Isolation and characterisation of DNA cytosine 5-methyltransferase from human placenta

DNA cytosine 5-methyltransferase has been extensively purified (about 2600-fold) from the soft tissue of human placenta by chromatography on DEAE-cellulose and hydroxyapatite, and by an affinity step on agarose-immobilized S-adenosylhomocysteine. The isolated enzyme has a molecular weight of 135000 and methylates DNA from various sources in native and heat-denatured forms. The synthetic copolymer poly(dG-dC)·poly(dG-dC) is methylated in B- and Z-conformation to about the same extent. DNA containing hemimethylated sites was isolated from P815 cells grown in the presence of 5-azacytidine. This P815 DNA was used to measure the ‘maintenance’ DNA methylase activity, whereas 5-methylcytosine-free procaryotic DNA served as a substrate for the ‘de novo’ DNA methylase activity in our enzyme preparation. The crude extract as well as the highly purified DNA methylase are capable of transferring methyl groups to these two types of substrate. The fact that both types of activity co-chromatograph during the isolation procedure suggests that one enzyme molecule may exercise both the ‘maintenance’ and ‘de novo’ activity.     

Molecular analysis of N6-methyladenine patterns in Tetrahymena thermophila nuclear DNA.

We have cloned two DNA fragments containing 5'-GATC-3' sites at which the adenine is methylated in the macronucleus of the ciliate Tetrahymena thermophila. Using these cloned fragments as molecular probes, we analyzed the maintenance of methylation patterns at two partially and two uniformly methylated sites. Our results suggest that a semiconservative copying model for maintenance of methylation is not sufficient to account for the methylation patterns we found during somatic growth of Tetrahymena. Although we detected hemimethylated molecules in macronuclear DNA, they were present in both replicating and nonreplicating DNA. In addition, we observed that a complex methylation pattern including partially methylated sites was maintained during vegetative growth. This required the activity of a methylase capable of recognizing and modifying sites specified by something other than hemimethylation. We suggest that a eucaryotic maintenance methylase may be capable of discriminating between potential methylation sites to ensure the inheritance of methylation patterns.     

Interaction of oligonucleotides containing 6-O-methylguanine with human DNA (cytosine-5-)-methyltransferase [published erratumm appears in Biochemistry 1992 Aug 4;31(30):7008

Thirty-base-pair synthetic oligonucleotide duplexes containing a single meG.C (meG = 6-O-methylguanine) or A.C base pair at the 16th position (i.e., 5'-CCCGTTTAAATATACXTATACCCGGGTACC-3', where X = A or meG) were used to study de novo methylation by the purified human DNA (cytosine-5)-methyltransferase isolated from CEM cells. Both duplexes containing meG.C and A.C base pairs show enhanced methyl group acceptor properties. Subsequent introduction of hemimethylated sites at the 15th position of the top strand (the C residue next to the abnormal base pair) and the 7th, 15th (which represents the C residue in the 6meG.C and A.C base pairs), and 27th positions of the bottom strand were used to study the maintenance methylation of the hemimethylated duplexes by the methylase. This revealed striking differences in the rate, amount, and sites of methylation, which are dependent on the position of the hemimethylated site in the duplex. The possible mechanism of action of the methylase is discussed. The data show that 6-O-methylguanine residues in DNA can have other genetic effects apart from their miscoding behavior and that meG.C and A.C base pairs exert different effects in terms of methylation.     

Mechanism of action of eukaryotic DNA methyltransferase: Use of 5-azacytosine-containing DNA

Hemimethylated DNA substrates prepared from cell cultures treated with 5-azacytidine are efficient acceptors of methyl groups from S-adenosylmethionine in the presence of a crude preparation of mouse spleen DNA methyltransferase. Partially purified methyltransferase was also capable of efficiently modifying single-stranded unmethylated DNA. The methylation of single-stranded DNA was less sensitive to inhibition by salt than duplex DNA. The presence of other DNA species in the reaction mix (duplex or single-stranded, methylated or unmethylated) inhibited the modification of the hemimethylated duplex DNA. The enzyme was specific for DNA, since the presence of RNA in reaction mixtures did not inhibit the methylation of DNA. DNA methyltransferase formed a tight-binding complex with hemimethylated duplex DNA containing high levels of 5-azacytosine, and this complex was not dissociated by high concentrations of salt. Treatment of cultured cells with biologically effective concentrations of 5-azacytidine and other cytidine analogs modified in the 5 position resulted in a loss of extractable active enzyme from the cells. The amount of extractable active enzyme recovered slowly with time after treatment. These results suggest that incorporation of 5-azacytidine into DNA inhibits the progress of DNA methyltransferase along the duplex, perhaps by the formation of a tight-binding complex. This complex formation might be irreversible, so that new enzyme synthesis might be required to reverse the block of DNA methylation.     

