Dr. Sang Woon Choi

Epigenetic connection

between nutrients and cancer





IFCC Advanced Summer School in Biochemistry and Molecular Cell Biology

Epigenetics: Molecular Mechanisms, Biology and Human Diseases



Shanghai, China, July 14, 2010

La Cathédrale de Rouen, le portail et la tour Saint-Romain



Claude Monet









effet du matin plein soleil, midi après-midi coucher du soleil

Epigenetics: DNA isn’t everything









The new science of epigenetics

reveals how your choices you

make can change your genes

---and those of your kids.







You are what your grandmother ate.

Identical twins,

Different Work-outs

Monozygotic twins









There are relatively few differences Changes between them may

between the twins when they’re first come about as a result of

born & begin to grow together: personal choice:

Twin study

Monozygotic twins share a common genotype.



Twins are epigenetically indistinguishable during the early years

of life but older monozygotic twins exhibited remarkable different

epigenetic patterns, affecting their gene-expression portrait.









PNAS 2005;102:10604

Honeybees







Honeybees grow to be either queens or

workers depending on whether they are

fed royal jelly or beebread.



Despite they are genetically identical at

the larvae level, honeybee queens fed

pure royal jelly are markedly different

from workers.



The different honeybee phenotype occurs

through epigenetic changes in DNA

methylation patterns induced by the

different type of honey.



Science 2008;319:1827

Agouti mouse model



Maternal methyl dietary contents affect the coat color of the rodent

offspring and alter the susceptibility of the animal to certain chronic

diseases, obesity and cancer.









Nature Genetics 1999:23:314

Tail kinking(AxinFu)mouse model





Methyl donor supplementation of

female mice during gestation period

increased DNA methylation at AxinFu,

silenced the expression from the cryptic

promoter, and decreased the incidence

of tail kinking in AxinFu /+ offspring.



Genesis 2006;44:401-406

A bridge that connects environmental factors to

our genes and bring the phenotype into being.

Epigenetics





The word ‘epigenetics’ refers to genome information that is ‘super’-

imposed on the DNA sequence.



In the early 1940s Waddington described epigenetics as ‘the

interactions of genes with their environment, which bring the

phenotype into being’.



Inheritable biological phenomena that modify DNA or chromatin

structures, thereby affecting gene expression without altering base

pairs.



Epigenetic phenomena are critical for the embryonic development,

aging, and the process of many diseases including cancers.



Epigenetic phenomena are reversible and can be modulated by

nutrients.

Epigenome



The epigenome is a catalog of the epigenetic modifications that

occur in the genome.



An epigenome can be described as the epigenomic profile of a

specific cell or tissue type which reflects its biological condition or

state and defines its transcriptional potential.







Epigenomics



Epigenomics is the study of epigenetic modification at a level

much larger than a single gene





Human Molecular Genetics 2006;15(Review Issue 1):R95

What is nutritional epigenetics?







Nutrients



Epigenetics







Genes









What your grandma didn’t tell you about nutrition

What is Cancer?









Abnormal accumulation of abnormal cells

with a loss of control to grow and spread

Homeostasis





Cell Proliferation Cell Death



Regulation of Cell Cycle: Control of Apoptosis

Cell Cycle Check points

Neoplasm



Cell Proliferation









Cell Death

Oncogene and tumor suppressor gene





Oncogenes, altered forms of normal cellular genes called

proto-oncogenes, increase the rate of transformation from a

normal to a cancerous cell by affecting the cell growth and

differentiation (e.g. c-myc, k-ras).



Tumor suppressor genes are present in normal cells and

suppress cancer development, either by controlling cell

proliferation and apoptosis or by controlling DNA repair and

genomic stability (e.g. p53, BRCA1, and RB1).

Neoplasm



Tumor suppressor gene



Cell Proliferation







Oncogene gene



Cell Death









Tumor suppressor gene

Molecular mechanisms to activate oncogenes and

inactivate tumor suppressor genes







Loss of heterozygosity: the loss of one of the two alleles at

one or more loci due to chromosome loss, deletion, or

mitotic crossing-over.



Mutation: changes in nucleotide sequence



Epigenetic silencing: epigenetic repression of tumor

suppressor or loss of imprinting

overview

Epigenetics and Nutrients



 DNA methylation

 Histone modifications

 Chromatin remodeling





Nutrients, epigenetics and cancer



 Inflammation and histone modifications

 Folate, epigenetics and cancer

 Rodent hepatocellular carcinoma model of chronic dietary

methyl deficiency

 Alcohol, epigeneticsand cancer

 Summary and future perspectives

DNA methylation

DNA methylation



DNA methylation is a unique modification of DNA and the most

common epigenetic phenomenon in eukaryotic cells.

DNA methyltransferases catalyze the transfer of methyl group from

S-adenosylmethionine (AdoMet) to the carbon-5’ position of

cytosine in CpG dinucleotides.

DNA methylation



3-5% of cytosine residues in genomic DNA are modified to 5-

methylcytosine in CpG dinucleotides.



70% of CpG dinucleotide sequences, which usually occur in

CpG islands, contain 5-methylcytosine.



DNA methylation is associated with gene expression and

integrity.



Aberrations in DNA methylation play a mechanistic role in

carcinogenesis.

(Cancer Res 2006;66:8462)









unmethylated

Promoter DNA methylation









Wong, J J L et al. Gut 2007;56:140-148

Progressive changes in promoter methylation at CpG

sites during cancer initiation and progression









Nephew &,Huang, Cancer Lett. 2003;190:125

The possible role of DNA methylation in

carcinogenesis



Mechanism

Initiation &  Epigenetic gatekeeper gene silencing

early  Activation of normally silenced allele by loss of

progression imprinting

 Activation of oncogene and chromosomal instability

 Interrelationship with histone modifications

Mutation  Inactivation of repair gene

 Spontaneous deamination at methylcytosine residue

Cancer  Epigenetic plasticity

progression  Spreading of aberrant methylation

Metastasis  Epigenetic plasticity

 Tumor microenvironment





Feinberg, Nat Rev 2006;7:21

One-carbon metabolism and DNA methylation



Methionine



MAT



THF

AdoMet





Methylation of MTs MS SHMT

DNA/histone/

protein/RNA/

lipids/small

molecules AdoHcy methylTHF methyleneTHF

MTHFR

SAHH





Homocysteine

CBS

AdoMet: S-adenosylmethionine

AdoHcy: S-adenosylhomoscysteine Cystathionine THF: tetrahydrofolate

MTs: methyltransferases MTHFR: methylenetetrahydrofolate reductase

CBS:cystathionine beta synthase MS: methionine synthase

MAT:methionine adenosyltransferase SHMT: serine hydroxymethyltransferase

SAHH: S-adenosylhomocysteine hydrolase

DNA methyltransferases

Dnmt1 is considered to be the major maintenance

methyltransferase in mammalian cells and to be responsible for

restoring the fully methylated status of CpG sites on the newly

synthesized daughter strand following replication.



