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DNA Methylation
David P Hornby, University of Sheffield, Sheffield, UK
DNA methylation occurs in most living organisms and is the enzyme-mediated transfer of a methyl group from S-adenosyl-L-methionine to specific cytosine or adenine bases within the genome of that organism. The biological consequences of DNA methylation are thought to be diverse, ranging from resistance to bacteriophage infection in microorganisms to the programming of gene expression in mammals.

Secondary article
Article Contents
. Introduction . Genetics . Biochemistry . Biological Roles of DNA Methylation

Introduction
Methylated deoxyribonucleic acid (DNA) is found in many species of bacteria, some fungi, plants and higher organisms: the notable exceptions being Drosophila melanogaster and the yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. These three organisms are popular model organisms in the sphere of classical and molecular genetics. Interestingly, the emergence of whole genome nucleotide sequences has revealed that genes that are at least very similar to those encoding bona de DNA methyltransferases (the enzymes responsible for this modication reaction) are present in some of the aforementioned organisms. The reaction catalysed by DNA methyltransferases involves the transfer of a single methyl group from the universal methyl donor S-adenosyl-l-methionine, to the C5 position or the nitrogen at C4 of cytosine or to the nitrogen at the C6 of adenine. There exist three distinct classes of enzymes that carry out these reactions, but all share the ability to ip the target base from within the double helical DNA substrate in order to perform the methylation reaction. The methylation of DNA in dierent organisms is central to a variety of biological phenomena. Classically, the methylation of specic DNA sequences in bacteria renders those sequences refractory to cleavage by restriction endonucleases (Wilson and Murray, 1991). In this way an organism can tolerate the expression of a potentially lethal enzyme activity in order to eliminate foreign (unmethylated) DNA molecules, such as those arising from bacteriophage infection. This phenomenon is referred to as biological restriction and modication and was the subject of one Nobel Prize and has been one of the driving forces behind contemporary experimental molecular cloning. Indeed, as a consequence of the advances in molecular cloning over the last two decades of the twentieth century, the nucleotide sequence of the rst mammalian gene specifying a DNA methyltransferase enzyme was obtained (Bestor et al., 1988). This has opened the way for a thorough molecular analysis of DNA methylation in mice and more recently in humans. It is clear, from experiments on transgenic mice decient in normal DNA methyltrans-

ferase activity, that this reaction is central to normal animal development (Li et al., 1992). More specically, DNA methylation is intimately associated with the phenomena of X-chromosome inactivation (in which expression of genes on the X-chromosome are selectively repressed) and genomic imprinting (by which expression of either maternal or paternal genes is selectively silenced). Finally, abnormal patterns of gene methylation and the uncoupling of DNA methyltransferases from other related activities during the transcription of genes, contribute to diseases such as cancer (Jones and Laird, 1999).

Genetics
The genetics of DNA methylation can be divided into two broad areas: the structure and organization of the genes encoding DNA methyltransferases and related proteins and the epigenetic phenomena associated with genomic methylation. In bacteria, the gene sequences of several hundred DNA methyltransferases have been determined and analysis of their open reading frames has revealed common features that enable C5 cytosine-specic DNA methyltransferases to be readily distinguished from those enzymes that modify the exocyclic nitrogens of adenines and cytosines (Dryden, 1999). Bacterial DNA methyltransferase genes are usually found in close association with restriction endonuclease genes, although there are some notable exceptions. These include the dam and dcm genes that encode isolated adenine and cytosine specic DNA methyltransferases, respectively. Mutants lacking the dam gene show defects in DNA replication and repair, whereas the dcm gene appears to be associated with the management of biological cytosine deamination, which is more prevalent when the base is unmodied, leading to genome damage. A less common class of cytosine C5-specic DNA methyltransferase genes has been found in a number of bacteriophage; these genes encode enzymes capable of methylating between two and ve specic DNA sequences, including the sequence modied by the product of the dcm gene (Noyer Weidner and Trautner, 1993).
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In one rationalization of their biological abundance, restriction and modication genes act in concert to provide a primitive immune response to bacteria. It has also been proposed that the coexistence of restriction and modication genes reects their selsh spread through the bacterial kingdom. Finally, ancillary roles for restriction and modication include stimulation of recombinational genetic exchange, thereby accelerating the evolutionary process. It is clear, nonetheless, that DNA methyltransferase genes are an essential adjunct to restriction endonuclease genes and that the two can work in concert to modulate a number of biological processes at the genetic level (Bird and Wole, 1999).

