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REVIEW

Folate and DNA Methylation: A Review of Molecular Mechanisms and the Evidence for Folate’s Role1,2
Krista S. Crider,3* Thomas P. Yang,4 Robert J Berry,3 and Lynn B. Bailey5
National Center on Birth Defects and Developmental Disabilities, Division of Birth Defects and Developmental Disabilities, Atlanta, GA, Department of Biochemistry and Molecular Biology, Center for Epigenetics, Division of Pediatric Genetics, University Florida College of Medicine, Gainesville, FL, University of Florida, Gainesville, FL, and 5Foods and Nutrition Department, University of Georgia, Athens, GA
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ABSTRACT

DNA methylation is an epigenetic modification critical to normal genome regulation and development. The vitamin folate is a key source of the one carbon group used to methylate DNA. Because normal mammalian development is dependent on DNA methylation, there is enormous interest in assessing the potential for changes in folate intake to modulate DNA methylation both as a biomarker for folate status and as a mechanistic link to developmental disorders and chronic diseases including cancer. This review highlights the role of DNA methylation in normal genome function, how it can be altered, and the evidence of the role of folate/folic acid in these processes. Adv. Nutr. 3: 21–38, 2012.

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). There are many critical cellular pathways dependent on folate as a 1-carbon source including DNA, RNA, and protein methylation as well as DNA synthesis and maintenance. Folate can be a limiting factor in all these reactions (Fig. 1). Epigenetics is the study of heritable changes in phenotype or gene expression that do not result from changes in the primary DNA sequence (4,5). Recognized mechanisms of epigenetic regulation in mammals include DNA methylation, post-translational modification of histones, chromatin remodeling, microRNAs, and long noncoding RNAs (6,7). These epigenetic regulatory mechanisms modulate chromatin structure and contribute to regulation of the major molecular processes in the nucleus including transcription, replication, repair, and RNA processing. DNA methylation

Author disclosures: The authors report no conflicts of interest. K.S. Crider and R.J. Berry performed this work as employees of the US government. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. 2 Supplemental Table 1 and Supplemental Figure 1 are available from the “Online Supporting Material" link in the online posting of the article and from the same link in the online table of contents at http://advances.nutrition.org. * To whom correspondence should be addressed. E-mail: kcrider@cdc.gov.

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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). DNA methylation patterns are stable and are retained in purified genomic DNA; therefore, studies of DNA methylation are amenable to a wide variety of cell-free assays and technologies including DNA methylation analysis at singlenucleotide resolution, next-generation sequencing, and genome-wide methylation profiling (10) (Table 1). Currently, new DNA sequencing technologies are beginning to provide novel insight into genome-wide patterns of DNA methylation (10–13). Although high-resolution truly genome-wide studies have been limited to a very small sample size, a recent study described the DNA methylation level at 1505 individual sites (loci) in 808 genes in 1628 human genomic DNA samples (14). Even this tremendous data set only covers a tiny portion of the genome in a limited number of samples. Despite these recent advances, a basic understanding of normal variation in genomic
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ã2012 American Society for Nutrition. Adv. Nutr. 3: 21–38, 2012; doi:10.3945/an.111.000992.

SAM. S-adenosylmethionine. intracisternal A particle. and folate. DNA methylation patterns are a product of the frequency of cytosine DNA methylation at specific sites along a strand of DNA. Although the importance of CpG methylation is established. DHFR. NTD. and 99. SAH. DNMT. 82. The methods needed to undertake these types of studies and the infrastructure to do these analyses are rapidly emerging (10. studies of cancer patients and folate status and global DNA methylation. 5-HMC. This review was written to provide the nutritional scientist with a synopsis of the mechanistic aspects of DNA methylation as a background for understanding the potential for the nutrient folate to affect these same molecular processes. with feedback loops and interactions not shown in the schematic. studies of folate in the fetus. SHMT. the potential role of folate in the DNA methylation process and its assessment as a folate status biomarker and link to disease outcomes are covered: folate’s role in 1-carbon metabolism related to DNA methylation. DNA methyltransferase. MS. S-adenosylmethionine. long interspersed element. tetrahydrofolate. methylenetetrahydrofolate reductase. studies of healthy adults and folate status. thymidylate. deoxyuridine monophosphate. THF. SINE. serine hydroxymethyltransferase. differentially methylated region. reprogramming of DNA methylation patterns during development and differentiation. neural tube defect. THF. Current status of knowledge Part I: overview of DNA methylation Introduction to DNA methylation. red blood cell. methionine synthase. 67. disease. Gene names for enzymes are in italics and cofactors are in parentheses.Figure 1 Folic acid metabolism.16). Recent genome-wide high-resolution DNA methylation analysis of a primary human fibroblast cell line demonstrated that 4. age. The MTHFR 677C/T variant reduces enzyme activity (175) and may help to divert the available methyl groups from the DNA methylation pathway toward the DNA synthesis pathway (176 –178). 5-methylTHF. the role of non-CpG methylation is a new active area of research. S-adenosylhomocysteine. the mechanisms of gene silencing by DNA methylation. BPA. Clearly there are many challenging research issues that need to be addressed to fill in the gaps in our knowledge related to the potential for folate to modulate DNA methylation and potentially have an impact on development and health maintenance.98% of DNA methylation occurs in CpG dinucleotides (10). TS. short interspersed element. dTMP. methylenetetrahydrofolate reductase. low folate status and DNA methylation. the frequency of CpG dinucleotides is lower than would be expected throughout most mammalian genomes (18). highlights of the following are covered: the roles and mechanisms of DNA methylation and demethylation. 5-methylcytosine. 5-hydroxymethylcytosine. tetrahydrofolate. S-adenosylhomocysteine. and the importance of changes in DNA methylation (hyper. MTHFR. DMR.7% of CpGs are methylated. . cancer. changes in DNA methylation in response to the environment and diet—the importance of the developmental timing of exposure.7% of CpG dinucleotides are methylated. This schematic shows the process by which folate/folic acid is used for DNA methylation. or environmental conditions (including dietary intakes) have not been well described.25% of total cytosines in genomic DNA are methylated. LINE. infants. This covalent modification most commonly occurs at cytosines within a 59-CpG-39 dinucleotide when a methyl group from Sadenosylmethionine (SAM)6 is enzymatically transferred to the 5 position of cytosine to generate 5-methylcytosine (5-MC) in genomic DNA. The pathway is complex and highly regulated. DNA methyltransferase. 22 Crider et al.and hypomethylation). dUMP. thymidylate synthase. patterns of DNA methylation in humans across tissues. Similar analysis of a human embryonic stem cell line showed that 5. and 25% of all cytosine methylation occurs at non-CpG sites (10). bisphenol A. dihydrofolate. Interestingly. the function of DNA methlyation. cord blood. and high folate and folic acid intake and DNA methylation. 5-MC. background of DNA methylation. base excision repair.83% of cytosines are methylated. However. SAM. SAH. Following this. an earlier study also reported high levels of non-CpG methylation in mouse embryonic stem cells in contrast to methylation patterns in mouse somatic cells (17). RBC. Specifically. DNMT. DHF. 5-methyltetrahydrofolate. populations. certain regions of the genome have approximately a 10-fold higher 6 Abbreviations used: BER. Methylation of cytosine is common throughout the human genome. This is believed to be due to the spontaneous deamination of 5-MC to yield thymine and a cytosine-tothymidine transition in DNA (19).15. dihydrofolate reductase. The review concludes with a focus on research issues including methodological considerations that are key in planning and interpreting research investigations related to assessing differences in DNA methylation in response to changes in folate status. IAP. MTHFR.

