Properties of a tobacco DNA methyltransferase, NtMET1 and its involvement in chromatin movement during cell division

BACKGROUND AND AIMS: Plants possess three types of DNA methyltransferase, among which methyltransferase type 1 (MET1) is considered to play a major role by maintaining the CpG methylation patterns. However, little information is available as to its enzymatic activity, interacting proteins and spatial and temporal behaviours during DNA replication. In the present study, one example, NtMET1 from tobacco plants, was selected and an analysis was made of its biochemical properties and cellular localization. METHODS: NtMET1 was expressed in Sf9 insect cells, and a purified sample was subjected to a standard in vitro methylation assay. Intramolecular interaction was examined by the yeast two-hybrid and pull-down assays. Transgenic tobacco plants (Nicotiana tabacum) over-expressing NtMET1 were constructed via Agrobacterium-mediated transformation. Cellular localization was examined by fluorescence protein fusion, which was expressed in tobacco bright yellow 2 cells. KEY RESULTS: In vitro assays showed no detectable methylation activity when both hemimethylated and unmethylated DNA samples were used as the substrate. In planta assays with over-expressing transgenic lines showed no hypermethylation but rather hypomethylation of genomc DNA. The inability of methylation was conceivably due to a tight intramolecular interaction between the N- and C-terminal regions with the catalytic domain residing on the C-terminus being completely masked. Cellular localization analyses indicated that NtMET1 localized to the nucleus in the resting stage and migrates to the cytoplasm during mitosis, particularly at metaphase. The pattern observed resembled that of Ran GTPase, and in vitro pull-down assays showed a clear interaction between NtMET1 and AtRAN3, an Arabidopsis orthologue of tobacco Ran GTPase, NtRan-A1. CONCLUSIONS: The results suggest that enzymatic activity of NtMET1 is well adjusted by its own intra/intermolecular interaction and perhaps by interactions with other proteins, one of which was found to be Ran GTPase. Results also revealed that NtMET1 becomes localized to the vicinity of chromatin with the aid of Ran GTPase during cell division, and may play an important role in progress through mitosis independently of methylation activity.     

Gestational intake of methyl donors and global LINE-1 DNA methylation in maternal and cord blood: Prospective results from a folate-replete population

Maternal diet affects offspring DNA methylation in animal models, but evidence from humans is limited. We investigated the extent to which gestational intake of methyl donor nutrients affects global DNA methylation in maternal and umbilical cord blood. Among mother-infant pairs in Project Viva, a folate-replete US population, we estimated maternal intakes of vitamin B12, betaine, choline, folate, cadmium, zinc and iron periconceptionally and during the second trimester. We examined associations of these nutrients with DNA methylation, measured as %5-methyl cytosines (%5mC) in Long Interspersed Nuclear Element-1 (LINE-1), in first trimester (n = 830) and second trimester (n = 671) maternal blood and in cord blood at delivery (n = 516). Cord blood methylation was higher for male than female infants {mean [standard deviation (SD)] 84.8 [0.6] vs. 84.4 [0.7]%}. In the multivariable-adjusted model, maternal intake of methyl donor nutrients periconceptionally and during the second trimester of pregnancy was not positively associated with first trimester, second trimester or cord blood LINE-1 methylation. Periconceptional betaine intake was inversely associated with cord blood methylation [regression coefficient = -0.08% (95% confidence interval (CI): -0.14,-0.01)] but this association was attenuated after adjustment for dietary cadmium, which itself was directly associated with first trimester methylation and inversely associated with cord blood methylation. We also found an inverse association between periconceptional choline [-0.10%, 95% CI: -0.17,-0.03 for each SD (~63 mg/d)] and cord blood methylation in males only. In this folate-replete population, we did not find positive associations between intake of methyl donor nutrients during pregnancy and DNA methylation overall, but among males, higher early pregnancy intakes of choline were associated with lower cord blood methylation.     

