Human Molecular Genetics

Human Molecular Genetics 2005 14(Review Issue 1):R139-R147; doi:10.1093/hmg/ddi100

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The impact of metabolism on DNA methylation

Clayton L. Ulrey¹, Liang Liu^1,2, Lucy G. Andrews¹ and Trygve O. Tollefsbol^1,2,3,*

¹Department of Biology, ²Center for Aging and ³Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA

^* To whom correspondence should be addressed at: Department of Biology, 175 Campbell Hall, 1300 University Boulevard, University of Alabama at Birmingham, Birmingham, AL 35294-1170, USA. Tel: +1 2059344573; Fax: +1 2059756097; Email: trygve{at}uab.edu

Received December 15, 2004; Revised February 7, 2005; Accepted February 23, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
Methylation of genomic cytosines is one of the best characterized epigenetic mechanisms, and investigation of its relationship with other biochemical pathways represents a critical stage in the elucidation of biological information processing. The field also has immense potential for the development of medical treatments for any number of conditions ranging from aging to neurological disorders. The DNA methylation status of genes is responsible for many heritable traits and varies more or less independently of the genetic code. This variation is often a result of cellular environmental factors including metabolism. A key metabolite in this regard is homocysteine. Knowledge of homocysteine metabolism continues to be amassed, yet the part played by aberrant DNA methylation in homocysteine-related pathologies is often, at best, conjectural. In this analysis, we will review recent insights and attempt to speculate meaningfully concerning the dynamics of the methionine cycle in relation to DNA methylation and disease.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
The methyl groups needed for all biological methylation reactions are derived from dietary methyl donors and from cofactors carrying 1-carbon units. A pathway which is key to many of these reactions is the metabolic cycling of methionine (Fig. 1). Briefly, methionine is converted to the methyl cofactor S-adenosylmethionine (SAM or AdoMet). Subsequent to methyl donation, the product S-adenosylhomocysteine (SAH) becomes homocysteine (Hcy), which is then either catabolized or remethylated to methionine. Here, we review recent literature on this topic with a special emphasis on DNA as the methyl acceptor.

View larger version (22K): [in this window] [in a new window]   Figure 1. The methionine cycle including some of the known modulators. Green, dashed arrows represent a tendency of the modulator to increase and red a tendency to decrease activity of the indicated reaction. Most of the modulation points are discussed in the text; otherwise, references are cited presently. It should be noted that the exact, individual roles of the hormonal modulators as activators/repressors have not necessarily been delineated: [1] MAT: MATIII is activated and MATI inhibited by SAM (83,84). One form is transcriptionally activated by glucocorticoids (85). Hydrolysis of ATP yields inorganic phosphate (Pi) and pyrophosphate (PPi); [2] guanidinoacetate methyltransferase; [3] GNMT; [4] general methyltransferase reaction as in Figure 2, shown in bold [e.g. X may represent cytosine in a CG palindrome being methylated by a DNMT]; [5] SAH hydrolase [6] betaine-homocysteine methyltransferase (this reaction uses a zinc cofactor); [7] methionine synthase (MS); [8] serine hydroxymethyltransferase [along with glycine and 5,10-methylenetetrahydrofolate (5,10-CH2THF), a molecule of H2O is produced]; [9] MTHFR; [10] CBS (high CBS mRNA levels in diabetic rat liver are reduced by insulin treatment) (86); [11] cystathionine -lyase: after cleavage of cystathionine, -ketobutyrate and cysteine can be converted, in several steps, to succinyl coenzyme A and pyruvate, respectively. Pyruvate is immediately available for glucose synthesis, whereas succinyl coenzyme A must first be converted to oxaloacetate; [12] L-arginine/glycine amidinotransferase: this reaction occurs in the kidney; creatine is subsequently produced in the liver.

 
The palindromic CpG dinucleotide often serves as substrate for DNA methyltransferases (DNMTs) targeting the 5-carbon position of the cytosine residues. The added methyl group can interfere with transcription factor binding, thereby regulating transcription (1). It can also designate a possible attachment site for methyl-CpG-binding proteins, which in turn effect further regulation by their association with the histone deacetylase-containing chromatin remodeling complexes. DNMT1, 3a and 3b are the most thoroughly studied DNMTs, and the activity of these enzymes is often described as being either maintenance or de novo methylation. The former process serves to maintain preexisting epigenetic control status in dividing cells by methylating hemimethylated sites on newly synthesized strands, whereas the latter methylates sites in which both strands are unmethylated, for example, during early embryonic development. The DNMT genes are considered to have lethal status in mammals as mice lacking the genes die during gestation or shortly after birth (2,3).

