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Epigenetic connection between nutrients and cancer Epigenetic connection between nutrients and cancer IFCC Advanced Summer School in Biochemistry and Molecular.

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Presentation on theme: "Epigenetic connection between nutrients and cancer Epigenetic connection between nutrients and cancer IFCC Advanced Summer School in Biochemistry and Molecular."— Presentation transcript:

1 Epigenetic connection between nutrients and cancer Epigenetic connection between nutrients and cancer IFCC Advanced Summer School in Biochemistry and Molecular Cell Biology IFCC Advanced Summer School in Biochemistry and Molecular Cell Biology Epigenetics: Molecular Mechanisms, Biology and Human Diseases Shanghai, China, July 14, 2010

2 après-midicoucher du soleil effet du matinplein soleil, midi La Cathédrale de Rouen, le portail et la tour Saint-Romain Claude Monet

3 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.

4 Monozygotic twins There are relatively few differences between the twins when they’re first born & begin to grow together: Changes between them may come about as a result of personal choice: Identical twins, Different Work-outs

5 Twin study PNAS 2005;102:10604 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.

6 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

7 Agouti mouse model Nature Genetics 1999:23:314 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.

8 Tail kinking(Axin Fu )mouse model Methyl donor supplementation of female mice during gestation period increased DNA methylation at Axin Fu, silenced the expression from the cryptic promoter, and decreased the incidence of tail kinking in Axin Fu /+ offspring. Genesis 2006;44:401-406 Genesis 2006;44:401-406

9 A bridge that connects environmental factors to our genes and bring the phenotype into being.

10 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. Epigenetics

11 Epigenomics Epigenomics is the study of epigenetic modification at a level much larger than a single gene 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. Human Molecular Genetics 2006;15(Review Issue 1):R95

12 What is nutritional epigenetics? Nutrients Genes Epigenetics What your grandma didn’t tell you about nutrition

13 What is Cancer? Abnormal accumulation of abnormal cells with a loss of control to grow and spread

14 Cell ProliferationCell Death Homeostasis Regulation of Cell Cycle: Cell Cycle Check points Control of Apoptosis

15 Cell Proliferation Cell Death Neoplasm

16 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).

17 Cell Proliferation Cell Death Neoplasm Tumor suppressor gene Oncogene gene

18 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

19 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 overview

20 DNA methylation

21 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.

22 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) DNA methylation unmethylated methylated

23 Wong, J J L et al. Gut 2007;56:140-148 Promoter DNA methylation

24 Progressive changes in promoter methylation at CpG sites during cancer initiation and progression Nephew &,Huang, Cancer Lett. 2003;190:125

25 The possible role of DNA methylation in carcinogenesis Mechanism Mechanism Initiation & early progression  Epigenetic gatekeeper gene silencing  Activation of normally silenced allele by loss of imprinting  Activation of oncogene and chromosomal instability  Interrelationship with histone modifications Mutation  Inactivation of repair gene  Spontaneous deamination at methylcytosine residue Cancer progression  Epigenetic plasticity  Spreading of aberrant methylation Metastasis  Epigenetic plasticity  Tumor microenvironment Feinberg, Nat Rev 2006;7:21

26 One-carbon metabolism and DNA methylation Methionine SAHH methylTHF THF methyleneTHF MTHFR MS CBS Homocysteine AdoMet AdoHcy SHMT Methylation of DNA/histone/ protein/RNA/ lipids/small molecules MAT MTs Cystathionine AdoMet: S-adenosylmethionine AdoHcy: S-adenosylhomoscysteine MTs: methyltransferases CBS:cystathionine beta synthase MAT:methionine adenosyltransferase SAHH: S-adenosylhomocysteine hydrolase THF: tetrahydrofolate MTHFR: methylenetetrahydrofolate reductase MS: methionine synthase SHMT: serine hydroxymethyltransferase

27 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. 1 415 Dnmt2 1859 Dnmt3b PWWP ATRX C- rich TRD 1 1620 Dnmt1 C- rich BAH Replicati on foci IIVIV VIVI VII I IXIX X NLS 1 908 Dnmt3a PWWPATRX 1 421 Dnmt3L

