DNA methylation in mammalian nuclei

A novel system to study the methylation of newly synthesized DNA in isolated nuclei was developed. Approximately 2.5% of cytosine residues incorporated into nascent DNA became methylated by endogenous methylase(s), and the level of DNA modification was reduced by methylation inhibitors. DNA synthesis and methylation were dependent on separate cytosol factors. The cytosol factor or factors required for DNA methylation were sensitive to trypsin digestion and were precipitable by (NH4)2SO4, suggesting that they were proteinaceous. Time-course experiments revealed a short lag of approximately 20 s between synthesis and methylation in nuclei. The DNAs produced in these nuclei were a mixed population of low molecular weight fragments and higher molecular weight fragments shown to be short extension of existing replicons. The methylation level found in low molecular weight DNA was lower than that found in bulk L1210 DNA, indicating that further methylation events might take place after ligation of small fragments. These data suggest that newly synthesized DNA is a good substrate for methylase enzymes and that nuclear cytoplasmic interactions may be important in controlling inheritance of methylation patterns.     

Pre-replicative association of multiple replicative enzyme activities with the nuclear matrix during rat liver regeneration

As a step toward the molecular elucidation of the putative replicational apparatus associated with the nuclear matrix, we have investigated the possible matrix association of several replicational related enzymes. In addition to the previously identified DNA polymerase alpha, DNA primase, 3'-5' exonuclease, RNase H, and DNA methylase were all recovered at significant levels (20-30% of total nuclear activity) in nuclear matrix isolated from regenerating rat liver during maximal in vivo replication (22 h post-hepatectomy). In contrast, DNA ligase was not detected on the nuclear matrix even though significant activity was present in isolated nuclei. Examination of the replicative dependency of these enzyme activities following partial hepatectomy revealed pre-replicative elevations which were distinct for each matrix-bound enzyme. A second late-replicative peak in DNA methylase is consistent with a role of this matrix-bound enzyme in the maintenance of the inheritable methylation pattern. Mild sonication resulted in a significant release of all of these activities except RNase H. A major portion of the matrix-solubilized DNA polymerase alpha, DNA primase, 3'-5' exonuclease, and DNA methylase activities cosedimented on sucrose gradients between approximately 8-12 S. Our results are consistent with the organization of at least a portion of these replicative enzymes into nuclear matrix-bound replicational complexes. We also propose a novel pre-replicative assembly model of the matrix-bound replicational apparatus in which DNA primase plays an initial and critical role.     

Effects of DNA binding proteins on DNA methylation in vitro

The inheritance of DNA methylation patterns may play an important role in the stability of the differentiated state. We have therefore studied the inhibitory effects of DNA binding proteins on DNA methylation in vitro. Mouse L1210 cells grown in the presence of 5-azacytidine acquire hemimethylated sites in their DNA. Purified hemimethylated DNA accepted methyl groups from S-adenosyl-L-methionine in the presence of a crude maintenance methylase more readily than purified DNA isolated from cells not exposed to 5-azacytidine. On the other hand, chromatin fractions isolated from cells grown in the presence or absence of 5-azacytidine were poor substrates for the maintenance methylase irrespective of the number of hemimethylated sites present in the DNA. Inhibition of DNA methylation was shown to be associated primarily with chromatin proteins bound to DNA, and trypsinization of nuclei increased their methyl accepting abilities. Methyl acceptance was increased by salt extraction of chromosomal proteins. These data suggest that association of histones with DNA may play a role in the modulation of methylation patterns.     

Cell cycle-dependent regulation of eukaryotic DNA methylase level

DNA methylase activity in the nuclei of somatic cells arrested at G0 increased markedly when the cells were subjected to a mitogenic stimulus. Treatment of mouse splenocytes with Concanavalin A resulted in about 20-fold increase in methylase activity within 20 h starting 12-15 h after Concanavalin A addition. The methylase level in rat liver was elevated approximately 3-fold at about 20-h posthepatectomy. A detailed time course of the increase in methylase activity with respect to the cell cycle revealed that the onset of this event coincided with the entry of the cells into S phase. In both systems, the extent of methylation in CpG sequences is not altered significantly even under conditions of active DNA synthesis which is induced by the mitogenic effect. These results suggest that the cell responds to the mitogenic stimulus by adjusting the DNA methylase activity to enable conservation of the methylation level in DNA.     