DNA methylation is critical for developmental changes in gene regulation, the classic model being promoter methylation in genes to be down-regulated and hypomethylation of promoters in genes associated with the succeeding developmental stage. Maintenance of X-chromosome inactivation is mediated by DNA methylation, as is genomic imprinting during germ line development, a process for which DNMT3a has recently been found to be essential (4). The promoters of housekeeping and most tissue specific genes contain CpG-rich segments. The methylation status of these so called CpG islands is often abnormal in cancers, e.g. of the prostate and gastrointestinal tract (reviewed in 5,6). Under most circumstances, methylation is associated with a decrease in transcription. Global hypomethylation with age and promoter-specific hypermethylation are considered to be associated with conditions such as aging, cancer and atherosclerosis (7–13). As metabolism plays a significant role in DNA methylation, it will likewise have an impact on such epigenetically influenced conditions. For a summary of recent findings on these matters, see Table 1.

View this table: [in this window] [in a new window]   Table 1. Summary of recent literature concerning metabolic effects on DNA methylation

 

	SAM AND DNA METHYLATION

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
SAM or AdoMet, the primary biochemical methyl donor, is a cosubstrate reacting with nucleophilic acceptors in association with various methyltransferases including DNMTs. The functionality of the labile methyl group is due to the energy-dependent adenosylation of methionine, which converts the inactive thiomethyl to an active sulfonium group. This is the first reaction of the methionine cycle (Fig. 1). The enzyme that catalyzes the reaction is methionine adenosyltransferase (MAT), isoforms of which are tissue-specific and differentially regulated according to metabolic conditions. Mice lacking the MAT1A isozyme have hypermethioninemia, reduced hepatic SAM concentrations and normal levels of global hepatic DNA methylation, as well as marked effects on the expression of many genes (14). Substrates for SAM methyl transfer reactions include DNA, RNA, proteins, neurotransmitters and phospholipids. The products of the methylation reaction (Fig. 2) are a methylated substrate and SAH.

View larger version (9K): [in this window] [in a new window]   Figure 2. Generalized SAM-dependent methyl transfer reaction with structures of cosubstrates. The figure shows the methyltransferase-catalyzed reaction of a methyl acceptor X with the methyl donor SAM and the products SAH and methylated substrate CH3-X. SAH often feeds back to inhibit the reaction, and its concentration may be the primary determinant of enzyme activity in certain tissues.

 

	SAM METABOLISM AND METHYL TRANSFER

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
SAH is favored in an equilibrium with adenosine and Hcy catalyzed by SAH hydrolase and inhibits the activities of most SAM-dependent methyltransferases (15). Thus, the efficient clearing of the reaction products is vital in terms of meeting methylation demand in the cell. This effect with regard to DNA methylation has been illustrated in a study on men with hyperhomocysteinemia and uremia in which the patients' peripheral mononuclear blood cells had increased hypomethylation when compared with controls (16). The principle has, perhaps more emphatically, been demonstrated in a study on peripheral lymphocytes of adult females (17). Lymphocyte SAH concentration was positively correlated with plasma SAH, which in turn was correlated with DNA hypomethylation. No such relationship was observed in the case of SAM concentration. In addition, in the previously mentioned MAT1A knockouts, SAM levels were decreased, whereas SAH and DNA methylation were not significantly altered. Such results have been taken to indicate that metabolite modulation of DNMTs likely occurs primarily through SAH in many cell types (18). However, there are other results that cannot be explained by this inhibitory scheme. Recently, a case of SAH hydrolase deficiency was reported in which the patient's leukocyte DNA was hypermethylated relative to controls despite extreme levels of plasma SAH (19). Likewise, DNA from rat colon with high SAH concentration has recently been noted to have methylation levels comparable to those in controls in three separate studies (20–22).

