Modification of p53 protein profile by gamma irradiation followed by methyl donor starvation

The possible beneficial radio-protective effects of one-carbon transfer agents namely folate, choline and methionine have been the subject of extensive investigation. Ionizing radiation is known to extensively damage the DNA. One-carbon transfer agents have been proposed to have important role in context of DNA repair via their role in purine and thymidylate synthesis and in DNA methylation. Sufficient dietary availability of one-carbon transfer agents therefore, might have ability to modify radiation effects. In present study modifications in level of tumor suppressor protein p53 by gamma irradiation followed by methyl donor starvation was observed. Experiments showed an increase in nuclear and cytoplasmic p53 protein concentration in liver, spleen and thymus. The overall rise in the level of p53 protein in liver was found to be less than that in spleen and thymus. Moreover significant heterogeneity in the basal level of expression of the p53 protein in liver, spleen and thymus was observed as the level of p53 protein in spleen and thymus was found to be 7–8 fold more than that in liver. Results indicated that radiation stress followed by methyl donor starvation could significantly induce p53 protein in spleen and thymus where there was a dramatic accumulation of p53 following irradiation, while in other tissues, particularly the liver, no such dramatic response was seen. Folate contribution of intestinal bacteria was found to influence p53 protein levels. Our observations indicated a prominent role played by the methyl donors in protecting the cell against harmful effects of ionizing radiation.     

Methyl‐donor deficiency in adolescence affects memory and epigenetic status in the mouse hippocampus

DNA methylation is one of the essential factors in the control of gene expression. Alteration of the DNA methylation pattern has been linked to various neurological, behavioral and neurocognitive dysfunctions. Recent studies have pointed out the importance of epigenetics in brain development and functions including learning and memory. Nutrients related to one‐carbon metabolism are known to play important roles in the maintenance of genomic DNA methylation. Previous studies have shown that the long‐term administration of a diet lacking essential one‐carbon nutrients such as methionine, choline and folic acid (methyl donors) caused global DNA hypermethylation in the brain. Therefore, the long‐term feeding of a methyl‐donor‐deficient diet may cause abnormal brain development including learning and memory. To confirm this hypothesis, 3‐week‐old mice were maintained on a folate‐, methionine‐ and choline‐deficient (FMCD) or control (CON) diet for 3 weeks. We found that the methyl‐donor deficiency impaired both novel object recognition and fear extinction after 3 weeks of treatment. The FMCD group showed spontaneous recovery of fear that differed from that in CON. In addition, we found decreased Gria1 gene expression and specific CpG hypermethylation of the Gria1 promoter region in the FMCD hippocampus. Our data suggest that a chronic dietary lack of methyl donors in the developmental period affects learning, memory and gene expressions in the hippocampus.     

Genomic hypomethylation is specific for preneoplastic liver in folate/methyl deficient rats and does not occur in non-target tissues

Chronic dietary insufficiency of the lipotropic nutrients choline and methionine is hepatocarcinogenic in male rats and certain mouse strains. Despite the fact that DNA hypomethylation is a hallmark of most cancer genomes, the tissue-specific consequences of this alternation with respect to tumorigenesis remain to be determined. In the present study, the folate/methyl deficient model of multistage hepatocarcinogenesis was used to evaluate in vivo alterations in DNA methylation in the liver, the carcinogenesis target tissue, and in non-target tissues, including pancreas, spleen, kidney, and thymus, of male F344 rats. By utilizing the HpaII/MspI-based cytosine extension assay, we demonstrated that the percent of CpG sites that lost methyl groups on both strands progressively increased in liver tissue after 9, 18, and 36 weeks of folate/methyl deficiency. The endogenous activity of DNA methyltransferase in liver of rats fed with folate/methyl deficient diet for the 36-week period gradually increased with time. In contrast, non-target tissues displayed no changes in DNA methylation level or activity of DNA methyltransferase. The failure of DNA methyltransferase to restore and maintain DNA methylation patterns in preneoplastic liver tissue may lead to the establishment of tumor-specific DNA methylation and DNA methyltransferase profiles that are not expressed in normal liver. These results provide additional information about alterations in DNA methylation during early preneoplastic stages of carcinogenesis. They also demonstrate that DNA hypomethylation is localized to tissue that undergoes carcinogenesis, and is not altered in non-target tissues.     

