Presentation on theme: "Epigenetic connection between nutrients and cancer"— Presentation transcript:
1 Epigenetic connection between nutrients and cancer IFCC Advanced Summer School in Biochemistry and Molecular Cell BiologyEpigenetics: Molecular Mechanisms, Biology and Human DiseasesShanghai, China, July 14, 2010
2 La Cathédrale de Rouen, le portail et la tour Saint-Romain Claude Moneteffet du matinplein soleil, midiaprès-midicoucher du soleil
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 Identical twins, Different Work-outs Monozygotic twinsIdentical twins, Different Work-outs 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:
5 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
6 HoneybeesHoneybees 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 modelMaternal 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.Figure 1. The Avy allele: map of the A locus and range of phenotypes in isogenic Avy mice. a, Avy has an IAP (subtype I I, striped box) inserted in the pseudoexon 1A of the locus, with the direction of transcription from the LTRs (arrowhead) opposite to that of the A promoters. Hair-cycle−specific non-coding exons (open boxes), coding exons (filled boxes) and an interrupted inverted repeat (grey bar arrow) are indicated. The locus is not shown to scale (100 kb separates the insertion site and hair-cycle−specific promoters). Transcription originating in a cryptic promoter (arrowhead) in the 3' LTR of the IAP in the Avy allele results in constitutive expression of agouti in multiple tissues1, 2, 4, 25. b, Isogenic C57BL/6 Avy/a mice show a continuum of phenotypes ranging from completely yellow, through degrees of yellow/agouti mottling, to completely agouti (termed pseudoagouti because the mice are isogenic with fully yellow mice and not genetically agouti). The extent of the yellow coat colour correlates closely with adult body weight. Yellow mice have pancellular agouti expression driven by the inserted IAP. Mottled mice are mosaics of cells that have or lack ectopic expression driven by the IAP. Pseudoagouti mice lack expression from the cryptic promoter, so that A is regulated by its hair-cycle promoters, and these mice have the wild-type coat colour and normal body weight1, 2, 3.Nature Genetics 1999:23:314
8 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:
9 A bridge that connects environmental factors to our genes and bring the phenotype into being.
10 EpigeneticsThe 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.While epigenetics refers to the study of single genes or sets of genes, epigenomics refers to more global analyses of epigenetic changes across the entire genome.Epigenetics is an emerging frontier of science that involves the study of changes in the regulation of gene activity and expression that are not dependent on gene sequence. For purposes of this program, epigenetics refers to both heritable changes in gene activity and expression (in the progeny of cells or of individuals) and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable.
11 Epigenome Epigenomics 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.EpigenomicsEpigenomics is the study of epigenetic modification at a level much larger than a single geneHuman Molecular Genetics 2006;15(Review Issue 1):R95
12 What is nutritional epigenetics? NutrientsEpigeneticsGenesWhat your grandma didn’t tell you about nutrition
13 What is Cancer?What is cancer? Yes, it is crab but what is the real cancer?Does anyone of you know about the real cancer?Cancer is an Abnormal accumulation of abnormal cells with a loss of control to grow and spreadThe body is made up of many types of cells. Normally, cells grow and produce more cells to keep the body healthy and functioning properly. Sometimes, however, the process goes wrong. Cells become abnormal and form more cells in an uncontrolled way. These extra cells form a mass of tissue which can be benign (non-cancer) or malignant (cancer).Collectively,Cancer develops when cells in a part of the body begin to grow out of control. Although there are many kinds of cancer, they all start because of out-of-control growth of abnormal cells.Abnormal accumulation of abnormal cells with a loss of control to grow and spread
14 Homeostasis Cell Proliferation Cell Death Regulation of Cell Cycle: Cell Cycle Check pointsControl of Apoptosis
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).
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 sequenceEpigenetic silencing: epigenetic repression of tumor suppressor or loss of imprintingHeterozygosity :The presence of different alleles at one or more loci on homologous chromosomes.Loss of heterozygosity: loss of allelic uniqueness, In a heterozygote, the loss of one of the two alleles at one or more loci in a cell lineage or cancer cell population due to chromosome loss, deletion, or mitotic crossing-over.
19 overview Epigenetics and Nutrients Nutrients, epigenetics and cancer DNA methylationHistone modificationsChromatin remodelingNutrients, epigenetics and cancerInflammation and histone modificationsFolate, epigenetics and cancerRodent hepatocellular carcinoma model of chronic dietary methyl deficiencyAlcohol, epigeneticsand cancerSummary and future perspectives
21 DNA methylationDNA 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.First, DNA methylation is a unique modification of DNA and the most common epigenetic phenomenon in eukaryotic cells.DNA methylation is associated with gene expression and gene integrity, therefore aberrations in DNA methylation play a mechanistic role in carcinogenesis.