Determination of DNA methylase activity in animal tissue extracts

An express method for measuring the level of in vitro DNA methylation in homogenates and nuclei from animal tissues as well as during initial steps of DNA methylase isolation and purification when methylase activity is low and hardly testable by other methods has been suggested. The method is based on the measuring the radioactivity incorporated in filter adsorbed DNA (acid-insoluble material) 3H-label from S-adenosile-L-methionine as a result of in vitro DNA methylation. The advantage of the method consists in the replacement of a long-duration repeated deproteinization procedure traditionally used by a relatively simple procedure (15 min incubation of the mixture at 80 degrees C with 10 volumes of the 8M urea, 5 mM EDTA, 5% n-butanol, 2% sodium dodecilsulfate, 1 M sodium chloride solution) and the absence of any loss of DNA. The method is fit for the fast serial assay of DNA methylase activity taking into consideration that about one third of the total acid-insoluble radioactivity is due to the radioactivity in 5-methylcytosine residues in DNA.     

Methylation of DNA of the brain and liver of young and old rats.

In vitro methylation of purified DNA and chromatin-DNA in nuclei of the liver and brain of young (18 week) and old (120 week) female rats has been studied using 3H-SAM as the -CH3 group donor. Incorporation of -CH3 group is higher in old liver and brain, but it is far higher in the latter. 5 mC is 11% lower in the old brain, but there is no difference in the liver. Methylation by Hpa II methylase does not show any difference in the incorporation of -CH3 group into DNA of the liver of the two ages. However, its incorporation is lower in the old brain. Methylation by Msp I methylase causes slightly higher incorporation of -CH3 groups in the old brain. This shows a higher percentage of unmethylated external cytosines in the 5'-CCGG-3' sequences. On the contrary, methylation by Eco RI methylase is considerably higher in the old brain. These studies show alterations in the methylation status of the DNA during ageing which may cause changes in the expression of genes.     

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.     

A possible role of chromatin and tightly-bound chromatin proteins on enzyme-catalyzed methylation of DNA

Upon extensive digestion with DNAaseI of placenta chromatin matrix, previously "stripped" from its loosely-bound components by high-salt extraction, a fraction is obtained that contains almost no endogenous DNA methylase activity but whose DNA, if still included in this whole fraction--not if it has been purified to a protein-free condition--is a good substrate for externally added enzyme. This chromatin matrix can even cause a significant stimulation of methylation of single-stranded Micrococcus luteus DNA by placental methylase. In vivo, this phenomenon may have possible counterparts in the existence of highly-methylated regions of chromatin loops that appear to be protected by tightly-bound protein components from digestion of the "stripped loops" with DNAaseI.     

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.     

Partial purification of a ribosomal ribonucleic acid methylase from rat liver nuclei and methylation of undermethylated nuclear ribonucleic acid from regenerating liver of ethionine-treated rat

S-Adenosylmethionine-dependent ribosomal RNA (rRNA) methylase has been purified approx. 90-fold from rat liver nuclei. The partially purified methylase catalyzes the methylation of base and ribose in hypomethylated nuclear rRNA prepared from the regenerating rat liver after treatment with ethionine and adenine. The enzyme has an apparent molecular weight of about 3 x 10(4) and a sedimentation coefficient of 3.0 S. The enzyme is optimally active at pH 9.5 and sensitive to p-chloromercuribenzoate. Thiol-protecting reagents, such as dithiothreitol, are necessary for its activity, and the enzyme requires no divalent cations for its full activity. This enzyme did not efficiently transfer the methyl group to nuclear rRNA from normal rat liver, compared with hypomethylated nuclear rRNA. Methyl groups were mainly incorporated into pre-rRNA larger than 28 S, and the extent of 2'-O-methylation of ribose by this enzyme was greater than that of base methylation in the hypomethylated rRNA. No other nucleic acids, including transfer RNA (tRNA) and microsomal RNA from normal as well as ethionine-treated rat livers, tRNA from Escherichia coli, yeast RNA, and DNA from rat liver and calf thymus, were significantly methylated by this methylase. These results suggest that partially purified rRNA methylase from rat liver nuclei incorporates methyl groups into hypomethylated pre-rRNA from S-adenosylmethionine.     