Gestational intake of methyl donors and global LINE-1 DNA methylation in maternal and cord blood: Prospective results from a folate-replete population

Maternal diet affects offspring DNA methylation in animal models, but evidence from humans is limited. We investigated the extent to which gestational intake of methyl donor nutrients affects global DNA methylation in maternal and umbilical cord blood. Among mother-infant pairs in Project Viva, a folate-replete US population, we estimated maternal intakes of vitamin B12, betaine, choline, folate, cadmium, zinc and iron periconceptionally and during the second trimester. We examined associations of these nutrients with DNA methylation, measured as %5-methyl cytosines (%5mC) in Long Interspersed Nuclear Element-1 (LINE-1), in first trimester (n = 830) and second trimester (n = 671) maternal blood and in cord blood at delivery (n = 516). Cord blood methylation was higher for male than female infants {mean [standard deviation (SD)] 84.8 [0.6] vs. 84.4 [0.7]%}. In the multivariable-adjusted model, maternal intake of methyl donor nutrients periconceptionally and during the second trimester of pregnancy was not positively associated with first trimester, second trimester or cord blood LINE-1 methylation. Periconceptional betaine intake was inversely associated with cord blood methylation [regression coefficient = −0.08% (95% confidence interval (CI): −0.14, −0.01)] but this association was attenuated after adjustment for dietary cadmium, which itself was directly associated with first trimester methylation and inversely associated with cord blood methylation. We also found an inverse association between periconceptional choline [−0.10%, 95% CI: −0.17, −0.03 for each SD (∼63 mg/day)] and cord blood methylation in males only. In this folate-replete population, we did not find positive associations between intake of methyl donor nutrients during pregnancy and DNA methylation overall, but among males, higher early pregnancy intakes of choline were associated with lower cord blood methylation.Key words: DNA methylation, pregnancy, cord blood, maternal diet, cadmium     

Gestational choline deficiency causes global and Igf2 gene DNA hypermethylation by up-regulation of Dnmt1 expression

During gestation there is a high demand for the essential nutrient choline. Adult rats supplemented with choline during embryonic days (E) 11-17 have improved memory performance and do not exhibit age-related memory decline, whereas prenatally choline-deficient animals have memory deficits. Choline, via betaine, provides methyl groups for the production of S-adenosylmethionine, a substrate of DNA methyltransferases (DNMTs). We describe an apparently adaptive epigenomic response to varied gestational choline supply in rat fetal liver and brain. S-Adenosylmethionine levels increased in both organs of E17 fetuses whose mothers consumed a choline-supplemented diet. Surprisingly, global DNA methylation increased in choline-deficient animals, and this was accompanied by overexpression of Dnmt1 mRNA. Previous studies showed that the prenatal choline supply affects the expression of multiple genes, including insulin-like growth factor 2 (Igf2), whose expression is regulated in a DNA methylation-dependent manner. The differentially methylated region 2 of Igf2 was hypermethylated in the liver of E17 choline-deficient fetuses, and this as well as Igf2 mRNA levels correlated with the expression of Dnmt1 and with hypomethylation of a regulatory CpG within the Dnmt1 locus. Moreover, mRNA expression of brain and liver Dnmt3a and methyl CpG-binding domain 2 (Mbd2) protein as well as cerebral Dnmt3l was inversely correlated to the intake of choline. Thus, choline deficiency modulates fetal DNA methylation machinery in a complex fashion that includes hypomethylation of the regulatory CpGs within the Dnmt1 gene, leading to its overexpression and the resultant increased global and gene-specific (e.g. Igf2) DNA methylation. These epigenomic responses to gestational choline supply may initiate the long term developmental changes observed in rats exposed to varied choline intake in utero.     

Plant-exuded choline is used for rhizobial membrane lipid biosynthesis by phosphatidylcholine synthase.

Phosphatidylcholine is a major lipid of eukaryotic membranes, but found in only few prokaryotes. Enzymatic methylation of phosphatidylethanolamine by phospholipid N-methyltransferase was thought to be the only biosynthetic pathway to yield phosphatidylcholine in bacteria. However, mutants of the microsymbiotic soil bacterium Sinorhizobium (Rhizobium) meliloti, defective in phospholipid N-methyltransferase, form phosphatidylcholine in wild type amounts when choline is provided in the growth medium. Here we describe a second bacterial pathway for phosphatidylcholine biosynthesis involving the novel enzymatic activity, phosphatidylcholine synthase, that forms phosphatidylcholine directly from choline and CDP-diacylglycerol in cell-free extracts of S. meliloti. We further demonstrate that roots of host plants of S. meliloti exude choline and that the amounts of exuded choline are sufficient to allow for maximal phosphatidylcholine biosynthesis in S. meliloti via the novel pathway.     

