DNA demethylation.

Cytosine 59 methylation of CpG dinucleotides within and around genes exerts a major influence on transcription in many plants and animals (1–3). DNA methylation can be causal for transcriptional silencing (4–5) and targets the machinery necessary to assemble specialized chromatin enriched in deacetylated histones (6–8). Once established in somatic cells, CpG methylation patterns within the genome are very stable and provide an attractive mechanism for segregating a large fraction of stably repressed chromatin (9–11). In contrast, DNA methylation is remarkably dynamic during early mammalian development and in certain tumor cells (12, 13). Alterations in the methylation status of the entire genome (14, 15), individual chromosomes (16), and specific genes (17–20) are essential for normal development (21, 22) and can promote tumorigenesis (23, 24). Understanding how these important transitions might be regulated requires the biochemical definition of the enzymatic processes that both methylate and demethylate the genome. Two mammalian DNA methyltransferases have been functionally defined (25, 26) from a family of related proteins (26, 27). Dnmt1 is essential for inactivation of the X chromosome and genomic imprinting in the mammalian embryo (21). This large enzyme (1,620 amino acids) is targeted to replication foci consistent with the rapid remethylation of DNA in somatic cells (28–30). Like the prokaryotic cytosine-5 methyltransferases with which it shares homology, the enzyme makes use of the Michael addition mechanism to carry out the reaction by first increasing the reactivity of the C-5 position of cytosine (31–33). An enzyme cysteinyl thiolate forms a covalent linkage with C-6 of cytosine, and the carboxyl group of an invariant glutamyl residue protonates the N-3 position to create a cytosine 4, 5 enamine. This reactive moiety attacks the sulfonium linked methyl group of S-adenosyl L-methionine. After transfer of the methyl group, the proton is abstracted from the C-5 of cytosine to reform the 5, 6 double bond and release the enzyme by b-elimination (34, 35). In contrast to the well defined molecular genetics, cell biology, and biochemistry of the Dnmt1 methyltransferase, the enzymatic basis of demethylation of 5-methylcytosine in vivo has been mysterious. Although strategies for demethylating DNA have been proposed (Figs. 1 and 2), none have yet been proven to operate under relevant physiological circumstances in vivo. This issue has now been brought into sharper focus by the recent reports of a mammalian protein with specific demethylase activity for methyl CpG dinucleotides (36), together with a demethylase enzyme complex that acts processively (37) and as published in this issue of Proceedings the finding that this complex converts 5-methylcytosine to cytosine and methanol (38).