The in vivo function of Dnmt2 is not yet clear.



The Dnmt3 family consists of two active de novo Dnmts, Dnmt3a

and Dnmt3b. Both Dnmt3a and 3b are highly expressed in

embryonic stem cells but their expression decreases as the cells

differentiate. Dnmt3L is a regulatory factor for de novo DNA

methylation and connects unmethylated lysine 4 of histone H3 to

de novo DNA methylation.

Replicati

NLS on foci C- BAH I I V VII I X

Dnmt1 rich V I I X

1 TRD 1620



Dnmt2

1 415

PWWP ATRX

Dnmt3a

1 908

PWWP ATRX

Dnmt3b

1 859

C-

Dnmt3L rich

1 421

Nutrients that may affect DNA methylation

Nutrients Action

B-vitamins Folate Methyl acceptors and donors in 1-C metabolism

Vitamin B-12 Coenzyme for MS

Vitamin B-6 Coenzyme for SHMT, CBS, and cystathionase

Vitamin B-2 Coenzyme for MTHFR



Dietary Methionine Precursor of AdoMet

methyl donor Choline Homocysteine remethylation by BHMT

nutrients Betaine Homocysteine remethylation by BHMT

Serine Methyl donor to tetrahydrofolate by SHMT



Micronutrients Retinoic acid Increases the activity of GNMT

Zinc Coenzyme for MAT

Selenium Increases the transsulfuration pathway



Bioactive food Genistein Inhibition of DNA methyltransferases

compounds Tea Polyphenols Inhibition of DNA methyltransferases



BHMT: betaine homocysteine methyltransferase, CBS: cystathionine ß-synthase,

GNMT: glycine N-methyltransferase, MAT: methionine adenosyltransferase, MS:

methionine synthase, MTHFR: methylenetetrahydrofolate reductase, SHMT:

serine hydroxymethyltransferase

Histone modifications

Chromatin Structure

Histone tail modification









Lysine acetylation

Arginine methylation

Lysine methylation

Phosphorylation

Ubiquitination





15 to 38 amino acids from each histone N terminus for the histone

“tails”, providing a plat form for posttranslational modifications

Histone acetylation



Histone acetylation is a reversible post-translational process.





Generally, acetylation of histone is pretty much linked to

transcriptional activation whereas hypoacetylated histones are

found in transcriptionally inactive regions.







Active Inactive





HDAC



HAT









(Georgopouos K, Nature Rev Immunol 2, 162, 2002)

Histone acetyltransferase (HAT) and

histone deacetylase (HDAC)





Levels of acetylation of the core histones result from the steady

balance between HAT and HDAC.

Histone acetylation





Since 1964 when discovered and proposed to regulate gene

expression, the most extensively studied histone modification is

histone acetylation that occurs at lysine residues located in tail

domains.

(Proc Natl Acad Sci USA 1974;51:786)



In general, increased histone acetylation such as histone H4-K5 or

H4-K8 is found in euchromatin regions, whereas acetylation of H4-

K12 is increased in heterochromatin regions.



Acetylation of H4-K16 is found along the transcriptionally

hyperactive male X chromosome and loss of acetylation at this

residue is a common hallmark of human cancer.

(Fraga, Nature Genetics 2005;37:391)

HDAC, a new target for cancer prevention



Until now most of epigenetic studies have focused on DNA

methylation and DNA methyltransferase inhibitors (5-aza dC)

have been considered as effective chemopreventive agents.



However, tumor cells harbor abnormalities not only in DNA but

also in the histone modification, suggesting their implication for a

target of cancer chemotherapy.



A whole new class of anticancer drugs called histone deacetylase

(HDAC) inhibitors is poised to be used clinically.









(Garcia-Manero, Cancer Invest 2005;23:635)

HDAC inhibiting nutrients



Dietary HDAC inhibitors such as butyrate, diallyl disulfide (DADS)

and sulforaphane modulate histone acetylation.



In general, these dietary agents are weak ligands and inhibit

HDAC activity at higher concentrations than pharmacological

HDAC inhibitor such as trichostatin A.



A pertinent question concerns the concentrations needed for

inhibition of HDAC activity by dietary compounds, and the

likelihood that these levels might be achieved under normal

physiological condition



(Dashwood, Carcinogenesis 2006;27:344)

Examples of dietary compounds able to modulate

HAT or HDAC activities

Plant sources Dietary components



modulators of classic HDAC

Allium sativum L. (garlic) Diallyl disulfide (DADS)

S-allylmercaptocysteine

Allyl mercaptan

Dietary fiber fermentation Butyrate

Brassicaceae family

broccoli sprouts Sulforaphane

japanese horseradish (wasabi) 6-methylsulfinylhexyl-isothiocyanate



modulators of SIRT

vitis vinifera (Red grapes, wines) Resveratrol

Rhus verniciflua (stems) Butein

Rhus toxicodendron (leaves) Fisetin

Apple, tea, onion, nuts, berries, Quercetin

Blueberries Piceatannol

Sweet red pepper, celery, parsley Luteolin



modulators of HAT

Curcuma longa (Tumeric roots) Curcumin

Garcina indica (fruit) Garcinol

Camellia sinensis (black and green tea) Theophylline

Calorie restriction and histone acetylation







Calorie restriction increases the life span of many organisms from

yeast to mammals and reduces the risk of cancer.



In yeast, calorie restriction induced extension of life span requires

Sir2 gene (equivalent to Sirt1 in mammals). This gene has

deacetylase activity that is dependent on NAD, an oxidized

coenzyme that is important for catabolic process.



Calorie restriction spares NAD due to less catabolism and enhances

deacetylating activity of Sir2 gene, resulting in repression of down-

steam genes related to aging.

