In vitro methylation of DNA with Hpa II methylase.

The enzyme Hpa II methylase extracted and partially purified from Haemophilus parainfluenza catalyzes the methylation of the tetranucleotide sequence CCGG at the internal cytosine. The enzyme will methylate this sequence if both DNA strands are unmethylated or if only one strand is unmethylated. Conditions have been developed for producing fully methylated DNA from various sources. In vitro methylation of this site protects the DNA against digestion by the restriction enzyme Hpa II as well as the enzyme Sma I which recognizes the hexanucleotide sequence CCCGGG. These properties make this enzyme a valuable tool for analyzing methylation in eukaryotic DNA where the sequence CCGG is highly methylated. The activity of this methylase on such DNA indicates the degree of undermethylation of the CCGG sequence. Several examples show that this technique can be used to detect small changes in the methylation state of eukaryotic DNA.     

Methylation of human ornithine decarboxylase gene before transfection abolishes its transient expression in Chinese hamster ovary cells

Different methylations of cloned human ornithine decarboxylase gene with restriction methylases in vitro before transfection greatly reduced the transient expression of ODC in Chinese hamster ovary cells. Single methylation of the gene with Hpa II (CCGG) methylase decreased the transiently expressed peak activity by about 50%, single methylation with Hha I (CCGG) methylase by about 80% whilst a double methylation at both Hpa II and Hha I restriction sites virtually abolished any transiently expressed ornithine decarboxylase activity. These results together with our earlier circumventing evidence indicate that the expression of mammalian ornithine decarboxylase is critically influenced by the methylation state of the gene.     

Isolation and characterization of two proteins possessing Hpa II methylase activity

Two proteins exhibiting Hpa II methylase activity have been purified to homogeneity from Haemophilus parainfluenzae and their physical and catalytic properties have been studied. Separation of the two Hpa II methylase activities was achieved by DEAE-Sephadex A-50 chromatography. In subsequent steps, each methylase was purified separately by chromatography on Sephacryl S-200, phosphocellulose, and hydroxylapatite. The proteins have molecular weights of 38,500 +/- 1,000 (Hpa II) and 41,500 +/- 1,000 (Hpa II') as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Sedimentation equilibrium analyses of the native proteins yield molecular weights of 38,800 +/- 3,000 and 42,200 +/- 3,000 for Hpa II and Hpa II', respectively, indicating that both enzymes are composed of a single subunit. Furthermore, both methylases exhibit identical specificity in the methylation of the nucleotide sequence dC-C-G-G in simian virus 40 (SV40) DNA and in a short synthetic oligonucleotide duplex. Although pH, temperature, and salt optima are the same for both enzymes, homogeneous Hpa II' methylase is more stable than Hpa II methylase. Preliminary peptide mapping indicates that the two enzymes are structurally related, suggesting the possibility that Hpa II' methylase may represent a precursor form of Hpa II methylase.     

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.     

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.     

Specific site methylation in the 5'-flanking region of CYP1A2 interindividual differences in human livers

Human cytochrome P4501A2 (CYP1A2) is involved in the metabolism of a large number of common drugs and is responsible for the metabolic activation of numerous promutagens and procarcinogens. Large interindividual differences exist in the expression of this enzyme. This variability has important implications for drug efficacy and cancer susceptibility. In this sudy, the methylation status of the CCGG site (bp -2759) located adjacent to an AP-1 site in the 5'-flanking region of the CYP1A2 gene was assessed in liver samples from a pool of 55 human donors. DNA methylation is an important epigenetic mechanism controlling gene expression and may be one of the molecular mechanisms underlying the interindividual variation. Analysis was conducted using Hpa II digestion and PCR. Results showed that individual samples varied in the methylation status at this site. The site was found to be hypermethylated in approximately 60% of the samples. To compare methylation status with level of CYP1A2 expression, results were analyzed by median test. In 44% of the samples with expression levels above the median the CCGG site was hypermethylated. However, for those samples with levels below the median hypermethylation of the site was found in 73% of the samples. The difference was statistically significant (p<0.05). These findings indicate that CpG methylation may be involved in controlling the expression of CYP1A2, with hypermethylation reducing expression. Moreover, this approach can be useful in assessing the role of site-specific DNA methylation in interindividual variation of CYP1A2. Analysis of other CpG sites in potentially important regulatory elements of the CYP1A2 gene will continue.     