Technical simplicity tion of genomic DNA by SssI methylase Standard molecular biology techniques Assesses methylation at specific restriction sites using Southern blotting Sensitivity. not full genome coverage. (195) Gu et al. sodium bisulfite genomic sequencing. 1500 > 450K sites per sample Determines methylation status of each cytosine in a region of Single-nucleotide resolution. etc.) CHARM. limited sequence coverage Herman et al. technically accessible RRBS Low resolution. HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction. (45) meDIP/meDNA pull-down/MIRA Determines methylation status of each cytosine in the genome after chemical conversion of unmethylated cytosines to uracils in genomic DNA Uses antimethylated cytosine antibody or methylated DNAbinding protein to enrich for methylated DNA Single-nucleotide resolution Single-nucleotide resolution. full genome coverage. methylation-sensitive cut counting.. accurate. retechnically straightforward stricted sequence coverage Quantitative. not restricted to CpG islands Folate DNA methylation 23 1 For sodium bisulfite conversion used in methylation-specific polymerase chain reaction. meDIP. (198) Ball et al. cost Lister et al. Nair et al. reveals DNA methinterest within a single DNA molecule ylation patterns of individual DNA molecules Highly quantitative. comprehensive high-throughput arrays for relative methylation. Fernandez et al. (131). technically straightforward. (186) Balaghi and Wagner (187) Methyl acceptance Digestion/hydrolysis of genomic DNA to nucleotides or ba. RRBS. accurate. weak reproducibility Digestion/hydrolysis of genomic DNA to nucleosides and fractionation by LC-MS Shellnut et al. HPLC. . (This table is not a comprehensive list of methods used to assay DNA methylation. (189) Determines level of methylation at specific CpG sites Tost and Gut (190) Bibikova et al. single-nucleotide resolution. meDNA. methylated CpG island recovery assay. lacks full genome coverage Weber et al. specific. Description Pros Cons References Representative list of commonly used methodologies for analyzing DNA methylation1 Global Liquid chromatography tandem mass spectrometry HPLC Highly sensitive. Requires costly instrumentation. pyrosequencing. Requires costly instrumentation. all cytosines except methylated cytosines in genomic DNA are chemically converted uracil by sodium bisulfite. (199) HELP MSCC A size-selected portion of the genome is isolated after restriction enzyme digestion and sequenced after sodium bisulfite treatment Uses restriction enzyme that specifically cleaves methylated DNA Compares the relative representation of MspI and HpaII fragments at individual loci Assesses methylation status at all recognition sites of a methylation-sensitive restriction enzyme Assays both CpG islands and non-CpG island sites. (191). (14) Clark et al. (193). methylated cytosines are resistant to conversion and can be detected by various methods.Highly quantitative. MSCC. and reproducible Requires costly instrumentation. technical simplicity Wolf and Migeon (188) Gene specific Methyl-sensitive restriction enzyme digestion Methylation-specific polymerase chain reaction Pyrosequencing Specifically amplifies methylated genomic DNA sequences after sodium bisulfite treatment of genomic DNA Limited to analysis of individual restriction sites. full genome sequencing. time-consuming. liquid chromatography-mass spectrometry. (197) Khulan et al. (137) Armstrong et al. not strictly quantitative Not quantitative. the most comprehensive method for genome-wide profiling of DNA methylation Successfully used in a wide variety of published studies. efficiency dependent on CpG density Biased toward analyzing CpG islands.Table 1. requires w1 μg amounts of DNA Requires costly instrumentation. and reproducible ses and fractionation on HPLC Global methylation determined by level of in vitro methyla. methylated DNA immunoprecipitation. HELP. indirect assessment of methylation by detecting unmethylated CpG sites. methylated DNA.5 μg DNA Labor intensive. requires μg amounts of DNA Lacks sensitivity. restricted sequence coverage Requires sequencing of full genome at w15-fold coverage. imprecise. Rauch et al. (196) CHARM Quantitative Irizarry et al. lacks full genome coverage Lacks full genome coverage Lacks full genome coverage Not strictly quantitative. RRBS. Christensen et al. reduced representation bisulfite sequencing. (194). high-performance liquid chromatography. quantitative High resolution. relarge number of CpGs at a time quires w0. MIRA. LC-MS. (192) Microarray Sodium bisulfite genomic sequencing Genome-wide profiling MethylC-seq Large numbers of individual CpG sites from specific genes are assayed using standard microarray equipment after bisulfite treatment. quantitative.

131). and certain shores have shown altered methylation patterns in colon cancer (13). including some tumor suppressor genes. CpGs throughout the genome are targeted for methylation by DNA methyltransferases (DNMT1. The numbered blue rectangles are exons. Mapping CpG islands has been used as a tool for gene discovery because >50% of all mammalian genes are associated with CpG islands (19. Each bar represents a 59-CpG -39 site. . and permanent changes in the phenotype distribution of the offspring (147). Methylated CpG are indicated by bars with balls at the end.6 (20). The sequence of the retrotransposon is a candidate for methylation in the early embryo. Cancer cells have aberrant DNA methylation patterns. Methylation of CpG island shores has been correlated with gene expression. CpG islands generally are thought to be actively protected from DNA methylation to allow for appropriate regulation of transcription (179). (B) Dysregulation of DNA methylation seen in cancer post-transformation.183). the extent to which it becomes methylated varies in genetically identical individuals based on maternal diet (methyl donors as well as other dietary factors) (146. CpG islands were originally defined as genomic regions w200 base pairs in size with a C+G content of 50% and an observed CpG/expected CpG >0. 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. (C) Maternal diet changes methylation levels at the metastable epiallele.” which are CpG-containing regions that are within 2 kb of a CpG island. 2). These regions are referred to as CpG islands and are often correlated with the location of genes. 24 Crider et al. a change in expression at nearby genetic loci. particularly promoter regions and other regulatory regions (20.21). The red X equals gene silencing.frequency of the CpG dinucleotide than the rest of the genome. DNMT3b). The green arrow indicates transcription start and active transcription. which can change transcription of a neighboring region. These islands compose <1% of the genome and are typically unmethylated (Fig. A subset of genetic sites is hypermethylated. 2). (A) DNA methylation patterns conducive to transcription:– patterns set during development.22–24). This is a cartoon of a hypothetical single section of double-stranded DNA. Metastable epialleles are regions of the genome that have a variable methylation patterns that are semiheritable across generations. DNA methylation and demethylation: roles and mechanisms.180–182). although this definition is somewhat arbitrary and has been refined more recently to correlate more closely with promoter regions and regulatory regions (21) (Fig. CpG islands are regions with a concentration of CpGs often (but not always) associated with the promoter regions of genes. Another functionally important region for DNA methylation are CpG island “shores. DNMT3a. In mice. contain low densities of CpG sites. Methylation Figure 2 DNA methylation patterns at different types of genetic loci and how the patterns change in cancer and in response to maternal diet. and have been shown in recent work to be a location of tissuespecific differential methylation in normal tissues (13). illustrated by the agouti mouse model (147). A recent study found candidate loci in the human genome that show differential methylation dependent on season of birth (148). a maternal diet that includes additional methyl donors can lead to increased methylation at the metastable epiallele (the intracisternal A particle retrotransposon). the specific loci hypermethylated vary among types of tumors (114. Overall DNA methylation levels are reduced (58. however.