In light of such conflicting results, it seems likely that theories regarding methyl metabolism and epigenetics will need to take a large range of variables into account. Some components of the mechanisms involved are tissue- and cell type-specific and may be dependent not only on genetics, but also on many behavioral characteristics such as diet. An illustration of this can be seen in a gene–nutrient interaction involved with folate metabolism, which is discussed subsequently. There are also numerous other issues involved, such as histone methylation (23), which represents another gene regulatory mechanism reliant on SAM. Consideration of such complicating factors indicates a potential for the use of mathematical models, such as the two that have recently been developed (24,25), to predict epigenetic and pathological outcomes given different genetic and nutritional/metabolic inputs. That is, future more advanced models could be extended to include epigenetics and become useful not only for hypothesis testing, but also for diagnostic and therapeutic purposes.

	GLYCINE N-METHYLTRANSFERASE

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
One variable that might be useful in such applications involves the tissue-specific modulation of SAM levels by glycine N-methyltransferase (GNMT). SAM inhibits the synthesis of 5-methyltetrahydrofolate (5-CH₃THF), which is an inhibitor of GNMT (26), a SAM-dependent enzyme present in liver, kidney and pancreas (27). Therefore, at sufficient concentrations, it is thought that SAM relieves the suppression of GNMT activity, inducing its own attrition and lowering the ratio of SAM to SAH. Studies in rats show that retinoid treatment induces GNMT in a tissue- and gender-specific manner (28), with subsequent hypomethylation of hepatic DNA (29). Methionine supplementation also up-regulates GNMT in rat liver and kidney (30). Glucocorticoid treatment induces GNMT in rat liver and in rat hepatoma cells (31), and a similar effect in normal liver is mediated by glucagon (32). In cell culture, induction by glucocorticoids is prevented by pretreatment with insulin (33). Further comment on this will be made subsequently.

	HOMOCYSTEINE METABOLISM

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
Hcy in the body is solely of metabolic origin, ultimately derived from methionine intake; however, in circulation it may exist as a free thiol or as homocystine or may be bound to cysteine or to albumin protein (80%) (34). Two processes are currently known that function in the removal of Hcy: remethylation and transsulfuration (Fig. 3). The former represents completion of the methionine cycle and will be discussed shortly. Entrance into the two-step transsulfuration pathway is irreversible and both reactions require pyridoxal 5'-phosphate (B6). Cystathionine ß-synthase (CBS), occurring in relatively few cell types, mediates the condensation of serine with Hcy to yield cystathionine. It is thought that 60% of Hcy is metabolized by this pathway (35). As a key intersection in the methionine cycle, CBS activity is regulated by several factors including hormones.

View larger version (27K): [in this window] [in a new window]   Figure 3. Overview of three independent pathways involved in the removal of Hcy. Hcy can be remethylated into methionine through the activity of methionine synthase or by a liver-specific betaine-Hcy methyltransferase (remethylation pathways). Alternatively, Hcy can be irreversibly converted into cysteine by transulfuration. Concerning methylation reactions, metabolism of Hcy represents a key intersection in the methionine cycle.

 
Transsulfuration links the methionine cycle to gluconeogenesis by producing cysteine and -ketobutyrate, which, after a number of steps, are converted to glucogenic pyruvate and succinyl coenzyme A, respectively. In the fasted state, insulin levels are low, and levels of glucagon and glucocorticoids are elevated, hormonal conditions similar to those found in diabetes. It was found that insulin treatment decreased CBS expression in diabetic rat liver (36), whereas glucagon has been shown to increase CBS activity and expression in rat liver (32). Glucocorticoids and cAMP, glucagon's second messenger, increased CBS activity in rat hepatoma cells (37). Such results could point to a relationship between caloric intake and biological methylation, as up-regulation of Hcy catabolism under restrictive conditions may to some extent alleviate SAH inhibition of methyltransferases. If, on the other hand, GNMT activity is elevated during caloric restriction, this may act as a counterbalance by depleting SAM. The epigenetic consequences of metabolism under differing dietary energy states are a possible complement to the current interest in diabetic metabolism and gene regulation.

Serine contributes to 1-carbon metabolism through the serine hydroxymethyltransferase reaction, in which tetrahydrofolate (THF) is reversibly converted to 5,10-methylenetetrahydrofolate (5, 10-CH₂THF) in the synthesis of glycine. Methylenetetrahydrofolate reductase (MTHFR) can then make the 1-carbon moiety available for methionine synthase activity. An alternate pathway for remethylation of Hcy is through betaine-Hcy methyltransferase in the liver, where betaine, from the diet or derived from choline, is the methyl donor. The role of betaine as a methyl donor has not been studied as extensively as that of the folates, but seems to be gaining greater attention in the field (reviewed in 38).

