Vitamin B9

Dietary methyl donors and DNA methylation

• What nutrients are considered methyl donors?

• Where does DNA methylation normally occur?

• What is the significance of the agouti mouse experiments?

• Why are dietary methyl donors important in cancer, what are the mechanisms?

• Higher eukaryotes use epigenetic modifications to reversibly suppress transcription of genes and repeat elements, often by stable silencing

• Epigenetic marks are retained through mitosis, allowing maintenance of characteristic cell types

• By their nature, epigenetic modifications are susceptible to environmental influence: the interposition of epigenetic modifications between genes and the environment provides a way in which the environment can exert heritable influences on phenotype

Methylation of DNA

• Promoter regions of many genes are rich in CpG dinucleotides

• Carbon 5 of cytosine in CpG dinucleotides is a prime target for methylation by DNA methyltransferases (DNMT)

• Depending on cell type or tissue, 3–4% of all cytosines in vertebrate DNA and 70% of cytosines in CpG dinucleotides are 5-methylated

• Methylation of cytosines is a heritable modification of DNA. However, the following mechanisms are available to remove methylation marks in vivo:

• enzyme-mediated removal of methyl groups

• passive removal of methyl groups if methylation is not maintained at the time of replication

• DNMT utilize S-adenosylmethionine (SAM) as a methyl donor

• Folate plays a unique role in the generation of SAM, methylentetrahydrofolate reductase catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5- methyltetrahydrofolate

• The latter donates a methyl group to convert homocysteine to methionine

• Methylenetetrahydrofolate reductase deficiency results in hypomethylation of DNA, consistent with a role for folate in DNA methylation

• Nutrients other than folate also play roles in one-carbon metabolism: choline and methionine are the major dietary sources of methyl groups

• whereas vitamins B-6 and B-12, riboflavin and zinc are coenzymes and cofactors, respectively, for various steps in one-carbon metabolism

• Methylation of CpG islands is associated with transcriptional repression and provides a mean to control gene expression

• DNA methylation plays a role in allele-specific gene expression (genomic imprinting), heritable transcriptional silencing of parasitic sequence elements and X-chromosome inactivation

• Impaired DNA methylation is associated with perinatal death, decreased fertility, abnormal fetal development and tumorigenesis

Histone and DNA Methylation

Effect of Methyl Donors on the Epigenetic Process

• Several observations point to the ability of several environmental factors, including diet, to be key regulators of epigenetic processes, and evidence exists for diet effects on both DNA methylation and histone posttranslational modulation

• Diet can influence the degree of methylation by influencing the availability of methyl donors, including folate, choline, and methionine

• Several dietary factors ranging from alcohol to zinc have been reported to influence the supply of methyl groups for the formation of SAM and thereby affect the methylation of CpGs

• Dietary methyl deficiency (of folate, choline, and methionine) in a rat model is known to alter hepatic DNA methylation patterns and induce hepatocarcinogenesis in the absence of a carcinogen

• Dietary components such as EGCG found in green tea can influence gene methylation

Gene Imprinting and Diet

• Genomic imprinting is an epigenetic modification that inactivates one allele of a gene in a parent-of-origin-dependent manner. It occurs primarily by allelic-specific methylation of cytosines in CpG dinucleotides during gametogenesis

• Although relatively few in number, imprinted genes are thought to be vital to human development, and their dysregulation appears to affect the risk of developing some diseases

• One example by which early dietary exposures may affect genomic imprinting involves the imprinted gene insulin-like growth factor 2 (IGF2)

• The maternally inherited allele of the imprinted gene encoding IGF-2 is normally epigenetically silenced, resulting in expression almost exclusively from the paternal allele

• Interestingly, loss of imprinting at the IGF2 locus has been shown to induce biallelic expression of this growth factor in 10% of normal human adults and is implicated in several types of cancer

• For the most part, the cause of the IGF2 loss of imprinting is unknown

• Mice weaned on either a synthetic control or a synthetic methyl donorcofactor deficient diet compared with a standard diet displayed hypomethylation of IGF2 and consequent dysregulation of IGF2 allelic expression

• The loss of imprinting caused by the deficient diet persisted during a subsequent 100-d recuperation period after the mice were switched to the control diet

• Thus, early nutritional influences may stimulate changes in cytosine methylation to which imprinted genes, such as IGF2, may be susceptible

• These findings in view of the relation between IGF-2, insulin, and metabolic syndrome suggest that early nutrition may influence susceptibility to adult obesity, diabetes, cardiovascular disease, and cancer

Dutch Hunger Winter Study - Heijmans B. T. et.al. PNAS 2008;105:17046-17049

Difference in IGF2 DMR methylation between individuals prenatally exposed to famine and their same-sex sibling. Periconceptional exposure: Difference in methylation according to the mother's last menstrual period (a common estimate of conception) before conception of the famine-exposed individual.