Enzymatic methylation of inverted DNA repeats in the vicinity of the mouse beta-major globin gene

This paper examines the extent of enzymatic methylation in 5'-CCGG sequences of inverted repeats in DNA isolated from adult liver and bone marrow of DBA/2 mice, with special attention to the methylation of such sequences in the vicinity of the beta-major globin gene. Two thirds of inverted repeats contain 5'-AGCT and 5'-CCGG sequences, as found by a method based on the capability of inverted repeats of forming intramolecular duplexes under the conditions of "zero-time" reassociation. Methylation in internal cytosines of 5'-CCGG sequences of inverted DNA repeats differs between bone marrow and liver tissues. The beta-major globin gene was found in DNA covalently linked to inverted repeats. The enzymatic methylation of inverted repeats neighbouring the beta-major globin gene differs at HpaII recognition sites; the DNA of bone marrow tissue, in which this gene is expressed, is less methylated at such sites as compared to liver DNA.     

Novel pathway for phosphatidylcholine biosynthesis in bacteria associated with eukaryotes

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesised by either of two pathways, the CDP-choline pathway or the methylation pathway. Many prokaryotes lack PC, but it can be found in significant amounts in membranes of distantly related bacteria such as Rhizobacteria and Spirochetes. Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria. However, a novel choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti. In this pathway, a novel enzymatic activity, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step. Surprisingly, genomes of some pathogens (Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) contain genes similar to the sinorhizobial gene for phosphatidylcholine synthase. We, therefore, suggest that the new PC synthase pathway is present in a number of bacteria displaying symbiotic or pathogenic associations with eukaryotes and that the eukaryotic host functions as the provider of choline for this pathway.     

In vitro methylation of the 5'-flanking regions of the mouse beta-globin gene

The enzymatic methylation of the 5'-flanking region of the mouse beta-globin (major) gene containing putative regulatory regions has been investigated. In vitro methylation of this 368-base pair regulatory DNA by a DNA methyltransferase obtained from mouse erythroleukemia cells yields an asymmetric methylation pattern. Of the 10 available CG pairs, only 5-6 are modified, leading to one hemimethylated site and two apparently fully methylated sites. Only CG pairs which are localized in a 29-base pair cluster are methylated. The data suggest that a CG cluster approximately 100 base pairs upstream from the CAP site may be the in vivo site of methylation in the 5'-regulator region of the mouse beta-globin gene.     

Effect of cobalamin derivatives on in vitro enzymatic DNA methylation: methylcobalamin can act as a methyl donor

5-Methylcytosine synthesis in DNA involves the transfer of methyl groups from S-adenosyl-methionine to the 5'-position of cytosine through the action of DNA (cytosine-5)-methyltransferase. The rate of this reaction has been found to be enhanced by cobalt ions. We therefore analyzed the influence of vitamin B12 and related compounds containing cobalt on DNA methylation. Vitamin B12, methylcobalamin, and coenzyme B12 were found to enhance significantly the de novo DNA methylation in the presence of S-adenosylmethionine for concentrations up to 1 microM, but at higher concentrations these compounds were found to inhibit DNA methylation. Methylcobalamin behaves as a competitive inhibitor of the enzymatic methylation reaction (Ki = 15 microM), the Km for S-adenosylmethionine being 8 microM. In addition, the use of radioactive methylcobalamin shows that it can be used as a methyl donor in the de novo and maintenance DNA methylation reactions. Thus, two DNA methylation pathways could exist: one involving methylation from S-adenosylmethionine and a second one involving methylation from methylcobalamin.     

The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1

Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo.     

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.     

Biosynthesis of major phospholipids inCandida albicans

Biosynthesis of major phospholipids was examined by identifying enzymes and in vitro uptake of specific labeled precursors through various pathways inCandida albicans. The presence of PS synthetase, choline kinase, and ethanolamine kinase was demonstrated in this organism. Phosphatidylcholine was found to be synthesized mainly through cytidine diphosphate-choline (CDP)-choline and methylation pathways. The presence of a methylation pathway was further confirmed by blocking methyltransferases with 2-hydroxyethyl hydrazine. Phosphatidylethanolamine was synthesized by all three, CDP-ethanolamine, Phosphatidylserine (PS)-decarboxylase, and base exchange pathways, while PS was formed by PS-synthetase and base exchange mechanisms.     