As more genome-wide methylation studies are reported. methylation of the CpG sequence on both strands of DNA) (as seen in Fig. transcription factor) to that sequence. Riggs (25) and Holliday and Pugh (26) proposed a scheme by which DNA methylation patterns at CpG dinucleotides could be restored in newly synthesized DNA after DNA replication. In vitro binding studies have identified sequence-specific DNA-binding transcription factors whose Folate DNA methylation 25 . especially those islands colocalized with promoters or other regulatory regions. Thus. reports demonstrating this mechanism have not been subsequently confirmed or reproduced (34–36). Although DNA methylation has usually been regarded as a relatively stable epigenetic mark. One mechanism of passive DNA demethylation involves inhibition or loss of maintenance methylation at CpG sites so that multiple rounds of DNA replication and cell division eventually result in the loss of DNA methylation at these sites in subsequent cell generations. the excised base would be replaced with an unmethylated cytosine by BER (37). They postulated that hemimethylated CpG sites generated by DNA replication could be specifically recognized by a maintenance DNA methyltransferase that would methylate CpG sites on the newly synthesized DNA strand immediately after DNA replication. methylation of a specific regulatory DNA sequence and the attendant insertion of the methyl group into the major groove of DNA may prevent stable binding of a regulatory protein (e. Most recently. and DNMT3b (32). DNMT3a and DNMT3b function as de novo methyltransferases to methylate fully unmethylated CpG sites.. after DNA replication. however. Nucleotide excision repair has also been reported to play a role in active DNA demethylation. although several mechanisms have been proposed. DNMT1 is the most abundant DNMT in cells and. Other potential mechanisms of active demethylation involve base excision repair (BER) of DNA after events such as removal of 5-MC bases in DNA by glycosylase activity or enzymatic deamination of 5-MC to thymidine and formation of a T:G mismatch. the 2 resulting daughter DNA molecules become hemimethylated with the parental template strand maintaining its methylation and the newly synthesized strand lacking methylation. the finding of variation in DNA methylation between the sexes and changes in methylation during aging suggests that there is more interindividual variation and variation in DNA methylation patterns than was previously expected (47–50). what was once regarded as a highly stable and terminal epigenetic state. Thus. in either case. In 1975. maintenance DNA methylation of hemimethylated substrates provides a clear example of a mechanism that allows the transmission and retention of epigenetic information through multiple cell generations.e. 2).g. Continued DNA replication of these hemimethylated daughter molecules would eventually result in the loss of DNA methylation patterns in subsequent generations. DNMT3a and DNMT3b methylate different subsets of DNA sequences in the genome as evidenced by the different phenotypes of mice carrying a knockout of either methyltransferase (32). However. It has been postulated that the availability of folate as a source of methyl groups may affect the ability to maintain DNA methylation patterns in replicating cells (30. First. studies of DNA demethylation have been controversial and mechanisms of demethylation in mammals still are not well understood (33). One potential mechanism of 5-HMC–mediated demethylation would involve recognition and removal of 5-HMC and replacement with an unmethylated cytosine by BER. although more recent evidence suggests that maintenance methylation alone may not be sufficient to fully perpetuate DNA methylation patterns genome-wide (29). suggesting the existence of mechanisms of active DNA demethylation (33). Mechanisms of gene silencing by DNA methylation. Several mechanisms of active DNA demethylation also have been postulated and reported.31). the general mechanism is now widely accepted as a mechanism for maintaining DNA methylation patterns after DNA replication and cell division. the appreciation that notable levels of 5-hydroxymethylcytosine (5-HMC) are present in mammalian genomic DNA and that conversion of 5-MC to 5-HMC in DNA can be catalyzed by mammalian TET (ten-eleven translocation) proteins has generated interest in the possible role of 5-HMC in mechanisms of DNA demethylation (41 – 43). In 1983. Furthermore. These de novo methyltransferases also appear to play a role in maintenance methylation to fully preserve parental DNA methylation patterns in daughter cells (29). Nonetheless. DNMT3a.of CpG dinucleotides results in symmetrically methylated CpG sites (i. as previously described. there are clear examples where DNA demethylation in mammalian cells occurs in the absence of DNA replication and cell division. thereby directly preventing gene activation by a transcription factor.28).. a DNA methyltransferase (DNMT) (now referred to as DNMT1) was identified that demonstrated the predicted preference for hemimethylated DNA (27. although other studies seem to contradict this mechanism (38–40). Mammals have 3 types of DNMT: DNMT1. there is evidence of both increases and decreases in DNA methylation on differentiation (44–46). They function primarily during mammalian development to establish DNA methylation patterns as epigenetic remodeling and reprogramming proceed during differentiation. However. but none have been conclusively demonstrated. DNA methylation is becoming increasingly viewed as a more dynamic process. is generally associated with gene repression. The mechanisms by which DNA methylation silences transcription are not fully understood. is believed to act as the primary maintenance methyltransferase to methylate hemimethylated DNA after DNA replication and preserve parental DNA methylation patterns in daughter cells. In contrast. Methylation of CpG islands. One potential mechanism of active demethylation involves direct removal of the methyl group via cleavage of a highly stable C-C bond. This would regenerate the patterns of symmetrically methylated CpG dinucleotides in daughter cells and maintain the epigenetic states and patterns of gene regulation that existed in parental cells.