Susceptibility to intestinal tumorigenesis in folate-deficient mice may be influenced by variation in one-carbon metabolism and DNA repair

Low dietary folate is associated with increased risk of colorectal cancer. In earlier work, we showed that folate deficiency induced intestinal tumors in BALB/c but not C57Bl/6 mice through increased dUTP incorporation into DNA with consequent DNA damage. To determine whether strain differences between one-carbon metabolism and DNA repair pathways could contribute to increased tumorigenesis in BALB/c mice, we measured amino acids and folate in the normal intestinal tissue of both strains fed a control diet or a folate-deficient diet. We also determined the expression of critical folate-metabolizing enzymes and several DNA repair enzymes. BALB/c mice had lower intestinal serine (major cellular one-carbon donor), methionine and total folate than C57Bl/6 mice under both dietary conditions. BALB/c mice had higher messenger RNA and protein levels of three folate-interconverting enzymes: trifunctional methyleneTHF (5,10-methylenetetrahydrofolate) dehydrogenase–methenylTHF cyclohydrolase–formylTHF (10-formyltetrahydrofolate) synthetase 1, bifunctional methyleneTHF dehydrogenase–methenylTHF cyclohydrolase and methylenetetrahydrofolate reductase. This pattern of expression could limit the availability of methyleneTHF for conversion of dUMP to dTMP. BALB/c mice also had higher levels of uracil DNA glycosylase 2 protein without an increase in the rate-limiting DNA polymerase β enzyme, compared with C57Bl/6 mice. We conclude that BALB/c mice may be more prone to DNA damage through decreased amounts of one-carbon donors and the diversion of methyleneTHF away from the conversion of dUMP to dTMP. In addition, incomplete excision repair of uracil in DNA could lead to accumulation of toxic repair intermediates and promotion of tumorigenesis in this tumor-susceptible strain.     

Disruption of choline methyl group donation for phosphatidylethanolamine methylation in hepatocarcinoma cells

Despite being widely hypothesized, the actual contribution of choline as a methyl source for phosphatidylethanolamine (PE) methylation has never been demonstrated, mainly due to the inability of conventional methods to distinguish the products from that of the CDP-choline pathway. Using a novel combination of stable-isotope labeling and tandem mass spectrometry, we demonstrated for the first time that choline contributed to phosphatidylcholine (PC) synthesis both as an intact choline moiety via the CDP-choline pathway and as a methyl donor via PE methylation pathway. When hepatocytes were labeled with d(9)-choline containing three deuterium atoms on each of the three methyl groups, d(3)-PC and d(6)-PC were detected, indicating that newly synthesized PC contained one or more individually mobilized methyl groups from d(9)-choline. The synthesis of d(3)-PC and d(6)-PC was sensitive to the general methylation inhibitor 3-deazaadenosine and were specific products of PE methylation using choline as a one-carbon donor. While the contribution to the CDP-choline pathway remained intact in hepatocarcinoma cells, contribution of choline to PE methylation was completely disrupted. In addition to a previously identified lack of PE methyltransferase, hepatocarcinoma cells were found to lack the abilities to oxidize choline to betaine and to donate the methyl group from betaine to homocysteine, whereas the usage of exogenous methionine as a methyl group donor was normal. The failure to use choline as a methyl source in hepatocarcinoma cells may contribute to methionine dependence, a widely observed aberration of one-carbon metabolism in malignancy.     

Enhanced one-carbon flux towards DNA methylation: Effect of dietary methyl supplements against γ-radiation-induced epigenetic modifications