Methyl Donors, Nutrition and Cancer

• In comparison to normal differentiated cells, tumor cells are characterized by a global hypomethylated state despite overexpression of DNA methyltransferase, although they have distinct regions of hypermethylation

• Even though this process and the influence of inadequate dietary supply of methyl donors and/or genetic predisposition to aberrant methylation have been extensively studied, the precise mechanisms underlying the methylation aberrations in cancer are poorly understood

• Neither is it known whether global hypomethylation and regional hypermethylation are causally linked or are completely independent events occurring in the same cellular context of malignant transformation

• The prevailing hypothesis is that hypermethylation of promoter-associated CpG islands of tumor suppressor genes leads to inactivation of these genes and development or progression of cancer

• Promoter hypermethylation has been observed in genes that play a role in cell cycle regulation, apoptosis, DNA repair and replication, angiogenesis, cell differentiation, and other processes that, when uncontrolled, can promote carcinogenesis

• Silencing these genes can be a second hit in the two-step carcinogenesis process

• Global DNA hypomethylation has been measured in many cancer cells and primary tumors from humans and animal models

• Chromosomal instability and DNA double-strand breaks have been demonstrated in laboratory rats fed hypomethylated diets, in people with methyl-donor deficient diets and in cancer cells

• Hypomethylation could lead to activation of oncogenes. In vivo studies of rats fed methyl-donor deficient diets showed that overexpression of oncogenes such as cmyc, c-fos, and c-ha-ras

• When diets were methyl-donor replete, the DNA methylation reverted to normal, but not for all cytosines that were examined. This suggests that DNA methylation defects can be irreversible after prolonged exposure to diets low in methyl donors

• Increased sensitivity of rats to known carcinogens has also been observed when diets are methyl-donor deficient

• In humans a good correlation between reduced tissue and plasma folate levels and hypomethylation of both normal and tumor tissues was demonstrated in patients with cervical cancer

• A large prospective study in humans also showed that methyl donor deficiency correlates with an increased risk for tumors of the liver and colon and demonstrated an inverse relationship between DNA methylation of colon adenomas and dietary folate

• Such studies further suggest that this risk may be aggravated by other risk factors, such as alcohol intake and smoking. Polymorphisms in the folate metabolic enzymes, e.g., the MTHFR 677C>T also influence cancer risk

• Because of the variability in methylation within one tumor or between tumor types, the potential benefit of methylation diets in cancer treatment or prevention, versus treatment with demethylating agents, may be highly variable

  
  

Folate and DNA Stability

Dietary Folate and DNA Stability

• Conversion of dUMP to dTMP is dependent on the presence of N5 N10 Methylene THF

• Folate deficient cultured cells

• 6X increase in dUTP/dTTP ratio after 11 days folate deficiency

• 3 days folate repletion corrected imbalance (Melnyk et al., 1999)

• Catastrophic repair cycle- (Dianov et al., 1991)

• Uracil removed by uracil-DNA glycosylase, strand break

• Uracil residues within 14 bp on opposing strands, double strand break

• DNA from folate deficient individuals

• 9X more uracil residues than folate sufficient individuals

• 50X more likely to have double strand breaks (Blount et al., 1997)

Dietary Folate and Cancer

• Nurses health study (N = 88,757): 15 yrs of 400 µg folate/day resulted in 75% reduction in colon cancer incidence (Giovanucci et al., 1998)

• At least 5 major case/control and 5 prospective studies have found lowered colorectal cancer risk with increased dietary folate (Giovannuci et al., 2002)

• Epidemiological evidence demonstrates fruit and vegetable consumption is inversely related to colon cancer is hypothesized to be related to folate intake (Ames, 1998)

• Folate intake is initially protective in animal colon cancer models (Song et al., 2000; Trasler et al., 2003)

The Avy allele and the Agouti mouse model

• The Avy allele carries an insertion of an IAP retrotransposon in an antisense direction in agouti pseudoexon

• The Avy transcript originates from a cryptic promoter in the 5'LTR of the IAP and is spliced to agouti coding exons 2, 3 and 4, which encode ASP

• When the IAP is silent, agouti is transcribed from hair-cycle-specific promoters in exons 1B and 1C

• Avy phenotypes are scored from 1 to 5 based on coat color. Fully yellow mice are scored as 1, and fully agouti mice are scored as 5. Phenotypes of mosaic mice range from mostly yellow (2) to mottled yellow/agouti (3) to mostly agouti (4).

  
  

• Avy/a mice vary in coat color phenotype from clear yellow (CY), to slightly mottled yellow (SMY), mottled yellow (MY), heavily mottled yellow (HMY), and pseudoagouti (PA)

• The top panel shows percentage methylation of 7 CpG sites in the Avy region in Avy/a mice according to the coat color classifications displayed in the bottom panel

• Methylation of the Avy locus is responsive to nutrition and tightly correlated with coat color phenotype, rendering the Avy/a mouse useful as an epigenetic biosensor.

Your Grandma's Diet & Epigenetics

• (F1) pseudoagouti Avy/a females that had been exposed to methyl donors in utero were mated to a/a males without any further methyl donor supplementation

• This strategy takes advantage of the tendency for the Avy epigenotype to be partially stable in the female germ line

• The phenotypes of the second generation (F2) offspring were compared with phenotypes of pups born to pseudoagouti females with no history of exposure to methyl donor supplementation

• Phenotypes of these F2 mice were significantly shifted toward the pseudoagouti

• Thus a pseudoagouti dam who was exposed to methyl donor supplementation only when she was in utero gives rise to phenotypically different offspring than does an otherwise (genetically and phenotypically) identical female who had no exposure to methyl donor supplementation

• This grandparental effect is directly attributable to the epigenetic state of the Avy allele.