in contrast to CpG island methylation. the function of methylation at CpG sites in these regions still is poorly understood. which contributes to the stable maintenance of transcriptional silencing of the associated genes on the inactive X chromosome (72). all mitotic progeny of that cell maintain inactivation of the same X chromosome throughout the remainder of development into adult tissues. transposons. the gene for telomerase has been shown to be activated by methylation (65). these same CpG islands are typically unmethylated on the active X chromosome. Function of DNA methylation. reports indicate that methylation in the body of genes has a role as a positive regulator of transcription (56) and may also have a role in transcription elongation (67). This differential monoallelic expression of the maternal or paternal allele is usually associated with differential DNA methylation of 1 or more regulatory regions of these genes.g.g. CpG island. which is associated with chromatin remodeling (56). only the paternally inherited allele is expressed. whether overall levels of hypermethylation within a CpG island govern silencing. recognize and bind to 1 or more methylated CpG dinucleotides.g..g. Genomic imprinting is another mammalian epigenetic system of monoallelic gene expression in which the 2 alleles of a particular gene within the same cell are differentially regulated. For example. imprinting.. extended blocks of tandemly repeated DNA sequences (e. One of the hallmarks of the inactive X chromosome is the hypermethylation of promoter CpG islands along the length of the inactive X. One such indirect mechanism is via DNA-binding proteins containing methylated DNA-binding domains (52). These . including transcriptional regulation. In contrast. DNA methylation also occurs within the body of genes and between genes. particularly for CpG islands associated with a gene promoter region (55).interaction with their cognate DNA recognition sequence is negatively affected by methylation of a CpG dinucleotide within the binding sequence (51). Therefore. correlated with transcriptional silencing of the associated gene. Once a female cell in early embryogenesis randomly chooses which X chromosome to inactivate (i. Mammalian genomes contain a large proportion of repetitive DNA sequences (e. Generally. etc. such as methyl CpG–binding protein 2 and methylated DNA–binding domain 2.. suggesting that the DNA methylation machinery also may be recruited to specific genes by other mechanisms (similar to those that recruit histone modification and remodeling activities) and that chromatin modification/remodeling and DNA methylation act cooperatively to silence transcription. promoter region). DNMTs have also been found as components of multisubunit chromatin modifying and remodeling complexes involved in transcriptional repression. the paternally inherited allele is stably silenced and only the maternally inherited allele is transcriptionally active. These methylated DNAbinding proteins.62. However. histone deacetylases) that act to form a transcriptionally repressive condensed chromatin structure at the associated gene (53–55). and tissue-specific gene expression (19. alternative promoter use (68). or whether both mechanisms contribute to silencing. X-chromosome inactivation. satellite DNA). and nucleosome positioning (69). This includes suppressing mobility and propagation of repetitive elements capable of transposition and suppressing illegitimate recombination between related repetitive elements (58–60). methylation of a CpG island does not necessarily lead to gene silencing.. retrotransposons long interspersed elements (LINEs). whereas the maternally inherited allele is stably silenced. DNA methylation is thought to maintain gene silencing versus initiating gene silencing (70) such that a gene is silenced initially by another mechanism and that silencing is stabilized by methylation of associated regulatory regions (e. Females randomly inactivate 1 of the 2 X chromosomes present in each and every somatic cell in a process that occurs in early female embryogenesis. a recent study suggests that this may occur by suppressing the repressive effects of the polycomb complex. other more indirect mechanisms must also be involved in repressing transcription by DNA methylation. suggesting that methylation in the body of genes is a positive regulator of gene expression. X-chromosome inactivation is the process by which female mammals compensate for the unequal dose of X-linked genes between male (XY) and female (XX) cells (71). However. it is not clear whether methylation of critical CpG sites within CpG islands triggers hypermethylation of the island and gene silencing (66). Also. for some imprinted genes. It is hypothesized that over time the function of DNA methylation has evolved and now plays additional roles. These functions serve to prevent widespread genome instability and rearrangement mediated by repetitive DNA sequences (61). in a parent-of-origin fashion (73). Thus. short interspersed elements (SINEs). Recently. nearly 50% of human genomic DNA) that include retroviral sequences. Hypermethylation of CpG islands is highly 26 Crider et al. 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). Conversely. intragenic DNA methylation of active genes is often higher than in promoter regions.e. in this case. These proteins in turn interact with or recruit transcriptional silencing complexes containing chromatin remodeling complexes and/or histone modification enzymes (e. 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). One clear example of this is the aberrant stable transcriptional silencing of certain tumor suppressor genes in cancer cells by the hypermethylation of their promoter CpG islands (64). resulting in monoallelic expression of genes subject to X-chromosome inactivation. for other imprinted genes.. many transcription factors are not sensitive to DNA methylation and not all DNA sequences bound by a sequence-specific transcription factor will contain a CpG dinucleotide.63). the maternally or paternally inherited X chromosome).