细胞DNA低甲基化是否为化学致癌启动过程的必经途径

There seems to be a current view that the inhibition of DNA methylation may be a mechanism of initiation of carcinogenesis or one of the steps required in the carcinogenic process. However, because of the deficiencies in research works on cellular level, there is a long way to make such a general relationship between carcinogen and the inhibition of cellular DNA methylation. In this paper the effects of some chemical carcinogens on methylation of newly replicated DNA in human FL cells was analyzed by comparing the weight average length (Lw) of the DNA digests after complete digestion by restriction endonuclease Hpa II. It was observed that two non-genotoxic carcinogens (5-azacytidine and L-ethionine) and two genotoxic carcinogens (MNNG and aflatoxin B1) used in this study all caused obvious cytotoxicity on FL cells at the test concentration. Five days after the termination of 5-azacytidine treatment (2 x 10(-6) M, 24 hours), Lw (kb) of the Hpa II digests of cellular DNA was smaller than that of control (8.0 +/- 0.1 vs 10.9 +/- 1.0, P less than 0.01), the Lw change rate was -27%. When DNA was analyzed from FL cells after 9 days continuous treatment of L-ethionine (2 x 10 M), the digestibility of Hpa II was also increased as compared with that of the control, the Lw values (kb) showed a decrease of about 8% (9.8 +/- 0.3 vs 10.6 +/- 0.3, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Reduced methyl group acceptance of 1-β-d-arabinofuranosylcytosine-containing DNA polymers

Previous studies have shown that 1-β-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 satellite sequences in mouse spermatogenic and somatic DNAs.

The distribution of 5-methyl cytosine (5-MeC) residues in a highly repetitive sequence, mouse major satellite, was examined in germinal versus somatic DNAs by digestion with the methylation sensitive isoschizomers Msp I and Hpa II and Southern blot analysis, using a cloned satellite probe. DNA from liver, brain, and a mouse fibroblast cell line, C3H 10T1/2, yielded a multimeric hybridization pattern after digestion with Msp I (and control Eco RI) but were resistant to digestion with Hpa II, reflecting a high level of methylation of the satellite sequences. In contrast, DNA from mature sperm was undermethylated at these same sequences as indicated by the ability of Hpa II to generate a multimeric pattern. DNAs from purified populations of testis cells in different stages of spermatogenesis were examined to determine when during germ cell differentiation the undermethylation was established. As early as in primitive type A, type A, and type B spermatogonia, an undermethylation of satellite sequences was observed. This suggest that this highly specific undermethylation of germ cell satellite DNA occurs very early in the germ cell lineage, prior to entry into meiosis.     

Purification and properties of the Hpa I methylase.

The purification and catalytic properties of the homogeneous Hpa I methylase is described. The enzyme exists as a single polypeptide chain with a molecular weight of 37,000 +/- 2,000 was shown by sedimentation equilibrium and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The Hpa I methylase transfers methyl groups of S-adenosylmethionine to adenine present in the recognition sequence d(G-T-T-A-A*-C), A* is the N6 methyl adenosine. An average of 2.1 methyl groups per recognition site are transferred by the Hpa I methylase.     

Demethylation of specific sites in the 5'-flanking region of the CYP17 genes when bovine adrenocortical cells are placed in culture.

DNA methylation of CYP17 (steroid 17 alpha-hydroxylase) was studied in bovine adrenocortical cells, which lose the capacity to express this tissue-specific gene in culture by phenotypic switching. Restriction enzyme digestions, and sequencing of a lambda clone of a second CYP17 gene (CYP17A2), showed that there are at least three CYP17 genes in the bovine genome. Southern blotting of DNA digested with Msp I or Hpa II together with Eco RI was used to investigate the methylation status of Hpa II sites at -1.0 kb (H1), -1.8 kb (H2), and -2.3 kb (H4) in CYP17A1 and CYP17A2 and at -0.7 kb (H0) in CYP17A3. In cells and tissues other than white blood cells, H0 was nonmethylated whereas H1 was always methylated; H2 and H4 showed variation in methylation status among different cells and tissues. In particular, whereas H4 was methylated in the bovine adrenal cortex in vivo, there was a rapid and complete demethylation at H4 when adrenocortical cells were placed in culture. Sites downstream from H4 did not change methylation over the first six passages in culture; additionally, the coding region of CYP17 remained fully methylated under all conditions. In contrast to adrenocortical cells, DNA from fibroblasts was nonmethylated at H2, whereas all downstream sites were fully methylated. Digestion with another methylation-sensitive enzyme, Bsa HI, which has a site between H2 and H4, showed that this region is methylated in intact adrenal cortex but nonmethylated both in cultured adrenocortical cells and in fibroblasts. The specific changes in methylation at this site and at H4 in adrenocortical cells indicate a reproducible, environmentally determined change in methylation in adrenocortical cells when they are placed in culture.     

