Contributed by Dr. A. Gratchev

Physiological DNA methylation - the only known covalent modification of DNA molecule - is accomplished by transfer of the methyl group from S-adenosyl methionin to 5 position of the pyrimidine ring of cytosine. DNA methylation is observed in most of the organisms at the different stages of evolution, in such a distinct species as E.coli and H.sapience. However some species, like Drosophilae melanogaster lack DNA methylation [1].

Function of DNA methylation in prokaryotes

The extensive research on methylation was conducted on bacteria. In this lower forms, both adenine and cytosine can be methylated, and this modification is involved in DNA replication and arrangement. A series of DNA methyltransferases (DNA-MTases) which can catalyse cytosine methylation in different sequence context were identified [2]. The main function of DNA methylation in bacteria is to provide a mechanism, which protects the cell from the effect of foreign DNA introduction. Restriction endonucleases discriminate between endogenous and foreign DNA by its methylation pattern. Introduced DNA which is not protected by methylation is then eliminated by cleavage [2].

Another function of DNA methylation in prokaryotes is the involvement in the control of replication fidelity. During DNA replication the newly synthesised strand does not get methylated immediately, but analysed for mismatches by the mismatch repair system. When a mutation is found the correction takes place on the nonmethylated strand [3].

Differences of DNA methylation between eukaryotes and prokaryotes

Eukaryotic DNA methylation affects only cytosine residues and specific for CpG sequence. However, the protective function of DNA methylation is similar in eukaryotes and prokaryotes. In humans and rodents inserted viral sequences can become methylated in association with silencing of the introduced genes [4]. The same mechanism is involved in silencing of transgenes in mice [5,6]. Thus function of DNA methylation machinery for recognition and/or eliminating of foreign DNA seem to be conserved in evolution.

The hypothesis on the involvement of DNA methylation in the repair process in eukaryotes was disproved by Araujo et al [7]. It was shown that methylation in eukaryotic cells occurs immediately after replication and even Okazaki fragments are already methylated [7].

Due to the much higher complexity of eukaryotic genome in comparison to prokaryotic one it is logical to presume some additional roles of methylated cytosine as a "fifth base". Indeed, there is number of experimental evidences for the involvement of cytosine methylation in the functional reorganisation of eukaryotic genome. The regions of the genome with a high number of methylated cytosine are usually transcriptionally inactive. The absence of DNA methylation is a prerequisite for transcriptionally active regions. Since DNA methylation is reversible and does not directly depend on the sequence context it was described as an epigenetic mechanism of gene regulation [8,9].

Regulation of DNA methylation in eukaryotic cell

There are two basic types of normal methylation processes known in eukaryotic cells. First is de novo methylation which is involved in the rearrangement of methylation pattern during embryogenesis or differentiation processes in adult cells [10,11]. Recently a family of enzymes was described, containing two methyltransferases DNMT3a and DNMT3b which show the de novo methylation activity [12,13]. The homologous genes were identified in mouse [14]. Gene targeting experiments showed that both DNMT3a and DNMT3b are essential for de novo methylation and have no effect of maintenance methylation [15].

The second methylation activity in eukaryotic cell is the so-called maintenance methylation which is responsible for maintaining the methylation pattern once established. The first mouse maintenance methyltransferase DNMT1 was described by Bestor et al [16]. The enzymes with high homology were found in human [17] and chicken [18]. The functional analysis of the enzyme showed that it has maintenance methylation activity and is vitally important for embryonic development in the mouse. The total homozygous knockout of mouse DNMT1 was lethal for the embryo [19,20]. During DNA replication DNMT1 is located in the replication complex where it recognises the normally methylated CpG sites in the parent strand and catalyses the addition of the methyl group in the corresponding CpG site in the daughter strand. Active localisation of the enzyme to sites of DNA replication in dividing cells may facilitate a maintenance role of DNMT1 [21]. One more methyltransferase - DNMT2 with unclear function was identified by Yoder and Bestor [22]. However already initial studies showed, that this enzyme is not essential for de novo methylation in eukaryotic cells [23].

To alter the established pattern of methylation there must be a mechanism responsible for the removal of existing methylation. There are two mechanisms known until now. First is a passive demethylation which occurs when DNMT1 fails to maintain the existing methylation pattern [24]. Second is active demethylation which is performed by recently described demethylase [25].