so the level of methylation at that site will either be 0% or 100%...75). 50% (symmetrically methylated on 1 chromosome and unmethylated on the other chromosome. thereby maintaining throughout embryogenesis the differential DNA methylation of the paternally and maternally inherited alleles in somatic cells characteristic of most imprinted loci (90).e. These cytidine analogs and other DNA-demethylating drugs have recently been introduced as potential therapeutic agents for the treatment of human diseases. Imprinting control regions that regulate imprinting across an entire domain of imprinted genes are typically DMRs (74..79).87. Imprinted genes are thought to comprise a small proportion of mammalian genomes with <100 currently confirmed in humans and mice (77). In humans there is a limited number of known imprinted loci (<80). 1). diet and nutrition) on the developing embryo to alter the normal process of establishing DNA methylation (and other epigenetic) patterns (91–93). 1 allele methylated and the other allele unmethylated). it has been shown that there is a wave of demethylation at the 4-cell stage followed by remethylation beginning as early as the late morula (89). Reprogramming of DNA methylation patterns during development and differentiation. a finding that a particular CpG site is 50% methylated in a population of cells could mean that this site is 50% methylated in every cell (i. or some other combination of methylation levels among cells in the population that yields a composite methylation level of 50%. particularly myelodysplastic syndromes (9). 1A) (76). that in mouse embryogenesis begins shortly after fertilization and continues to the blastocyst stage (86). During the course of gametogenesis. DNA methylation patterns are erased (i.g. What does a change in DNA methylation reflect? Hypermethylation versus hypomethylation. demethylated) within each developing germ cell (primordial germ cells) and then remethylated and reset to the genomic DNA methylation patterns specific to either the sperm or the egg as gametogenesis proceeds (81. a recent study of imprinting in embryonic and adult mouse brains found parent-of-origin bias in the expression of 1300 genes and transcript isoforms and. Thus. However.88).or paternal-specific methylation patterns before fertilization (although some parent-of-origin methylation patterns may also continue to develop post-zygotically) (Supplemental Fig. the level of methylation at a given CpG site in these assays reflects the collective methylation at this site in multiple cells (and multiple DNA molecules) and the variation in methylation seen at individual CpG sites.g. or 100% (symmetrically methylated on both chromosomes). most commonly cytidine analogs such 5-azadeoxycytidine. It is also possible to alter DNA methylation levels and patterns within intact mammalian cells by treatment with various chemical inhibitors. In early embryogenesis. and only a subset of these are shared with the mouse (78. the methylation level of a given CpG site on 1 strand of DNA can be 0% (symmetrically unmethylated on both chromosomes). The different functional consequences of these 3 different scenarios are important. would suggest that imprinting (or parent-of-origin effects) may be more widespread and complex than originally believed (80). This process is particularly important for establishing the gamete-specific methylation patterns of imprinted genes in sperm and egg before fertilization. even if these changes in methylation occur in only a subset of cells of a given tissue. if confirmed. When considering the implications of changes in the percentage of methylation at a specific CpG site (e. as often seen in DMRs of imprinted genes). therefore. thereby leading to an increased risk of diseases later in life. However. DNA methylation patterns undergo remodeling during the course of mammalian differentiation and development to eventually generate the cell type–specific methylation patterns found in adult somatic cells. Dynamic changes in methylation are particularly pronounced during gametogenesis and early embryogenesis (Supplemental Fig. within a single diploid cell. Current data suggest that other nonimprinted loci may also be subject to at least partial erasure. The differential parent-of-origin DNA methylation patterns at imprinted genes are established during gametogenesis where DNA of the male and female gametes acquire either maternal. In small studies of human embryos.e.so-called differentially methylated regions (DMRs) are generally (but not always) hypermethylated on the transcriptionally silenced allele and hypomethylated on the expressed allele. DNA methylation patterns also undergo a genome-wide process of erasure. Any CpG site in a single DNA molecule can either be methylated or unmethylated. that this site is unmethylated in 50% of the cells in the population and fully methylated in the other 50% of cells.82). and resetting of DNA methylation patterns during gametogenesis (83– 85). These differential gametic methylation imprints are then maintained and propagated during subsequent somatic development to facilitate the parent-of-origin patterns of monoallelic transcription associated with imprinted genes. it is important to consider Folate DNA methylation 27 . These periods of dynamic reprogramming of DNA methylation patterns during gametogenesis and embryogenesis may also present windows of opportunity for environmental influences (e. in response to dietary conditions). This wave of demethylation erases the methylation patterns of the gametes and is followed by DNA remethylation during the remaining course of embryogenesis to establish the genome-wide and cell-type specific DNA methylation patterns found in fully differentiated adult somatic cells (44. DNA methylation assays are usually performed on a population of cells. resulting in hypomethylation of the genome in treated cells. Such changes in DNA methylation patterns could affect normal patterns of gene expression or alter genome stability.. An important exception to this dynamic reprogramming of DNA methylation patterns during embryogenesis occurs with imprinted genes that escape this process of erasure and resetting. 5-Azadeoxycytidine (and its ribonucleotide analog 5-azacytidine) functions as a demethylating agent (9).

1). Methionine is the substrate for SAM or AdoMet. In some cases. Folic acid is normally first reduced to dihydrofolate by dihydrofolate reductase and subsequently to THF to enter the folate pool (Fig. such that uracil may be substituted in the DNA sequence (Fig. after which it behaves identically to natural dietary folate. therefore. Low folate status (as defined by various measures including blood folate concentrations. MTHFR activity and thus 5-methylTHF formation may also be modified by the common genetic variant. and neural tube defects (103–105). Under the condition of high SAM concentration. functions under tight regulatory controls. During DNA replication. ensuring normal 28 Crider et al. the capacity of dihydrofolate reductase is exceeded and folic acid may appear in the circulation in the oxidized form (94). and other small molecules. The 1-carbon pathway. site-specific methylation can result in gene silencing if the methylated CpG blocks the binding of a DNA methylation-sensitive transcription factor. In addition to folate. Once the THF coenzyme is formed from either folic acid or dietary folate. folate intake. In the case of CpG islands. absorbed folate is metabolized to 5-methyltetrahydrofolate (5-methylTHF. SAM formation. Conversely. When folic acid is consumed in fortified foods or supplements. For this reason. Moderate elevations in plasma homocysteine concentration have been reported to be associated with increased concentration of SAH. Inadequate folate availability during cell division can result in the compromised production of thymidine. In tissue culture.108) (Fig. it is first converted to 5. gene silencing is generally associated with multiple methylated CpG sites across a strand of DNA (Fig. monoglutamyl form) in the intestine and/or liver. RNA. A number of SAM-dependent reactions have regulatory roles by affecting both genome stability and gene transcription (55). which then must be polyglutamated for cellular retention and 1 carbon cycle coenzyme function.that a 10% change in DNA methylation level at a site could be distributed across an entire population of cells (and not appreciably affect transcription). 5-MethylTHF is the primary folate constituent taken up by nonhepatic tissues. w5% of genes were found to be hypermethylated in nonrandom patterns specific to the type of tumor (109). . These nutrients include vitamin B-6 (serine hydroxymethyltransferase activity).111). which reduces the synthesis of 5-methylTHF and hence remethylation of homocysteine. Early in the study of DNA methylation. it is important to consider the exact location assayed and the potential of methylation at that site or region to change gene expression or other biological processes of interest.23. 1). including histones (95). SAM is the major regulator of folate-dependent homocysteine remethylation because it is a potent inhibitor of MTHFR. and folate. It was determined that these changes in DNA methylation in tumors resulted in silencing of tumor suppressor genes and chromosome instability (Fig. At most genetic loci. The mechanisms by which low folate status contributes to these disorders remain unclear. and choline (betaine precursor as a hepatic methyl source via betaine:homocysteine methyltransferase) (98). 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. It is also recognized that S-adenosylhomocysteine (SAH) functions as a potent product inhibitor of SAM-dependent methyltransferases (100). and increased SAH concentration has been associated with global DNA hypomethylation (102). a cofactor and methyl group donor for numerous methylation reactions including the methylation of DNA. or something in between. and/or folic acid intake) has been associated with an increased risk of cardiovascular disease. 2B). Under normal dietary conditions. vitamin B-12 (methionine synthase function). 5-methylTHF must be converted to THF via the methionine synthase reaction (Fig. Part II: Folate and DNA methylation Folate’s role in 1-carbon metabolism related to DNA methylation. DNA methylation. When evaluating the impact of changes in DNA methylation. it has been shown that low folic acid results in the formation of micronuclei (indicative of chromosome breakage) and that the MTHFR TT genotype leads to increased micronuclei formation under low folate conditions (106). and DNA methylation. However. Additionally. 1). folate depletion can have destabilizing consequences. 2B) (110. it is metabolized primarily to 5-methylTHF during intestinal absorption and/or first pass in the liver. continual hydrolysis of SAH to homocysteine is essential to maintain normal DNA methylation (101). but not SAM.58. DNA methylation patterns are widely dysregulated in human cancer (Fig. 2B). localization of protein (96). 677C/T. 2). Low folate status and DNA methylation. which reduces enzyme activity (99). homocysteine remethylation. and proteins. multiple cancers. genome-wide hypomethylation was found in tumor tissues compared with matched healthy tissues (58). cancer. Global hypomethylation and targeted hypermethylation are considered defining characteristics of human tumors (14. phospholipids. when SAM concentrations are low. MTHFR is inhibited. Tetrahydrofolate (THF) is the most effective substrate for polyglutamate synthetase. it is unknown which CpG sites or how many along the strand of DNA must be methylated to silence any given gene.107. This mutagenic event may trigger attempts to repair the defect and increases the frequency of chromosomal breaks (104). riboflavin (MTHFR stability).10-methyleneTHF by the vitamin B-6–dependent enzyme serine hydroxymethyltransferase and subsequently irreversibly reduced to 5-methylTHF by methylenetetrahydrofolate reductase (MTHFR). Each of these potential scenarios has different implications for the impact of the same 10% change in methylation level. reflect a 100% change in a subpopulation of cells (and represent gene silencing or activation in these cells). and small molecule degradation (97). remethylation of homocysteine is favored. and thus DNA methylation. a number of other dietary nutrients are required to maintain 1 carbon flux. neurotransmitters.