A model for dietary calorie, histone acetylation,

and longevity









(Hasty, Mech Ageing Develop 2001;122:1651)

Histone methylation





Methylation occurs on lysine and arginine on histones N-terminal

by histone methyltransferases (HMTs).



Histone methylation can result in either transcriptional activation

or repression, depending on the modified residue and the pattern

of other modifications.

Histone lysine methylation



Methylation of lysine in the histone tails of H3 and H4 appears to be

mono-, di- or tri-methylated and is found at the K4, K9, K27, K36,

and K79 of histone H3 and K20 of histone H4.









(Zhang, Genes & Development 2001;15:2343)

Histone lysine methylation and tumor suppressor

gene silencing in colon cancer



Deacetylation and methylation of H3-K9 are related with

promoter DNA methylation-associated hMLH1 silencing in colon

cancer cells

(Fahrner, Cancer Res 2002;62:7213)





Reduced H3-K4 methylation and increased H3-K9 methylation

play a critical role in the maintenance of promoter DNA

methylation-associated gene silencing (p16, MLH1, and MGMT)

in colon cancer cells.

(Kondo, Mol Cell Biol 2003;23:206)

Histone methyltransferases (HMTs)





HMTs transfer the methyl group from AdoMet to the arginine or

lysine residues in histone.



HMTs can be divided into three classes, protein arginine

methyltransferases, SET domain containing lysine

methyltransferases, and Dot1 class lysine methyltransferase.



SET domain-containing histone methyltransferases is a family

of protein that contain the evolutionary conserved SET domain

and play a fundamental role in epigenetic regulation of gene

activation and silencing in all eukaryotes. They also interact

with DNMT3A and DNMT3B.



Dot1-mediated H3K79 methylation is associated with telomere

silencing, meiotic checkpoint control, and DNA damage

response.



(Nature Reviews 2002;2:469, Nature Reviews Genetics 2009;10:295)

Histone demethylase





PADI4 (Petidylarginine deiminase 4) is the first to be identified.



LSD1 (lysine-specific demethylase 1) is the second class of

enzyme that directly reverse histone H3K4 or H3K9 modifications

by an oxidative demethylation reaction.



The third class of demethylase enzymes contain JmjC domain and

catalyze lysine demethylation through an oxidative reaction.

(Tan H et al. Mol Biol Rep 2008;35:551)

Chromatin remodeling

ATP-dependent chromatin remodeling complexes

Polycomb Group: PRC & E(Z) complexes

Spreading, H3K9&27me, Deacetylation, Ubiquitination



H3K9Ac K9Ac Deacet.

H3K9

K9/27me K9/27me

&27me







H3K4&K20 me K4/20me

H2AK119

Ubiq

PRE



Trithorax Group: ALL/Trithorax, Ash1 & SWI/SNF

Activating methylation, remodeled chromatin







PRC2: Histone methylation at H3-K27

EZH2







PRC1: p16

Bmi-1 repression









Histone ubiquitination at H2A-K116

Dietary modulation of polycomb repressive

complexes



The polycomb repressive complex 1 (PcG complex 1), which

contains the protein Bmi-1, binds to the K27me3 in histone H3

and catalyzes the ubiquitinylation of Histone H2A.



Bmi-1 is overexpressed in some human cancers, including

colorectal cancer, and human non-small cell lung cancer and

epidermal squamous cell carcinoma cells.



EGCG (40 μM) was found to suppress Bmi-1 levels and reduce

Bmi-1 phosphorylation, resulting in displacement of the Bmi-1

polycomb protein complex from chromatin and reducing survival

of transformed cells.



The importance of the polycomb repressive complexes in the

development of cancer is currently an active research area.



Br J Cancer 2001;84:1372 Nutrition Rev, in press

Dietary modulation of polycomb repressive

complexes-1





Retinoic acid (RA) is known to be involved in differentiation of ES

cells as well as differentiation of various cancer cells in culture.



Global levels of the enzyme which mediates the K27me3 (histone

K27 methyltransferase EZH2) also decreased with RA treatment.



A loss of EZH2 binding and K27me3 was observed locally on PcG

complex 2 target genes induced after 3 days of RA.



In contrast, direct RA-responsive genes that are rapidly induced,

such as Hoxa1, showed a loss of EZH2 binding and K27me3 after

only a few hours of RA treatment.





Stem Cells 2007;25:2191

Inflammation and histone

modifications

Chronic inflammation and cancer



Inflammation appears to be a risk factor for a great number of

cancers.



Some conditions, such as infection by the bacteria Helicobacter

pylori or ulcerative colitis, illustrate the role of inflammation in

the occurrence of digestive cancers.



J Clin Gastroenterol, 2008

Anti-inflammatory agents and histone acetylation





Glucocorticoids are highly efficient at inhibiting inflammation in a

number of chronic inflammatory disorders, such as asthma,

rheumatoid arthritis, and inflammatory bowel diseases. Histone

deacetylation is required for glucocorticoid mediated-

transcriptional suppression.



A natural compound extracted from tea leaves (Camellia sinensis),

theophylline (also called dimethylxanthine), was first recognized

as a phosphodiesterase inhibitor and has long been used in the

treatment of respiratory diseases like asthma. Recently,

theophylline has been reported to enhance HDAC activity.

Theophylline was able to potentiate the glucocorticoid-induced

increase in HDAC activity.



Am J Respir Crit Care Med 2004:170:141, PNAS 2002;99:8921

Figure 1. Regulation of chromatin structure influences the expression of pro-inflammatory

genes. The recruitment of co-factors with HAT activity, stimulated by pro-oxidants, increases

NFκB transcriptional activity and pro-inflammatory gene expression. In contrast,

glucocorticoids and natural chromatin-modifying agents trigger HDAC recruitment and HAT

inhibition, which results in NFκB inactivation, histone deacetylation and blockade of the

inflammatory process.

Resveratrol and inflammation







Resveratrol, found in the skin of red grapes and in red wine (vitis

vinifera), is an antioxidant with potential anti-cancer, anti-

inflammatory, and anti-aging properties.



The therapeutic interest in resveratrol has been mainly attributed to

its ability to control oxidative stress and to activate the NAD+-

dependent sirtuins.



A recent study revealed that SIRT1 and SIRT2 were dramatically

decreased in monocyte-macrophage cells in vitro and rat lungs

exposed to cigarette smoke. A similar reduction of SIRT1 was

reported in lungs of smokers and COPD patients.