Isolation and characterization of DNA cytosine 5-methyltransferase from human placenta

DNA cytosine 5-methyltransferase has been extensively purified (about 2600-fold) from the soft tissue of human placenta by chromatography on DEAE-cellulose and hydroxyapatite, and by an affinity step on agarose-immobilized S-adenosylhomocysteine. The isolated enzyme has a molecular weight of 135,000 and methylates DNA from various sources in native and heat-denatured forms. The synthetic copolymer poly(dG-dC) . poly(dG-dC) is methylated in B- and Z-conformation to about the same extent. DNA containing hemimethylated sites was isolated from P815 cells grown in the presence of 5-azacytidine. This P815 DNA was used to measure the "maintenance' DNA methylase activity, whereas 5-methylcytosine-free procaryotic DNA served as a substrate for the "de novo' DNA methylase activity in our enzyme preparation. The crude extract as well as the highly purified DNA methylase are capable of transferring methyl groups to these two types of substrate. The fact that both types of activity co-chromatograph during the isolation procedure suggests that one enzyme molecule may exercise both the "maintenance' and "de novo' activity.     

DNA methylation in wheat. Purification and properties of DNA methyltransferase

The origin and function of the large amount of 5-methylcytosine in plant DNA is not well understood. As a tool for in vitro studies of methylcytosine formation in plants we have isolated and characterized the DNA methyltransferase present in germinating wheat embryo. An enzyme fraction enriched 300-fold over the tissue homogenate was obtained by salt extraction of nuclei, chromatography on DEAE-cellulose, Sephadex G-75, blue Sepharose and on DNA immobilized on cellulose. It catalyzes the methylation of cytosine residues in double-stranded DNAs isolated from wheat, maize, calf thymus or bacteria using S-adenosylmethionine as methyl donor. The efficient methylation of both an unmethylated plasmid DNA and its hemimethylated derivative indicate that the wheat DNA methylase can function de novo and in maintenance methylation. A relative molecular mass of 50,000-55,000 was estimated by gel permeation chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis showed the presence of a protein of Mr = 50,000 and one other component (Mr = 35,000). The preference for endogenous, double-stranded DNA as substrate and the lower molecular mass distinguish wheat DNA methyltransferase from the DNA methylases obtained from mammalian sources. The properties of the wheat enzyme resemble, however, those of the DNA methylase isolated from the alga Chlamydomonas reinhardii, suggesting that plant cells possess their own type of DNA methyltransferase for the biosynthesis of their high methylcytosine content in DNA.     

Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase

DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during replication. Dnmt1 is targeted to replication foci, interacts with PCNA, and favors methylating the hemimethylated form of CpG sites. To understand the underlying mechanism of its maintenance function, we purified recombinant forms of full-length Dnmt1, a truncated form of Dnmt1-(291-1620) lacking the binding sites for PCNA and DNA and examined their processivity using a series of long unmethylated and hemimethylated DNA substrates. Direct analysis of methylation patterns using bisulfite-sequencing and hairpin-PCR techniques demonstrated that full-length Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of over 95%, but unmethylated DNA with much less processivity. The truncated form of Dnmt1 showed identical properties to full-length Dnmt1 indicating that the N-terminal 290-amino acid residue region of Dnmt1 is not required for preferential activity toward hemimethylated sites or for processivity of the enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA during a single processive run. Our findings suggest that these inherent enzymatic properties of Dnmt1 play an essential role in the faithful and efficient maintenance of methylation patterns in the mammalian genome.     

Sequence specificity of cytosine methylation in the DNA of the Chinese hamster ovary (CHO-K1) cell line

We have determined the DNA renaturation kinetics for those DNA sequences of the Chinese hamster ovary (CHO-K1) cells in which enzymatic cytosine methylation occurred immediately after strand synthesis and for those in which methylation was delayed after strand synthesis. DNA sequences showing immediate or delayed methylation were found to be distributed throughout all repetition classes of the DNA of these cells, with a slight concentration of immediate methylation in moderately repetitive sequences and with delayed methylation being slightly over-represented in the highly repetitive fraction. However, DNA sequences showing both classes of methylation were represented equally in unique DNA sequences. We interpret these data to mean that the methylase acting near the replication forks (the 'immediate' methylase) is a relatively inefficient enzyme, missing some 20% of hemimethylated sites produced by DNA replication in these cells. We suggest that the methylase performing maintenance methylation at sites remote from the replication forks (the 'delayed' methylase) is simply a back-up enzyme for the first and that it has no true sequence specificity. The implications of this for the function(s) of DNA methylation in mammalian cells are discussed.     

