The 5-methylcytosine content of highly repeated sequences in human DNA.

Previously, we found much tissue- or cell-specificity in the levels of 5-methylcytosine (m5C) in the total human genome as well as in DNA fractions resolved by reassociation kinetics. We now report that there were even greater differences in the m5C content of the highly repeated, tandem EcoRI family of DNA sequences from different human organs or cell populations. The ratio of m5C levels in this DNA fraction from brain, placenta, and sperm was 2.0:1.2:1.0. At a HhaI site in this repeat family, sperm DNA was 5-10 fold less methylated than somatic DNAs. In contrast, the highly repeated Alu family, which is approximately 5% of the genome, had almost the same high m5C content in brain and placenta despite marked tissue-specific differences in m5C levels of the single copy sequences with which these repeats are interspersed. These data show that very different degrees of change in methylation levels of various highly repeated DNA sequences accompany differentiation.     

Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells.

Analysis of the total base composition of DNA from seven different normal human tissues and eight different types of homogeneous human cell populations revealed considerable tissue-specific and cell-specific differences in the extent of methylation of cytosine residues. The two most highly methylated DNAs were from thymus and brain with 1.00 and 0.98 mole percent 5-methylcytosine (m5C), respectively. The two least methylated DNAs from in vivo sources were placental DNA and sperm DNA, which had 0.76 and 0.84 mole percent m5C, respectively. The differences between these two groups of samples were significant with p less than 0.01. The m5C content of DNA from six human cell lines or strains ranged from 0.57 to 0.85 mole percent. The major and minor base composition of DNA fractionated by reassociation kinetics was also determined. The distribution of m5C among these fractions showed little or no variation with tissue or cell type with the possible exception of sperm DNA. In each case, nonrepetitive DNA sequences were hypomethylated compared to unfractionated DNA.     

Effect of tamponade on the nucleotide composition and cytosine methylation level of DNA in the heart muscle of dogs

Base composition and quantitative changes in the 5-methylcytosine (m5c) content in DNA isolated from male dogs' heart muscle under the acute tamponade have been studied. Due to the effect of tamponade statistically reliable decrease (15%) in m5c content in DNA has been found to take place in experiment in comparison with control. Possible mechanisms of DNA methylation decrease by cytosine are under discussion.     

Expression of various genes is controlled by DNA methylation during mammalian development

Despite thousands of articles about 5-methylcytosine (m(5)C) residues in vertebrate DNA, there is still controversy concerning the role of genomic m(5)C in normal vertebrate development. Inverse correlations between expression and methylation are seen for many gene regulatory regions [Heard et al., 1997; Attwood et al., 2002; Plass and Soloway, 2002] although much vertebrate DNA methylation is in repeated sequences [Ehrlich et al., 1982]. At the heart of this debate is whether vertebrate DNA methylation has mainly a protective role in limiting expression of foreign DNA elements and endogenous transposons [Walsh and Bestor, 1999] or also is important in the regulation of the expression of diverse vertebrate genes involved in differentiation [Attwood et al., 2002]. Enough thorough studies have now been reported to show that many tissue- or development-specific changes in methylation at vertebrate promoters, enhancers, or insulators regulate expression and are not simply inconsequential byproducts of expression differences. One line of evidence comes from mutants with inherited alterations in genes encoding DNA methyltransferases and from rodents or humans with somatically acquired changes in DNA methylation that illustrate the disease-producing effects of abnormal methylation. Another type of evidence derives from studies of in vivo correlations between tissue-specific changes in DNA methylation and gene expression coupled with experiments demonstrating cause-and-effect associations between DNA hyper- or hypomethylation and gene expression. In this review, I summarize some of the strong evidence from both types of studies. Taken together, these studies demonstrate that DNA methylation in mammals modulates expression of many genes during development, causing major changes in or important fine-tuning of expression. Also, I discuss previously established and newly hypothesized mechanisms for this epigenetic control.     