Nature 2003;425:191, Am J Physiol Lung Cell Mol Physiol 2007;292:L567

Curcumin and inflammation





Curcumin (diferuloymethane) is a polyphenolic plant compound,

found in the rhizome of the Indian curry spice, turmeric (Curcuma

longa L.).



Curcumin was shown to interfere with NFkB activation and activity in

a significant number of inflammatory diseases and may potentially

increase the efficacy of glucocorticoids. Curcumin could impair NFkB

translocation to the nucleus through inhibition of IKKa

phosphorylation and IkBa degradation.



Curcumin specifically inhibits HAT p300 enzymatic activity.









Med Chem 2006;2:169, J Biol Chem 2003;278:2758 , J Clin Immunol 2007; 27:19,

Carcinogenesis 2003;24:1269

Dietary HDAC inhibitors and inflammation



Other dietary approaches with chromatin modifying agents, such

as the isothiocyanate sulforaphane (Brassica family members,

such as broccoli) or organosulfur compounds diallyl disulfide and

its derivative allyl mercaptan from garlic (Allium sativum L.), may

also alter NFkB function and markedly attenuate aberrant

activation of inflammatory processes.

Nutr Neurosci 2005;8:101



HDAC inhibitors differentially impact inflammatory pathways

depending on the nature of the compound used, which may affect

other biological targets (e.g., oxidants and regulators of cell

cycle).

Br J Pharmacol 2004;141:874



In addition, although dietary HAT and HDAC modulators can affect

NFkB proinflammatory function in several inflammatory diseases,

the mechanisms of action still need to be more carefully

examined.

J Clin Immunol 2007;27:19

Folate and cancer

Epidemiologic evidence







More than 30 epidemiologic studies indicate that diminished

folate status, measured by dietary folate or blood

concentrations, leads to an increased risk of cancer.



Evidence suggests the association of folate with cancers of

the colon, pancreas, esophagus, stomach, lung, liver, blood,

cervix, breast and prostate.

Evidence from animal studies







Chemical Carcinogen Model:

Cravo et al. reported that folate depletion increases the

development of colonic tumor in dimethylhydrazine-treated rats.

(Cancer Res 1992;52:5002)





Genetically Engineered Mouse Model:

Kim et al. also reported that folate depletion also increases the

development of intestinal neoplasia in genetically engineered

mice (min).



(Cancer Res 2000;60:5434)

Evidence from animal studies-1



Animal models that develop tumors with diet alone:



• Methyl deficient diet

Diets that are deficient in methionine, choline, folate and

B-12 lead to spontaneous development of liver cancer

with hepatic DNA hypomethyation.

(Cancer Res 1989;49:4094)



• Western-style diet

Western-style diet containing low levels of calcium,

vitamin D, fiber, folate, methionine, and choline as well as

increased fat content has been shown to induce colonic

neoplasms in normal mice over a period of 18 months.

(Carcinogenesis. 2001;22:1871)



• Folate deficient diet

Mthfr+/+ and Mthfr+/- mice developed intestinal tumors

after 1 year of low dietary folate.

(Knock, Cancer Res 2006;66:10349)

Folate for DNA methylation



Folate is also essential for the synthesis of S-adenosylmethionine

(AdoMet, SAdoMet, SAM, SAMe), the universal donor for biological

methylation reactions.



Folate depletion diminishes the cellular pool of S-

adenosylmethionine, but the more consistent consequence of

depletion is the rise in S-adenosylhomocysteine (AdoHcy, SAdoHcy,

SAH), an inhibitor of DNA methylation reactions.



(J Biol Chem 2000;275:29318)









SAM

one-carbon metabolism



Purine

Synthesis





AdoMet Methionine* THF DHF 10-formyl

THF

Dimethyl Serine*

glycine SHMT

MS B12 B6 Thymidylate

Choline* Synthesis 5,10-

Methylation of methenyl

DNA/histone Betaine* glycine TS THF

B2, B3

5-methyl 5,10-

AdoHcy Homocysteine THF MTHFR methylene

THF

CBS B6

B6 Glutathione

Cystathionine Cysteine

Taurine

Methylation pathway Nucleotide synthesis pathway



*one-carbon donor nutrients

J Nutr 2000;130:129

Major changes in the study regarding the

association between folate and colon cancer



Low folate status increases the risk of colon cancer and

supplementation of folate may decrease the risk.









Low folate status may increase the risk of colon cancer but

it is not important anymore because folate deficiency is

quite rare in the US after the folate fortification era. On the

other hand, folate fortification or supplementation,

especially with folic acid, may increase the risk of colon

cancer.

Colorectal cancer: age-adjusted incidence in the

United States and Canada









Age-adjusted CRC incidence from

1986 to 2002 in the United States (A)

and Canada (B) based on nationally

representative databases.





Mason J B et al. Cancer Epidemiol Biomarkers Prev

Animal Study



Aim:



To determine the effect of aging and dietary folate on DNA

methylation status in the colon



Methods:



Young (4 month old, n=32) and 18 month old (n=34) male

C57BL/6 mice were randomly divided into three different diets

with different folate levels:

1) 0 mg folate/kg: folate-deplete state

2) 2 mg /kg: basal requirement of folate

3) 8 mg /kg: folate-supplemented state



 Mice were killed at 20 wk.

 Genomic DNA methylation was measured by LC/MS method

and the 16 promoter methylation was measured by

methylation specific PCR



(J Nutr 2007; 137:1713)

Genomic DNA methylation in the young and old

mice colon at 20 weeks







p for trend=0.023

Genomic DNA methylation (%)





6





** *

* old

4.5 young









3

0mg 2mg 8mg



Dietary folate groups



old vs young: * p<0.001, **p=0.032

p16 promoter methylation in the young and old

mice colon at 20 weeks





p for trend=0.009



100 *

p16 promoter methylation (%)









80 *

60

*

old

young

40



20



0

0mg 2mg 8mg



Dietary folate groups

*old vs young: p<0.001

Discussion for folate and aging study







Aging decreases genomic DNA methylation and increase p16

promoter methylation.



Dietary folate further modifies these age-associated changes in

DNA methylation.



The altered methylation pattern observed in the old mouse colon

recapitulates the pattern observed in cancer, suggesting that

aging provides an epigenetic milieu that is conducive to cancer

development.