Sequence specificity of cytosine methylation in the DNA of the Chinese hamster ovary (CHO-K1) cell line

We have determined the DNA renaturation kinetics for those DNA sequences of the Chinese hamster ovary (CHO-K1) cells in which enzymatic cytosine methylation occurred immediately after strand synthesis and for those in which methylation was delayed after strand synthesis. DNA sequences showing immediate or delayed methylation were found to be distributed throughout all repetition classes of the DNA of these cells, with a slight concentration of immediate methylation in moderately repetitive sequences and with delayed methylation being slightly over-represented in the highly repetitive fraction. However, DNA sequences showing both classes of methylation were represented equally in unique DNA sequences. We interpret these data to mean that the methylase acting near the replication forks (the ‘immediate’ methylase) is a relatively inefficient enzyme, missing some 20% of hemimethylated sites produced by DNA replication in these cells. We suggest that the methylase performing maintenance methylation at sites remote from the replication forks (the ‘delayed’ methylase) is simply a back-up enzyme for the first and that it has no true sequence specificity. The implications of this for the function(s) of DNA methylation in mammalian cells are discussed.     

DNA methylation. Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides

A partially purified HeLa cell DNA methylase will methylate a totally unmethylated DNA (de novo methylation) at about 3-4% the rate it will methylate a hemimethylated DNA template (maintenance methylation). Our evidence suggests that many, if not most, dCpdG sequences in a natural or synthetic DNA can be methylated by the enzyme. There is a powerful inhibitor of DNA methylase activity in crude extracts which has been identified as RNA. The inhibition of DNA methylase by RNA may indicate that this enzyme is regulated in vivo by the presence of RNA at specific chromosomal sites. The pattern of binding of RNA to DNA in the nucleosome structure and the DNA replication complex may determine specific sites of DNA methylation. An even more potent inhibition of DNA methylase activity is observed with poly(G), but not poly(C), poly(A), or poly(U). The only other synthetic polynucleotides studied which inhibit DNA methylation as well as poly(G) are the homopolymers poly(dC).poly(dG) and poly (dA).poly(dT). These results point out the unique importance of the guanine residue itself in the binding of the DNA methylase to dCpdG, the site of cytosine methylation. The surprising inhibition of the methylation reaction by poly(dA).poly(dT), which is itself not methylated by the enzyme, suggests the possible involvement of adjacent A and T residues in influencing the choice of sites of methylation by the enzyme.     

Induction of alpha-fetoprotein synthesis in differentiating F9 teratocarcinoma cells is accompanied by a genome-wide loss of DNA methylation.

F9 teratocarcinoma cells can be grown as monolayers or aggregates, and upon treatment with retinoic acid they will differentiate into parietal or visceral endoderm, respectively. Visceral endoderm specifically synthesizes alpha-fetoprotein and albumin mRNAs, which are not found in parietal endoderm. In contrast, both endoderms produce enhanced levels of the major histocompatibility antigen (H2) mRNA compared with F9 cells. F9 cells contain highly methylated DNA as judged by restriction enzyme digestion. However, upon differentiation into visceral endoderm, there is a genome-wide loss of methylation in induced, silent, and constitutively expressed genes. Experiments in which methylation loss is induced via the methyltransferase inhibitor 5-azacytidine result in no induction of alpha-fetoprotein mRNA and no morphological differentiation, suggesting that methylation loss alone is not sufficient to induce the visceral endoderm phenotype. Likewise, 5-azacytidine treatment of differentiated cells does not result in enhanced expression of alpha-fetoprotein mRNA. However, the patterns of loss of DNA methylation at all sites examined after differentiation or 5-azacytidine treatment were remarkably similar, suggesting that the two occur by a similar mechanism, the inhibition of DNA methyltransferase activity. These results argue that the specificity for methylation loss at a given site is an inherent property of aggregated F9 cell chromatin. This system provides a model for studying a tissue-specific change in DNA methylation upon differentiation.     

Effect of 5-azacytidine on DNA methylation in Ehrlich's ascites tumor cells

5-Azacytidine inhibited in vivo DNA methylation in Ehrlich's ascites tumor cells depending upon the dose at which 5-azacytidine did not inhibit DNA synthesis significantly. This drug did not inhibit DNA methylation in vitro. The DNA methylase activity in ascitic cells decreased with the increasing dose of 5-azacytidine. Hypomethylated DNA was obtained from the 5-azacytidine treated ascitic cells.     