Biochemistry
5-Methylcytosine is present at a level of between 2 and 7% in mammalian genomic DNA, but no detectable adenine methylation has been observed to date. In contrast, bacterial DNA is generally methylated at adenines in GATC sites (the target for the product of the dam gene) and additionally at those adenines that are modied by any host-encoded restriction and modication genes. However, it should be noted that the detection of DNA methylation in genomic DNA is only as good as the sensitivity of base detection after separation of modied from unmodied bases, typically via chromatography. The uneven distribution of methylated cytosines in CpG sequences is a dominant feature of mammalian genomes and is thought to provide a linkage between chromosomal structure and packaging and the regulated expression of genes. Indeed, the combination of the DNA methylation apparatus, comprising at least four distinct enzymes and a family of CpG-binding proteins, is critical for normal epigenetic gene function (Wole and Matzke, 1999). The widespread adoption of DNA methylation as means of modulating gene function in vivo arises for two main reasons. The machinery responsible for DNA replication is largely insensitive to chemical substitutions at the C5 and C4 position of pyrimidines and at the N6 position of adenines (at least in bacteria); moreover, once a methylation signature is incorporated into genomic DNA, it is faithfully duplicated at each subsequent round of chromosomal replication. Secondly, in contrast, the anities with which many gene regulatory proteins and nucleic acid-modifying enzymes interact with specic nucleic acid sequences can be modulated by site-specic DNA methylation. For example, site-specic methylation of restriction endonuclease recognition sites renders those sites catalytically inert, and methylation of certain gene promoter element prevents the assembly of competent transcription complexes, thereby facilitating gene silencing. It should also be made clear at this point that the presence of an additional hydrophobic methyl group in a
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given DNA sequence context can also stimulate protein interactions with DNA. Indeed, restriction endonucleases exist that require methylation for their activity; and, in higher organisms, methylated DNA-binding proteins, as their name suggests, are able to discriminate between methylated and unmethylated CpG sequences by virtue of the gain in interaction energy aorded by the presence of a pair of major groove methyl groups. The catalytic transfer of a methyl group to DNA is best understood for C5 cytosine-specic methylation. Through a combination of synthetic nucleotide chemistry and X-ray crystallography, the remarkable pathway of DNA methyl transfer has been elucidated and provided signicant new insight into how many enzymes modify nucleic acids (Chen et al., 1991; Klimasauskas et al., 1994). Nucleotide analogues containing substitutions in the pyrimidine ring, such as 5-azacytidine and 5-uorocytidine, when incorporated into DNA at specic sequences, seed the formation of covalent complexes between the DNA and C5 cytosine-specic DNA methyltransferases. By synthesizing such inhibitory DNA duplexes and analysing crystals of the DNA in complex with a bacterial methyltransferase M. Hhal, Xiaodong Cheng and Richard Roberts were able to capture a DNA methyltransferase half way through its catalytic cycle. The most surprising result of this physicochemical analysis was the realization that this type of enzyme (and subsequently many others) ips out a nucleotide from the DNA double helix, in order to access that base that is targeted for methylation. This spectacular reaction enables all of the S-adenosyl-l methionine-dependent catalytic steps to take place in a conventional active site cleft, before the product, methylated cytosine, is returned to its resting state in double helical DNA. Subsequent crystal structures of related DNA methyltransferases and DNA repair enzymes point to base ipping as a universal mechanism employed by proteins to facilitate DNA modication in vivo.

Biological Roles of DNA Methylation

Table 1 lists some examples of the diverse uses to which living organisms have put the DNA methylation reaction. Given that enhanced hydrophobicity in the major groove coupled with a sequence-specic context eect can modulate protein binding, it is not surprising to nd methylation being adopted as an epigenetic device. In bacteria, the avoidance of restriction endonuclease attack through site-specic methylation in restriction driven immunity contrasts with the nding that the same modication can render some DNA sequences prey to methyl-dependent restriction enzymes. This demonstrates how proteins have evolved as sensors of the biological methylation status of genes, with the accompanying switch operating in both on and o modes. Similarly in higher

