How well do you methylate? Where do these methyl groups come from? And why should you care? Well, Tro Nation, today’s article explores methylation in detail because, like Hansel from Zoolander, methylation is just “so hot right now”.
So first a definition: methylation is the addition of a methyl group (consisting of one carbon atom bonded to three hydrogen atoms) to a substrate and impacts physiology in a number of fundamental ways including the regulation of gene expression (i.e. epigenetics), RNA processing, modifying heavy meals, modulating the function of an array of different proteins, and much much more.
Where Do Methyl Groups Come From?
Methyl groups are obtained from the diet, mainly from foods that contain methionine (an essential amino acid), one-carbon units (serine and glycine primarily), and choline (or the choline metabolite betaine) . Humans eat around 50 mmol of methyl groups every day, with 60% of these coming from their choline intake.
To give you some examples, foods high in methionine include turkey, beef, tuna, pork, cheese, brazil nuts, and quinoa. Foods high in one-carbon units include bone broth, chicken skin, animal protein, shellfish, legumes, spinach, dried seaweed, and watercress. And foods high in choline include whole eggs, organ meat, caviar, fish, shiitake mushrooms, beef, and soy.
The process of methylation itself is driven by the folate and methionine cycles – pathways that rely on an adequate supply of important micronutrients such as folate (vitamin B9) and cobalamin (vitamin B12) [2,3]. Folate is a limiting factor in the methylation cycle, and therefore inadequate dietary intake of this essential micronutrient can alter the epigenetic function of the organism due to limited folate availability .
Vitamin B12 is a key cofactor for enzymes such as methionine synthase that help catalyze the methylation of homocysteine to methionine within the methionine cycle; a reaction that is indispensable to form S-adenosylmethionine, the most important cellular methyl-donor .
There is, however, flexibility in the synthesis pathways, such that if choline intake isn’t high enough, more methyltetrahydrofolate (methyl-THF) is used to re-methylate homocysteine in the liver, which increases the need for folate from the diet. Likewise, if folate intake is insufficient, more choline is needed to supply methyl groups.
Vitamin B12 can be found in meat, fish, milk, cheese, eggs, whereas folic acid can be obtained from dark green leafy vegetables, whole grains, liver, and seeds. Both these essential micronutrients can also be found in certain fortified and processed foods as well but best to get them in their “natural state” when possible!
What are the different types of Methylation?
There are several different types of methylation depending on the target, or substrate, that the methyl group is being added to. Examples include the conversion of heavy metals, such as arsenic, into derivative compounds that can enter the food chain and cause potential harm.
The same biochemical process applies to ribonucleic acid (RNA), deoxyribonucleic acid (DNA) and other molecules such as proteins (as a post-translational modification), neurotransmitters, hormones, and other substrates.
Proteins can undergo methylation as a type of post-translational modification, as in the addition of a methyl group (usually to an arginine or lysine amino acid residue) which alters the biological function of the protein. As an example, the addition of a methyl group to histones – proteins that are abundant in lysine and arginine residues found in the cell nucleus – is catalyzed by methyltransferases. Histone methylation can act in an epigenetic fashion to block or activate gene expression in this manner .
DNA methylation is a fundamentally important biological process that can alter the activity of a given section of DNA without changing its sequence. It is a major epigenetic modification that is used by cells to control gene expression. As a mechanism, it is of principal concern within the settings of health and disease, as dysregulated DNA methylation is linked to several adverse outcomes in humans. Below, we will investigate how this important process turns genes on and off. We will also consider its epigenetic mode of action, its role in aging and disease, and its modulation by pharmacological and other means.
How Does DNA Methylation Work?
The field of genetics focuses on the study of the heritable changes in gene activity or function that occur due to the direct alteration of the DNA sequence , the set of genetic instructions that is required for the growth, survival, and reproduction of an organism (such as you for example!).
