By Traci Pantuso ND, MS,
Bastyr University, Seattle, Washington Dr.
Pantuso reports no financial relationships relevant to this field of study.
A popular field that is being discussed in medical research, in popular science, and by the media, epigenetics is broadly defined as the study of the changes in gene expression that occur on the DNA sequence and are heritable. These changes, or epigenetic marks, occur as modifications on the DNA sequence itself or to associated proteins that affect the transcription of the genome during normal development and in response to environmental factors.1,2 Part of the excitement of epigenetics is that it followed in the steps of genome-wide association studies (GWAS). There have been numerous GWAS investigating the relationships between DNA variants and diseases.2 The results of these GWAS have not been very fruitful in identifying gene variants as the causes of disease; however, epigenetic studies are yielding more results along these lines.2
Epigenetic marks have been demonstrated to be involved in a wide variety of biological processes from X-chromosome inactivation and imprinting to the state of health and disease in humans.3 With epigenetic regulation being involved in diabetes, cancer, cardiovascular disease (CVD), schizophrenia, autism, and numerous other illnesses, it is no wonder that it is an important field and a "hot topic.2-6
It has long been recognized that there is an association between fetal nutrition and chronic diseases in later life, and epigenetics is beginning to shed some light on this relationship. Epigenetic mechanisms also have been able to demonstrate effects of the environment (including diet) on gene expression in animal models and humans.4
Due to the rapidly expanding field of epigenetics, information overload is a common phenomenon. A basic understanding of both the history of the field and the mechanisms of regulation are required to fully understand the magnitude of the discoveries in epigenetics.
This article will review fundamental epigenetic concepts, relevant research (especially regarding nutrition), and the implications for the field of integrative medicine.
There is confusion in the field of epigenetics, both from the definition of epigenetics itself and from the sheer volume of data. The term epigenetics traces back to the 1940s when C.H. Waddington, a British embryologist, first used the word to describe how a cell is able to maintain the same observable characteristics or phenotype through cell division.7 It is interesting to note that DNA was not identified as the transforming particle until 1943, through the Avery-Macleod-McCarty experiment, and was not discovered to be a hereditary unit until the Hershey-Chase experiment in 1952.8 Waddington's term epigenetics was not widely used until the discovery of DNA methylation in 1975 by Holliday and Pugh.9 Holliday and Pugh proposed that DNA methylation was a molecular mechanism that explained Waddington's epigenetic concept.
The explosion of studies focusing on epigenetic regulation appears to have started in the early 2000s. Prior to that time, there were less than 100 published articles with epigenetics in the title, compared to 1300 articles by 2010.9 Much of this increase in epigenetic research occurred with the advent of Next Generation Sequencing, which allows for more samples to be sequenced in a shorter period of time at a decreased cost. The first commercial device was available in 2005.2
Currently, epigenetics encompasses the mechanisms of DNA methylation, histone modifications, and regulation by noncoding RNAs in DNA transcription. Researchers are using these epigenetic mechanisms, or marks, to understand the effects of both the fetal and adult environment on gene expression. The most discussed and researched type of epigenetic mark is known as DNA methylation.10 Although DNA methylation is the classic example of epigenetics, many other post-translational mechanisms regulate transcription, such as modifications to the histone proteins that keep the DNA tightly wound into chromatin and noncoding RNAs.