Extent of equilibrium perturbation of the DNA helix upon enzymatic methylation of adenine residues

The extent of equilibrium perturbation of the DNA helix associated with enzymatic methylation of dA residues has been determined by the agarose gel electrophoresis band-shift method. Utilization of EcoRI methylase under conditions of reduced specificity together with Escherichia coli dam methylase permitted modification of up to 300 dA residues/plasmid pBR322 dimer. A conformational change associated with methylation was observed, with the magnitude of the transition being linear with extent of modification of relaxed DNA circles. The conformational change corresponds to an unwinding of the DNA helix by 0.5 degrees/methyl group transferred to relaxed molecules. The magnitude of the effect was independent of temperature from 5-37 degrees C indicating that it is not the consequence of a thermal transition within this range.     

Nuclear matrix-associated DNA methylase

Previous procedures for the extraction of DNA methylase (EC 2.1.1.37) from nuclei of mouse ascites cells have involved the use of buffers containing 0.2M NaCl. Whilst such 'soluble' methylase accounts for the bulk (70-80%) of DNA methylase activity a further portion of activity is detectable in a 'bound' form firmly associated with 2 M NaCl-resistant nuclear matrix-like structures. This association, which in part requires continuing DNA replication and protein synthesis, can, however, be disrupted in vitro with high concentrations of ammonium sulphate, and the enzymic properties of the 'bound' form of DNA methylase are similar to those described for the 'soluble' form.     

Substrate recognition and selectivity in the type IC DNA modification methylase M.EcoR124I.

The type I DNA modification methylase M.EcoR124I binds sequence specifically to DNA and protects a 25bp fragment containing its cognate recognition sequence from digestion by exonuclease III. Using modified synthetic oligonucleotide duplexes we have investigated the catalytic properties of the methylase, and have established that a specific adenine on each strand of DNA is the site of methylation. We show that the rate of methylation of each adenine is increased at least 100 fold by prior methylation at the other site. However, this is accompanied by a significant decrease in the affinity of the methylase for these substrates according to competitive gel retardation assays. In contrast, methylation of an adenine in the recognition site which is not a target for the enzyme results in only a small decrease in both DNA binding affinity and rate of methylation by the enzyme.     

鼠肝细胞癌变中DNA甲基化作用的研究

Activity of DNA methylase and DNA methylation level were measured from normal mouse liver, mouse liver charged with H22a ascitic hepatoma and H22a ascitic hepatoma cell by measuring incorporation of H3-methyl. S-Adenosyl-3H-methyl-methionine (3H-SAM) was used as methyl donor. DNA methylation level of different cells were measured by HP-LC. DNA methylase activity and DNA methylation level of H22a ascitic hepatoma, mouse liver charged with H22a ascitic hepatoma are lower than normal mouse liver. Treatments of antitumor drugs lead to a rising of DNA methylase activity of tumor cell, however, the DNA methylation level of tumor cell has not rised after such treatments.     

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.     

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.     

DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase.

Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (greater than 65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivo.     

Selenite: a good inhibitor of rat-liver DNA methylase

DNA methylase from rat liver was partially purified through a DEAE sephacel column and characterized in an in vitro assay with respect to time, protein, DNA and S-adenosylmethionine curves. The Km for S-adenosylmethionine was 2.5 microM. Sodium selenium inhibited the methylation of DNA in a dose dependent fashion when added to the assay. It was also demonstrated that selenite non-competitively inhibits rat-liver DNA methylase with a Ki of 6.7 microM. Dithiothreitol had no effect on selenite inhibition and increasing amounts of DNA did not alter the inhibition. However, increasing amounts of protein overcame the inhibition, suggesting that selenite is reacting with the DNA methylase protein. DNA methylase isolated from selenite treated animals had only 43% of the activity as enzyme from control rats. It appears that selenite is a good inhibitor of DNA methylase.