Quite differently, the study of epigenetics attempts to unravel the heritable changes in gene activity or function that are not associated with any change in the DNA sequence itself. Although pretty much all cells in a given organism contain the same genetic information, not all genes are expressed by all cell types at the same time . These tissue-specific differences are brought about thanks to epigenetic processes. In an individual the genome is largely consistent throughout the lifespan, whereas the epigenome is dynamically influenced by factors within the environment as well as developmental processes .
DNA methylation is a major epigenetic mechanism that involves direct chemical modification to the DNA, and was discovered as early as the 1940s . It is one of the most well-known epigenetic alterations and has been linked with numerous biological processes, yet it is distinct due to its relative stability . It wasn’t until 1975 that DNA methylation was found to influence gene expression, thanks to the independent work of Holliday and Pugh, as well as Riggs .
Biochemically, the enzymes that regulate DNA methylation have been referred to intuitively as “writers”, “erasers”, and “readers” . The enzymatic methylation of a DNA base (usually cytosine) at the 5-carbon position forms 5-methylcytosine. In other words, gene expression is regulated by the presence or absence of a methyl group (i.e., one carbon atom bonded to three hydrogen atoms; chemical formula CH3) on cytosine-phosphate-guanine (CpG) dinucleotides . This modification is catalyzed by DNA methyltransferases (or “writers”) and brings about the silencing of gene expression  by blocking the binding of transcription factors to DNA or by recruiting proteins involved in gene repression .
The ”erasers” (i.e., DNA methylases) modify and remove the methyl group from the cytosine residue in an active or passive manner [6,8], replacing the 5-methylcytosine in a given DNA sequence with cytosine.
Lastly, the “readers” consist of proteins that recognize DNA methylation, and comprise MBD proteins, UHRF proteins, and the zinc-finger proteins . These proteins have a high affinity for 5-methylcytosine and support or maintain DNA methylation to varying degrees and/or repress gene transcription by inhibiting the binding of transcription factors.
DNA Methylation in Health and Disease
If epigenetic processes are fundamental in specializing different groups of cells for optimal function - with DNA methylation an integral component - then it stands to reason that epigenetic deregulation would be implicated across many disease settings .
Cancer is perhaps one of the most intuitive diseases when it comes to genetics and epigenetics. Although it was originally thought to be a genetic disease, due to early work in familial cancer, evidence from the previous two decades suggests that genetics cannot fully explain cancer and epigenetic alterations instead appear to be at the forefront [12,13]. It now seems that in the early stages of tumor growth epigenetic changes occur, such as widespread DNA methylation within the cell, and these are highly pervasive across a given tumor type . Other evidence suggests that cancer-associated DNA hypomethylation can decrease genomic stability, whereas promoter hypermethylation silences genes involved in tumor suppression, DNA repair, and cell cycle progression . It is difficult to assume cause and effect relationships, however.
In addition to cancer, epigenetic modifications are associated with aging of the brain and impaired memory, changes in synaptic plasticity, as well as certain neurodegenerative diseases such as Parkinson’s and Huntington's Disease .
There also exist polymorphisms affecting theMTHFR (methylenetetrahydrofolate) genes that reduceMTHFRenzyme activity, which effectively impairs DNA methylation capacity and, in some cases, can induce folate deficiency amongst other biochemical derangements .
DNA Methylation and Aging
As we age, we accumulate a history of epigenetic changes that may eventually result in multiple age-related diseases. Older monozygotic (i.e., identical) twins exhibit global differences in DNA methylation patterns when compared to younger counterparts, and the methylome of centenarians displays reduced DNA methylation levels relative to that of a newborn [17,18].
Measures of DNA methylation have been used to build accurate composite biomarkers of chronological age, so-called epigenetic age estimators. These tools are capable of predicting lifespan after adjusting for chronological age and other risk factors, and are also associated with a wide array of age-associated conditions . The GrimAge epigenetic clock is one such composite biomarker that has excellent predictive ability for time-to-death, time-to-coronary disease, and time-to-cancer. It’s expected to have many applications in anti-aging research moving forward .