DNA methylation is the process where methyl groups are added to a cytosine preceding a guanine at the 5' end of the DNA; these are called CpG sites. The methyl groups are covalently attached to the cytosine by the enzyme DNA methyltransferase (DNMT) from the methyl donor SAM. Although a number of enzymes are involved in DNA methylation, DNMTs are the best studied. CpG islands are regions of DNA that are rich in CpG sites and occur near gene promoters. Currently, it is understood that methylations at CpG islands will turn off gene transcription by preventing the transcription machinery from binding to the promoter region of the gene.10
Between 70-80% of the cytosines in DNA are methylated in the healthy human genome. Changes in the percentages of DNA methylation are associated with both health and disease.2 Decreased methylation of the genome (hypomethylation) is associated with increasing age and increased cancer risk.11,12 However, there is also evidence to suggest hypermethylation at tumor suppressor gene promoter sites also may lead to increased carcinogenesis. It is becoming more clear that the particular gene promoter affected by the methylation status may be more important than the global methylation status of the genome. There is controversy over whether there is an enzyme that actively removes methyl groups from the DNA or if hypomethylation is the result of decreased methyl donors or decreased activity of DNMTs when DNA replication occurs. 10,11
The preeminent studies demonstrating the importance of DNA methylation were performed in mice; these studies demonstrated that offspring from mothers supplemented with methyl donors were brown in color and lean (Agouti phenotype) compared to non-supplemented mothers who produced offspring that were yellow in color, obese, and had increased incidence of diabetes and tumors.11
DNA is tightly packed into the nucleus as chromatin, which consists of 147 base pairs of DNA wrapped around a histone octamer. When DNA transcription occurs, the histones have to open to allow access to the DNA. Modifications are made to histone proteins to control transcription through phosphorylation, acetylation, sumoylation, methylation, ubiquitylation, prolyl-isomeryzation, and ADP-ribosylation, and these enzymes rely on a number of cofactors and metabolites (see Table 1).13,14 In general, increased histone tail acetylation signifies areas of active transcription while decreased acetylation is associated with less activity.14 In contrast to DNA methylation, histone tail methylation can signify increased or decreased transcription. Due to the number of different modifications that can occur on histones regulating DNA transcription, a lot of research still remains to be done to determine clinical relevance.
|Table 1. Epigenetic Modification and Sources of Cofactors 13,14|
|Modification||Cofactor Required||Sources of Cofactors|
|DNA methylation||SAM||Folate, choline, B-group vitamins, methionine|
|5-methyl-cytosine oxidation||Oxygen á-ketoglutarate, Fe(II)||Fe(II) Glutamic acid, Fe|
|Histone methylation||SAM||Folate, choline, B-group vitamins|
|Histone demethylation||Oxygen, á-ketoglutarate, Fe(II) FADH||Glutamic acid, Fe, vitamin B2|
|Histone O-linked glycosylation||UDP-GlcNAc|
|Histone deacetylation||NAD+||Dietary tryptophan, niacin|
|Histone ADP-ribosylation||NAD+||Dietary tryptophan, niacin|
|Histone isomerization||None identified|
Noncoding RNAs (ncRNA) are characterized by size as micro-RNAs, piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNA), and long ncRNAs (lncRNAs), and have numerous regulatory functions in transcription. This is a broad topic beyond the scope of this review article.2,15
The communication between all of these epigenetic pathways is still being elucidated. For example, the enzymes that catalyze histone and DNA methylation are supplied with methyl donors by related pathways and may be in competition.14 In addition, there are data supporting that modifications to histones may increase DNMT enzyme activity, further repressing transcription. Relevant to the dietary and supplement effects on these interactions, there is active in vitro cell research investigating dietary compounds involved in histone modifications.4,16
Epigenetic research relevant to the field of integrative medicine is mostly centered on the effects of the environment on DNA expression. One example is the effect of diet on gene expression. One hypothesis is that DNA methylation patterns are established early in development but that alterations, such as from diet, lead to diseases in adulthood; this has actually been documented in animal and human models. One interesting observation is that identical twins have similar epigenetic patterns in childhood, but DNA methylation and histone acetylation differences are noted in later life.2,4,17
Fetal nutrition. The concept of the fetal nutritional environment having long-term effects on the adult is now being referred to as the Developmental Origins of Health and Disease Hypothesis, which is based on the pivotal work of a few research groups that demonstrated low birth weight is associated with CVD, insulin resistance, and hypertension.17 Other epidemiological studies also have demonstrated the link between low birth weight and CVD, insulin resistance, cancer, obesity, and some behavioral disorders.2,4,17 There may be an epigenetic basis for these findings.