Synchronous synthesis of DNA in coleoptiles and the initial leaf of developing etiolated wheat shoots: the nature and correlation of nuclear and mitochondrial DNA syntheses

In etiolated coleoptiles and initial leaf of developing wheat shoots the DNA synthesis is periodical and synchronous. In the initial leaf each step of DNA synthesis results in a stepwise increase of DNA content and is doubled at the first three steps. During the leaf plane formation the synthesis of nuclear DNA (nDNA) is decreased, while that of mitochondrial DNA (mitDNA) continues in synchronous cycles. This is the cause of relative stabilization of DNA content per unit of leaf plane length. The DNA increase in this organ occurs due to synchronous synthesis of nDNA and mitDNA in intercalary meristem cells. In coleoptiles a marked replication of nDNA is observed at the first three steps of the synthesis; in each cycle nDNA synthesis precedes that of mitDNA. With completion of coleoptile formation the nDNA synthesis in it practically ceases, whereas that of mitDNA continues in synchronous cycles. MitDNA is non-methylated and its composition (56 mol.% GC) differs significantly from that of the newly synthesized nDNA (44 mol.% GC; 100 X m5C/(C + m5C) = 16-17%). It may be concluded that in various organs of wheat shoots the composition and methylation of newly synthesized DNA depend on the age of the shoot and on the ratio of nDNA/mitDNA syntheses.     

Global 5-methylcytosine alterations in DNA during ageing of Quercus robur seeds

Background and Aims Epigenetic regulation plays an important role in the management of plant growth, development and response to stress factors, and several reports have indicated that DNA methylation plays a critical role in seed development and viability. This study examines changes in 5-methylcytosine (m⁵C) levels in the DNA of seeds during ageing, a process that has important implications for plant conservation and agriculture.Methods Changes in the global level of m⁵C were measured in mature seeds of oak, Quercus robur. The extent of DNA methylation was measured using a protocol based on two-dimensional thin-layer chromatography. Viability of seeds was determined by germination and seedling emergence tests.Key Results An ageing-related decrease in total m⁵C during storage of recalcitrant seeds was highly and significantly correlated with a decrease in seed viability, as reflected by a reduction in germination (r = 0·8880) and seedling emergence (r = 0·8269).Conclusions The decrease in viability during ageing of Q. robur seeds is highly correlated with a global decline in the amount of m⁵C in genomic DNA, and it is possible that this may represent a typical response to ageing and senescence in recalcitrant seeds. Potential mechanisms that drive changes in genomic DNA methylation during ageing are discussed, together with their implications for seed viability.     

Unique structure in the intergenic and 5' external transcribed spacer of the ribosomal RNA gene from the pea aphid Acyrthosiphon pisum.

We analyzed the DNA sequence of the 5' external transcribed spacer (ETS) and part of the intergenic transcribed spacer (IGS) of the aphid ribosomal RNA gene (rDNA). The 5' ETS of aphid rDNA consists of 843 nucleotides with a G/C content of 69 mol/100 mol, far higher than that of any other known 5' ETS for insect rDNA. The IGS of aphid rDNA contained a characteristic array of repeated sequences of 247 nucleotides. The repeated sequences were identical. It was shown that the number of the repeating sequence is heterogeneous.     

5-Methylcytosine content of rat hepatoma DNA substituted with bromodeoxyuridine.

The aim of these experiments was to test whether incorporation of bromodeoxyuridine into DNA affects DNA methylation. Rat hepatoma (HTC) cells in culture were labeled for two generations with [14C]bromodeoxyuridine and [3H]thymidine to yield DNA which was 2.1, 20.6, 52.6, and 95.0% bromodeoxyuridine-substituted in the newly made strands. The DNA then was fractionated into highly repetitive, moderately repetitive, and single copy sequences. As determined by a comparison of 14C and 3H counts per min, the percentage of substitution with bromodeoxyuridine was found to be the same in each repetition class. The 5-methylcytosine content of each fraction was determined using high pressure liquid chromatography. It was found that bromodeoxyuridine, even at a level of substitution into newly mad DNA of 95%, has no effect on the 5-methylcytosine content of DNA. At all levels of bromodeoxyuridine substitution, highly repetitive DNA has slightly more 5-methylcytosine (3.0% of total cytosine) than does single copy DNA or moderately repetitive DNA (2.3%). The 5-methylcytosine content of whole HTC DNA is the same as that of rat liver DNA (2.4%).     

