Methylation of parental and progeny DNA strands in Physarum polycephalum

Although 5-methylcytosine comprises 4 to 8% of the cytosine residues in the major nuclear DNA of Physarum polycephalum (Evans &; Evans, 1970), only 1 % of the cytosine residues of progeny DNA become methylated during replication. Further methylation occurs during the same and subsequent mitotic cycles, so that 6 to 7 cycles after its synthesis, 5-methylcytosine comprises 5 to 7% of the DNA-cytosine residues of a single generation of DNA. The extent of methylation occurring during the S period has been measured by the determination of the specific activity of the precursor (S-adenosylmethionine) and the product (DNA-5-methylcytosine) and by comparison of the radioactivity in DNA-cytosine and DNA-5-methylcytosine after incorporation of [¹⁴C]deoxycytidine. Continuing methylation of parental DNA has been shown, by density shift experiments and by the conversion of prelabeled DNA-cytosine to DNA-5-methylcytosine. The DNA-5-methylcytosine once formed was found to be stable.     

Effect of trypsin on DNA methylation in isolated nuclei from developing sea urchin embryos

Nuclei isolated from the developing sea urchin embryo $\text{Paracentrotus lividus}$ and incubated in the presence of [³H-methyl] S-adenosylmethionine methylate their own DNA. Addition of small amounts of trypsin produces a 20-fold increase in DNA methylation. The time kinetics and the specificity of the trypsin activation of DNA methylation are described. The only products of the reaction are 5-methylcytosine and thymine. DNA adenine, guanine and cytosine are not labeled. The distribution of the counts between 5-methyl-cytosine and thymine is variable. While 5-methylcytosine originates by enzymatic methylation of DNA cytosines, the origin of the labeled thymine cannot be inferred with certainty.     

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts

The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m⁵C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m⁵C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.     

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.     

5-Methylcytosine content and methylation status in six millet DNAs

High performance liquid chromatographic analysis of the total nuclear DNAs of 6 millets plant species indicates that the 5-methylcytosine content ranges from 3% in barn yard millet to 9.6% in great millet while the fraction of cytosines methylated varies between 14% in little millet to 31 % in pearl millet. Digestion of millet DNAs with MspI/HpaII suggests that CpG methylation is more in great millet DNA while CpC methylation is more in the other 5 millet DNAs. Digestion of millet DNAs with MboI, Sau3AI andDpnI indicates that some of the^5’ GATC^3’ sequences are methylated at adenine and/or cytosine residues except in little millet where adenine methylation of the^5’GATC^3’ sequences is insignificant and there is a predominance of cytosine methylation in these sequences.     

Correlation between synthesis and methylation of DNA in HeLa cells

For the whole cell cycle the methylation of DNA was studied in synchronized $\text{HeLa}$ cells and in nuclei isolated from them. In the intact cells the methylation of DNA cytosine runs parallel to DNA synthesis. The pattern of DNA cytosine methylation by the isolated nuclei is almost identical to that obtained with the whole cells. Since the isolated nuclei do not synthesize DNA, it is shown that DNA methylation continues for at least 30 min after DNA synthesis is over. No DNA minor thymine is found in the isolated nuclei.     

Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging

The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as ¹⁵N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HpaII (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HpaII methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HpaII methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered.     

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.     

Distribution of N<Superscript>6</Superscript>-Methyladenine in DNA of T2 Phage and its Host <Emphasis Type="Italic">Escherichia coli</Emphasis> B

N⁶-METHYLADENINE (6-MeAde) and 5-methylcytosine occur as minor bases in bacterial and phage DNA^1–7 and seem to result from the selective methylation of adenine and cytosine residues by specific DNA methylases⁸. Methylation is the final stage in DNA synthesis and is essential for the phenomenon of host modification of phages^9–11; it is one of the mechanisms controlling DNA replication in the cell^{12, 13}. A study of the distribution of minor bases in DNA is therefore important not only for the elucidation of the specificity and mechanism of action of DNA methylases but also for an understanding of the purpose of this methylation. We believe that in Escherichia coli, DNA methylase exerts its action on adenine residues in chain terminating triplets: 6-MeAde may serve as a signal for gene termination in this system.     

Structure of animal mitochondrial DNA: nucleotide composition, pyrimidine clusters, and methylation character.