Moreover, L-arginine/glycine amidinotransferase mediates synthesis of ornithine and guanidinoacetate in the kidney. Guanidinoacetate is methylated by SAM in the liver to make creatine. Creatine synthesis significantly influences the SAM/SAH ratio as it represents the bulk of SAM consumption (39). Supplemental arginine may contribute to Hcy production due to an increase in this pathway (40). Rats supplemented with creatine had a 27% reduction in plasma Hcy levels, whereas guanidinoacetate supplementation produced a 49% increase (41). If a pathological condition is being worsened owing to competition of DNMTs with numerous other methyl transfer pathways as well as the associated inhibitory effects of SAH, lightening the burden of creatine synthesis may have ameliorating effects. As this supplement is readily available, human trials including assays of DNA methylation could prove a practical next step.

Remethylation of Hcy is catalyzed by methionine synthase with a cobolamin (B12) cofactor and 5-CH₃THF donating a methyl group to become THF. Recycling of this cofactor by MTHFR is a key reaction in 1-carbon metabolism, which is present in most cells and influences DNA methylation. The consequent relation to colorectal cancer has spurred great interest (42), especially in relation to alcohol consumption (43,44). Interestingly, the frequently observed 677CT transition in the gene can cause a reduction to 30% of normal activity in homozygotes (T/T) or 60% in heterozygotes. In a population of young American females, DNA methylation in leukocytes of T/T subjects was hypermethylated relative to normals (C/C) during folate repletion (following depletion) (45). A Scottish population showed no interaction between either plasma methyl metabolites or MTHFR genotype and global DNA methylation in lymphocytes (46). Analysis of subjects from northern Italy (47) revealed a strong, direct relationship between red blood cell and plasma folate levels and genomic DNA methylation in the peripheral blood mononuclear cells of T/T individuals. This relationship was not significant for those of C/C genotype.

Several studies in transgenic mice have also consistently demonstrated the importance of the MTHFR gene in development. Homo- and heterozygous Mthfr knockouts have either significantly decreased SAM or increased SAH levels with coincident global DNA hypomethylation. The heterozygotes appear normal, whereas the homozygotes are smaller and show developmental retardation with cerebellar pathology (48). It has also been demonstrated that mice heterozygous for the defect are vulnerable to hyperhomocysteinemia when fed with low folate diets and have altered tissue methylation capacity and impaired endothelial function in cerebral microvessels (49). Related pathologies will be discussed subsequently.

	FOLATE IN THE DIET

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
A study on rats found that normal dietary folate levels with supplemental selenium yielded greater methylation of DNA in colon and liver than when diets contained only one or neither of the two nutrients (20). DNMT activity in liver was elevated after provision of either nutrient, and activity in colon was elevated by supplemental selenium. Another study found no significant differences in colonic p53 gene methylation that were relatable to gene expression and levels of dietary folate (21). Unexpectedly, at one point in the study, folate-deficient rats had increased genomic DNA methylation in the colon. Folate/methyl deficiency leads to hepatocarcinogenesis in male rats with the associated paradoxical hypomethylation of hepatic DNA and simultaneous increase in liver DNMT activity and expression. Evidence indicates that these effects are specific to the liver (50). Folate therapy has also been noted to normalize the expression of certain genes in patients with hyperhomocysteinemia, evidently by restoring normal DNA methylation patterns (16).

In as much as B12 is required in folate methyl donation, inadequacy may in some ways resemble folate deficiency. Methionine synthase activity can determine the extent of DNA methylation when B12 is deficient (51). Methylcytosine content of colonic epithelial DNA was reduced 35% after 10 weeks of moderate B12 deficiency in rats (52), indicating a possible role in colorectal cancer. An earlier study had demonstrated no effect of either dietary folate or age on genomic DNA methylation in the colon (22).

	ATHEROSCLEROSIS AND ALZHEIMER'S DISEASE

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
Both clinical and epidemiological studies have established an association between Hcy and the incidence of cardiovascular disease. Hyperhomocysteinemia produces several conditions that are potentially harmful to the vasculature including increased oxidant stress, impaired endothelial function, induction of thrombosis and increase of arterial pressure (53,54). The mechanisms underlying these conditions have not been elucidated, but may be related to the impairment of vascular endothelial and smooth muscle cell functions. Many teams are now devoted to investigating metabolic/epigenetic involvement. Notably, in one group of vascular disease patients, a relationship was discovered between both plasma Hcy and SAH (but not SAM/SAH ratio) and hypomethylation in leukocytes (55). Specific genes with altered methylation include extracellular superoxide dismutase in rabbits (56) and human estrogen receptor- (57), which have roles in vasoprotection and cell proliferation.