28 Nutrients that may affect DNA methylation NutrientsAction B-vitaminsFolate Vitamin B-12 Vitamin B-6 Vitamin B-2 Methyl acceptors and donors in 1-C metabolism Coenzyme for MS Coenzyme for SHMT, CBS, and cystathionase Coenzyme for MTHFR Dietary methyl donor nutrients MethionineCholineBetaineSerine Precursor of AdoMet Homocysteine remethylation by BHMT Methyl donor to tetrahydrofolate by SHMT Micronutrients Retinoic acid ZincSelenium Increases the activity of GNMT Coenzyme for MAT Increases the transsulfuration pathway Bioactive food compounds Genistein 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

29 Histone modifications

30 Chromatin Structure

31 Histone tail modification 15 to 38 amino acids from each histone N terminus for the histone “tails”, providing a plat form for posttranslational modifications Lysine acetylation Arginine methylation Lysine methylation Phosphorylation Ubiquitination

32 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. HDAC HAT ActiveInactive (Georgopouos K, Nature Rev Immunol 2, 162, 2002)

33 Histone acetyltransferase (HAT) and histone deacetylase (HDAC) Levels of acetylation of the core histones result from the steady balance between HAT and HDAC.

34 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) (Fraga, Nature Genetics 2005;37:391)

35 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)

36 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)

37 Examples of dietary compounds able to modulate HAT or HDAC activities Plant sourcesDietary components modulators of classic HDAC Allium sativum L. (garlic)Diallyl disulfide (DADS) S-allylmercaptocysteine Allyl mercaptan Dietary fiber fermentationButyrate Brassicaceae family broccoli sproutsSulforaphane 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 BlueberriesPiceatannol Sweet red pepper, celery, parsleyLuteolin modulators of HAT Curcuma longa (Tumeric roots)Curcumin Garcina indica (fruit)Garcinol Camellia sinensis (black and green tea)Theophylline

38 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.

39 A model for dietary calorie, histone acetylation, and longevity (Hasty, Mech Ageing Develop 2001;122:1651)

40 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.

41 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)

42 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)

43 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)

44 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)

45 Chromatin remodeling

46 ATP-dependent chromatin remodeling complexes

47 PRE H3K9Ac K9Ac H3K9 &27me K9/27me H3K4&K20 meK4/20me H2AK119 Ubiq Deacet. Trithorax Group: ALL/Trithorax, Ash1 & SWI/SNF Activating methylation, remodeled chromatin Polycomb Group: PRC & E(Z) complexes Spreading, H3K9&27me, Deacetylation, Ubiquitination PRC1: Bmi-1 Histone methylation at H3-K27 Histone ubiquitination at H2A-K116 p16 repression PRC2: EZH2

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

49 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 Stem Cells 2007;25:2191

50 Inflammation and histone modifications

51 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 J Clin Gastroenterol, 2008

52 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

53 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.

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

55 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 Curcumin and inflammation

56 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

57 Folate and cancer

58 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.

59 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) (Cancer Res 2000;60:5434)

60 Evidence from animal studies-1 Animal models that develop tumors with diet alone: Methyl deficient diet 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 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) (Carcinogenesis. 2001;22:1871) Folate deficient diet Folate deficient diet Mthfr+/+ and Mthfr+/- mice developed intestinal tumors after 1 year of low dietary folate. (Knock, Cancer Res 2006;66:10349) (Knock, Cancer Res 2006;66:10349)

61 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

62 Methylation of DNA/histone one-carbon metabolism Homocysteine Methionine* AdoMet AdoHcy 5-methyl THF 5,10- methylene THF Betaine* Dimethyl glycine DHF Thymidylate Synthesis B12 5,10- methenyl THF 10-formyl THF Purine Synthesis Choline* MS MTHFR Cystathionine B6 Serine* glycine B6 SHMT Methylation pathway Nucleotide synthesis pathway CBS TS Cysteine Glutathione Taurine *one-carbon donor nutrients J Nutr 2000;130:129 B6 B2, B3

63 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.

64 Colorectal cancer: age-adjusted incidence in the United States and Canada Mason J B et al. Cancer Epidemiol Biomarkers Prev Age-adjusted CRC incidence from 1986 to 2002 in the United States (A) and Canada (B) based on nationally representative databases.