Radiation exposure poses a major risk for workers in the nuclear power plants and other radiation related industry. In this context, we demonstrate that γ-radiation is an efficient DNA demethylating agent and its injurious effect can be minimized by dietary methyl supplements (folate, choline and vitamin B12). To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on normal control diet (NCD) and methyl-supplemented diet (MSD). After 2 weeks of NCD and MSD dietary regimen, we exposed the animals to γ-radiation (2, 4 and 6 Gy) and investigated the profile of downstream metabolites and activity levels of one-carbon (C₁) flux generating enzymes. In MSD fed and irradiated animals, hepatic folate levels increased (P < 0.01), while hepatic homocysteine levels decreased (P < 0.01) compared to NCD fed and irradiated animals. Although hepatic folate level increased significantly in MSD fed animals (P < 0.01), it showed a decrease in response to high doses of γ-irradiation. Under these conditions, a marked suppression of S-adenosylmethionine (SAM) levels occurred in NCD fed and irradiated animals, suggesting reduced conversion of homocysteine to SAM. Concomitant with decline in liver SAM Pool, activities of DNA methyltransferase (Dnmt, that methylates DNA) and methionine synthase (MSase, that regenerates methionine from homocysteine) were both decreased in NCD fed and irradiated mice. However, in MSD fed and irradiated mice, they were increased. These results strongly indicated that increased levels of dnmt and MSase may enhance C₁ flux towards DNA methylation reactions in MSD fed animals. These results were confirmed and further substantiated by measuring genomic DNA methylation levels, which were maintained at normal levels in MSD fed and irradiated mice compared to NCD fed and irradiated animals (P < 0.01). In conclusion, our results suggest that maintenance of genomic DNA methylation under γ-radiation stress might be a very dynamic, progressive diet dependent process that could involve increased one-carbon flux through various C₁ metabolites.     

Nutrition and epigenetics: an interplay of dietary methyl donors, one-carbon metabolism and DNA methylation

DNA methylation is the most extensively studied mechanism of epigenetic gene regulation. Increasing evidence indicates that DNA methylation is labile in response to nutritional and environmental influences. Alterations in DNA methylation profiles can lead to changes in gene expression, resulting in diverse phenotypes with the potential for increased disease risk. The primary methyl donor for DNA methylation is S-adenosylmethionine (SAM), a species generated in the cyclical cellular process called one-carbon metabolism. One-carbon metabolism is catalyzed by several enzymes in the presence of dietary micronutrients, including folate, choline, betaine and other B vitamins. For this reason, nutrition status, particularly micronutrient intake, has been a focal point when investigating epigenetic mechanisms. Although animal evidence linking nutrition and DNA methylation is fairly extensive, epidemiological evidence is less comprehensive. This review serves to integrate studies of the animal in vivo with human epidemiological data pertaining to nutritional regulation of DNA methylation and to further identify areas in which current knowledge is limited.     

Methyl-donor nutrients inhibit breast cancer cell growth

Lipotropes (methyl group containing nutrients, including methionine, choline, folate, and vitamin B(12)) are dietary methyl donors and cofactors that are involved in one-carbon metabolism, which is important for genomic DNA methylation reactions and nucleic acid synthesis. One-carbon metabolism provides methyl groups for all biological methylation pathways and is highly dependent on dietary supplementation of methyl nutrients. Nutrition is an important determinant of breast cancer risk and tumor behavior, and dietary intervention may be an effective approach to prevent breast cancer. Apoptosis is important for the regulation of homeostasis and tumorigenesis. The anti-apoptotic protein Bcl-2 may be a regulatory target in cancer therapy; controlling or modulating its expression may be a therapeutic strategy against breast cancer. In this study, the effects of lipotrope supplementation on the growth and death of human breast cancer cell lines T47D and MCF-7 were examined and found to inhibit growth of both T47D and MCF-7 cells. Furthermore, the ratios of apoptotic cells to the total number of cells were approximately 44% and 34% higher in the lipotrope-supplemented treatments of T47D and MCF-7 cancer cells, respectively, compared with the control treatments. More importantly, Bcl-2 protein expression was decreased by approximately 25% from lipotrope supplementation in T47D cells, suggesting that lipotropes can induce breast cancer cell death by direct downregulation of Bcl-2 protein expression. Cancer treatment failure is often correlated with Bcl-2 protein upregulation. These data may be useful in the development of effective nutritional strategies to prevent and reduce breast cancer in humans.     