Much recent speculation has focused on atherosclerosis as being analogous to cancer in that it involves similar phenotypic alterations and widespread hypomethylation in affected tissues (13). Some have suggested that the role of Hcy in atherosclerotic hypomethylation may consist more in its hyperproliferative effects than in DNMT inhibition (58). An interesting parallel to this idea has been noted in colon cancer cells (59), which can be stimulated to hyperproliferate by treatment with Hcy. This effect is retarded by treatment with 5-CH₃THF.

The apolipoprotein E-deficient mouse is prone to atherosclerosis and harbors global DNA hypomethylation in aorta and peripheral blood mononuclear cells preceding the animal's characteristic development of atherosclerotic lesions (60). In the cited study, it was also shown that global DNA hypermethylation in human monocytic THP-1 cells may be induced by high levels of atherogenic lipoproteins. The investigators point out that this is consistent with the tendency of the more salubrious high density lipoproteins to contribute to gene activation. Theoretically then, following a hypermethylated state in the initial stages, the condition worsens as hyperproliferation of smooth muscle cells induces progressive attrition of methylcytosine. This result is significant as a causative role of these lipoproteins in epigenetic alterations is implied. In addition, the authors advance the idea of using DNA methylation polymorphisms as markers of disease.

Recently, evidence has also established a connection between Hcy metabolism and cognitive function. Abnormal levels of Hcy have been related to multiple cognitive dysfunctions including age-related memory loss, vascular dementia and Alzheimer's disease (61–63). Deficiencies in folic acid and B12 are often observed in the elderly population with a resultant increase in Hcy. This deficiency is proposed to be owing to an increasing prevalence of atrophic gastritis type B, which occurs with a frequency of 20–50% in elderly subjects (64).

One study confirmed an Hcy-related effect with regard to two methyltransferases (65). The investigators found 26% higher SAH levels in prefrontal cortices of Alzheimer patients relative to controls. The high SAH levels also correlated with brain homogenate inhibition of rat liver catechol O-methyltransferase (COMT), and inhibition was 15% greater with Alzheimer homogenates than in controls. COMT and phenylethanolamine N-methyltransferase (PNMT) activities were at least 30% lower than in controls, and there was a negative correlation of methyltransferase inhibition and SAH with COMT and PNMT activities. In addition, concentrations of SAH comparable to those in the homogenates were demonstrated to inhibit COMT and PNMT extracted from human brain. COMT and PNMT are involved in neurotransmitter metabolism and are believed to affect cognitive function. Such studies should be extended to investigate the modulating effects of SAH and other metabolites on brain DNMTs, which may influence synaptic plasticity and long-term memory (66). Simultaneous assays for methylation status of key putative Alzheimer gene promoters may also be of value.

One such gene product, presenilin 1 (PS1), increases -secretase activity, which, along with ß-secretase, is responsible for production of ß-amyloid peptide from amyloid precursor protein (APP). Scarpa et al. (67) studied the effects of SAM treatment on PS1 promoter methylation and activity in differentiating neuroblastoma cells and demonstrated the inhibition of demethylation and decreased expression of PS1 with subsequent decline in ß-amyloid production. Another study utilized bisulfite sequencing to compare methylation status in a GC-rich region of the APP gene in cerebral cortices from human autopsies (68). It was discovered that a negative relationship exists between age and methylation in this region. As plasma Hcy levels are known to increase with age (69), it might be useful to perform assays for SAH and DNA methylation in parallel to test the hypothesis that key gene promoters are demethylated due to SAM and SAH methyl modulation.

	MATERNAL METABOLISM AND NUTRITION

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
Aberrant methyl metabolism in utero is linked with disorders such as intrauterine growth retardation (IUGR), neural tube defects (NTDs) and adult onset disease. Insight into the role of epigenetic etiology in this field continues to grow. IUGR induced in rats increases hepatic levels of SAH and methionine and decreases expression of MAT and CBS with contemporaneous genomic hypomethylation in the liver (70).