65 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 )

66 Dietary folate groups Genomic DNA methylation (%) * ** * p for trend=0.023 old vs young: * p<0.001, ** p=0.032 Genomic DNA methylation in the young and old mice colon at 20 weeks

67 Dietary folate groups p16 promoter methylation (%) * * * p for trend=0.009 * old vs young: p<0.001 p16 promoter methylation in the young and old mice colon at 20 weeks

68 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.

69 Rodent hepatocellular carcinoma model of chronic dietary methyl deficiency

70 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.

71 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

72 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.

73 Pogribny, I. P. et al. J. Nutr. 2007;137:216S-222S Figure 1 Western blot analysis of histone H3 and H4 modifications in liver of control rats and rats fed methyl-deficient diet 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).

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

75 Molecular Carcinogenesis 2008;48:479 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.

76 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.

77 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

78 MicroRNA biogenesis (a) miRNAs are transcribed by RNA 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.

79 Alcohol, epigenetics and cancer

80 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).

81 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. Effects of alcohol on one-carbon metabolism (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)

82 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 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 alcohol on one-carbon metabolism-1

83 Bailey, L. B. J. Nutr. 2003;133:3748S-3753S Effect of total folate intake and alcohol on the relative risk of breast cancer in the Nurses' Health Study

84 Bailey, L. B. J. Nutr. 2003;133:3748S-3753S Relative risk of colon cancer in participants in the Physician's Health Study

85 Methylation of DNA/protein (histone)/RNA/ lipids Homocysteine Methionine AdoMet AdoHcy 5-methyl THF 5,10- methylene THF Betaine Dimethyl glycine Thymidylate and purine synthesis B12 Choline MS MTHFR Cystathionine B6 Serine glycine B6 SHMT Methylation pathway Nucleotide synthesis pathway Alcohol and one-carbon metabolism CBS (J Nutr 2000;130:129, Biochem Pharm 1994;47:1561) ? MAT GlutathioneCysteine B6 MTs

86 Hypothesis Alcohol disturbs methyl transfer in one-carbon metabolism Aberrations in DNA methylation and histone modifications Alters carcinogenesis

87 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

88 Effect of chronic alcohol consumption on one-carbon metabolism in rats 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 Plasma LiverGroups 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 ± SD, n=10, All values are mean ± SD, n=10, *Significantly different from control rats, p<0.05 Alcohol Clin Res Exp, 2000;24:259

89 Methyl acceptance (kBq/2 ug DNA) * 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 Genomic DNA methylation in the colonic DNA from alcohol- fed rats

90 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

91 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. OldYoung Diet5 week10 week5 week10 week Control4.58 ± 0.064.37 ± 0.08*4.37 ± 0.154.75 ± 0.08 18% EtOH4.52 ± 0.064.60 ± 0.064.67 ± 0.074.44 ± 0.10# 18% EtOH and low folate 4.22 ± 0.12†4.31 ± 0.114.61 ± 0.094.63 ± 0.07 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

92 p16 promoter methylation * * * * * * 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).

93 Effect of alcohol on histone acetylation Figure 2. H3-K9 acetylation in the rat liver after binge drinking (Alcohol Alcohol 2006;41:126) (Alcohol Alcohol 2006;41:126) Figure 1. Alcohol dose-dependent and time-dependent acetylation of histone H3 lysine 9 (H3-K9) acetylation in rat hepatic stellate cells (Alcohol Alcohol 2005 40:367) Alcohol modulates H3K9 acetylation via increasing HAT activity. (Alcohol Clin Exp Res 2008;32:1)

94 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)

95 NCM460, Human colonic epithelial cell line DMEM + 10% fetal bovine serum Added 100mmol/L Ethanol Cultured cells for 0, 24, 48, 72h Reduced methyl availability and inhibition of one- carbon metabolism by alcohol

96 Time-dependent trimethylation of histone H3 at Lys4 (H3K4me3) by ethanol p<0.005 H3K4me3 H4 Control Ethanol for 24h Ethanol for 48h Ethanol for 72h TSA for 24h

97 Time-dependent acetylation of histone H3 at Lys9 (H3K9ac) by ethanol 5 5.5 4 p<=0.0001 H3K9ac H4 Control Ethanol for 24h Ethanol for 48h Ethanol for 72h TSA for 24h 4.5

98 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.