Pharmacological Modulation of DNA Methylation and Behavior
Epigenetic modifications, such as those that can occur during infancy, can have lasting effects across the lifespan. For example, rodent studies have shown that adverse maternal care brings about transient and long-term modifications to the epigenome, which can have functional consequences by altering gene expression and protein products in areas of the brain that control behavior . In short, maltreated rats tend to mistreat their offspring.
Interestingly, the administration of zebularine, a drug known to alter DNA methylation, to maltreated rats brought about lower levels of adverse care towards their offspring, and conversely increased levels of adverse care in controls . These findings illustrate two important facts: (1) epigenetic alterations are causally related to behavior, at least in rodents, and (2) it is possible to modulate this behavior using pharmacological drugs.
Schizophrenia and bipolar disorder are characterized by gene expression deficits as shown from postmortem brain studies in patients. These genes include GAD67, reelin, brain-derived neurotrophic factor and others involved in telecephalic GABAergic and glutamatergic neurons . The downregulation of these genes is associated with the enrichment of 5-methylcytosine and 5-hydroxymethylcytosine (i.e., hypermethylation). The administration of drugs that can reduce this hypermethylation, such as clozapine (a second-generation antipsychotic drug) could offer promise for correcting these gene expression deficits .
The pharmacological modulation of DNA methylation also has relevance for cancer treatment. For example, the DNA methyltransferase inhibitors such as decitabine and azacitidine are used in the treatment of myelodysplastic syndrome and acute myeloid leukemia and are being explored as therapeutic options for multiple solid cancers .
So to sum up, methyl groups come from your diet, the process of methylation itself is driven by the folate and methionine cycles, and then these methyl groups enter circulation and have a myriad of functions including epigenetic modification. As we age, all of this is likely to go pear shaped (great Scottish expression!) so the key is to measure your levels of what you need to keep your methylation optimized. For more information on how to do this, check out homehope.org and learn about HOMeHOPE, our nonprofit organization educating doctors and practitioners on how to optimize health rather than treat disease.
 M.D. Niculescu, S.H. Zeisel, Diet, methyl donors and DNA methylation: interactions between dietary folate, methionine and choline, J Nutr. 132 (2002) 2333S-2335S.https://doi.org/10.1093/jn/132.8.2333S.
 I.H.R. Abbasi, F. Abbasi, L. Wang, M.E. Abd El Hack, A.A. Swelum, R. Hao, J. Yao, Y. Cao, Folate promotes S-adenosyl methionine reactions and the microbial methylation cycle and boosts ruminants production and reproduction, AMB Express. 8 (2018) 65.https://doi.org/10.1186/s13568-018-0592-5.
 D.S. Froese, B. Fowler, M.R. Baumgartner, Vitamin B12 , folate, and the methionine remethylation cycle-biochemistry, pathways, and regulation, J Inherit Metab Dis. 42 (2019) 673–685.https://doi.org/10.1002/jimd.12009.
 R.E. Irwin, K. Pentieva, T. Cassidy, D.J. Lees-Murdock, M. McLaughlin, G. Prasad, H. McNulty, C.P. Walsh, The interplay between DNA methylation, folate and neurocognitive development, Epigenomics. 8 (2016) 863–879.https://doi.org/10.2217/epi-2016-0003.
 J. Nakayama, J.C. Rice, B.D. Strahl, C.D. Allis, S.I. Grewal, Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly, Science. 292 (2001) 110–113.https://doi.org/10.1126/science.1060118.
 L.D. Moore, T. Le, G. Fan, DNA Methylation and Its Basic Function, Neuropsychopharmacol. 38 (2013) 23–38.https://doi.org/10.1038/npp.2012.112.
 S. Kumar, V. Chinnusamy, T. Mohapatra, Epigenetics of Modified DNA Bases: 5-Methylcytosine and Beyond, Front. Genet. 9 (2018) 640.https://doi.org/10.3389/fgene.2018.00640.
