Download Trans-HHS Workshop: Diet, DNA Methylation

Trans-HHS Workshop:
Diet, DNA Methylation Processes and Health
Gene-Nutrient Interactions and DNA Methylation1,2
Simonetta Friso3 and Sang-Woon Choi
Vitamin Metabolism Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research
Center on Aging, Tufts University, Boston, MA, 02111
KEY WORDS:
●
MTHFR
●
C677T
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folate
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DNA methylation
Nutrition research has recently highlighted the role of
several nutrients in regulating the genome machinery. A number of vitamins and micronutrients are substrates and/or cofactors in the metabolic pathways that regulate DNA synthesis
and/or repair and the expression of genes (1). It has been
documented that the deficiency of such nutrients may result in
the disruption of genomic integrity and alteration of DNA
●
gene expression
methylation, thus linking nutrition with modulation of gene
expression. The discovery of polymorphic enzymes involved in
critical steps of nucleic acids metabolic pathways contributed
to new insights into the interplay of genetics and nutrition for
the phenotypic expression of a defect. The response to a
nutrient status seems in many cases to be specific for each
genotype, and specific nutrient impairment results in different
gene expression, depending on each genotype. The field of
gene-nutrient interactions, therefore, seems to be a fascinating
model to explain the different response to environmental/diet
exposure at the molecular level.
Recently, the interaction between nutrients and DNA
methylation has been emphasized. DNA methylation, a characteristic feature of many eukaryotic genomes, consists in the
addition of a methyl group at the carbon 5⬘ position of cytosine within the cytosine-guanine (CpG)4 dinucleotide (2) in a
complex reaction that probably involves the flipping of the
cytosine base out of the intact double helix (3). Typically,
DNA methylation occurs in CpG-dinucleotide-rich regions,
1
Presented at the “Trans-HHS Workshop: Diet, DNA Methylation Processes
and Health” held August 6 – 8, 2001, in Bethesda, MD. This meeting was sponsored by the National Center for Toxicological Research, Food and Drug Administration; Center for Cancer Research, National Cancer Institute; Division of Cancer Prevention, National Cancer Institute; National Heart, Lung and Blood
Institute; National Institute of Child Health and Human Development; National
Institute of Diabetes and Digestive and Kidney Diseases; National Institute of
Environmental Health Sciences; Division of Nutrition Research Coordination,
National Institutes of Health; Office of Dietary Supplements, National Institutes of
Health; American Society for Nutritional Sciences; and the International Life
Sciences Institute of North America. Workshop proceedings are published as a
supplement to The Journal of Nutrition. Guest editors for the supplement were
Lionel A. Poirier, National Center for Toxicological Research, Food and Drug
Administration, Jefferson, AR and Sharon A. Ross, Nutritional Science Research
Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD.
2
This material is based upon work supported by the U. S. Department of
Agriculture, under agreement No. 581950-9-001. Any opinions, findings, conclusions or recommendations expressed in this publication are those of the authors
and do not necessarily reflect the view of the U. S. Department of Agriculture.
3
To whom correspondence should be addressed.
E-mail: [email protected].
4
Abbreviations used: C677T, cytosine-to-thymine transition at position 677;
CC, cytosine/cytosine; CpG, cytosine guanine dinucleotide; DNase, deoxyribonuclease; Dnmt1, DNA methyltransferase 1; methyl-THF, 5-methyltetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase; RBC, red blood cell; SAM,
S-adenosylmethionine; TT, thymine/thymine.
0022-3166/02 $3.00 © 2002 American Society for Nutritional Sciences.