there was not a consistent association between global DNA methylation and folate status (intake or blood folate) across studies (121–125). There are many techniques available to assess “global” DNA methylation level including those that look at repetitive element methylation level LINE. and types of tissues examined. 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..g. resulting in global hypomethylation.Hypomethylation of repetitive elements may be predictive of increased risk of cancer and increased mortality from cancer (112).2–2620.g. As tumors increase in severity.122. Folic acid intake has been reported to prevent loss of heterozygosity of a tumor suppressor gene in a small supplementation trial in humans (115).0% methylated P-trend = 0. (49) evaluated DNA methylation levels at 2 CpG island–containing genes in colon tissue of persons with a previous adenoma after a placebo-controlled folic acid supplementation trial (1 mg/d for 3 y). cohort and case-control) of the association of folate status (measured as folate/folic acid intake and or blood folate concentration) and cancer.9 mg/L) vs. 5 studies examined the global DNA methylation level (121–125) (Supplemental Table 1). and it was limited to 2 loci (49). This assay provides an estimate of global DNA methylation by using an enzyme to methylate purified genomic DNA with radiolabeled tritium. The magnitude of DNA methylation changes observed in cancer tissues is substantially greater than any differences in DNA methylation between types of normal tissue (14). Additionally.117) but also targeted hypermethylation of the H-cadherin locus (117). In recent large-scale DNA methylation studies of multiple types of primary tumors (thousands of loci in hundreds of tumors). Indeed.3% vs.3% methylated. In tumors. highest RBC folate quintile (Q5 1081. or unmethylated (<20% methylated) depending on the exact location of the CpG sites (130). Methyl acceptance is limited in precision and necessitates intact DNA (129) so that the use of damaged DNA (which may occur as a result of the outcome of interest or during preparation or handling) will result in inaccurate results. however.8). SINE. only 1 trial examined the effect of folic acid supplementation (1 mg/d for 3 y) on sitespecific DNA methylation. intake.2% methylated vs. global hypomethylation) as rapidly dividing tissue tumors may be susceptible to low folate availability..3–520. In these studies. Three clinical trials examined the impact of folic acid supplementation (0. The association of global hypomethylation with folate status (e. There was no association of DNA methylation at either of these loci Folate DNA methylation 29 . The changes in DNA methylation observed in association with variation in red blood cell (RBC) folate concentration in humans are much smaller than those present in tumors. 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. 11. There has been great interest in reversing epigenetic changes seen in early tumorigenesis (e. Q5.119).126).4–10 mg/d) on global DNA methylation in colon cancer patients (30.01].114).120). the functional impact of these small changes in DNA methylation (<2%) on gene transcription or longer term health outcomes is unclear at this time. DNA methylation changes in tumors were associated with concomitant changes in gene expression and tumor characteristics (113. Among the observational studies (e. Studies of cancer patients and folate status and global DNA methylation. 10.03 and SFRP1: Q1 vs. Careful consideration of assay methodology is critical for study design to ensure appropriate interpretation and reproducibility of the data. 23. The DNA methylation changes in cancer do not appear to be random because tumors of specific cell lineages show similar DNA methylation changes that are distinct from other tumor types and normal tissues.119) (Supplemental Table 1). However.118. each of the early trials of global DNA methylation in colon cancer patients used the methyl acceptance assay (30. They found a trend of greater methylation at each genetic locus as the quintile of RBC folate increased [ERa: lowest RBC folate quintile (Q1 43.118. 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. This is not unexpected given the variety of study designs.g. This is not necessarily due to the accuracy of the measure but due to the fact that although they can be thought of as global. 21. recent work suggests that there are different changes in DNA methylation between types of tumors and types of repetitive DNA elements often used as proxies for global methylation (128). P-trend = 0. especially colon cancer (Supplemental Table 1). and Alu and those that examine various restriction sites throughout the genome. they are actually measuring different types and subsets of DNA sequences in disparate regions of the genome that may be regulated differently. different global DNA methylation assays. global hypomethylation was found in tumors and even normal colon cells and blood of cancer patients (121. blood folate concentration) has been inconsistent among studies of cancer patients (Supplemental Table 1). Folate depletion of human tissue culture cells can result not only in the expected global hypomethylation (116.. a progressive number of loci show increased DNA methylation primarily at CpG islands and decreased DNA methylation in non-CpG islands sites (14). Although statistically significant. the correlation between these measures is actually quite low (127). Across studies outlined in Supplemental Table 1. Wallace et al. For patients with a previous adenoma of the colon. methylation of tumor suppressor CpG islands is generally either fully methylated and silenced (>80% methylated). Studies of cancer patients and folate status and sitespecific DNA methylation. an association was found between cancer and global DNA hypomethylation in circulating blood and/or tumor tissue.124. to date.