The nucleotide composition, relative concentration of pyrimidine clusters, and the degree of methylation of the mitochondrial and nuclear DNA's of various vertebrates and the protozoan Crithidia oncopelti have been studied. With respect to the relative concentration of GC pairs, the mtDNA of animals (bull, rat) does not differ from the corresponding nDNA. The relative concentration of GC pairs in the mtDNA of certain fish and birds is 1.5-2.5 mole% higher than in the respective nDNA. The kinetoplast DNA of the protozoan C. oncopelti (where the relative concentration of the GC pairs is 42.9 mole %) differs very sharply in composition from the nDNA (where the relative concentration of GC pairs is 51.3 mole %). The mtDNA's and kDNA's studied are distinguished from the respective nDNA'S by a lower degree of clustering of pyrimidine nucleotides. The proportion of mono- and dipyrimidine fragments in the mtDNA and kDNA is 30 mole %, while in the nDNA it does not exceed 23 mole %. The relative concentration of long pyrimidine clusters (hexapyrimidine clusters of larger) in the mtDNA is smaller than in the nDNA by a factor of 2-5. The low degree of clustering of the pyrimidine nucleotides is apparently characteristic of all the known mtDNA's and may support the fact that they have a single type of organization and are of a single origin. All the vertebrate mtDNA's studied contain 5-methylcytosine as a minor base (1.5-3.15 mole %), and their level of methylation is 1.5-2 times greater than that in the respective nDNA's. It has been shown that animals display species specificity with respect to the 5-methylcytosine content in the mtDNA. Its distribution among the pyrimidine clusters in the bovine heart mtDNA differs substantially from that in the nDNA. This suggests that the methylation specificities of nuclear and mitochondrial DNA are different. A DNA methylase, which effects the in vitro methylation of cytosine residues both in the homologous mtDNA and in different heterologous DNA's, has been found in rat liver and bovine heart mitochondria. The specificity of the in vitro methylation of the cytosine residues in the same heterologous Escherichia coli B DNA by the nuclear and mitochondrial enzymes is different: The mitochondrial enzyme methylates predominantly in monopyrimidine fragments, and the nuclear enzyme methylates mostly in di- and tripyrimidine fragments. They, therefore, recognize different nucleotide sequences.     

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.     

The kinetics of DNA methylation in cultures of a mouse adrenal cell line

Direct measurements of the methylation of newly-synthesized DNA were made in cultures of a clonal mouse adrenal cortex cell line, Y129OS3, by (1) following the incorporation of radioactivity from methionine-(methyl)-C¹⁴ into a segment of DNA which had been density-labeled with bromouracil and (2) labeling DNA cytosine with C¹⁴-deoxycytidine and then following the appearance of radioactivity in DNA 5-methylcytosine. The results establish that during exponential growth the DNA of this cell line is methylated entirely within a few minutes of its synthesis. Using the second technique described above accurate, sensitive measurements of DNA methylation levels can be made by comparing radioactivity in 5-methylcytosine to radioactivity in cytosine plus 5-methylcytosine. In this cell line 5-methylcytosine accounts for 4.3 ± 0.2% of the DNA cytosine. Some apparent contradictions between these results and those of other workers are discussed.     

Characteristics of enzymatic DNA methylation in cultured cells of human and hamster origin,and the effect of DNA replication inhibition

In mammalian cells, inhibitors of DNA replication have been shown to induce chromosomal aberrations, cell death and changes in gene control. Inhibition of DNA synthesis has been reported to induce hypermethylation of mammalian DNA (enzymatic postsynthetic formation of 5-methylcytosine). These 5-methylcytosines in mammalian DNA have variously been suggested to be important in gene control, DNA repair, and control of DNA replication. In establishing the normal characteristics of enzymatic DNA methylation, we have demonstrated that, in asynchronously growing cells of both human and hamster origin, some cytosine methylation is delayed for several hours after strand synthesis and that this delayed methylation is completed before the DNA strand acts as a template for DNA replication in the next S-phase. Further, in testing whether the deleterious effects on mammalian cells of DNA synthesis inhibitors might be mediated via changes in enzymatic DNA methylation, we have found, contrary to some previous findings, no evidence for any change in the level of DNA methylation in DNA strands synthesized during 6 h of treatment of cells of human origin with high concentrations of four different inhibitors of DNA replication or during the 4 h following the 6 h treatment. Almost totally blocking DNA replication had no effect on the small amount of delayed methylation of DNA strands not involved in semi-conservative replication during the time of the experiment. This lack of effect on DNA methylation was obtained when the labelling medium contained normal, undialysed serum. In contrast, if dialysed serum was used in the labelling medium in order to maximize l-[Me-³H]methionine utilization, highly variable, totally irreproducible patterns of apparent DNA hypermethylation were obtained.     

Sequence specificity of DNA methylases from Bacillus amyloliquefaciens and Bacillus brevis