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ABSTRACT Many micronutrients and vitamins are critical for DNA synthesis/repair and maintenance of DNA
methylation patterns. Folate has been most extensively investigated in this regard because of its unique function
as methyl donor for nucleotide synthesis and biological methylation. Cell culture and animal and human studies
showed that deficiency of folate induces disruption of DNA as well as alterations in DNA methylation status. Animal
models of methyl deficiency demonstrated an even stronger cause-and-effect relationship than did studies using
a folate-deficient diet alone. Such observations imply that the adverse effects of inadequate folate status on DNA
metabolism are mostly due to the impairment of methyl supply. Recently, an interaction was observed between
folate status and a common mutation in the gene encoding for methylenetetrahydrofolate reductase, an essential
enzyme in one-carbon metabolism, in determining genomic DNA methylation. This finding suggests that the
interaction between a nutritional status with a genetic polymorphism can modulate gene expression through DNA
methylation, especially when such polymorphism limits the methyl supply. DNA methylation, both genome-wide
and gene-specific, is of particular interest for the study of cancer, aging and other conditions related to cell-cycle
regulation and tissue-specific differentiation, because it affects gene expression without permanent alterations in
DNA sequence such as mutations or allele deletions. Understanding the patterns of DNA methylation through the
interaction with nutrients is fundamental, not only to provide pathophysiological explanations for the development
of certain diseases, but also to improve the knowledge of possible prevention strategies by modifying a nutritional
status in at-risk populations. J. Nutr. 132: 2382S–2387S, 2002.
GENE-NUTRIENT INTERACTIONS AND DNA METHYLATION
DISCUSSION
Effect of nutrients on genomic integrity and DNA
methylation
Reduced dietary intake or low tissue/plasma levels of several
nutrients have been associated with higher risk for developing
cancer. Interestingly, many micronutrients and vitamins are
indispensable in DNA metabolic pathways (1,11).
Although most studies have been conducted in vitro and in
animal models and there is no clear evidence for the optimal
dietary ranges able to protect against DNA damage, roles in
maintaining genomic stability have been documented for several nutrients. For example, vitamin C and E deficiencies are
known to cause DNA oxidation and chromosome damage
(12,13). Vitamin D exerts an antioxidant activity, stabilizes
chromosomal structure and prevents DNA double strandbreaks (14). Magnesium is an essential cofactor in DNA
metabolism, and its role has been recognized in maintaining
high fidelity in DNA transcription (15). Iron may cause DNA
breaks (16). A carotenoid-rich diet has been shown to reduce
DNA damage (17). Vitamin B-12 deficiency is associated with
micronuclei formation (1,18), and reduced transcobalamin II
is associated with chromosomal abnormalities (19).
The roles of nutrients in DNA methylation, especially in
genome-wide methylation, have also been described. Zinc
deficiency can reduce the utilization of methyl groups from
S-adenosylmethionine (SAM) in rat liver and results in
genomic DNA hypomethylation as well as histone hypomethylation (20,21). Dietary deficiency in selenium decreased
genomic DNA methylation in Caco-2 cells and in the rat liver
and colon (22,23). Vitamin C deficiency has been associated
with DNA hypermethylation in lung cancer cells (24,25).
Interestingly, niacin, precursor of NAD⫹, is required to maintain the unmethylated state of CpG dinucleotides by inhibiting the enzymatic DNA methylation (26,27), because it is
necessary for the synthesis of poly-ADP–ribose polymerase-1,
which converts histone H1 to poly-ADP–ribosylated forms.
The poly-ADP–ribosylated forms of histone H1 are responsible
for the enzymatic inhibition of DNA methylation (26,27).
Nevertheless, folate and/or methyl group dietary supply
provides the most compelling data for the interaction of nutrients and DNA methylation, because these dietary elements
are directly involved in DNA methylation via one-carbon
metabolism. The sole metabolic function of all coenzymatic
forms of folate is to transfer one-carbon units. Within the
scope of this function is the synthesis of SAM, universal
methyl donor for several biological methylation reactions, and
the de novo deoxynucleoside triphosphate synthesis. Methionine is regenerated from homocysteine by methionine synthase in a reaction in which 5-methyltetrahydrofolate (methyl
THF) serves both as a cofactor and as a substrate. The reduced
availability of methyl-THF, the main circulating form of folate, decreases the biosynthesis of SAM, thus limiting the
availability of methyl groups for methylation reactions. Therefore, not only can dietary folate depletion decrease genomic
DNA methylation in both human (28,29) and animal models
(30), but, as described in the study by Rampersaud and colleagues, a folate replete diet also may restore the DNA methylation status (29).