Rodent hepatocellular carcinoma model of

chronic dietary methyl deficiency

Prolonged intake of diets deficient in sources of methyl groups

leads to development of hepatomas in rats and promotes

chemical carcinogenesis in both rats and certain strains of mice.

Rodent hepatocellular carcinoma model of chronic

dietary methyl deficiency

Diets that are deficient in methionine, choline, folate and B-12

lead to spontaneous development of liver cancer with hepatic

DNA hypomethylation.

Cancer Res 1989;49:4094



During the first 36 weeks of methyl deficient diet a

progressive loss of methyl groups at most CpG sites was

demonstrated. However, after 54 weeks of deficiency, the

majority of CpG sites in the DNA of tumor were remethylated.

Both p53 gene-specific and genomic DNA methylation were

also increased.



In the preneoplastic lesions, the level of p53 mRNA was

increased in association with hypomethylation in the gene. On

the other hand, in tumor tissues, p53 mRNA was decreased

along with relative hypermethylation in the gene.

Cancer Lett 1997;115:31

Feeding animals with the methyl-deficient diet led to

progressive loss of histone H4 lysine 20 trimethylation

(H4K20me3), H3 lysine 9 tirmethylation (H3K9me3), and

histone H3 lysine (H3K9ac) and histone H4 lysine 16

(H4K16ac) acetylation.

Figure 1 Western blot analysis of histone H3 and H4 modifications in liver of control rats and

rats fed methyl-deficient diet

Pogribny, I. P. et al. J. Nutr. 2007;137:216S-222S









Acid extracts of total histones were separated by SDS-PAGE and subjected to

immunoblotting using primary antibodies against H3K9me3, H3K9me2, H3K9me1,

H3K9 ac, and H3S10ph (A) and H4k20me3, H4K20me2, H4K20me1, and H4K16 ac

(B), respectively. Results are presented as change relative to age-matched control

rats. * Significantly different from control at the same (n = 5, means ± SEM).

Figure 2 Expression of Suv39h1, Suv4-20h2, and PRDM/Riz1 HMTs and HAT1 in liver of control

rats and rats fed a methyl-deficient diet

Pogribny, I. P. et al. J. Nutr. 2007;137:216S-222S









Immunoblotting using primary antibodies against Suv4–20h2, Suv39h1, PRDM/Riz1,

and HAT1. The lower part of the figure shows a quantitative evaluation of the Suv4–

20h2, Suv39h1, PRDM/Riz1, and HAT1 expression in liver of methyl-deficient rats

relative to those of control rats.

* Significantly different from control at the same time (n=5, means±SEM).

Altered expression of microRNAs (miRNAs) has been reported

in diverse human cancers.



In the rat model of liver carcinogenesis induced by a methyl-

deficient diet, the development of hepatocellular carcinoma

(HCC) is characterized by prominent early changes in

expression of miRNA genes, specifically by inhibition of

expression of microRNAs miR-34a, miR-127, miR-200b, and

miR-16a involved in the regulation of apoptosis, cell

proliferation, cell-to-cell connection, and epithelial-

mesenchymal transition.









Molecular Carcinogenesis 2008;48:479

qRT-PCR of differentially expressed miRNA genes in the livers

of control rats and rats fed methyl-deficient diet.









Expression changes of miR-34a, miR-127, miR-200b, miR-16a, miR-17-5p, and

miR-19b, in the livers during rat hepatocarcinogenesis induced by methyl

deficiency. The miRNA expression data presented as average fold change of each

miRNA normalized to that of 5S RNA in liver of methyl-deficient rats compared to

control rats.

MicroRNAs, small noncoding RNAs with regulatory

functions, in cancer



MicroRNAs (miRNAs) are a new class of non-protein-coding,

endogenous, small RNAs that regulate gene expression by

translational repression, mRNA cleavage, and mRNA decay initiated by

miRNA-guided rapid deadenylation.



Some miRNAs regulate cell proliferation and apoptosis processes by

playing roles as oncogenes or tumor suppressor genes.



miRNAs can play important roles in controlling DNA methylation and

histone modifications.



Small RNA mediated transcriptional gene silencing is associated with

changes in chromatin structure at the targeted promoter.



The expression of miRNAs is different in normal and tumor tissues.



Developmental Biology 2007;302:1, Cell Cycle 2008;7:602, Mol Carcinog

2009;48:479

(a) miRNAs are transcribed by RNA

MicroRNA biogenesis polymerase II (pol II) into long

primary miRNA transcripts of

variable size (pri-miRNA), which are

recognized and cleaved in the

nucleus by the RNase III enzyme

Drosha, resulting in a hairpin

precursor form called pre-miRNA.

(b) Pre-miRNA is exported from the

nucleus to the cytoplasm by

exportin 5 and is further processed

by another RNase enzyme called

Dicer (c), which produces a

transient 19–24-nt duplex. Only one

strand of the miRNA duplex (mature

miRNA) is incorporated into a large

protein complex called RISC (RNA-

induced silencing complex). (d) The

mature miRNA leads RISC to cleave

the mRNA or induce translational

repression, depending on the

degree of complementarity between

the miRNA and its target.

Alcohol, epigenetics and cancer

Many facets of alcohol



In chemistry, alcohol is any organic compound in which a hydroxyl

group is bound to a carbon atom of an alkyl or substituted alkyl

group.



In pharmacology, alcohol is a weak drug which has an enormous

variety of effects on biochemical systems throughout the body, not

only in the brain and liver.



Alcohol has been used medicinally throughout recorded history.

There was evidence that moderate consumption of alcohol was

associated with a decrease in the risk of heart attack.



Alcohol is the dirtiest drug we have. It permeates and damages all

tissue. No other drug can cause the same degree of harm that it

does. (National Institute on Alcohol Abuse and Alcoholism)



In nutrition, alcohol is a nutrient and alcoholic beverages are foods

(with great potential for abuse).

Effects of alcohol on one-carbon metabolism



Alcohol impairs folate absorption across intestinal brush border

membrane and decreases the hepatic uptake and renal

conservation of circulating folate and diminishes methionine

synthase (MS) activity in the liver, increasing the proportion of

methylated THF (methyl folate trap).



In chronic alcoholics PLP serum levels are lower than in non-

alcoholics. Acetaldehyde impairs the net formation of PLP from

pyridoxal, pyridoxine, and pyridoxine phosphate.



Vitamin B-12 deficiency, assessed as low circulating

concentrations, is less common in chronic alcoholics.

