukaryotes. However, further evidence for the existence of such an epigenetic system has been lacking. In this issue of PNAS, Malone et al. describe the presence of a subclass of CWG methylation, namely that of the internal cytosine within the sequence CCWGG (CmCWGG), in an endogenous mammalian gene (8). The authors studied a group of primary effusion lymphoma (PEL) and myeloma cell lines that no longer express many B lineage-specific genes. To test whether the silent state of such genes is associated with DNA methylation, several cell lines in which the B cell-specific B29 gene is silent were analyzed. Whereas bisulfite genomic sequencing of the B29 promoter revealed dense methylation exclusively at CG sites in several of these lines, a significant number of CCWGG sites were methylated in the B29 promoter region in one PEL and one myeloma line. Evidence for this class of cytosine methylation in mammalian cells comes from several previous studies. Bisulfite analysis of genomic DNA from murine erythroleukemia (MEL) cells infected with a Moloney murine leukemia virus vector revealed that several proviral CCWGG sites were methylated de novo shortly after infection (9). Additionally, by using the CmCWGG-sensitive restriction enzyme EcoRII and Southern blotting of genomic DNA from normal peripheral blood leukocytes (PBL), Franchina et al. reported a high level of CCTGG methylation flanking the CpG island of the myogenic myf-3 gene, although no CCAGG methylation was detected (10). Moreover, the study describing efficient maintenance of CWG methylation in mammalian cells involved the introduction of plasmid DNA isolated from Escherichia coli expressing the Dcm m5C-MTase, which methylates CCWGG sites (7). Thus, the observed maintenance of CWG methylation may have been the result of recognition of the CCWGG pentanucleotide. The observation of CCWGG methylation in several systems suggests that this site may be the target sequence of a eukaryotic epigenetic modification system. Which enzymes are responsible for establishment and maintenance of CCWGG methylation? The diversity of target recognition sites already described for bacterial members of the m5C-MTase family (11) suggest that one or more of the mammalian m5C-MTase homologs may be capable of methylating CCWGGs. Four known or putative mammalian m5CMTases have been identified thus far: Dnmt1, Dnmt2, Dnmt3A, and Dnmt3B. Analysis of the human genome project database reveals no additional genes in the highly conserved m5C-MTase family, suggesting that the proteins encoded by the known m5C-MTase genes are responsible for the constellation of cytosine methylation in the mammalian genome. Dnmt1, the first mammalian m5C-MTase cloned, shows a strong preference for CGs, particularly when hemi-methylated (12), suggesting that this enzyme is responsible for the maintenance of CG methylation. The closely related Dnmt3A and Dnmt3B proteins on the other hand show strong de novo CG MTase activity (13), apparently confirming the hypothesis that the establishment and maintenance of CG methylation are carried out by distinct m5C-MTases. Recently however, several groups have reported that Dnmt3A also methylates non-CG cytosines. As determined by nearest neighbor analysis and bisulfite sequencing, mCW represent '15% of the total number of methylated cytosines in embryonic stem (ES) cells (14), which express Dnmt3A at high levels. Expression of dnmt3A in Drosophila, which lack a Dnmt3 homolog and show no cytosine methylation in adult tissues, revealed that Dnmt3A is capable of methylating CW dinucleotides (14). Whereas the non-CG methylated sites showed no clear flanking consensus sequence in vivo, the internal cytosine of CCAGG is efficiently methylated by Dnmt3A in vitro (15). Analysis of CCWGG methylation in dnmt3A2/2 (16)