CpG islands

The distribution of CpG sites in the genome is as important as the role of DNMT1 activity. During the evolution, the sequence CpG has been progressively eliminated from the genome due to deamination of methylcytosines to thymines. For example in humans this dinucleotide is present only 5 to 10% of its predicted frequency. In 70 to 80% these CpG dinucleotides are methylated. These methylated regions are typical of the bulk chromatin that constitutes most nontranscribed DNA (for review 26)[27].

In contrast to the rest of the genome, smaller regions of DNA termed CpG islands, ranging in size from 0.5 to about 4 to 5 kb [27] have maintained the expected frequency of CpG content. A CpG island is defined as a sequence with a G+C content of greater than 60% and ratio of CpG to GpC of at least 0.6 [28]. Most frequently these islands are located within 5' regulatory regions of genes. This may result from the fact that during evolution these regions were not methylated and, therefore, not depleted through the C to T transitions [26].

These two types of DNA methylation patterns determined by either low content of CpG sequence or CpG islands represent two types of regulatory regions. Genes which contain CpG island in their promoter are usually "housekeeping" genes, which have a broad tissue pattern of expression. Many relatively tissue specific genes are also regulated by CpG island methylation [26]. It is important to note that nonmethylated CpG island within the promoter region is not always associated with actively transcribed gene. However the lack of methylation of the CpG island within the promoter region of the gene is required for transcription of the gene. This modulatory role of methylation is reflected by the fact that chemically induced demethylation of CpG islands associated with inactivated genes leads to their partial reactivation [29].

CpG dinucleotides within the promoters without CpG island are usually methylated in a tissue-specific manner and can reflect the transcriptional status of the genes. In many cases these CpG sites are not methylated if the gene is actively expressed and methylated in cells with little or no transcription of the gene [27].

Regulation of transcription by methylation

In the case of CpG island-containing promoters the lack of methylation is usually associated with the chromatin pattern of actively transcribed genes, as characterised by an opened nucleosome configuration, reduces amount of histone H1 and presence of acetylated histones [30]. The ability of methylation to silence genes with CpG islands was studied on inactivated genes on X-chromosome [29]. Transfection studies showed, that this silencing is mainly a result of chromatin condensation which makes DNA less accessible for transcription factors [31]. The role of single methyl groups, preventing binding of specific factors appears to be less important in this case.

In contrast to this, genes without CpG islands are dependent on the methylation of single sites within their promoter regions. This observation can be explained by the property of some transcription factors whose binding to DNA is methylation dependent, i.e. the protein binds to its binding site only in the case of nonmethylated DNA. Such methylation dependency was described for transcription factor AP-2 [32]. For another well characterised transcription factor - Sp1 the data are contradictory [33,34].

Another mechanism of transcription regulation by methylation of single sites involves methylation dependent binding proteins (MDBPs). MeCP1 described by Meehan et al. needs at least 7 methylated CpGs for efficient DNA binding and therefore is less important for genes without CpG islands in the promoters [35]. MeCP2 (MDBP-2) binds to a single methylated CpG and can inhibit the transcription of the gene [36]. In addition to this two proteins recently Hendrich and Bird described a family of MDBP which have high homology with MeCP2. All of them contain DNA binding domain as well as transcription inhibitory domain [37].

Role of DNA methylation in cancer

Methylation dependent mutations

CpG sites are hotspots for mutation in the human germline [38]. More recently it has become clear that they can be also hotspots for inactivating mutations in tumour suppresser genes [39,40]. About 25% of all mutations in p53 gene in all human cancers studied occur at CpG sites, and almost 50% occur at methylation sites in colon cancer [41]. Since no endogenous chemicals have been found to increase directly the rate at which these mutations occur [42], they should be considered as part of an endogenous process.

The conventional explanation for the existence of the hotspots has been spontaneous hydrolytic deamination of 5-mCyt to T [43]. However, errors made during the methylation process may also contribute to mutagenesis. DNA methyltransferases can catalyse the deamination of C to U when S-adenosylmethionine is limiting [44]. Experiments of Yebra and Bhagwat [45] have shown that cytosine methyltransferases are also capable of the direct conversion of 5-mCyt to T, thus extending the repertoire of side reactions that could contribute to C->T transition.

The direct involvement of the DNA methyltransferase in mutagenesis at CpG sites would be expected to be facilitated by higher levels of enzyme expression, lower level of S-adenosylmethionine and decreased levels of specific repair enzymes. Although there is evidence for 4-3000 fold increase of methyltransferase activity in tumour cell lines [46], the existence of biochemical conditions favouring a C->U->T pathway in human colon tumours was not shown [47].