Epigenetic regulation of left–right asymmetry by DNA methylation

DNA methylation is a major epigenetic modification; however, the precise role of DNA methylation in vertebrate development is still not fully understood. Here, we show that DNA methylation is essential for the establishment of the left–right (LR) asymmetric body plan during vertebrate embryogenesis. Perturbation of DNA methylation by depletion of DNA methyltransferase 1 (dnmt1) or dnmt3bb.1 in zebrafish embryos leads to defects in dorsal forerunner cell (DFC) specification or collective migration, laterality organ malformation, and disruption of LR patterning. Knockdown of dnmt1 in Xenopus embryos also causes similar defects. Mechanistically, loss of dnmt1 function induces hypomethylation of the lefty2 gene enhancer and promotes lefty2 expression, which consequently represses Nodal signaling in zebrafish embryos. We also show that Dnmt3bb.1 regulates collective DFC migration through cadherin 1 (Cdh1). Taken together, our data uncover dynamic DNA methylation as an epigenetic mechanism to control LR determination during early embryogenesis in vertebrates.     

Feedback regulation of DNA methyltransferase gene expression by methylation.

This paper tests the hypothesis that expression of the DNA methyltransferase, dnmt1, gene is regulated by a methylation-sensitive DNA element. Methylation of DNA is an attractive system for feedback regulation of DNA methyltransferase as the final product of the reaction, methylated DNA, can regulate gene expression in cis. We show that an AP-1-dependent regulatory element of dnmt1 is heavily methylated in most somatic tissues and in the mouse embryonal cell line, P19, and completely unmethylated in a mouse adrenal carcinoma cell line, Y1. dnmt1 is highly over expressed in Y1 relative to P19 cell lines. Global inhibition of DNA methylation in P19 cells by 5-azadeoxycytidine results in demethylation of the AP-1 regulatory region and induction of dnmt1 expression in P19cells, but not Y1 cells. We propose that this regulatory region of dnmt1 acts as a sensor of the DNA methylation capacity of the cell. These results provide an explanation for the documented coexistence of global hypomethylation and high levels of DNA methyltransferase activity in many cancer cells and for the carcinogenic effect of hypomethylating diets.     

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.     

DNA甲基化及其对植物发育的调控

DNA甲基化属于一种表观遗传修饰,主要发生在CpG双核苷酸序列中的胞嘧啶上,是在DNA甲基转移酶催化下,以S-腺苷甲硫氨酸为甲基供体,将甲基转移到胞嘧啶上,生成5-甲基胞嘧啶的一种反应。DNA甲基化在植物生长过程中具有极其重要的作用。综述了植物DNA甲基化的特征、调控机制,及其对植物基因表达影响的研究进展。     

DNA methylation,an epigenetic mechanism connecting folate to healthy embryonic development and aging

Experimental studies demonstrated that maternal exposure to certain environmental and dietary factors during early embryonic development can influence the phenotype of offspring as well as the risk of disease development at the later life. DNA methylation, an epigenetic phenomenon, has been suggested as a mechanism by which maternal nutrients affect the phenotype of their offspring in both honeybee and agouti mouse models. Phenotypic changes through DNA methylation can be linked to folate metabolism by the knowledge that folate, a coenzyme of one-carbon metabolism, is directly involved in methyl group transfer for DNA methylation. During the fetal period, organ-specific DNA methylation patterns are established through epigenetic reprogramming. However, established DNA methylation patterns are not immutable and can be modified during our lifetime by the environment. Aberrant changes in DNA methylation with diet may lead to the development of age-associated diseases including cancer. It is also known that the aging process by itself is accompanied by alterations in DNA methylation. Diminished activity of DNA methyltransferases (Dnmts) can be a potential mechanism for the decreased genomic DNA methylation during aging, along with reduced folate intake and altered folate metabolism. Progressive hypermethylation in promoter regions of certain genes is observed throughout aging, and repression of tumor suppressors induced by this epigenetic mechanism appears to be associated with cancer development. In this review, we address the effect of folate on early development and aging through an epigenetic mechanism, DNA methylation.     