double-crossover study.with treatment with 1 mg/d folic acid for 3 y. there were no statistically significant associations of methylation at either of these loci with hyperplasic polyps or adenomas after 3 y of treatment. In mice it is clear that maternal dietary changes in 1carbon availability can affect the DNA methylation patterns and phenotype of offspring (87). genotypes. DNA methylation varies between the sexes and changes during aging (47–50). (138) did not find DNA methylation changes in response to depletion or repletion. ERb.g. In this study. the studies highlighted in Supplemental Table 1 show a number of loci that warrant examination in future studies.139) (Supplemental Table 1). and particulate pollution (141) have been linked to changes in DNA methylation (e. In healthy adults. Thus. 1C).7 mg/L vs. resulting in misregulation of this complex system. This may be because dividing tissues are susceptible to the same insults or that the change that resulted in the predisposition to cancer happened in the embryonic period and is reflected in many tissues (Supplemental Fig. whereas no changes in methylation were found at the ERa. In humans. and race were strongly associated with DNA methylation at these loci. global hypomethylation was reversed and monoallelic expression of imprinted genes restored in patients given folic acid therapy (140) (Supplemental Table 1).124). these findings suggest that there may be a differential global DNA methylation response to folate depletion and repletion dependent on age. Although the results from the controlled feeding trials of healthy adults are somewhat inconsistent.144. Some of these changes appear to precede transformation (initiation of cancer). The best-studied example is the agouti mouse. or dietary folate intake. these studies are limited by small sample size (n = 8–33 individuals per 30 Crider et al. and these changes may be modulated by environmental exposures. however. (126) observed a trend toward lower DNA methylation with lower serum folate concentration. repetitive element hypomethylation) in adults. In addition. and magnitude of exposure. Taken together. Changes in DNA methylation in response to the environment and diet: the importance of the developmental timing of exposure. This type of study examining thousands of loci simultaneously will likely produce a better understanding of the interaction between DNA methylation and folate. (132) examined DNA methylation at 3 genetic loci and found that high plasma folate concentration among cases ($6. an increasing number of environmental exposures in humans such as air pollution (141). None of these studies of healthy adults examined site-specific DNA methylation changes. duration.. Both Jacob et al. < 4. It is important to note that low folate status can lead to both hypo.132). and others may be a consequence of transformation. Studies of cancers have found that there are changes in DNA methylation in the blood that reflect the cancer risk in the tissue of interest (119. certain environmental exposures have been associated with changes in DNA methylation and changes in 1-carbon metabolites. (136). plasma folate concentration. protein intake. and p16 genes. There is increasing evidence of more interindividual variation and variation in DNA methylation patterns across the life span than was previously expected. the study was restricted to those with the MTHFR CC genotype. (139) found an MTHFR genotype-dependent association between lower global DNA methylation and lower plasma folate concentration. dividing tissues such as the blood and the gut mucosa are likely to be most susceptible to low folate in the diet. In the Shelnutt et al. There has been increasing interest in in utero effects of environmental exposures (from toxins to nutrition) on long-term health outcomes. group). (137) observed considerable increases in methylation with folate intake during the repletion period. (131) recently examined a cohort of women with breast cancer and the DNA methylation level at 1413 sites in 733 genes. different initial folate status. Friso et al. Large-scale genome-wide analysis may provide additional candidates. the increases were limited to those with the MTHFR TT genotype (see Fig. Two additional observational studies of cancer examined site-specific DNA methylation and folate status (131. (143) found that estrogen replacement resulted in lowering of plasma homocysteine concentration and increases in global methylation in a small (n = 13 postmenopausal women) double-blind. placebo-controlled.134). and it has been hypothesized that disease risk may in part be determined by maternal and paternal diet (87.and hypermethylation. it is unclear whether subsets of genetic loci are susceptible to DNA methylation changes in response to folate/folic acid intake in cancer patients.145). They found that dietary folate intake was associated with DNA methylation class membership in primary breast tumors (131). 1 for location in the folic acid cycle). and varying lengths of intervention. in a study of men with hyperhomocystinemia. benzene exposure (142). Folate depletion resulted in reductions in global DNA methylation in older women in controlled feeding studies (135. Kim et al. Although age.136). Friso et al. and Shelnutt et al. The changes observed in cancer do not appear to be random such that not all CpG islands are susceptible to hypermethylation (133. (137) study. The agouti mouse strain has an insertion of an intracisternal A particle (IAP) sequence in an upstream . Furthermore. a finding that was not observed by Shelnutt et al.1 mg/L) was associated with an increased methylation at p73 but not p16in4A or hMLH1. differences in age groups. (137) in younger female adults. Studies of healthy adults and folate status. At this time. Pufulete et al. although substantial analytic and informatics challenges exist. 3 studies examined the effects of controlled folate depletion and repletion on global DNA methylation in leukocytes (135–137) (Supplemental Table 1). Christensen et al. Axume et al. Two additional observational studies examined global DNA methylation and folate status in patients without cancer (126. however.

Interestingly. Thus.5% vs. 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). Recently. counteract the epigenetic effects of these environmental conditions on offspring.5% increase in IGF2 DMR methylation (49.014) in children.148). site-specific methylation (27. This would suggest the potential for a complex interplay between the effects of maternal and paternal environments and diets on the epigenome of offspring. The association between maternal use of folic acid and DNA methylation in offspring was examined in 1 study. In both of these studies. In rats. with altered expression of genes involved in lipid and cholesterol biosynthesis.7% methylation per SD birth weight. The agouti gene in this strain is now regulated by the promoter activity of the IAP. Waterland et al. Tobi et al. Steegers-Theunissen et al. early development could be particularly susceptible to folate intake. Heijmans et al. Although highly significant.578 genetic loci) (163). and GNASAS. This increase in methylation in response to the low-protein diet was prevented by dietary supplementation of the lowprotein diet with folic acid (151). (161) compared the DNA methylation level in various tissues from terminated fetuses (18–28 wk) affected by NTDs with matched normal controls. (160) found that maternal use of folic acid was associated with a 4. depending on the precise timing of the exposure and the sex of the fetus (153–158). and both IGF2 and H19 (165) Folate DNA methylation 31 . metabolic syndromes.region of the agouti gene that encodes a paracrine signaling molecule. in some cases. and increased risk of schizophrenia. compared with control offspring of fathers fed a normal protein diet (149). In the agouti mouse strain. cord blood. ABCA1. Epigenetic effects of other dietary components are of interest as well. IGF2 DMR methylation in the child was associated with maternal SAM level and inversely with birth weight (21. 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. 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. the identity of the causative agent for changes in DNA methylation is unclear because there were a pool of methyl donors supplied in the agouti mouse model. the absolute changes observed in these studies were a <3% change in the DNA methylation level of loci that are normally unmethylated. (153) examined 15 loci important for growth and metabolic disease and found that methylation was lower at INSIGF and higher at IL10. (159) found methylation changes in IGF2 associated with prenatal exposure to prenatal famine. 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). LEP. There was hypomethylation in the brain of fetuses with NTDs compared with controls (P < 0.034). Studies in fetus. infants. Increasing the availability of methyl donors in the mother’s diet during pregnancy resulted in increased DNA methylation of the IAP and phenotypic changes associated with altered regulation of the agouti gene in offspring (146. changes in DNA methylation at the agouti locus in response to BPA exposure in utero (via maternal dietary consumption of BPA during pregnancy) were negated by supplementation of the BPA-containing diet with folic acid (152). LEP. (148) found loci in the human genome that also may act as metastable epialleles (similar to the agouti locus in the agouti mouse) that show differential methylation in response to season of birth in a small human study. epigenetic effects of certain environmental conditions in utero appear to be abrogated by dietary folic acid supplementation. P = 0. In addition to the lower mean serum folate concentration in mothers with NTD-affected pregnancies. IGF2 promoter methylation (164). certain environmental conditions and diets of parents may have epigenetic effects on their offspring and dietary folic acid can.4%. In men only LEP was significantly associated with later exposures. It is unknown whether these DNA methylation changes would result in functionally relevant transcriptional changes. However. and MEG3 in persons exposed to famine periconceptionally compared with their sex-matched siblings (all P < 0. Exposure to famine at later gestational ages resulted only in changes to GNASAS (P < 0. 47. 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. A recent study by Chang et al. a correlation between a woman’s serum folate concentration and DNA methylation in the brain tissue of NTD-affected fetuses was found (161) (Supplemental Table 1). and folate status. Extreme exposures can be used as proof-of-principle to show plausibility of a cause-and-effect relationship. An interaction with sex was found for INSIGF. Three recent studies examined the relationship between maternal folic acid supplementation in cord blood on methylation of the LINE-1 repetitive element (162). A similar outcome was reported for prenatal exposure to the widespread environmental contaminant bisphenol A (BPA). P = 0. There were differences in the level and patterns of global DNA methylation between the NTD-affected and control fetuses.01). it is not clear which macronutrient or micronutrient deficiency or combination thereof may be responsible for these health outcomes. a maternal lowprotein diet has been reported to increase DNA methylation of the imprinting control region of the imprinted Igf2/H19 domain in the livers of offspring compared with control offspring whose mothers were fed a normal protein diet. 1B).001). and multiple dietary factors were altered in the small human study.001). GNASAS. It has been hypothesized that epigenetic changes as a result of starvation exposure may be responsible for these effects later in life. Paternal high-fat diets were reported to reduce DNA methylation at the Il13ra2 gene in mouse pancreatic islet cells of female offspring (150).