Overall decrease of DNA methylation in cancer cells

More than a decade ago it was shown that global genomic levels of DNA methylation are lower in cancer cells than in normal tissue [48-50]. In the number of experimental models of carcinogenesis, this decrease in numbers of methyl groups appears to begin early in tumour progression and before the tumour formation [51,52]. A possible direct role for DNA hypomethylation in the neoplastic process has been proposed from experimental data showing that in rodents depletion of methyl donor from the diet results in liver carcinogenesis and in DNA hypomethylation [53].

Despite the clear association of DNA hypomethylation with both spontaneous and experimentally derived tumours, the exact role of this change is poorly understood. In 1983 Feinberg and Vogelstein reported a decrease of methylation in the promoter regions of c-Ha-ras and c-Ki-ras in lung and colon carcinomas [54]. Therefore activation of oncogenes was proposed as a possible role of decrease in DNA methylation in carcinogenesis. However no significant data was collected to test this hypothesis.

Schmidt et al. observed the abnormalities in chromosomal division during cell replication after decrease of overall methylation induced by 5-aza-2-deoxycytidine treatment [55]. This suggests that demethylation may influence the structural integrity of chromosomes leading to cell transformation [56]. However more studies are required to establish the consequence of DNA demethylation in neoplastic cells.

Regional hypermethylation in cancer

The same tumour cells which were described to have the overall genomic hypomethylation frequently have regions of dense hypermethylation. The fact that most of nonmethylated cytosines are located within CpG islands suggests that the normally nonmethylated CpG islands within 5' regulatory regions are the primary targets for aberrant hypermethylation in tumour cells [27].

Baylin et al described that a CpG island in the promoter region of the calcitonin gene at chromosome 11p, which was unmethylated in all normal tissues tested, was densely methylated in human solid tumours [57], leukemias [58] and cells transformed with various viruses [58,59]. Additionally some other CpG-rich regions on 11p, which is known to contain multiple potential tumour suppresser genes [60], were simultaneously hypermethylated [59]. It was suggested that 11p region is a hot spot for CpG island methylation in neoplasia and that this DNA methylation change could be an important potential mechanism for inactivation of tumour suppresser genes [59].

At the same time Antiquera and Bird showed that multiple CpG islands, some associated with genes, were hypermethylated in immortalised human and murine cells [26]. It was postulated that as many as half of the CpG islands in the genome might be so altered in such cells [26,61]. A concept of methylation -associated gene inactivation (MAGI) was suggested [61].

Later loss of the expression, associated with hypermethylation of promoter CpG island was shown for retinoblastoma (Rb) gene in 10% of patients with sporadic form of retinoblastoma [62]. Several publications have documented de novo methylation of the CpG island for the cyclin dependent kinase inhibitor p16 in both cancer cell lines and primary tumours [63,64]. The aberrant hypermethylation correlated with the lack of p16 expression in these cells. Treatment of the cell lines with 5-aza-2'-deoxycytidine resulted in the demethylation of p16 promoter and reactivation of p16 expression [65].

In the case of colon cancer methylation of promoters of several genes was analysed. The particular site of interest in the case of colon carcinoma is the short arm of chromosome 11. Promoters of two genes located there, WT1 and calcitonin appeared to be hypermethylated in the majority of colon carcinomas (68-74%) [66]. Further, the promoter region of APC gene (Chromosome 5q21-q22) appears to be hypermethylated in more than half of patients with sporadic colorectal carcinoma, leading to the loss of the gene expression [67]. This hypermethylation was observed on the later stages of colon carcinoma progression [67], suggesting the possible role of this effect not in the initiation, but in the progression of carcinoma. Expression of hMLH1 gene was shown to be suppressed by methylation [68]. In this case methylation of the hMLH1 promoter can cause the same effect as inactivating mutation leading to loss of functional gene [69]. Additional evidence for late role of methylation in the progression of colon carcinoma given by the results of Issa et al. [70] showing the increased activity of DNMT1 in carcinoma but not in adenoma.

One of the most striking system used for the analysis of the role of DNA methylation in carcinogenesis was an APC^MIN mouse model analysed by Laird et al. [71]. APC^MIN mouse carries a germline mutation in APC gene and develop hundreds of intestinal polyps. The inhibition of methyltransferase with 5-aza-2'-deoxycytidine in these mouse resulted in the reduction of polyp number from 113 to only 2. Although the mechanism by which demethylation reduces the polyp formation remains unclear, these results provide an important evidence for the involvement of methylation in carcinogenesis [71].

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