Mythylation of human HLA-D/DR genes: derangement in chronic lymphocytic leukemia

HLA-DR antigens are expressed as differentiation markers in certain human leukemias. To investigate whether DNA methylation plays a role in expression of DR genes in leukemia, we analyzed methylation patterns of the DR-alpha and D/DR-beta genes in the DR antigen-positive and -negative B-cell lines, in normal adults and in chronic lymphocytic leukemia (CLL) patients using Southern blot hybridization of DNA digested with Msp I and Hpa II. The DR-alpha and D/DR-beta genes of a DR antigen positive B-cell line, T5-1, were heavily methylated, while those of DR antigen-negative variant, 6.1.6, were hypomethylated. Blood cells collected from four normal adults contained different levels of DR-alpha and D/DR-beta mRNAs, but their relative amounts were about the same among the individuals. By contrast, the relative amounts of these mRNAs in CLL cells varied widely, indicating aberrant expression of one or both of these genes in CLL. The DR-alpha gene in four normal adults and six CLL patients produced only a 3 kb hybridizable band after Msp I digestion. Normal adult DR-alpha genes were resistant to Hpa II digestion, suggesting that all Hpa II sites are methylated. In contrast, digestion of CLL DNA with Hpa II yielded various bands of larger sizes which differed among the CLL patients, suggesting that Hpa II sites are differentially methylated in the CLL DNA. In the case of D/DR-beta genes, normal adult DNA gave Msp I bands which were slightly polymorphic among four individuals tested. In contrast, CLL DNA showed a high degree of restriction fragment length polymorphism (RFLP) on Msp I digestion. We speculate that the high RFLPs in the CLL DNA may result from differential methylation in CpG clusters in the D/DR-beta genes, and that this characteristic may be of use for diagnosis of CLL.     

DNA methylation controls the inducibility of the mouse metallothionein-I gene in lymphoid cells

The W7 mouse thymoma cell line does not express the metallothionein-I (MT-I) gene in the presence of either cadmium or glucocorticoids, unlike most other cell lines. This cell line was therefore used as a model system for studying the role of DNA methylation on MT-I gene expression. The extent of DNA methylation within the MT-I gene and its flanking regions was determined by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I. In W7 cells, all of the Hpa II sites in the vicinity of the MT-I gene are methylated, whereas in cells that have an expressible MT-I gene (for example, Friend erythroleukemia cells) all of these Hpa II sites are unmethylated. When W7 cells are treated for a few hours with 5-azacytidine, the MT-I gene becomes inducible by both cadmium and glucocorticoids. Addition of hydroxyurea along with 5-azacytidine prevents MT-I gene induction, suggesting that incorporation of 5-azacytidine into DNA is required before this gene can be activated. To determine whether 5-azacytidine treatment changes the methylation pattern near the MT-I gene, we treated W7 cells with 5-azacytidine and selected inducible cells in 10 μM cadmium. All of the Hpa II sites within the MT-I gene are unmethylated in these cadmium-resistant W7 cells. In addition, flanking DNA sequences are also undermethylated in a pattern similar to that seen in Friend erythroleukemia cells that express the MT-I gene. The possible significance of methylation as a mechanism of gene commitment during cell differentiation is discussed.     