The Dutch Cohort Study is one of the classic research trials supporting the concept that epigenetic changes occurring during fetal development are responsible for long-term health and disease. The Dutch Cohort study investigated the effects of a 6-month famine (Dutch Hunger Winter) on pregnant mothers and fetuses who suffered malnutrition from the World War II Nazi blockade. The adult children of mothers pregnant during this episode show increased incidence of CVD, metabolic disorders, obesity, and breast cancer, as well as alterations in epigenetic marks. It is widely accepted that these genetic changes and diseases were the result of nutritional imbalances in utero. Specifically, the Dutch Cohort Study demonstrated lower methylation status of genes involved in growth, metabolism, and CVD; some of the genes involved are IGF2, IL10, GNAsAS, INSIGF, LEP, ABCA1, and MEG3.18
According to other research, dietary factors influence the epigenome during both "critical windows" and "dietary transitions." Critical windows are identified as periods of fetal or neonatal development, while dietary transitions occur over longer time periods in adults.4
A number of different nutrients, including folate, choline, betaine, and vitamins B2, B6 and B12, have been shown to be important in epigenetic regulatory pathways. The most well-studied nutrient is folate, which has a role in the one-carbon metabolism pathway. Vitamins B2, B6, and B12 are used as cofactors in the one-carbon metabolism, which maintains the cellular methyl donor SAM used in DNA methylation and histone methylation.17-19 Many of the enzymes responsible for the epigenetic marks in DNA methylation, histone modification, and noncoding RNAs also are required as dietary cofactors (see Table 1). Also, in vitro cell culture studies have demonstrated that botanical extracts and phytochemicals have epigenetic effects on DNA transcription.4,17
Folate. The folate story is both fascinating and complicated. Some data show that DNA hypomethylation may be the result of low folate status and is associated with increased cancer risk; this points to the importance of dietary folate. Also, well known is the necessity of folate during pregnancy to prevent neural tube defects. Futhermore, folate is required to prevent homocysteinemia, a complex mechanism of action. Briefly, folate's role in homocysteine metabolism starts with the reduction of folate to tetrahydrofolate (THF), then a conversion to 5,10-methylene THF, and on to 5-methyl THF via the enzyme methyltetrahydrofolate reductase (encoded by the MTHFR gene). Methyl groups from 5-methyl THF are dontated to homocysteine, which coverts it to methionine in the one-carbon metabolism pathway. The one-carbon pathway uses the cofactors vitamins B2, B6, and B12 as mentioned above. With the discovery of the MTHFR gene and its numerous variants, which have varying degrees of methyltetrahydrofolate reductase activity, methylated folate has become a popular supplement. However, the effects on specific gene expression of these supplements or additional folate supplementation in folate-replete populations are unclear and require further investigation.18-21 Hopefully, further research will identify how and why a particular gene promoter is selected to be methylated. Until then, many experts caution about supplementing with too much folate.
Choline and betaine. Dietary choline is oxidized to betaine, which is a methyl group donor in the conversion of homocysteine to methionine. Animal studies have demonstrated a complex relationship between choline/betaine supplementation; there appears to be methylation that occurs at specific gene regions compared to non-supplementation.4
Amino acids. Methionine is the precursor to SAM and is one of the essential amino acids, in that it is required through dietary intake. Data suggest that methionine supplementation causes hypermethylation at specific gene regions. Research studies on methionine supplementation demonstrate that there is an alteration in the one-carbon metabolism; however, it is unclear how methionine effects DNA methylation. Diets low in homocysteine, which is the precursor to methionine, may lead to hypomethylation.2
Botanicals. In vitro cell line studies investigating the effects of Allium tuberosum L. and Artemisia dracunuculus L. reduced DNMT expression.4,16 Epigallocatechin-3-gallate (EGCG), found in green tea and soybeans, inhibits DNMT activity in a cell line. Genistein also has been found to have effects on DNA methylation in mice; however, more research is needed to further unravel the effects on the epigenome.
IMPLICATIONS FOR THE FIELD OF INTEGRATIVE MEDICINE
Epigenetic research is still expanding and much research needs to occur to detail the interconnectedness of epigenetic regulation. Despite the promise of being able to theoretically manipulate epigenetic mechanisms to treat disease in humans, there are still many unanswered questions. For example, how do individual nutrients (i.e., folic acid) affect the global methylation status as well as the methylation of single target genes? Some experts point out that until more is known about the interconnectedness of these pathways, simply supplementing everyone above-and-beyond a nutritionally sound diet may be contraindicated because the dietary cofactors could affect the epigenome in unknown ways.
Epigenetic research may help show the mechanism of action of integrative therapeutics and the interactions between diet and gene expression. This area of research, known as nutrigenomics and metabolomics, may be able not only to offer patients knowledge about their nutritional and metabolic gene expression but also to identify potential targets for mechanisms of action for diet, lifestyle, and other integrative medicine treatments. With a basic understanding of the field of epigenetics, providers eventually may be better able to guide patients through this advancing field and apply diagnostic and treatment approaches appropriately.