Introduction
Folate is an essential water-soluble vitamin occurring naturally in select foods as well as in the synthetic form (folic acid) used in supplements and in food fortification programs (1–3).
DNA methylation is a covalent modification of genomic DNA that modifies gene expression and provides a mechanism for transmitting and perpetuating epigenetic information through DNA replication and cell division. The role of DNA methylation in cellular regulation has also provided the potential for a new paradigm of disease intervention and treatment. The development of various inhibitors of DNA methylation that alter methylation patterns within intact mammalian cells has led to the clinical use of some inhibitors in experimental therapies for human diseases such as hematological malignancies (8) and myelodysplastic disorders (9).
Currently, new DNA sequencing technologies are beginning to provide novel insight into genome-wide patterns of DNA methylation (10–13).
Current status of knowledge
Part I: overview of DNA methylation
Introduction to DNA methylation.
Methylation of cytosine is common throughout the human genome. This covalent modification most commonly occurs at cytosines within a 5′-CpG-3′ dinucleotide when a methyl group from S-adenosylmethionine (SAM)6 is enzymatically transferred to the 5 position of cytosine to generate 5-methylcytosine (5-MC) in genomic DNA.
DNA methylation and demethylation: roles and mechanisms.
Methylation at CpG dinucleotides provides a mechanism for transmitting DNA methylation patterns after DNA replication and perpetuating patterns of epigenetic regulation through subsequent cell generations. Methylation of CpG dinucleotides results in symmetrically methylated CpG sites (i.e., methylation of the CpG sequence on both strands of DNA) (as seen in Fig. 2).
Mechanisms of gene silencing by DNA methylation.
Methylation of CpG islands, especially those islands colocalized with promoters or other regulatory regions, is generally associated with gene repression. The mechanisms by which DNA methylation silences transcription are not fully understood, although several mechanisms have been proposed.
Function of DNA methylation.
Cytosine methylation has been hypothesized to be an ancient component of the immune system designed to recognize and inactivate parasitic viral DNA sequences that infiltrate the genome (57). Mammalian genomes contain a large proportion of repetitive DNA sequences (e.g., nearly 50% of human genomic DNA) that include retroviral sequences, transposons, retrotransposons long interspersed elements (LINEs), short interspersed elements (SINEs), extended blocks of tandemly repeated DNA sequences (e.g., satellite DNA), etc. Most of the DNA methylation present in mammalian genomes is associated with these repetitive DNA sequences and serves to suppress the potential activity and deleterious effects of repetitive DNA (58–60). This includes suppressing mobility and propagation of repetitive elements capable of transposition and suppressing illegitimate recombination between related repetitive elements (58–60). These functions serve to prevent widespread genome instability and rearrangement mediated by repetitive DNA sequences (61).
What does a change in DNA methylation reflect? Hypermethylation versus hypomethylation.
Any CpG site in a single DNA molecule can either be methylated or unmethylated, so the level of methylation at that site will either be 0% or 100%. However, within a single diploid cell, the methylation level of a given CpG site on 1 strand of DNA can be 0% (symmetrically unmethylated on both chromosomes), 50% (symmetrically methylated on 1 chromosome and unmethylated on the other chromosome, as often seen in DMRs of imprinted genes), or 100% (symmetrically methylated on both chromosomes).
Part II: Folate and DNA methylation
Folate's role in 1-carbon metabolism related to DNA methylation.