Methyl Donor-Deficient Diet during Development Can Affect Fear and Anxiety in Adulthood in C57BL/6J Mice

DNA methylation is one of the essential factors in the control of gene expression. Folic acid, methionine and choline (methyl donors)–all nutrients related to one-carbon metabolism–are known as important mediators of DNA methylation. A previous study has shown that long-term administration of a diet lacking in methyl donors caused global DNA hypermethylation in the brain (Pogribny et al., 2008). However, no study has investigated the effects of a diet lacking in methyl donors during the developmental period on emotional behaviors such as fear and anxiety-like behavior in association with gene expressions in the brain. In addition, it has not been elucidated whether a diet supplemented with methyl donors later in life can reverse these changes. Therefore, we examined the effects of methyl donor deficiency during the developmental period on fear memory acquisition/extinction and anxiety-like behavior, and the relevant gene expressions in the hippocampus in juvenile (6-wk) and adult (12-wk) mice. We found that juvenile mice fed a methyl-donor-deficient diet had impaired fear memory acquisition along with decreases in the gene expressions of Dnmt3a and Dnmt3b. In addition, reduced anxiety-like behavior with decreased gene expressions of Grin2b and Gabar2 was observed in both the methyl-donor-deficient group and the body-weight-matched food-restriction group. After being fed a diet supplemented with methyl donors ad libitum, adult mice reversed the alteration of gene expression of Dnmt3a, Dnmt3b, Grin2b and Gabar2, but anxiety-like behavior became elevated. In addition, impaired fear-memory formation was observed in the adult mice fed the methyl-donor-deficient diet during the developmental period. Our study suggested that developmental alterations in the one-carbon metabolic pathway in the brain could have effects on emotional behavior and memory formation that last into adulthood.     

Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis

To produce a severe choline-methionine deficiency, a synthetic L-amino acid diet, free of choline, methionine, vitamin B12, and folic acid and supplemented with guanidoacetic acid, a methyl group acceptor, was fed to female rats for 2 weeks. The in vitro activity of liver microsomal phosphatidylethanolamine methyltransferase was stimulated twofold when compared with basal diet controls. The activity of choline phosphotransferase was depressed by 86%; thus, the contribution of the methyltransferase in the overall synthesis of phosphatidylcholine apparently increased. However, measurement of the in vivo methylation of phosphatidylethanolamine by incorporation of [1,2-14C]ethanolamine into phosphatidylcholine indicates that the methylation pathway is markedly depressed in methyl deficiency. Hepatic concentrations of the methyltransferase substrate, S-adenosylmethionine, and the inhibitory metabolite, S-adenosylhomocysteine, were significantly altered such that an unfavorable environment for methylation was present in the deficient animal. The ratio of substrate to inhibitor was depressed from 5.2:1 in the controls to 1.7:1 in the livers of methyl-depleted rats. Control of transmethylation in accordance with the availability of substrates, phosphatidylethanolamine, or S-adenosylmethionine, and the level of S-adenosylhomocysteine is discussed.     

Impact of folic acid fortification on global DNA methylation and one-carbon biomarkers in the Women's Health Initiative Observational Study cohort

DNA methylation is an epigenetic mechanism that regulates gene expression and can be modified by one-carbon nutrients. The objective of this study was to investigate the impact of folic acid (FA) fortification of the US food supply on leukocyte global DNA methylation and the relationship between DNA methylation, red blood cell (RBC) folate, and other one-carbon biomarkers among postmenopausal women enrolled in the Women's Health Initiative Observational Study. We selected 408 women from the highest and lowest tertiles of RBC folate distribution matching on age and timing of the baseline blood draw, which spanned the pre- (1994–1995), peri- (1996–1997), or post-fortification (1998) periods. Global DNA methylation was assessed by liquid chromatography-tandem mass spectrometry and expressed as a percentage of total cytosine. We observed an interaction (P = 0.02) between fortification period and RBC folate in relation to DNA methylation. Women with higher (vs. lower) RBC folate had higher mean DNA methylation (5.12 vs. 4.99%; P = 0.05) in the pre-fortification period, but lower (4.95 vs. 5.16%; P = 0.03) DNA methylation in the post-fortification period. We also observed significant correlations between one-carbon biomarkers and DNA methylation in the pre-fortification period, but not in the peri- or post-fortification period. The correlation between plasma homocysteine and DNA methylation was reversed from an inverse relationship during the pre-fortification period to a positive relationship during the post-fortification period. Our data suggest that (1) during FA fortification, higher RBC folate status is associated with a reduction in leukocyte global DNA methylation among postmenopausal women and; (2) the relationship between one-carbon biomarkers and global DNA methylation is dependent on folate availability.