Distribution of repetitious sequences in chick nuclear DNA

By an improved method of hydroxylapatite chromatography, the reassociated sequences of chick nuclear DNA were isolated, and their base composition analysed. By increasing the amount of reassociation, the G + C content of the renatured sequences decreased progressively to reach a mean value corresponding to that of the total DNA. In order to study the distribution of the families, or group of families having different amount of reassociation, DNA was fractionated by CsC1 density gradient centrifugation. Fractions having different G + C content were obtained, and their reassociation rates analysed. At high C(o)t value of renaturation (C(o)t=50) the amount of reassociated sequences included in the high or in the low buoyant density DNA fractions was approximately the same, but their G + C content was as expected different. At lower C(o)t values of renaturation (between C(o)t of 0.2 and the C(o)t of 10), the results indicated an heterogeneity of the repeated sequences in the A + T rich DNA fractions, as compared to the G + C rich ones.     

Tissue specificity of the decrease of cattle lymphocyte DNA methylation during chronic lymphoid leukemia]

It has been found that the content of m5C in the DNA preparations tested have been revealed. The DNAs from normal and leukemic lymphocytes of blood, lymphonodi and spleen differ in ther acceptor ability in the reaction of heterologous methylation in vitro, induced by DNA-methylase from Enterobacter cloacea in the presence of [3H-methyl]S-adenosyl methionine: the ratio of radioactivities in methylated cytosine and adenine residues (m5C/m6A) in leukemic lymphocyte DNA is much lower than in healthy animals' lymphocytes. The decrease in the methylation of DNAs from various lymphoid organs of animals with chronic lymphoid leukemia is well correlated with the impairment. No significant changes of the m5C level and the acceptor ability of the in vitro reaction of heterologous methylation of cow lymph lymphocyte DNA have been observed. The data obtained may be interpreted in terms of tissue (cell) specificity or differences in the degree of DNA methylation under conditions of chronic lymphoid leukemia. It is assumed that the changes in DNA methylation may underlie the disturbances in the regulation of activity of the leukemic cell genetic mechanisms.     

Polyamine accumulation in aged wheat seeds

The present work was conducted to evaluate the content of the main polyamines (Spm, Spd, Put) in a series of naturally aged durum-wheat seeds as well as the activities of the enzymes ODC and ADC involved in their biosynthesis. In dry seeds the content of polyamines, especially that of Spd, rose during ageing till 6 years and then declined sharply. However, an increase of PA content upon imbibition was observed only with the youngest seeds, while a decrease was found in the older ones. The activities of ODC and ADC differed in aged seeds, the ODC activity being constant and lower than the ADC in the course of seed ageing. The ADC increased till the early ageing and decreased then in the very old, ungerminating seeds. Imbibition increased both enzyme activities in the youngest seeds only, in the older ones rather a decrease and changed ADC/ODC ration was found. The obtained results are discussed in relation to the participation of these enzymes in the biosynthesis of polyamines during seed ageing and in the course of plant senescence or stress.     

Tissue-specific differences in DNA methylation in various mammals

The only naturally occurring modified base in vertebrate DNA is 5-methylcytosine. Using a precise high-performance liquid chromatographic analysis of DNA enzymatically digested to deoxynucleosides, we have shown that rats, mice and four types of monkey display tissue-specific as well as species-specific differences in the extent of methylation of their cytosine residues. Several similarities in the patterns of tissue-specific DNA methylation in these mammals and in the previously studied human samples were observed. Compared to most other types of DNA examined, brain and thymus DNAs were hypermethylated, which suggests that this hypermethylation is a determinant or a necessary byproduct of mammalian differentiation. In all of the studied rodents and primates, the highly repeated DNA sequence fraction was more methylated than the moderately repetitive or single copy fractions. The tissue-specific differences in overall DNA methylation showed no correlation with what is known about average cell turnover rates nor with the percentage of the genome that is transcribed. Liver regeneration in the rat following partial hepatectomy did not detectably alter 5-methylcytosine levels in liver DNA. A considerable increase in the extent of methylation of total liver DNA was observed during normal development of the rat. The latter phenomenon may be due to a major change in the cellular composition of the liver.     