Nonetheless, tissue deficiencies of this vitamin may still occur,

suggesting that chronic alcohol consumption may impair the

availability of B-12 in tissues.



(FEBS journal 2007;274:6317, Hepatology 1993;18:984, Alcohol Clin Exp Res

2005;29:2188, Biochem Pharmacol 1994; 47:1561, Nutrition 2000;16:296, JCI

1974;53:693, Alcohol 1998;15:305)

Effect of alcohol on one-carbon metabolism-1





Alcohol stimulates catabolism of methionine to generate cysteine

and replenish glutathione (transsulfuration pathway).

At the same time, the cell attempts to conserve methionine

through the choline oxidation pathway which remethylates

homocysteine using betaine homocysteine methyltransferase

This results in a drastic waste of betaine as well as increased

AdoHcy and homocysteine.

Hepatology 1993;18:984

Effect of total folate intake and alcohol on the relative

risk of breast cancer in the Nurses' Health Study









Bailey, L. B. J. Nutr. 2003;133:3748S-3753S

Relative risk of colon cancer in participants in the

Physician's Health Study









Bailey, L. B. J. Nutr. 2003;133:3748S-3753S

Alcohol and one-carbon metabolism



MAT

AdoMet Methionine THF ?

Thymidylate

MTs Dimethyl and purine

glycine Serine synthesis

SHMT

Methylation of MS B12 B6

DNA/protein Choline

(histone)/RNA/ glycine

lipids Betaine



5-methyl 5,10-

AdoHcy Homocysteine THF MTHFR methylene

CBS B6 THF

B6

Cystathionine Cysteine Glutathione









Methylation pathway Nucleotide synthesis pathway



(J Nutr 2000;130:129, Biochem Pharm 1994;47:1561)

Hypothesis



Alcohol disturbs methyl transfer in one-carbon metabolism









Aberrations in DNA methylation and histone modifications









Alters carcinogenesis

Chronic alcohol consumption induces genomic

DNA hypomethylation in the rat colon.









Animal Study



To determine the effect of chronic alcohol consumption on DNA

methylation in the colon



Twenty male Sprague Dawley rats were fed either Lieber-DeCarli

diet with alcohol (36% of total calorie) or control diet. Colonic

mucosal DNA was extracted and the extent of genomic DNA

methylation was assessed.

J Nutr 1999; 129:1945

Effect of chronic alcohol consumption on

one-carbon metabolism in rats

Plasma

Groups Alcohol-fed rats Control rats



Homocysteine (µmol/L) 17.23 ± 4.63* 10.73 ± 2.76



Folate (nmol/L) 250.8 ± 61.5 295.1 ± 38.4



PLP (nmol/L) 276.16 ± 85.15 325.80 ± 89.09



Vitamin B-12 (pmol/L) 62.91 ± 21.46 76.58 ± 12.54



Liver

Groups Alcohol-fed rats Control rats

AdoMet (nmol/g liver) 34.1 ± 5.6* 48.8 ± 13.9



AdoHcy (nmol/g liver) 14.8 ± 1.7* 10.4 ± 2.7



Folate (nmol/g liver) 29.3 ± 17.0 35.6 ± 12.3



All values are mean ± SD, n=10, *Significantly different from control rats, p<0.05



Alcohol Clin Res Exp, 2000;24:259

Genomic DNA methylation in the colonic DNA from alcohol-

fed rats





4

Methyl acceptance (kBq/2 ug DNA)



*

3







2







1







0

alcohol-fed control





Figure: Genomic DNA methylation was significantly decreased

in the colonic DNA from alcohol-fed rats compared with the

control group (p<0.05).

J Nutr 1999;129:1945

Mice study





Young and old C57B6 mice (n=10 per group) were fed with

Lieber-DeCarli control diet, Lieber-DeCarli alcohol diet (18% of

total calorie, 3.1% v/v) or Lieber-DeCarli alcohol diet with reduced

folic acid (0.25mg/L). During the 3 weeks of liquid diet adaptation

period, alcohol concentrations were gradually increased.



Animals were harvested after 5 and 10 week of diet.



Genomic DNA methylation and p16 promoter methylation were

analyzed by LC/MS and methylation-specific PCR from the colon

Genomic DNA methylation of colonic mucosa (% methylation)

in old and young mice fed control diet, 18% EtOH-containing

diet and 18% EtOH+low folate level for 5 and 10 weeks.







Old Young

Diet 5 week 10 week 5 week 10 week

Control 4.58 ± 0.06 4.37 ± 0.08* 4.37 ± 0.15 4.75 ± 0.08

18% EtOH 4.52 ± 0.06 4.60 ± 0.06 4.67 ± 0.07 4.44 ± 0.10#

18% EtOH 4.22 ± 0.12† 4.31 ± 0.11 4.61 ± 0.09 4.63 ± 0.07

and low folate



DNA methylation is significantly lower in overall old mice compared to the all

young mice (4.43±0.04% vs 4.58±0.04, p<0.02).

* Significantly different from corresponding young mice (p<0.02)

† Different tendency from corresponding young mice (p=0.08)

# Different tendency from young control mice (p=0.08)

All values are means ± SEM (%).



Sauer, Br J Nutr in press

p16 promoter methylation



5 weeks 10 weeks

60 60

old old

young

p16 promoter methylation (%)









young









p16 promoter methylation (%)

*

* * *

*

40 40

*





20 20









0 0

Control 18% EtOH 18% EtOH+low f olate Control 18% EtOH 18% EtOH+low f olate









Promoter methylation of p16 in the colon of old (18 mo) and young (4 mo)

mice fed control, 18% EtOH or 18% EtOH+low folate for 5 and 10 weeks.

Values are means±SEM (*p old vs. young <0.001).

Effect of alcohol on histone acetylation









Figure 1. Alcohol dose-dependent and Figure 2. H3-K9 acetylation

time-dependent acetylation of histone in the rat liver after binge

H3 lysine 9 (H3-K9) acetylation in rat drinking

hepatic stellate cells (Alcohol Alcohol 2006;41:126)

(Alcohol Alcohol 2005 40:367)





Alcohol modulates H3K9 acetylation via increasing HAT activity.