In the very limited number of studies to date. region of the genome. these relationships are complex and can involve other components of the diet and/or substrates from 1-carbon metabolism.(Supplemental Table 1). (164) found that maternal and cord blood vitamin B-12 concentrations were associated with P3 (third promoter CpG site) methylation (both P < 0. 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. In addition. Even in experimental animal models that show changes in DNA methylation after increased intake of 1-carbon sources. indeed the conflicting results presented in Supplemental Table 1 illustrate the complexity of the process. It remains an open question whether folate/folic acid intake is associated with changes in DNA methylation and whether there is an interaction with other micronutrients or genetic variation. At this time.specific DNA methylation (162–164).019) (163). .01). Additionally. DNA methylation is a highly regulated process dependent on time. Part III: Future research Although several studies have begun to examine the relationship between DNA methylation and folate (Supplemental Table 1). randomized. betaine. tissue to test. P2 was also associated with maternal weight gain and exposure to smoking (P = 0. However. specific tissue type. DNA sequence. These studies showed no association of folate status (as defined as serum folate concentration or folic acid intake) with global or site. the folate/folic acid exposure of interest. (165) examined the DNA methylation level in the cord blood of newly delivered infants at the H19 DMR and found significantly decreased DNA methylation level (toward the expected 50% level) with increasing maternal folic acid intake in pregnancy.157.001) (162).168.02). controlled trial has examined the effects of folic acid on DNA methylation levels and patterns in healthy humans. many studies are underpowered in terms of the number of participants.688. there was an association of increasing maternal plasma homocysteine concentration and decreasing percentage of methylation of LINE-1 (r = 20.038). No population-based.169). Measurement of DNA methylation is not indicative of a single process or phenomenon. The purpose of many of the controlled feeding trials was to attempt to reverse the aberrant DNA methylation observed in cancer. a fourth study by Hoyo et al.131. suggesting to Hoyo et al. there is no direct evidence of aberrant DNA methylation and change in gene expression in response to “high” levels of folate/folic acid intake. (165) found an association between increased folic acid intake and decreased DNA methylation at H19. 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. there are insufficient data to determine whether there is an effect of higher doses of folic acid at any particular locus. and number of epigenetic loci examined. the previously held dogma surrounding aspects of DNA methylation and folate status may require modification.028). suggesting no definitive linear associations of folate/folic acid intake in the mother with increases in DNA methylation in the child. As previously discussed. it is unknown whether the effects are due to folic acid or one of the other 1-carbon sources (e. Additionally. Because folate is not the only vitamin in the 1carbon cycle and DNA methylation is not the only methylation reaction that may affect health. DNA methylation patterning.125). methylation and silencing of CpG island–associated tumor suppressor genes observed in cancer among adults (49) or children (160). that folic acid intake in a fortified population may provide additional benefit. studies designed to evaluate interactions are needed. P = 0. Enumerable questions about the relationship between folate status and DNA methylation need further exploration and include the following: What is the normal variation in DNA methylation level and pattern across the genome and between tissues and developmental stages? High folate and folic acid intake and DNA methylation. Additionally. However.167). genomic region. LINE-1 methylation (P = 0.166.170. folic acid supplement use was not associated with the complete 32 Crider et al. Ba et al. However..114. In planning new studies.03 and P = 0. and birth weight percentiles (P = 0. a recent study by Hoyo et al. recent focus has shifted to concern about folic acid supplementation resulting in progression of existing tumors and altering normal DNA methylation patterns (31. Even in tumors. Currently.171). loci susceptible to hypermethylation vary among tumor types (108. As technology enables the greater use of genome-wide analyses of DNA methylation at single nucleotide resolution. tissues types. and significant correlation between methylation patterns (cluster in the modeling of the site-specific methylation) and plasma homocysteine concentration (P = 0. None of the findings associating folate status (indicated by folate/folic acid intake or blood folate concentration) with changes in global or site-specific DNA methylation have been consistently replicated. and a multitude of other regulated enzymes and proteins. Different regions of the genome are differentially regulated and would not be expected to respond similarly to any given exposure. or developmental state and whether the change would result in increased risk or benefit.g.113. These studies show the possibility that folate/folic acid in the maternal diet could change DNA methylation levels in the offspring. and the timing of the folate/folic acid exposure (Table 2). it is important to consider the appropriate assay. Simple correlations. there is no consensus on a dose of folic acid or a blood folate concentration that would be associated with potential adverse health outcomes. tissue. type of DNA methylation change anticipated. are unlikely to apply broadly. such as high folate status leading to increases in DNA methylation. choline) or a combination thereof (147). Promoter regions of genes are highly regulated and CpG islands in promoters generally remain protected from DNA methylation under normal circumstances.

tissue tested. assays. timing of exposure. and amount of folate/folic acid intake. and. the evidence suggests that low folate status is Folate DNA methylation 33 . not surprisingly. The human studies of DNA methylation and folate/ folic intake acid vary widely in their study design. including cancer. imprinting disorders. and developmental disabilities (172– 174). the findings.Are there systematic differences in DNA methylation between different populations that vary with micronutrient intake? Are there health consequences due to the small changes in DNA methylation levels/patterns observed in many of the existing studies? How does folate status interact with other nutrients to affect DNA methylation level/ patterning and does this vary depending on the developmental timing or tissue assayed? How does DNA methylation vary in relation to timing and dose of folate or specifically folic acid intake? Conclusions Appropriate DNA methylation patterns are critical to normal genome function. Aberrant DNA methylation patterns are present in many human diseases. At this time.

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