DNA modification, differentiation, and transformation

Substantial evidence has accumulated over the last 5 years that the methylation of cytosine residues in vertebrate DNA is implicated in the control of gene expression. We have used analogs of cytidine, modified in the 5 position, as specific inhibitors of DNA methylation to probe the relationship between this process and cellular differentiation. 5-Azacytidine effected marked changes in the differentiated state of cultured cells and induced the formation of biochemically differentiated muscle, fat, and chondrocytes from mouse fibroblast cell lines. Since the analog is a powerful inhibitor of DNA methylation, we suggest that this inhibition is causally related to the mechanism of phenotypic conversion. DNA extracted from cells treated with 5-azacytidine was hemimethylated and was used as an efficient acceptor of methyl groups in an in vitro reaction in the presence of eukaryotic methylases. In vitro methylation was inhibited if the substrate DNA was preincubated with a diverse range of chemical carcinogens including benzo(a)pyrene diolepoxide. Thus, chemical carcinogens may induce changes in gene expression by alteration of cellular methylation patterns. Recent experiments have also demonstrated that freshly explanted diploid fibroblasts from mice, hamsters, and humans lose substantial quantities of 5-methylcytosine during cell division and aging in culture. Taken together, these experiments suggest that the genomic distribution of 5-methylcytosine might have importance in normal differentiation and also in the aberrant gene expression found in cancer and senescence in culture.     

Methylation of hen erythrocyte DNA

We have analysed the 5-methylcytosine content of hen erythrocyte DNA and found it to be lower than that of DNA from other chick tissues analysed. Erythrocyte DNA is also a better substrate for DNA methylase having a five-fold lower Km than DNA from white blood cells. This is probably because it contains a large number of hemimethylated sites. Thus the inverse correlation between methylation and gene expression does not apply to the chick red blood cell.     

Substitutions of a cysteine conserved among DNA cytosine methylases result in a variety of phenotypes.

The proposed mechanism for DNA (cytosine-5)-methyltransferases envisions a key role for a cysteine residue. It is expected to form a covalent link with carbon 6 of the target cytosine, activating the normally inactive carbon 5 for methyl transfer. There is a single conserved cysteine among all DNA (cytosine-5)-methyltransferases making it the candidate nucleophile. We have changed this cysteine to other amino acids for the EcoRII methylase; which methylates the second cytosine in the sequence 5'-CCWGG-3'. Mutants were tested for their methyl transferring ability and for their ability to form covalent complexes with DNA. The latter property was tested indirectly with the use of a genetic assay involving sensitivity of cells to 5-azacytidine. Replacement of the conserved cysteine with glycine, valine, tryptophan or serine led to an apparent loss of methyl transferring ability. Interestingly, cells carrying the mutant with serine did show sensitivity to 5-azacytidine, suggesting the ability to link to DNA. Unexpectedly, substitution of the cysteine with glycine results in the inhibition of cell growth and the mutant allele can be maintained in the cells only when it is poorly expressed. These results suggest that the conserved cysteine in the EcoRII methylase is essential for methylase action and it may play more than one role in it.     

Reduced methyl group acceptance of 1-beta-D-arabinofuranosylcytosine-containing DNA polymers

Previous studies have shown that 1-beta-D-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.     

Methylation of DNA in NaCl-adapted cells of potato

Salt-adapted and control cells of the cultivated potato, Solanum tuberosum cultivar Russet Burbank, untreated or treated with 5-azacytidine (an inhibitor of DNA methylation), were compared with respect to: a) % of cytosine methylation in total nuclear DNA, as determined by HPLC; b) fresh and dry weight. Adapted and control cells were compared also with respect to % of cytosine methylation in DNA, which was purified from DNaseI-partially-digested chromatin and size fractionated by electrophoresis in agarose gels. The growth (represented by dry weight) of the NaCl-adapted cells in saline medium lacking 5-azacytidine was similar to that of control cells in standard medium. The adaptation of the cells was correlated with some increase (+16%) of methylation in total DNA and with a much greater increase in the lower molecular weight DNA fractions which were obtained from the presumably more active chromatin. As expected, the treatment of the cells with the methylation inhibitor induced a decrease in the level of methylation. The decrease of methylation, however, was much greater in the adapted cells, whose dry weight, unlike in the control, was not affected by this treatment.Abbreviations 5-azaCyt 5-azacytidine - C cytidine - 2,4-D 2,4 dichlorophenoxyacetic acid - DW dry weight - EDTA ethylenediaminetetraacetic acid - FW fresh weight - HPLC high performance liquid chromatography - m⁵Cyt 5 methyl cytidine - RB Russet Burbank - SDS sodium dodecyl sulfate - TE 10 mM Tris and 1 mM EDTA - Tris Tris [hydroxymethyl] aminomethane