Gene-nutrient interactions in one-carbon metabolism
Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20)
is considered a key enzyme in the one-carbon metabolism
because it catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to methyl-THF (31). In 1988 Kang and
colleagues identified a variant of the MTHFR that causes
enzyme thermolability and reduced activity (32). The mutant
enzyme was associated with elevated plasma homocysteine
levels, which is to be expected because the conversion of
homocysteine to methionine is impaired (31). Not only did
the mild hyperhomocystinemia appear as an indicator of altered one-carbon metabolism, but the higher levels of this
sulfur-containing amino acid also were recognized to be an
independent risk factor for cardiovascular disease (33,34).
The thermolabile variant of the MTHFR is due to a common missense mutation, a cytosine-to-thymine transition at
base pair 677 (C677T) (35) that results in an alanine-tovaline substitution in the MTHFR amino acid sequence. The
prevalence of the valine-valine substitution is rather common,
with a frequency in homozygous persons of up to 20% in
certain populations (35,36,37). By determining plasma total
homocysteine levels, a strong gene-nutrient interaction was
demonstrated in the phenotypic expression of this polymorphism in MTHFR (37,38). Only those affected homozygous
persons with inadequate folate status, as indicated by blood
folate levels, showed elevated plasma homocysteine concentrations (38). An intermediate effect has also been observed in
heterozygous persons (37). These findings contributed to the
opening of a new field of interest for both nutrition and
genetics, especially because the relationship of the MTHFR
polymorphism with plasma folate levels was implicated as the
likely link between the C677T genetic defect and cardiovascular disease (34) as well as neural tube defects (39).
The MTHFR C677T polymorphism also provides a paradigm of gene-nutrient interaction in carcinogenesis (40,41).
The mutant thymine/thymine (TT) genotype is associated
with a lower risk of developing colorectal cancer; however,
this protective effect is observed only in persons with adequate
folate status. Among those persons with low systemic folate
status, the protection associated with the mutation is eliminated (42) and an even higher risk of developing colorectal
cancer is reported (43).
The biological significance of the MTHFR C677T mutation is predominantly related to the reduced availability of
methyl-THF. Also consistent with this concept is the recent
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the so-called “CpG islands” that, in contrast to the overall
genome, are highly represented in gene promoter regions or
initial exons of genes (4). DNA methylation is a fundamental
mechanism for the epigenetic control of gene expression and
the maintenance of genomic integrity (5,6). Therefore, an
evaluation of genomic DNA methylation status is important
for the study of cell growth regulation, tissue-specific differentiation (2,4,7) and carcinogenesis (6).
Most recently, an interaction was described between folate
status and a common mutation in a key enzyme of the onecarbon metabolism that is responsible for the availability of
methyl groups for biological methylation reactions, including
that of DNA (8,9). These findings suggest that modulating
DNA methylation by nutrition may be a fascinating new field
of gene and nutrient interaction. Therefore, it is of considerable interest to identify the factors that determine the patterns
of methylation, not only to provide evidence for the mechanisms of several pathological conditions, but also to identify
at-risk populations in which to conduct appropriate diet-based
interventions (10).
The purpose of this review is to discuss the most recent
knowledge about the effects of nutrients on gene expression
and integrity, with an emphasis on gene-nutrient interactions
in the modulation of DNA methylation.
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SUPPLEMENT
observation that the distribution of different coenzymatic
forms of folate is altered in MTHFR TT homozygotes (44).