 S.C. Wu, Y. Zhang, Active DNA demethylation: many roads lead to Rome, Nat Rev Mol Cell Biol. 11 (2010) 607–620.https://doi.org/10.1038/nrm2950.
 A. Harrison, A. Parle-McDermott, DNA Methylation: A Timeline of Methods and Applications, Front. Gene. 2 (2011).https://doi.org/10.3389/fgene.2011.00074.
 M.P. Campagna, A. Xavier, J. Lechner-Scott, V. Maltby, R.J. Scott, H. Butzkueven, V.G. Jokubaitis, R.A. Lea, Epigenome-wide association studies: current knowledge, strategies and recommendations, Clin Epigenet. 13 (2021) 214.https://doi.org/10.1186/s13148-021-01200-8.
 J. Newell-Price, A.J. Clark, P. King, DNA methylation and silencing of gene expression, Trends Endocrinol Metab. 11 (2000) 142–148.https://doi.org/10.1016/s1043-2760(00)00248-4.
 J.-P.J. Issa, H.M. Kantarjian, Targeting DNA methylation, Clin Cancer Res. 15 (2009) 3938–3946.https://doi.org/10.1158/1078-0432.CCR-08-2783.
 W.J. Locke, D. Guanzon, C. Ma, Y.J. Liew, K.R. Duesing, K.Y.C. Fung, J.P. Ross, DNA Methylation Cancer Biomarkers: Translation to the Clinic, Front. Genet. 10 (2019) 1150.https://doi.org/10.3389/fgene.2019.01150.
 G.P. Pfeifer, Defining Driver DNA Methylation Changes in Human Cancer, Int J Mol Sci. 19 (2018) E1166.https://doi.org/10.3390/ijms19041166.
 F. Sallustio, L. Gesualdo, A. Gallone, New findings showing how DNA methylation influences diseases, World J Biol Chem. 10 (2019) 1–6.https://doi.org/10.4331/wjbc.v10.i1.1.
 I.T.S. de Arruda, D.C. Persuhn, N.F.P. de Oliveira, The MTHFR C677T polymorphism and global DNA methylation in oral epithelial cells, Genet. Mol. Biol. 36 (2013) 490–493.https://doi.org/10.1590/S1415-47572013005000035.
 Y. Salameh, Y. Bejaoui, N. El Hajj, DNA Methylation Biomarkers in Aging and Age-Related Diseases, Front. Genet. 11 (2020) 171.https://doi.org/10.3389/fgene.2020.00171.
 S. Gonzalo, Epigenetic alterations in aging, J Appl Physiol (1985). 109 (2010) 586–597.https://doi.org/10.1152/japplphysiol.00238.2010.
 A.T. Lu, A. Quach, J.G. Wilson, A.P. Reiner, A. Aviv, K. Raj, L. Hou, A.A. Baccarelli, Y. Li, J.D. Stewart, E.A. Whitsel, T.L. Assimes, L. Ferrucci, S. Horvath, DNA methylation GrimAge strongly predicts lifespan and healthspan, Aging (Albany NY). 11 (2019) 303–327.https://doi.org/10.18632/aging.101684.
 S.M. Keller, T.S. Doherty, T.L. Roth, Pharmacological manipulation of DNA methylation normalizes maternal behavior, DNA methylation, and gene expression in dams with a history of maltreatment, Sci Rep. 9 (2019) 10253.https://doi.org/10.1038/s41598-019-46539-4.
 A. Guidotti, D.R. Grayson, DNA methylation and demethylation as targets for antipsychotic therapy, Dialogues in Clinical Neuroscience. 16 (2014) 419–429.https://doi.org/10.31887/DCNS.2014.16.3/aguidotti.
 A.K. Giri, T. Aittokallio, DNMT Inhibitors Increase Methylation in the Cancer Genome, Front. Pharmacol. 10 (2019) 385.https://doi.org/10.3389/fphar.2019.00385.