Loss of CpG dinucleotides from DNA. VI. Methylation of mitochondrial and chloroplast genes

From nucleotide sequences of mitochondrial and chloroplast genes the probable frequency of the CpG----TpG + CpA substitutions was determined. These substitutions may indicate the level of prior DNA methylation. It was found that the level of this methylation is significantly lower in mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) than in nuclear DNA (nDNA) of the same species. The species (taxon) specificity of mtDNA and chDNA methylation was revealed. A correlation was found between the level of CpG methylation in nDNA, and mtDNA and chDNA in different organisms. It is shown that cytosine residues in CpG were not subjected to significant methylation in the fungi and invertebrate mtDNA and also in the algae chDNA. In contrast, the vertebrate mtDNA bears the impress of CpG-supression, which is confirmed by direct data on methylation of these DNA. Here the first data on the possible enzymatic methylation of the plant mtDNA and chDNA were obtained. It was shown that the degree of CpG-suppression in the 5S rRNA nuclear genes of lower and higher plants is significantly higher in the chloroplast genes of 4,5S and 5S rRNA. From data on pea chDNA hydrolysis with MspI and HpaII it was established that in CCGG sequences this DNA is not methylated. The role of DNA methylation in increasing the mutation rate and in accelerating the evolutionary rates of vertebrate mtDNA is discussed.     

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

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

Regulation of phosphatidylcholine biosynthesis in Plasmodium-infected erythrocytes

Plasmodium knowlesi-infected erythrocytes efficiently incorporated choline and metabolize it into phosphatidylcholine via the de novo Kennedy pathway. No formation of either betaine or acetylcholine was detected. At physiological concentrations of external choline, isotopic equilibrium between intracellular choline and phosphocholine was reached in less than 1 h, whereas labeled phosphatidylcholine accumulated constantly, until at least 210 min. During this time, intracellular CDP-choline remained quite low compared to phosphocholine, which suggests that choline-phosphate cytidylyltransferase (EC 2.7.7.15) is the rate-limiting step of the Kennedy pathway. However, this activity was probably not saturated in situ by phosphocholine, since the external choline concentration, up to 100 microM, can regulate phosphatidylcholine biosynthesis via the level of intracellular phosphocholine. This was corroborated by the respective velocities and affinity characteristics of the three enzymatic steps involved in the Kennedy pathway. These results, together with the localization of both choline metabolites and enzyme activities, provide a precise scheme of the dynamics of de novo phosphatidylcholine biosynthesis. Concerning the alternative pathway for phosphatidylcholine biosynthesis via the methylation of phosphatidylethanolamine, we show that an increase in de novo phosphatidylcholine biosynthesis could instigate a concomitant decrease in the steps of phosphatidylethanolamine methylation, indicating that the parasite is able to modulate its phosphatidylcholine biosyntheses.     

Differential inhibition of mitogen induced T cell proliferation by 5-azacytidine and cytosine-arabinoside

The cytotoxic drugs 5-azacytidine and cytosine-arabinoside influence the enzymatic methylation of DNA in opposite ways (1,2). The in vitro effects of these two drugs on Con A induced proliferation of thymic and splenic rat lymphocytes were investigated. Cytosine-arabinoside was found to inhibit mitogen induced proliferation already at a concentration of 0.001 microM, whereas 5-azacytidine was inhibitory only above concentrations of 1 microM. A stimulation of mitogen induced T cell proliferation was consistently seen with 5-azacytidine, but not with cytosine-arabinoside, at concentrations lower than the cytotoxic concentration. The results show that 5-azacytidine and cytosine-arabinoside interfere with mitogen stimulated lymphocyte proliferation by different mechanisms and suggest that hypomethylated DNA plays a role in the proliferation of T cells.     

In vitro incorporation of [14CH3]methionine into liver phosphatidylcholine in rats with vitamin E deficiency

The content of phosphatidyl choline, a terminal product of phosphatidyl ethanolamine methylation as well as in intensity in vitro incorporation of [14CH3]methionine into phosphatidyl choline of rat liver with E-hypovitaminosis are found to considerably decrease as compared to the norm. In this case the total content of phospholipids is practically the same.