(Alcohol Clin Exp Res 2008;32:1)

Distinct methylation patterns in histone H3-K4 and H3-K9

correlate with up- & down-regulation of genes by ethanol in

rat hepatocytes









Figure 3. Treatment of hepatocytes with alcohol reduces H3-K9 dimethylation

with subsequent increase of H3-K4 dimethylation in the upregulatory genes

(adh and GST-Yc2), whereas in down regulatory genes (lsdh and CYP2C11) the

dimethyl H3-K9 accumulated at the promoter.

(Life Sci. 2007;81:979)

Reduced methyl availability and inhibition of one-

carbon metabolism by alcohol









NCM460, Human colonic epithelial cell line

DMEM + 10% fetal bovine serum

Added 100mmol/L Ethanol

Cultured cells for 0, 24, 48, 72h

Time-dependent trimethylation of histone H3 at Lys4

(H3K4me3) by ethanol



Ethanol Ethanol Ethanol TSA

Control for 24h for 48h for 72h for 24h



H3K4me3





H4





p<0.005

2.5

Relative level of methylation (H3K4me3/H4)









2







1.5







1







0.5







0

Control 100mmol/L Ethanol 100mmol/L Ethanol 100mmol/L Ethanol 300nmol/L TSA for

for 24h for 48h for 72h 24h

Time-dependent acetylation of histone H3 at Lys9 (H3K9ac)

by ethanol



Ethanol Ethanol Ethanol TSA

Control for 24h for 48h for 72h for 24h



H3K9ac



H4





5.5

3.5

Relative level of acetylation (H3K9ac/H4)









53





2.5

4.5



2

4



1.5 p<=0.0001





1





0.5





0

Control 100mmol/L 100mmol/L 100mmol/L 300nmol/L TSA f or

Ethanol f or 24h Ethanol f or 48h Ethanol f or 72h 24h

Summary for epigenetics





Epigenetics is a (heritable) phenomenon that affects gene

expression without base pair changes. Epigenetic phenomena

include DNA methylation, histone modifications, and chromatin

remodeling.



Chromatin is much more than neutral system for packaging and

condensing genomic DNA. Modifications to chromatin can give rise

to a variety of epigenetic effects. It is a critical player in

controlling the accessibility of DNA for transcription and other

reactions.



During our whole life nutrients can modify our physiologic and

pathologic processes through epigenetic phenomena that are

critical for gene expression and integrity. Modulation of those

processes through diet or specific nutrients may prevent diseases

and maintain our health.

Future perspectives in nutritional epigenetics



We knew that nutrients and bioactive food components can

modulate epigenetic phenomena but only a few of them were

tested. Since those interact with genes and other lifestyle factors, it

is very hard to find out the precise effects of nutrients or bioactive

food components on each epigenetic phenomenon and their

associations with physiologic and pathologic processes in our body.



However, it is still worth while to test more nutrients or functional

compounds to find better ones for our health. That will be helpful to

find the better way to protect our health with nutritional modulation

that is more physiologic than using other pharmacological agents.



Exploring this area of research may open up a greater

understanding of the role of diet in altering epigenetic patterns and

guide research to develop new strategies for disease prevention.



Epigenomic approaches will characterize genome-wide epigenetic

marks that are targets for dietary regulation.

sang.choi@tufts.edu

Methods for epigenetic study

ChIP assay



Chromatin immunoprecipitation (ChIP) is a powerful tool

for identifying proteins, including histone proteins and

non-histone proteins, associated with specific regions of

the genome by using specific antibodies that recognize a

specific protein or a specific modification of a protein.



The technique involves crosslinking of proteins with DNA,

fragmentation and preparation of soluble chromatin

followed by immunoprecipitation with an antibody

recognizing the protein of interest. The segment of the

genome associated with the protein is then identified by

PCR amplification of the DNA in the immunoprecipitates.

How do we measure

distribution of histone

modifications at

specific loci?







Chromatin

immunoprecipitations

(ChIPs)

Schematic of ChIP







Crosslinking

500 bp

with

formaldehyde Sonication





Chromatin

purification



Immuno-

NF-E2

USF II

TF II I

No AB









preciptation

Input









with specific

PCR of

antibody

genomic

fragments

Reverse of

Crosslinking

DNA

purification

K562

Chromatin Immunoprecipitation (ChIP)



Grow cells and formaldehyde treat:

This treatment crosslinks the proteins

to the DNA ensuring co-precipitation of

the DNA with the protein of interest.



Lysis and sonication of the cells: Cells

are broken open and sonication is

performed to shear the chromatin to a

manageable size (200-1000bp).



Immunoselection: Immunoprecipitation

by using a primary antibody of choice

followed by Protein A/G-conjugated

agarose beads as the secondary

reagent. This enriches for the protein

of interest and the DNA that it is

specifically complexed with.



Purification of the DNA: Protein-DNA

crosslinks are reversed during

incubation at 65C° and DNA is purified

to remove the chromatin proteins and

to prepare the DNA for the detection

step.



Detection: PCR.

p16 gene specific histone modifications

(an example for ChiP assay)









No Ab control



triM3H3K4



triMeH3K9

Input DNA









AcH3K9

CONTROL



100uM Adox for 24hr



100uM Adox for 48hr



100uM Adox for 72hr



5uM ADC for 72hr

ratio (precipitated DNA/input DNA)

1

No Ab control









0.8

Input DNA









H3K4me3





H3K9me3





0.6





0.4

1

0.2

2

0

3

H3K4me3 H3K9me3 H3K4me3 H3K9me3 H3K4me3 H3K9me3 H3K4me3 H3K9me3



4

CONTROL 100uM Adox for 100uM Adox for 100uM Adox for

24hr 48hr 72hr



1 2 3 4









Figure 10. Changes in H3-K4 and H3-K9 trimethylation in p16 gene after incubating NCM 460 cells with 100 μM Adox. The ChiP assay

demonstrates a decreased pattern in trimethylation at both H3-K4 and H3-K9 residues in the p16 promoter region.

Histone H3 lysine ChiP assay

H3K9 acetylation of p16 promoter









H3K9 methylation of p16 promoter









H3K4 methylation of p16 promoter









DNA methylation of p16 promoter









(Kondo, Mol Cell Biol 2003;23:206)

Global histone modificaitons









Feeding animals with the methyl-deficient diet led to progressive

loss of histone H4 lysine 20 trimethylation (H4K20me3), H3 lysine

9 tirmethylation (H3K9me3), and histone H3 lysine (H3K9ac) and

histone H4 lysine 16 (H4K16ac) acetylation.



