Genetic and epigenetic aspects of DNA methylation on genome expression, evolution, mutation and carcinogenesis.

Jean-Marc Zingg and Peter A.Jones1 dromic sequences of two to six base pairs (3). The eukaryotic enzymes on the other hand are believed to be mainly ‘mainDepartment of Biochemistry and Molecular Biology, USC/Norris tenance’ enzymes that recognize and methylate hemi-methylComprehensive Cancer Center, University of Southern California, School of ated target sites generated by DNA replication. Since shortly Medicine, Los Angeles, CA 90033, USA after replication the parental strand is methylated whereas the 1To whom correspondence should be addressed newly synthesized is not, DNA methylation serves as a mechanism of strand discrimination for mismatch repair (4). Methylation occurs in mammals mainly at the dinucleotide Introduction CpG, although recent reports indicate that the sequence CpNpG DNA methylation has at least two important roles in tumorican also be methylated albeit with a lower efficiency (5). genesis. The target cytosines (C*) of (cytosine-5)-DNA methylAbout 70% of all CpG dinucleotides and ~3–6% of all transferase (Mtase) are mutated to thymine (T) in ~30% of cytosines in the genome are methylated in vertebrates. The inherited diseases and cancer, and genome-wide alterations of eukaryotic enzymes contain a large domain at their amino DNA methylation patterns occur at early stages of tumor terminus in addition to the catalytic domain and this is development. Insight into the normal function of DNA most likely involved in nuclear localization, targeting to the methylation will provide the knowledge to understand the replication fork and in various aspects of enzyme regulation origins of these aberrations and their importance for disease (6,7). initiation and progression. Originally the aberrations seen in The ‘maintenance’ methyl-transferase also has a low de tumors were attributed to the higher spontaneous deamination novo activity that can be stimulated by proteolytic cleavage rate of 5-methylcytosine (5-mC) as compared with C and to of the amino terminal domain (8) and by specific target DNAs misregulation of the Mtase gene. Recently, it has become clear such as oligonucleotides containing looped and mismatched that the Mtase can actively participate in mutagenesis by bases (9). It is still unclear whether this low de novo methylation enzymatically increasing both the rate of genetic and epigenetic activity is sufficient to account for the rapid genome-wide alterations. Proteins that recognize and repair these alterations de novo DNA methylation that occurs during embryonic determine the frequency of their fixation as disease causing development or whether there is another methyl-transferase mutations. In addition, alterations in the metabolism of Sthat has high de novo methylation activity. Homozygous adenosylmethionine can disturb DNA methylation by depleting deletion of the mouse Mtase gene leads to embryonic lethality, the cofactor S-adenosylmethionine or by increasing the level proving that DNA methylation is essential for embryonic of metabolites acting as inhibitors of DNA methylation. This development (10). Residual 5-mC in these mice suggests that review concentrates on the normal role of DNA methylation there might be another gene in mice with Mtase activity (10). in mammals and on aberrations of DNA methylation in Methylation of cytosine changes the structural characteristics inherited disease and cancer. of DNA in several ways. Methylation of DNA in the common B form facilitates a conformational change to the Z form, Function of DNA methylation increases the helical pitch of DNA and alters the kinetics of The only naturally occurring modification of DNA in higher cruciform extrusion (11,12). The methyl group of cytosine eukaryotes is the methylation of the 5 position of cytosine (C) sterically extends into the hydrophilic major groove of B DNA leading to the formation of 5-methylcytosine (5-mC). The and introduces hydrophobicity, two changes that may be reaction is catalyzed by the enzyme (cytosine-5)-DNA methylresponsible for the altered specificity of proteins interacting transferase (Mtase), which has been isolated from prokaryotes with DNA. and eukaryotes. Several aspects of the catalytic mechanism Regulation of gene expression by DNA methylation have been clarified mainly for prokaryotic Mtases (1). The Cytosine methylation can influence transcription directly by crystal structure of the bacterial M.HhaI enzyme revealed an interfering with the binding of positively or negatively acting intermediate in which the target cytosine is flipped out of the transcription factors or indirectly by the formation of inactive DNA helix and covalently attached to the enzyme (2). The chromatin (13). Since 5-mC is structurally similar to T, the enzyme first binds to its target sequence, removes the target methylation of cytosine might lead to the generation of new cytosine into its catalytic pocket and covalently attaches to the consensus sequences for some transcription factors. Indeed, C6 position of cytosine (1). This generates a covalently bound cytosine methylation has recently been found to convert a low enzyme-cytosine intermediate with the cytosine activated at affinity AP-1 binding site (CGAGTCA) into a high affinity C5, which can then accept the methyl group from the cofactor site (mCGAGTCA), which is more similar to the consensus S-adenosylmethionine. AP-1 binding site (TGAGTCA) (14). Several DNA binding Most prokaryotic enzymes are ‘de novo’ enzymes recognizproteins that contain a CpG in their recognition sequence are ing and methylating specific unor hemi-methylated palininhibited when the CpG is methylated (Table I). Interestingly, the transcription factor Sp1 required to tran*Abbreviations: C, cytosines; Mtase, methyltransferase; T, thymine; 5-mC,