Folic acid is known to improve conditions in pregnancies affected by NTDs (71,72). Reduced risk of NTD complications is also associated with increased methionine (73), B12 (74) and choline/betaine intakes (75). NTDs have been noted in mouse embryos treated in vitro with chemicals that impair choline metabolism (76). MTHFR-deficient mice had reduced mortality and improved somatic development when their mothers received betaine supplementation during pregnancy and lactation (77). Compared with controls, brain and liver transsulfuration were increased in the experimental group. Hippocampal and cerebellar growth and differentiation were also improved.

At present, there can be little doubt that DNA methylation has some influence on such occurrences, and experiments in the field of development are now more often designed to include it. Along these lines, an experiment employing a human neuroblastoma cell culture model showed that the cells developed global DNA hypomethylation when grown in choline deficient medium (78). The promoter of the cyclin-dependent kinase inhibitor 3 gene was also hypomethylated, apparently resulting in increased expression with a concomitant decrease in cell proliferation. Further experimentation with this and similar models may help delineate the negative effects of maternal malnutrition on fetal brain development.

The impact of maternal nutrition likewise extends into adulthood as DNA methylation patterns are reset in the postimplantation embryo (79). This has been elegantly illustrated in a murine model: the viable yellow agouti (A^vy) mouse (80). Mating of homozygous recessive males with heterozygous females produced offspring with varying distribution of coat color, according to maternal methyl nutrition (supplemental betaine, choline, folic acid and B12). Correlation of diet with both coat color and methylation status in portions of the A^vy loci in offspring demonstrated the potential for maternal metabolic influence on epigenotype. Conclusions drawn from these results have been further supported by a similar study in which A^vy methylation was shown to mediate the correlation of diet with coat color (81). Furthermore, A^vy methylation in tail DNA was strongly correlated with that from other tissues representing all three germ layers. This implies that the observed methylation patterns are set in early embryonic development and are maintained through subsequent stages, affecting all tissues.

	SAM AND DNA DEMETHYLATION

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
The histone deacetylase inhibitor, trichostatin A, was recently employed to stimulate demethylase activity in HEK 293 cells transiently transfected with a methylated reporter plasmid (82). Methylation-sensitive isoschizomer assays revealed that the percentage of methylated plasmid 72 h posttransfection increased with SAM treatment in a dose-dependent manner, whereas SAH treatment resulted in no significant effect. The mechanism implied by these results is in contrast with the more conventional model involving SAM-mediated activation of DNMTs because western blot analysis demonstrated no effect on reporter expression in cells transfected with unmethylated plasmids; i.e. no sign of de novo methylation was observed subsequent to SAM treatment. This model is controversial because of questions raised by results from other research which are beyond the scope of this review. If valid, it may have potential implications concerning the role of DNMT inhibitors as cancer drugs given the possibility that demethylases may also be an important target for therapy. If invalid, it can serve as an illustration of how little is known at the present juncture in the highly complex fields of metabolism and epigenetics.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 
Nutrition and metabolism are the sources of methyl groups that are used to methylate DNA, a process that influences chromatin structure and gene expression. In so far as a physiological condition is affected by genes that are under the control of this mechanism, understanding of the condition will be facilitated by investigation of the biochemistry underlying any implicated epigenetic influence. Elevated Hcy is the cause of several damaging effects. The extent of involvement of DNA methylation in Hcy-related pathologies is only beginning to be uncovered. In some cases, the involvement may be great, whereas in others small or absent. Perspectives on the putative effectors of the DNMTs and even the role of these enzymes continue to shift. Further efforts in the field will continue to enrich the understanding and improve the manipulation of these mechanisms.

	ACKNOWLEDGEMENTS

 
The authors apologize for the omission of relevant articles due to space and time limitations. This work was supported in part by grants from the American Institute for Cancer Research, the Purdue-UAB Botanicals Center, the Ovarian SPORE Program and a UAB Postdoctoral Career Development Award to L.L.

	REFERENCES

TOP ABSTRACT INTRODUCTION SAM AND DNA METHYLATION SAM METABOLISM AND METHYL... GLYCINE N-METHYLTRANSFERASE HOMOCYSTEINE METABOLISM FOLATE IN THE DIET ATHEROSCLEROSIS AND ALZHEIMER'S... MATERNAL METABOLISM AND... SAM AND DNA DEMETHYLATION CONCLUSIONS REFERENCES

 

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