Under normal dietary conditions, absorbed folate is metabolized to 5-methyltetrahydrofolate (5-methylTHF, monoglutamyl form) in the intestine and/or liver. 5-MethylTHF is the primary folate constituent taken up by nonhepatic tissues, which then must be polyglutamated for cellular retention and 1 carbon cycle coenzyme function. Tetrahydrofolate (THF) is the most effective substrate for polyglutamate synthetase; therefore, 5-methylTHF must be converted to THF via the methionine synthase reaction (Fig. 1). When folic acid is consumed in fortified foods or supplements, it is metabolized primarily to 5-methylTHF during intestinal absorption and/or first pass in the liver, after which it behaves identically to natural dietary folate. Folic acid is normally first reduced to dihydrofolate by dihydrofolate reductase and subsequently to THF to enter the folate pool (Fig. 1). In some cases, the capacity of dihydrofolate reductase is exceeded and folic acid may appear in the circulation in the oxidized form (94). Once the THF coenzyme is formed from either folic acid or dietary folate, it is first converted to 5,10-methyleneTHF by the vitamin B-6–dependent enzyme serine hydroxymethyltransferase and subsequently irreversibly reduced to 5-methylTHF by methylenetetrahydrofolate reductase (MTHFR). This reaction is key to maintaining the flux of methyl groups for the remethylation of homocysteine to methionine via the vitamin B-12–dependent methionine synthase reaction. Methionine is the substrate for SAM or AdoMet, a cofactor and methyl group donor for numerous methylation reactions including the methylation of DNA, RNA, neurotransmitters, and other small molecules, phospholipids, and proteins, including histones (95). A number of SAM-dependent reactions have regulatory roles by affecting both genome stability and gene transcription (55), localization of protein (96), and small molecule degradation (97).
In addition to folate, a number of other dietary nutrients are required to maintain 1 carbon flux, ensuring normal homocysteine remethylation, SAM formation, and DNA methylation. These nutrients include vitamin B-6 (serine hydroxymethyltransferase activity), riboflavin (MTHFR stability), vitamin B-12 (methionine synthase function), and choline (betaine precursor as a hepatic methyl source via betaine:homocysteine methyltransferase) (98).
The 1-carbon pathway, and thus DNA methylation, functions under tight regulatory controls. SAM is the major regulator of folate-dependent homocysteine remethylation because it is a potent inhibitor of MTHFR. Under the condition of high SAM concentration, MTHFR is inhibited, which reduces the synthesis of 5-methylTHF and hence remethylation of homocysteine. Conversely, when SAM concentrations are low, remethylation of homocysteine is favored. MTHFR activity and thus 5-methylTHF formation may also be modified by the common genetic variant, 677C→T, which reduces enzyme activity (99).
DNA methylation, cancer, and folate.
Global hypomethylation and targeted hypermethylation are considered defining characteristics of human tumors (14, 23, 58, 107, 108) (Fig. 2B). DNA methylation patterns are widely dysregulated in human cancer (Fig. 2B). Early in the study of DNA methylation, genome-wide hypomethylation was found in tumor tissues compared with matched healthy tissues (58). Additionally, ∼5% of genes were found to be hypermethylated in nonrandom patterns specific to the type of tumor (109). It was determined that these changes in DNA methylation in tumors resulted in silencing of tumor suppressor genes and chromosome instability (Fig. 2B) (110, 111). Hypomethylation of repetitive elements may be predictive of increased risk of cancer and increased mortality from cancer (112).
It is unclear whether increases in dietary folate and/or folic acid result in changes in healthy tissues that can predispose to carcinogenesis. Many studies of the effects of folic acid supplementation have focused on either preventing or promoting cancer, especially colon cancer (Supplemental Table 1).
Studies of cancer patients and folate status and global DNA methylation.
There has been great interest in reversing epigenetic changes seen in early tumorigenesis (e.g., global hypomethylation) as rapidly dividing tissue tumors may be susceptible to low folate availability, resulting in global hypomethylation. Three clinical trials examined the impact of folic acid supplementation (0.4–10 mg/d) on global DNA methylation in colon cancer patients (30, 118, 119) (Supplemental Table 1). These studies have shown that DNA methylation might be a biomarker for colorectal cancer and that methylation of DNA in the colon and leukocytes can be increased by supplementation with folic acid in human patients (118, 120). Among the observational studies (e.g., cohort and case-control) of the association of folate status (measured as folate/folic acid intake and or blood folate concentration) and cancer, 5 studies examined the global DNA methylation level (121–125) (Supplemental Table 1). In these studies, an association was found between cancer and global DNA hypomethylation in circulating blood and/or tumor tissue; however, there was not a consistent association between global DNA methylation and folate status (intake or blood folate) across studies (121–125). This lack of a definitive association between low folate status and global DNA hypomethylation could be due to a limited sample size or imprecision of the assays. Across studies outlined in Supplemental Table 1, global hypomethylation was found in tumors and even normal colon cells and blood of cancer patients (121, 122, 124, 126).
