The Essentials of DNA Methylation Minireview

Adrian Bird Institute of Cell and Molecular Biology University of Edinburgh King’s Buildings Edinburgh EH9 3JR Scotland An obstacle to progress in understanding eukaryotic DNA methylation has been the lack of a good genetic system- the most favorable eukaryotes for genetic analysis(yeasts, Caenorhabditis, Drosophila) have no detectable methyla- tion in their genomes. Not only has this hampered prog- ress, but the clear evidence for life without methylation that these organisms provide has raised questions about the relevance of the whole phenomenon. Li et al. (1992) have responded to the need for genetics by creating a methylation mutant and, in doing so, have shown that mice almost certainly have a requirement for DNA methylation. Transgenic approaches were used to obtain mouse em- bryos homozygous for disruption of the DNA methyltrans- ferase. In a world where gene knockouts can so often be asymptomatic, the effect is notably unambiguous. Homo- zygous mutant embryos are stunted and die at midgesta- tion. The result is all the more striking since it appears that inactivation of the gene is incomplete (approximately 30% of DNA methyltransferase activity remains in the homozy- gous disruptants). Evidently, embryogenesis cannot be completed when levels of DNA methyltransferase are re- duced. The gene encoding DNA methyltransferase is therefore essential. Death by Leaky Repression? Why is DNA methylation essential? (For the purposes of this article, it is assumed that the mutant phenotype is due to loss of the ability to methylate DNA and not some other as yet unknown function of this protein.) We do not know the answer to this question, but there are some clues. Since DNA methylation can stably repress transcription, it could be that repression is leaky in the absence of DNA methyltransferase, leading to premature death. Repres- sion of what? One possible answer is based on the evi- dence that cytosine methylation in both eukaryotes and prokaryotes is primarily a system for neutralizing invading DNA (see Bestor, 1990). Failure to repress transposable elements, proviruses, or other potentially damaging se- quences could be lethal. Another possibility is that methylation-mediated repres- sion of genes is somehow built into the developmental program. This popular idea could account for the lethality of DNA methyltransferase mutants and is supported by some evidence. The stability of gene repression on the inactive X chromosome in somatic cells of eutherian (but not marsupial) mammals clearly owes much to the methyl- ation of CpG islands (see Riggs and Pfeifer, 1992). More difficult to assess is the wealth of correlative data relating DNA methylation to transcription (e.g., current evidence relating DNA methylation to imprinting), as methylation levels have a known tendency to adapt the transcrip- tional status quo. Once they are silenced, genes can sub- sequently acquire methylation (e.g., Enver et al., 1988, and references therein), once active, they can subse- quently demethylate (e.g., Sullivan et al., 1989). Is methylation dancing to the piper or calling the tune? In one case it may be the latter. Transfection experiments have been used to show that the muscle-specific rat a-actin promoter is repressed by partial methylation in both muscle and nonmuscle cells (Paroush et al., 1990). Activation occurs specifically in myoblasts once demethyl- ation has taken place. An unexplained observation is that constructs in which all CpGs are methylated fail to activate the gene, although they do undergo myoblast-specific de- methylation (Yisraeli et al., 1986). 5-Azacytidine May Not Mimic an In Vivo Mechanism Apparent evidence for the involvement of DNA methyla- tion in development came from the early finding that lOTi/ cells differentiate into muscle and other cell types when treated with the methylation inhibitor Qzacytidine (Taylor and Jones, 1979). It seemed that genes were natu- rally repressed by methylation, and demethylation by 5-azacytidine was activating them. Unfortunately, doubt has been cast on this simple scenario by studies of DNA methylation in cultured cells. In many permanent cell lines, tens of thousands of genes have been stably inactivated, owing to de novo methylation of CpG-island promoters that are normally methylation free (Antequeraet al., 1990). Only genes that are essential for life in culture conditions seem to have been spared (presumably because of selec- tion). Methylation of these promoters is specific to cultured cells; in the animal, their repression (where it occurs) must be due to some other mechanism, since methylation apparently absent (see below and Figure 2). The effects of 5azacytidine in culture can be explained by removal of the “unnatural” methylation from genes whose expression triggers differentiation. Thus the myo- genesis-promoting gene MyoDl has been found to pos- sess a CpG island that is methylated in lOT1/2 cells but demethylates upon differentiation of these cells into mus- cle under the influence of 5azacytidine (Jones et al., 1990). In tissues of the mouse, the MyoDl CpG island is not methylated as expected, indicating that methylation is not involved in normal regulation of this gene. Many of the best-documented examples of gene reactivation induced by 5-azacytidine appear to involve reversal of unnatural methylation of CpG islands (Gounari et al., 1987; Sneller and Gunter, 1987). (The only exceptions are genes on the inactive X chromosome, whose CpG islands are de novo methylated naturally.) There is no evidence that 5-azacyti- dine can directly activate naturally methylated, CpG- deficient promoters (see below and Weih et al., 1991). Understanding Methylation-Mediated Repression Uncertainties in accounting for the results of the DNA methyltransferase knockout underline the need for a bet- ter understanding of the DNA methylation system. New