DNA methylation in Bacillus amyloliquefaciens strain H (Bam)² and Bacillus brevis (Bbv) has been examined by a variety of techniques. In vivo labelling studies revealed that Bam DNA contains no N⁶-methyladenine (MeAde), but contains 5-methylcytosine (MeCyt); approximately 0·7% of the cytosine residues are methylated.DNA methylase activity was partially purified from both Bam and Bbv; the Bam enzyme preparation transferred methyl groups from S-adenosyl-l-[methyl-³H]methionine ([³H]AdoMet) to specific DNA cytosine residues only; in agreement with Vanyushin & Dobritsa (1975), the Bbv enzyme preparation methylated both DNA adenine and cytosine residues. The (partial) sequence specificity of the methylases was determined by analyzing [³H]methyl-labelled dinucleotides obtained from enzymatic digests of DNA methylated in vitro. Bam and Bbv each contain a DNA-cytosine methylase with overlapping sequence specificity; e.g. both enzymes produce G-C1, C1-A and C1-T. This is consistent with a single, twofold symmetrical methylation sequence of 5′ … G-C1-(A or T)-G-C … 3′; this was observed by Vanyushin & Dobritsa (1975) for a different Bbv strain. Bam contains a second DNA-cytosine methylase (not present in Bbv), which produces T-C1 and C1-T. We propose that this methylase is the BamI modification enzyme, and that the modified sequence is 5′ … G-G-A-T-C1-C … 3′.Bbv appears to contain two DNA-adenine methylases which produce the (partial) methylated sequences, 5′ … G-A1-T … 3′ and 5′ … A-A1-G … 3′, respectively; in the former case, all the G-A-T-C sites on Bbv DNA appear to be methylated.     

DNA cytosine methylation and heat-induced deamination

The heat-induced conversion of 5-methylcytosine (m⁵C) residues to thymine residues and of cytosine to uracil residues in single-stranded DNA was studied. The calculated rates for deamination at 37°C and pH 7.4 were 9.5×10^–10 and 2.1×10^–10 sec^–1, respectively. N⁴-Methyldeoxycytidine, which is in the DNA of certain thermophilic bacteria, was more heat-resistant than was deoxycytidine and much more than was 5-methyldeoxycytidine. Thermophilic bacteria which contain N⁴-methylcytosine rather than m⁵C in their genomes may thereby largely avoid heat-induced mutation due to deamination, which is incurred by the many organisms that contain m⁵C in their DNA.     

Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax

Background

Cytosine methylation of DNA is conserved across eukaryotes and plays important functional roles regulating gene expression during differentiation and development in animals, plants and fungi. Hydroxymethylation was recently identified as another epigenetic modification marking genes important for pluripotency in embryonic stem cells.

Results

Here we describe de novo cytosine methylation and hydroxymethylation in the ciliate Oxytricha trifallax. These DNA modifications occur only during nuclear development and programmed genome rearrangement. We detect methylcytosine and hydroxymethylcytosine directly by high-resolution nano-flow UPLC mass spectrometry, and indirectly by immunofluorescence, methyl-DNA immunoprecipitation and bisulfite sequencing. We describe these modifications in three classes of eliminated DNA: germline-limited transposons and satellite repeats, aberrant DNA rearrangements, and DNA from the parental genome undergoing degradation. Methylation and hydroxymethylation generally occur on the same sequence elements, modifying cytosines in all sequence contexts. We show that the DNA methyltransferase-inhibiting drugs azacitidine and decitabine induce demethylation of both somatic and germline sequence elements during genome rearrangements, with consequent elevated levels of germline-limited repetitive elements in exconjugant cells.

Conclusions

These data strongly support a functional link between cytosine DNA methylation/hydroxymethylation and DNA elimination. We identify a motif strongly enriched in methylated/hydroxymethylated regions, and we propose that this motif recruits DNA modification machinery to specific chromosomes in the parental macronucleus. No recognizable methyltransferase enzyme has yet been described in O. trifallax, raising the possibility that it might employ a novel cytosine methylation machinery to mark DNA sequences for elimination during genome rearrangements.     

Characterization of DNA methylation in the rat

In the rat, differentiation and cell proliferation both affect DNA methylation. We studied 5-methylcytosine at the inner cytosine of the sequence C-C-G-G, a common methylation site, using endonuclease MspI (which cleaves C-C-G-G- and C-^mC-G-G), and its isoschizomer HpaII (which cleaves only C-C-G-G). DNA from all tissues and cell lines studied was methylated at C-C-G-G, at levels ranging from 45 to 80%, but the methylation sites were not distributed uniformly. Our analysis suggests a model in which cells contain variable amounts of three DNA methylation states, averaging 30–40, 70–80 and 95–100% methylation, respectively. One biological parameter that alters methylation is the prolferative state of the cell. We observed that NRK, a non-transformed cell line, increased its DNA methylation from 45 to 67% when monolayer cultures became confluent and non-dividing. We also observed that a class of repetitive DNA was completely methylated in DNA from all sources except a transformed cell line.     

Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry

A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2-(13)C]cytosine and [2-(13)C]5-methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 microg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%.     

Toxoplasma gondii and Cryptosporidium parvum lack detectable DNA cytosine methylation

Epigenetic factors play a role in the expression of virulence traits in Apicomplexa. Apicomplexan genomes encode putative DNA cytosine methylation enzymes. To assess the presence of cytosine methylation of Toxoplasma gondii and Cryptosporidium parvum DNA, we used mass spectrometry analysis and confirmed that these organisms lack detectable methylcytosine in their DNA.