99 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.





104 Methods for epigenetic study

105 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.

106 How do we measure distribution of histone modifications at specific loci? Chromatin immunoprecipitations (ChIPs)

107 Schematic of ChIP Immuno- preciptation with specific antibody Chromatin purification Reverse of Crosslinking DNA purification PCR of genomic fragments 500 bp Sonication Y Y Y K562 Input No AB  USF II  TF II I  NF-E2 with formaldehyde Crosslinking

108 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.

109 Input DNA No Ab control p16 gene specific histone modifications (an example for ChiP assay) triM3H3K4triMeH3K9 AcH3K9 CONTROL 100uM Adox for 24hr 100uM Adox for 48hr 100uM Adox for 72hr 5uM ADC for 72hr

110 0 0.2 0.4 0.6 0.8 1 H3K4me3H3K9me3H3K4me3H3K9me3H3K4me3H3K9me3H3K4me3H3K9me3 CONTROL100uM Adox for 24hr 100uM Adox for 48hr 100uM Adox for 72hr ratio (precipitated DNA/input DNA) Input DNAH3K9me 3 H3K4me3 No Ab control 1 2 3 4 123 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.

111 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)

112 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 Global histone modificaitons

113 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 H 2 0.

114 Pogribny, Carcinogenesis 2006;27:1180 Western blot analysis of methylation status of histone H3-Lys9 and histone H4-Lys20


116 A method to assess genomic DNA methylation using HPLC/ESI/MS HPLC DNA LC/MS interface ESI-SOURCE ION TRAP MS hydrolysis CH3 m/z=112m/z=126 CH3 HH (Friso, Analyt Chem 2002;74:4526)

117 Four ion peaks

118 Genomic DNA methylation in the young and old mice colon at 20 weeks * * p for trend=0.04 *p<0.05 old vs young Keyes et al. J Nutr in press

119 Genomic DNA methylation (mCyt ng /μg DNA) * P<0.042 (n=13) Friso et al. Br J Nutr 2007;97:617

120 0 100 300 400 200 0 100 300 400 200 Abundance (x10 3 ) m/z 112.1 m/z 114.9 m/z 126.1 m/z 130.1 0 2 48 10 Time (min) 6 0 2 48 10 6 12 A B N NH 2 O H+H+ N C N O H+H+ N D H3CH3C m/z 112.1 m/z 126.1 Question? Choi et al. Mol & Biochem Parasitology 2006;150:350

121 no peak at 126

122 Gene-specific DNA methylation

123 Principle of bisulfite modification ggg gcg gac cgc gggguggauugu ggg gc m g gac m c m gc m bisulfite modification Unmethylated DNA Methylated DNA

124 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. 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.

125 p16 promoter methylation in mice colon. p16 promoter methylation in young mice colon after 20 weeks of folate deplete diet: unmethylated p16 promoter methylation in old mice colon after 20 weeks of folate deplete diet: partially methylated U M U M U M

126 p16 promoter methylation in the young and old mice colon at 20 weeks p=0.03p=0.05 p for trend=0.04 * * * * p<0.05 old vs young

127 New Promoter methylation assay (Panomics, Methylation Promoter PCR Kit) 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.

128 Input DNA No Ab control p16 gene specific histone modifications (an example for ChiP assay) triM3H3K4triMeH3K9 AcH3K9 CONTROL 100uM Adox for 24hr 100uM Adox for 48hr 100uM Adox for 72hr 5uM ADC for 72hr

129 ChIP-chip Protocol 1. Sample cross-linking and fragmentation 2. Immunoprecipitation 3. Enrichment 4. Amplification 5. Labeling 6. Hybridization 7. Data Analysis

130 DNA methylation microarray 1. Sample fragmentation 2. DNA denaturation 3. Immunoprecipitation 4. Enrichment 5. Amplification 6. Labeling 7. Hybridization

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