After extracting histone, western blot is performed using Anti-

trimethyl-histone H3-Lys 9 and anti-trimethyl-histone H4-Lys 20

primary antibodies

Histone extraction





The acid cell extracts were prepared from frozen liver tissues

using lysis buffer containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl2,

10 mM KCl, 0.5 mM DTT, 1.5 mM PMSF, followed by the addition

of HCl to a final concentration of 200 mM.



Cell lysates were centrifuged at 14,000 xg for 10 min at 4°C, and

the acid-insoluble pellets were discarded. The supernatant

fractions, which contain the acid-soluble proteins, were purified by

sequential dialysis against 100 mM acetic acid and H20.

Western blot analysis of methylation status of histone

H3-Lys9 and histone H4-Lys20









Pogribny, Carcinogenesis 2006;27:1180

A method to assess genomic DNA methylation using

HPLC/ESI/MS



DNA hydrolysis HPLC LC/MS interface

CH3



ESI-SOURCE

CH3



ION TRAP MS





CH3

m/z=112 m/z=126

H H



(Friso, Analyt Chem 2002;74:4526)

Four ion peaks



MSD1 112, EIC=111.7:112.7 (IT1006B\004-0401.D) API-ES, Pos, SIM, Frag: 230

1500000

1250000

1000000

750000

500000

250000

0

3.5 4 4.5 5 5.5 6 6.5 7 7.5 min

MSD1 115, EIC=114.7:115.7 (IT1006B\004-0401.D) API-ES, Pos, SIM, Frag: 230



600000



400000



200000



0

3.5 4 4.5 5 5.5 6 6.5 7 7.5 min

MSD1 126, EIC=125.7:126.7 (IT1006B\004-0401.D) API-ES, Pos, SIM, Frag: 230



100000

80000

60000

40000

20000

0

3.5 4 4.5 5 5.5 6 6.5 7 7.5 min

MSD1 130, EIC=129.7:130.7 (IT1006B\004-0401.D) API-ES, Pos, SIM, Frag: 230



400000

300000

200000

100000

0

3.5 4 4.5 5 5.5 6 6.5 7 7.5 min

Genomic DNA methylation in the young and old

mice colon at 20 weeks









p for trend=0.04

6.5

Percent methylation









6.0



5.5

* OLD

5.0 * YOUNG

4.5



4.0

0 2 8

Dietary Folic Acid mg/kg

*p<0.05 old vs young



Keyes et al. J Nutr in press

Genomic DNA methylation 3 * P<0.042

(mCyt ng /μg DNA)



2.75





2.5





2.25





2

placebo phase ERT

(n=13) (n=13)







Friso et al. Br J Nutr 2007;97:617

Question?



A

400









Abundance (x103)

300



200 m/z 112.1

100

m/z 114.9

0

0 2 4 6 8 10 12





B

400

Abundance (x103)









300



200 m/z 126.1

100

m/z 130.1

0

0 2 4 6 8 10 Time (min)







C NH2 D NH2



H3C

N N m/z 126.1

m/z 112.1

N O N O

H+ H+







Choi et al. Mol & Biochem Parasitology 2006;150:350

no peak at 126



MSD1 112, EIC=111.7:112.7 (KYS323A\002-0201.D) API-ES, Pos, SIM, Frag: 230



6000000



4000000



2000000



0

3.5 4 4.5 5 5.5 6 6.5 7 7.5 min

MSD1 115, EIC=114.7:115.7 (KYS323A\002-0201.D) API-ES, Pos, SIM, Frag: 230



2500000

2000000

1500000

1000000

500000

0

3.5 4 4.5 5 5.5 6 6.5 7 7.5 min

MSD1 126, EIC=125.7:126.7 (KYS323A\002-0201.D) API-ES, Pos, SIM, Frag: 230





25000

20000

15000

10000



3.5 4 4.5 5 5.5 6 6.5 7 7.5 min

MSD1 130, EIC=129.7:130.7 (KYS323A\002-0201.D) API-ES, Pos, SIM, Frag: 230



1000000

800000

600000

400000

200000

0

3.5 4 4.5 5 5.5 6 6.5 7 7.5 min

Gene-specific DNA methylation

Principle of bisulfite modification



Unmethylated DNA

ggg gcg gac cgc

bisulfite modification



ggg gug gau ugu





Methylated DNA

ggg gcmg gacm cmgcm



bisulfite modification



ggg gcmg gacm cmgcm

Methylation specific PCR









 Methylation Specific PCR (MSP) of the p16 gene in two invasive

carcinomas, a squamous intraepithelial lesion (SIL), and an

adenocarcinoma of the cervix. Each numbered set are paired MSP

reactions specific for both the unmethylated (U) and methylated

(M) alleles of the p16 CpG island.

p16 promoter methylation in mice colon





U M U M U M







p16 promoter methylation in old mice colon after 20

weeks of folate deplete diet: partially methylated





U M U M U M



.





p16 promoter methylation in young mice colon after 20

weeks of folate deplete diet: unmethylated

p16 promoter methylation in the young and

old mice colon at 20 weeks





p for trend=0.04



120% p=0.03 p=0.05 *

100%

% methylation









80% *

* OLD

60%

YOUNG

40%

20%

0%

0 2 8

Dietary folate mg/kg

* p<0.05 old vs young

New Promoter methylation assay



 Genomic DNA is digested with MseI,

and the resulting DNA fragments

are incubated with the methylation

binding protein MeCP2.



 The methylated DNA fragments are

isolated with a spin column and the

amplified with promoter specific

primers.



 Agarose gel electrophoresis is used

to visualize the PCR products.



 The presence of a band on the gel

indicates that a specific promoter is

methylated in your genomic DNA

sample.







(Panomics, Methylation Promoter PCR Kit)

p16 gene specific histone modifications

(an example for ChiP assay)









No Ab control



triM3H3K4



triMeH3K9

Input DNA









AcH3K9

CONTROL



100uM Adox for 24hr



100uM Adox for 48hr



100uM Adox for 72hr



5uM ADC for 72hr

ChIP-chip Protocol



1. Sample cross-linking and fragmentation

2. Immunoprecipitation

3. Enrichment

4. Amplification

5. Labeling

6. Hybridization

7. Data Analysis

DNA methylation microarray



1. Sample fragmentation

2. DNA denaturation

3. Immunoprecipitation

4. Enrichment

5. Amplification

6. Labeling

7. Hybridization