Changes in DNA methylation in response to the environment and diet: the importance of the developmental timing of exposure.
There is increasing evidence of more interindividual variation and variation in DNA methylation patterns across the life span than was previously expected, and these changes may be modulated by environmental exposures. DNA methylation varies between the sexes and changes during aging (47–50).
Epigenetic effects of other dietary components are of interest as well. The offspring of male mice fed a low-protein diet (from weaning to sexual maturity) showed numerous although modest (typically 10–20%) changes in DNA methylation in the liver, with altered expression of genes involved in lipid and cholesterol biosynthesis, compared with control offspring of fathers fed a normal protein diet (149).
Extreme exposures can be used as proof-of-principle to show plausibility of a cause-and-effect relationship. Studies of prenatal starvation have shown an association with many adverse health outcomes including both short-term and long-term consequences: neural tube defects (NTDs), metabolic syndromes, and increased risk of schizophrenia, depending on the precise timing of the exposure and the sex of the fetus (153–158). The period around conception may be one of the more sensitive periods and corresponds to the time when the epigenetic patterns are reset (Supplemental Fig. 1B). It has been hypothesized that epigenetic changes as a result of starvation exposure may be responsible for these effects later in life. However, it is not clear which macronutrient or micronutrient deficiency or combination thereof may be responsible for these health outcomes.
Studies in fetus, infants, cord blood, and folate status.
As a result of the erasure and resetting of DNA methylation patterns in early development and differentiation as well as the rapid growth rate in early development, early development could be particularly susceptible to folate intake. Two studies showed epigenetic changes in individuals prenatally exposed to famine during the Dutch Hunger Winter at the end of World War II (which might be indicative of extreme folate deficiency) (153, 159) (Supplemental Table 1). Heijmans et al. (159) found methylation changes in IGF2 associated with prenatal exposure to prenatal famine.
High folate and folic acid intake and DNA methylation.
The purpose of many of the controlled feeding trials was to attempt to reverse the aberrant DNA methylation observed in cancer. The effect of folate insufficiency is clearly detrimental both to the embryo and its short-term risk of NTDs and the possible longer term risks of diabetes or other health outcomes (156, 157, 166, 167). Additionally, high blood folate concentrations (121) and high global DNA methylation have been shown in a number of studies to reduce the risk of cancer (123, 125). However, recent focus has shifted to concern about folic acid supplementation resulting in progression of existing tumors and altering normal DNA methylation patterns (31, 168, 169). Currently, there is no direct evidence of aberrant DNA methylation and change in gene expression in response to “high” levels of folate/folic acid intake. In addition, there is no consensus on a dose of folic acid or a blood folate concentration that would be associated with potential adverse health outcomes.
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Conclusions
Appropriate DNA methylation patterns are critical to normal genome function. Aberrant DNA methylation patterns are present in many human diseases, including cancer, imprinting disorders, and developmental disabilities (172–174). The human studies of DNA methylation and folate/folic intake acid vary widely in their study design, timing of exposure, and amount of folate/folic acid intake, tissue tested, assays, and, not surprisingly, the findings. At this time, the evidence suggests that low folate status is associated with decreases in global DNA methylation, which has in some studies has been associated with an increased risk of cancer. However, it is unclear how specific regions of the genome respond to higher or lower folate intakes. To date, the majority of studies have only examined a limited number of genetic loci and/or a small number of samples. There is no direct evidence that high dietary folate or folic acid intake leads to aberrant DNA methylation, changes in gene expression, or disease state. Given the conflicting results to date, it is clear that the current research does not support a linear relationship or dose response between folic acid supplementation and global or site-specific DNA methylation level. This is not unexpected given that DNA methylation is part of a complex, highly regulated system. Additional research is needed to elucidate the relationship between folate and DNA methylation.
Referência :
Adv Nutr. 2012 Jan;3(1):21-38.