The effect of 5-azacytidine on the cell cycle and chromosome structure in cell cultures of swine embryonal kidney tissue

Incubation of pig kidney cells (PK-cells) in the presence of 5-azacytidine (5-azaC), a DNA enzymatic methylation inhibitor, at concentration of 20 microM for 6 or 24 h results in a dramatic decrease in the DNA methylation level (5mC/C + m5.100) - from 3.0 in control to 1.0 in experiment. This is accompanied by a virtually complete arrest of mitosis and a decrease in the ratio of labeled interphase cells upon simultaneous introduction of 3H-deoxycytidine. The incubation with 5-azaC block PK-cells mainly in the G2-period. The inhibitory action is reversible, for the cells enter into mitosis after removal of the inhibitor. Metaphase chromosomes, whose DNA was replicated in the presence of the 5-azaC, exhibit certain ultrastructural differences from normal ones. The results are being discussed in connection with the earlier data on the anomalous structure of interphase chromatin formed in the course undermethylated DNA replication.     

Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B]

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.     

Limited accessibility of DNA methylation sites for bacterial methylases M. Eco RII and M.Eco dam in chromatin at different levels of organization

The bacterial methylases M. Eco RII and M. Eco dam can methylate DNA in rat liver chromatin to form the 5-methylcytosine (m5C) and N6-methyladenine (m6A) residues, respectively. The CH3-accepting capacity of DNA in chromatin (mono- and dinucleosomes, mono- and dinucleomers) is 15 - 30 times less than that of free total DNA in rat liver. Such a low level of DNA methylation in chromatin in vitro suggests that the accessibility and recognition of methylation sites by DNA-methylases are decreased in comparison with free DNA both in the core-particle DNA and in the internucleosomal DNA. The degree of DNA methylation in chromatin particles depends on the ionic strength and Mg2+; when the former is decreased from 0.515 down to 0.176, the DNA methylation by both enzymes is increased 2-fold. An addition of Mg2+ (1 - 2 mM) decreases the CH3-accepting capacity of nucleomeric DNA, that of nucleosomal DNA remains unchanged. Thus, the accessibility of DNA for methylases is variable depending on the conformational changes of chromatin. The values of the m6A to m5C ratio for free and nucleosomal DNAs formed by methylation with a methylation of nucleomeric DNA, i. e. 1.01, 0.92 and 0.51, respectively. As Mg/4 concentration rises, the m6A/m5C ratio for nucleosomal and nucleomeric DNA is increased. It seems therefore that at different levels of organization and upon certain conformation changes the number and, probably, the nature of exposed DNA methylation sites in chromatin are different. Bacterial DNA-methylases can be used as an effective probe for a fine analysis of chromatin ultrastructure, in particular at its different functional states.     

The effect of C(5) cytosine methylation at CpG sequences on mitomycin-DNA bonding profiles

Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C (1) adduction. The reports differ on the extent and uniformity of 1 modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug DNA modification was accomplished with I and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) condilions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the lambda-exonuclease stop assay provided a qualitative estimation of drug-DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m5C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sites at the nucleotide sequence level revealed that guanine (G) was the only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m5CpG* (G* = mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m5CpG* adduction patterns provided distinctive sequence-selective bonding profiles for 1-3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part, by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed.     

Methylated DNA-binding protein is present in various mammalian cell types.

A DNA-binding protein from human placenta, methylated DNA-binding protein (MDBP), binds to certain DNA sequences only when they contain 5-methylcytosine (m5C) residues at specific positions. We found a very similar DNA-binding activity in nuclear extracts of rat tissues, calf thymus, human embryonal carcinoma cells, HeLa cells, and mouse LTK cells. Like human placental MDBP, the analogous DNA-binding proteins from the above mammalian cell lines formed a number of different low-electrophoretic-mobility complexes with a 14-bp MDBP-specific oligonucleotide duplex. All of these complexes exhibited the same DNA methylation specificity and DNA sequence specificity. From the extracts of rat and calf tissues, oligonucleotide protein complexes formed that also had the same specificity as human placental MDBP although they had a higher electrophoretic mobility probably due to digestion by proteases in the nuclear extracts. Although MDBP activity was found in various mammalian cell types, it was not detected in extracts of cultured mosquito cells and so may be associated only with cells with vertebrate-type DNA methylation.