The red blood cells (RBC) of TT homozygous mutants show
variable amounts of formylated tetrahydrofolate polyglutamates at the expense of methylated tetrahydrofolates. In contrast, cells from the cytosine/cytosine (CC) wild-type persons
contain exclusively methylated tetrahydrofolate derivatives
(44).
A common mutation that results in an altered activity of
methionine synthase (5-methyltetrahydrofolate– homocysteine S-methyltransferase; EC 2.1.1.13), another important
enzyme of the homocysteine/methionine metabolic pathway,
has been described as interacting with levels of vitamin B-6
(45). This polymorphism, an adenine-to-guanine transition at
nucleotide position 2,756 which results in substitution of
glycine for aspartic acid at amino acid position 919, has been
observed to cause elevation of plasma total homocysteine
levels in persons with low levels of vitamin B-6 (45). However,
more evidence is needed to demonstrate a clear gene-nutrient
interaction in determining the biochemical expression of this
genotype.
A recent study investigated whether the mutant MTHFR,
in association with folate status, affected methylation of DNA.
(8). It was observed that subjects homozygous for the MTHFR
C677T polymorphism possessed a lower degree of genomic
DNA methylation in peripheral lymphocytes compared with
the CC wild-type persons (8) and also that there was an
inverse correlation between RBC folate and DNA methylation status. Because of the small number of subjects and the
indirect method used to assess genomic DNA methylation, a
large cohort of persons was subsequently studied using a newly
developed quantitative and highly specific liquid chromatography/electrospray ionization-mass spectrometry assay in
which the previous observations were reproduced and extended (9). The results showed that genomic DNA methylation in peripheral blood mononuclear cells directly correlated
with folate status and inversely correlated with plasma homocysteine levels. MTHFR TT genotypes had a diminished level
of DNA methylation compared with those with the CC wildtype. When analyzed according to folate status, however, only
the TT subjects with low levels of folate accounted for the
diminished DNA methylation (as shown in a model in Fig. 1).
Moreover, in TT subjects, DNA methylation status correlated
with the methylated proportion of RBC folate and was inversely related to the formylated proportion of RBC folates
that are known to be solely represented in TT persons (9).
These findings indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status.
Nutrients and gene-specific DNA methylation
Gene-specific DNA methylation at the promoter region.
Approximately one-half of human genes have CpG islands in
their 5⬘-promoter regions or within their first exons (46). CpG
islands usually are unmethylated (47), and the methylation of
these CpG-rich sequences induces inhibition of their expression. The patterns of DNA methylation, therefore, distinguish
at a molecular level the genes to be expressed selectively (48).
Alterations in DNA methylation have been described as regulating a differentiation path in a particular tissue. DNA in
germ line cells usually is fully methylated, and demethylation
usually is observed in a tissue-specific fashion, except that most
of the housekeeping genes usually are maintained in a completely unmethylated state in both the germ line and in
tissue-specific sites (49).
Although the exact molecular mechanism by which DNA
methylation represses the transcription is not yet clear, data
demonstrating an active role of promoter methylation in gene
silencing are quite convincing. In vitro methylation of promoter-reporter constructs inhibits their subsequent expression
in transfected cells (50). Demethylation by 5-azadeoxycytidine, a DNA methyltransferase inhibitor, leads to re-expression of previously methylated genes (51). Homozygous embryos with a germline deletion of the DNA methyltransferase
1 (Dnmt1; EC 2.1.1.37) gene, on which the prototypical
mammalian cytosine DNA methyltransferase is encoded, reexpress a number of genes, including the normally silent alleles
of several imprinted genes and the abundant but normally
repressed endogenous retroviral sequences that are methylated
and silent in heterozygous littermates (52).