DNA-methylase Sau 3A: isolation and various properties

DNA-methylase Sau 3A has been isolated for the first time from Staphylococcus aureus 3A cells and purified by column chromatography on phosphocellulose PII, heparin-Sepharose and blue Sepharose. The purified enzyme methylates the GATC sequence with the formation of GATm5C as can be evidenced from the protection of DNA from digestion with restrictases Sau 3A and Bam HI, the lack of the C3H3-group incorporation into Sau 3A DNA-restricts and the formation of a single methylated base m5C. Sau 3A methylase modifies only a two-filament (but not one-filament) DNA. Thus, methylase Sau 3A modifies the both DNA chains in the recognition site during a single binding act. The 5-azacytidine-containing DNA inhibits by 95% the activity of methylase Sau 3A. Ado-met is the single methyl group donor for methylase Sau 3A. The presence of m6A in the recognition site does not affect the activity of methylase Sau 3A. The practical recommendations for the use of M. Sau 3A, alongside with M. Eco dam, for the study of dam methylation by additional methylation of the DNA in vitro in the presence of [methyl-3H]-S-adenosyl-methionine are given.     

Relative levels of methylation in human growth hormone and chorionic somatomammotropin genes in expressing and non-expressing tissues.

It has been shown that the extent of methylation of cytosine in vertebrate DNA is inversely correlated with gene expression. We studied cytosine methylation in and around the homologous human growth hormone (GH) and chorionic somatomammotropin (CS) genes to determine if these genes are undermethylated in DNA from tissues in which they are expressed (pituitary and placenta, respectively) compared to other tissues. Hpa II and Hha I (which cleave only unmethylated 5' CCGG 3' and 5' GCGC 3' respectively) and Msp I (which cleaves CCGG and CmeCGG) were used to digest DNA samples followed by gel electrophoresis, Southern transfer and hybridization with a GH cDNA probe. The extent of methylation of Hpa II and Hha I sites in the GH and CS genes was leukocyte much greater than pituitary greater than placenta = hydatidiform mole. Taken as a whole, our data support the hypothesis that undermethylation is a necessary but not sufficient condition for gene expression since placental and pituitary DNAs are less methylated than leukocyte DNA in this region. However, the correlation between gene expression and undermethylation is imperfect since (1) hydatiform mole DNA has a very similar methylation pattern compared to placental DNA even though moles make little or no CS and (2) the level of methylation of the GH gene compared to the CS gene does not vary in a tissue-specific manner.     

Direct visualization of the sites of DNA methylation in human,and mosquito chromosomes

Human and mosquito fixed chromosomes were digested with restriction endonucleases that are inhibited by the presence of 5-methylcytosine in their restriction sites (Hha I, Hin PI, Hpa II), and with endonucleases for which cleavage is less dependent on the state of methylation (Taq I, Msp I). Methylation-dependent enzymes extracted low DNA amounts from human chromosomes, while methylation-independent enzymes extracted moderate to high amounts of DNA. After DNA demethylation with 5-azacytidine the isoschizomers Hpa II (methylation-dependent) and Msp I (methylation-independent) extracted 12-fold and 1.4-fold amounts of DNA from human chromosomes, respectively. These findings indicate that human DNA has a high concentration of Hpa II and Msp I restriction sites (CCGG), and that the internal C of this sequence is methylated in most cases, while the external cytosine is methylated less often. All the enzymes tested released moderate amounts of DNA from mosquito chromosomes whether or not the DNA was demethylated with 5-azacytidine. Hpa II induced banding in the centromere chromosome regions. After demethylation with 5-azacytidine this banding disappeared. Mosquito DNA has therefore, moderate to high frequencies of nonmethylated CpG duplets. The only exception is the centromeric DNA, in which the high levels of C methylation present produce cleavage by Hpa II and the appearance of banding. Centromere regions of human chromosomes 1 have a moderately low concentration of Hpa II-Msp I restriction sites.     

A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA

About 1% of the mouse genome is cleaved by Hpa II to give a discrete fraction on gels. The nonmethylated fraction is present in all tested tissues, including sperm, and contains Hpa II sites at about 15 times their frequency in bulk DNA. About 80% of the fraction is composed of sequences that occur once or a few times per genome; the remainder is largely rDNA. Unlike bulk DNA, the fraction is not deficient in CpG, and this may be directly due to the lack of methylation. Genomic mapping of three nonribosomal fragments showed that they are part of islands of DNA within which nonmethylated Hpa II and Hha I sites are highly concentrated. We estimate about 30,000 islands per haploid genome and discuss evidence that many may be associated with genes.