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DNA Methylation

Table 1

The diverse biological roles of DNA methylation Comments Exclusively prokaryotic process in which DNA methylation affords protection against endonuclease cleavage In bacteria, dam-mediated methylation coordinates synchronous replication and in certain bacteria methylation synchronizes the cell cycle. In mammals, an interaction between the cell replication protein, PCNA, has been reported In fungi, methylation has been demonstrated as central to duplication-induced epigenetic phenomena The mobilization of transposable elements is associated with DNA methylation Inactivation of genes on the X-chromosome is associated with methylation Parental-specific allele expression is associated with differential DNA methylation

DNA methylation-associated phenomena Restriction and modification

Cell cycle regulation

Recombination

Transposition Gene silencing Imprinting

organisms, methylation of clusters of CpG sites can render those regions of a genome transcriptionally dormant, by recruitment groups of methylcytosine-specic DNA binding proteins, often in concert with ancillary gene silencing proteins. One of the most unusual roles of methylation is in the fungal phenomena of RIP (repeat induced point mutation) in Neurospora and MIP (methylation induced premeiotically) in Ascobolus (Selker, 1999). In both cases, methylation of cytosines in DNA is used as a means of protecting the genome from potentially damaging change. During the RIP process, a cascade of G-C to A-T transition mutation is accompanied by subsequent cytosine methylation of these so called RIP-modied sequences. This process serves to inactivate expression of any unwanted duplicated genes and minimizes downstream homologous recombination. In the MIP phenomenon, as with RIP, methylation of duplicated sequences occurs, but it is not accompanied by widespread mutation. Perhaps the most fascinating aspect of DNA methylation is that a cluster of enzymes in mammals, bearing similarities to the C5 cytosine-specic DNA methyltransferases found in bacteria and plants, mediate complex regulatory events during animal development. Moreover, the failure to establish a normal epigenetic programme during development underlies several specic inherited disorders and is emerging as a key factor in tumour progression. Methylation of CpG sequences can lead to the selective silencing of maternal or paternal alleles, giving

rise to the phenomenon of genetic imprinting. Abnormal imprinting is thought to provide a molecular explanation for Angelman and PraderWilli syndromes. In addition, experimental disruption of the gene encoding one of the several murine DNA methyltransferase genes results in abortive embryonic development. It is perhaps not surprising then that, given the prevalence of epigenetic gene regulation in higher organisms, predisposition towards certain cancers appears to correlate with anomalous patterns of genomic methylation (Baylin et al., 2000). These links between disease and aberrant DNA methylation are currently providing the impetus for novel highthroughput diagnostic tests for gene specic and genomic methylation.

References
Baylin SB, Belinsky SA and Herman JG (2000) Aberrant methylation of gene promoters in cancer concepts, misconcepts, and promise. Journal of the National Cancer Institute 92: 14601461. Bestor T, Laudano A, Mattaliano R and Ingram V (1988) Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases. Journal of Molecular Biology 203: 971983. Bird AP and Wole AP (1999) Methylation-induced repression belts, braces, and chromatin. Cell 99: 451454. Chen L, MacMillan AM, Chang W et al. (1991) Direct identication of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase. Biochemistry 30: 1101811025.

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Dryden D (1999) Bacterial DNA methyltransferases. In: Cheng X and Blumenthal RM (eds) S-Adenosylmethionine-dependent Methyltransferases: Structure and Function, pp. 283340. Singapore: World Scientic Press. Jones PA and Laird PW (1999) Cancer epigenetics comes of age. Nature Genetics 21: 163167. Klimasauskas S, Kumar S, Roberts RJ and Cheng X (1994) Hhal methyltransferase ips its target base out of the DNA helix. Cell 76: 357369. Li E, Bestor TH and Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69: 915 926. Noyer-Weidner M and Trautner TA (1993) Methylation of DNA in prokaryotes. In: Jost J-P and Saluz HP (eds) DNA Methylation: Molecular Biology and Biological Signicance, pp 95103. Basel: Birkhauser.

Selker EU (1999) Gene silencing: repeats that count. Cell 97: 157160. Wilson GG and Murray NE (1991) Restriction and modication systems. Annual Review of Genetics 25: 585627. Wole AP and Matzke MA (1999) Epigenetics: regulation through repression. Science 286: 481486.

Further Reading
Kornberg A and Baker TA (1992) DNA Replication, 2nd edn. New York: WH Freeman. Noyer-Wiedner M and Trautner TA (1993) Methylation of DNA in prokaryotes. In Jost J-P and Saluz HP (eds) DNA Methylation: Molecular Biology and Biological Signicance, pp. 39108. Basel: Birkhauser.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net