The mechanism for the CpG island-associated gene silencing seems to involve the link of specific methylated DNA
binding proteins, followed by the recruitment of a silencing
complex that includes histone deacetylases (53,54). The de
novo methylation, by itself, has a minimal effect on gene
expression. However, methylated DNA recruits methyl-binding proteins, which also attracts a protein complex that con-
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Effect of folate and MTHFR gene interaction in genomic
DNA methylation
FIGURE 1
Model illustrating the gene-nutrient interaction between the MTHFR C677T variant and folate levels for genomic DNA
methylation status. The enzyme MTHFR is responsible for the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (CH3-THF). The MTHFR C677T variant encodes for a thermolabile enzyme with lower activity. As shown in this scheme, under folate
deficiency conditions, subjects carrying in homozygosity the mutant
allele for the thermolabile MTHFR (TT) (right) have a decreased formation of CH3-THF that results in lower production of S-adenosylmethionine and consequent lower availability of methyl groups (CH3) for the
methylation reactions including DNA methylation. The reduced availability of CH3-THF also is reflected by diminished homocysteine remethylation with consequent higher total plasma homocysteine levels
(tHcy). On the contrary, subjects wild-type for the C677T genotype (CC)
are not affected by folate deficiency (left), because the synthesis of
CH3-THF for methylation reactions and for the conversion of homocysteine to methionine is preserved. The MTHFR genotype does not alter
the availability of CH3-THF when folate status is adequate. The size of
the arrows indicates the different entity in enzyme activity and the flux
through the folate pools. The size of the font for the metabolic products
indicates approximately the relative change in the amount of metabolites among the MTHFR genotypes.
GENE-NUTRIENT INTERACTIONS AND DNA METHYLATION
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FIGURE 2 Molecular effects of nutrients on gene
expression and integrity through modulation of DNA
methylation.
c-fos, c-Ha-ras and c-myc were correlated with hypomethylation at specific sites within these genes (61,62). These observations suggest that nutrients may affect gene transcription by
exon-specific DNA methylation.
Hypomethylation of the coding regions of critical genes can
lead to instability either because this region becomes more
susceptible to endogenous nucleases (63) or because the site of
hypomethylation is likely to undergo enzymatic deamination
to uracil (64,65). The latter situation is particularly prone to
occur in conditions where intracellular S-adenosylmethionine
levels are low, such as in folate depletion (65). In a folatedeficient rat model, Kim and colleagues observed that hypomethylation of hypermutable sites (exon 5 through 8) on the
p53 gene was related to increased DNA strand breaks in the
same region (66). Furthermore, hypomethylation within the
exon 8 of the colonic p53 gene was shown to be induced in an
animal model of chemical carcinogenesis (67), and hypomethylation of this site in peripheral mononuclear cell DNA
was highly associated with the development of lung cancer in
a nested case-control study (68). Cell culture studies indicate
that these foci of aberrant methylation may serve as initiators
of mutations (64) and may induce susceptibility to breakage of
the DNA backbone (69) by increased uracil insertion and
strand breaks (70). Collectively, these studies suggest that
folate deficiency might induce DNA strand breaks and subsequent mutations through exon site hypomethylation. On the
other hand, increasing levels of dietary folate effectively overrode the induction of hypomethylation in a dose-responsive
manner in an animal model of chemical carcinogenesis (67),
which suggests that folate supplementation can reduce gene
disruption by reversing the site-specific DNA hypomethylation.
This review discusses recent data in the field of genenutrient interactions and DNA methylation, a fundamental
epigenetic feature of DNA that affects gene expression and
genomic integrity (Fig. 2). Several nutrients are involved in
the maintenance of DNA metabolism, however most convincing data indicate a critical role for folate, an essential vitamin
for DNA metabolism because it is involved in both DNA
synthesis/repair and DNA methylation. The observation of an
interaction between a common mutation in MTHFR, a key
enzyme of the one-carbon metabolic pathway, and DNA
methylation provides the basis for research on the potential
role of nutrients in modulating an epigenetic feature of DNA
as well as in possible future prevention strategies.
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tains histone deacetylases. Through the action of methylbinding proteins and histone deacetylases, the DNA structure
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