22 DNA methylation (Cancer Res 2006;66:8462) 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)unmethylatedmethylated
23 Promoter DNA methylation Figure 4 Organisation and consequences of CpG methylation in normal and cancer cells. The upper panel shows a normal cell, in which a cluster of CG dinucleotides (CpG island) remains unmethylated (pale pins), where as scattered cytosines elsewhere are methylated (red pins). In the absence of methylation of this CpG island, DNA in the promoter region remains accessible to transcription factors, and the gene is expressed. In the lower panel, a cancer cell shows characteristic CpG island methylation, with concomitant compact chromatin structure in the promoter region, causing silencing of gene expression.Wong, J J L et al. Gut 2007;56:
24 Progressive changes in promoter methylation at CpG sites during cancer initiation and progression De novo methylation occurs at the flanking CpG sites and progressively spreads into the core of a CpG island, resulting in silencing of the corresponding gene. In general, the number of methylated loci increase in more advanced stages of cancer. ●; methylated CpG dinucleotide; , ○;unmethylated CpG dinucleotide.Nephew &,Huang, Cancer Lett. 2003;190:125
25 The possible role of DNA methylation in carcinogenesis MechanismInitiation & early progressionEpigenetic gatekeeper gene silencingActivation of normally silenced allele by loss of imprintingActivation of oncogene and chromosomal instabilityInterrelationship with histone modificationsMutationInactivation of repair geneSpontaneous deamination at methylcytosine residueCancer progressionEpigenetic plasticitySpreading of aberrant methylationMetastasisTumor microenvironmentFeinberg, Nat Rev 2006;7:21
26 One-carbon metabolism and DNA methylation MethionineMATTHFAdoMetMethylation of DNA/histone/protein/RNA/lipids/small moleculesMTsMSSHMTAdoHcymethylTHFmethyleneTHFMTHFRSAHHHomocysteineCBSAdoMet: S-adenosylmethionineAdoHcy: S-adenosylhomoscysteineMTs: methyltransferasesCBS:cystathionine beta synthaseMAT:methionine adenosyltransferaseSAHH: S-adenosylhomocysteine hydrolaseCystathionineTHF: tetrahydrofolateMTHFR: methylenetetrahydrofolate reductaseMS: methionine synthaseSHMT: 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.TRD11620Dnmt1C-richBAHReplication fociIIVVIVIIIIXXNLS1415Dnmt21908Dnmt3aPWWPATRX1859Dnmt3bPWWPATRXC-rich1421Dnmt3L
28 Nutrients that may affect DNA methylation ActionB-vitaminsFolateVitamin B-12Vitamin B-6Vitamin B-2Methyl acceptors and donors in 1-C metabolismCoenzyme for MSCoenzyme for SHMT, CBS, and cystathionaseCoenzyme for MTHFRDietary methyl donor nutrientsMethionineCholineBetaineSerinePrecursor of AdoMetHomocysteine remethylation by BHMTMethyl donor to tetrahydrofolate by SHMTMicronutrientsRetinoic acidZincSeleniumIncreases the activity of GNMTCoenzyme for MATIncreases the transsulfuration pathwayBioactive food compoundsGenisteinTea PolyphenolsInhibition of DNA methyltransferasesBHMT: betaine homocysteine methyltransferase, CBS: cystathionine ß-synthase, GNMT: glycine N-methyltransferase, MAT: methionine adenosyltransferase, MS: methionine synthase, MTHFR: methylenetetrahydrofolate reductase, SHMT: serine hydroxymethyltransferase
31 Histone tail modification Lysine acetylationArginine methylationLysine methylationPhosphorylationUbiquitination15 to 38 amino acids from each histone N terminus for the histone “tails”, providing a plat form for posttranslational modifications
32 (Georgopouos K, Nature Rev Immunol 2, 162, 2002) Histone acetylationHistone 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.HDACHATActiveInactiveHistone acetylation is a reversible post-translational process, the dynamic equilibrium of acetylation and deacetylation is controlled by HAT (histone acetylatrnasferase) and HDAC (histone deacetylase).Generally, acetylation of histone is pretty much linked to transcriptional activation whereas hypoacetylated histones are found in transcriptionally inactive regions.Hyperacetylation of histone tails influence transcriptional activity b/c 1) HAT proteins bind to DNA-binding transcriptional activators and 2) negatively charged acetyl group neutralizes the positive charge of the histone tails and decreases their affinity for negative charged DNA.(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 acetylationSince 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)
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 componentsmodulators of classic HDACAllium sativum L. (garlic)Diallyl disulfide (DADS)S-allylmercaptocysteineAllyl mercaptanDietary fiber fermentationButyrateBrassicaceae familybroccoli sproutsSulforaphanejapanese horseradish (wasabi)6-methylsulfinylhexyl-isothiocyanatemodulators of SIRTvitis vinifera (Red grapes, wines)ResveratrolRhus verniciflua (stems)ButeinRhus toxicodendron (leaves)FisetinApple, tea, onion, nuts, berries,QuercetinBlueberriesPiceatannolSweet red pepper, celery, parsleyLuteolinmodulators of HATCurcuma longa (Tumeric roots)CurcuminGarcina indica (fruit)GarcinolCamellia 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 Fig. 1. The dual competition model for longevity and senescence. Energy levels determine the activity of a protein that possesses a function important for longevity and another function important for amelioration of senescence. Sir2 (green circle) in yeast cells is illustrated in an environment of low-energy consumption (Sparta) and in an environment of high-energy consumption (Rome). Mother yeast cells are large faces that become larger with each generation. Daughter cells are small faces and start off as buds (protuberances from the mother cell). (A) Sparta. In Sparta, little food is consumed and there are two consequences at the cellular level. First, low levels of NAD are reduced so little NADH is produced and second, low levels of ROS (lightning bolt) are produced. NAD levels are sufficient to maximize Sir2-dependent histone deacetylase activity (filled green circle). Chromatin becomes more compact at specific genomic locations including telomeres and the mating locus because histone (orange balls) tails are deacetylated (acetyl groups, blue squares). Replication forks in rDNA are stabilized to reduce Rad52 epistasis group (red circle)—dependent formation of ERCs. In addition, there are only a few DNA double-strand breaks generated in the general genomic DNA since there are only scant levels of ROS. The Rad52 epistasis group can repair almost all of these breaks with high fidelity such that mutations slowly accumulate and not much Sir2 is translocated to these breaks; thus, it is available to protect rDNA. Therefore, due to modest food consumption Spartan yeast cells expend little energy, reproduce over an extended period of time, look young for their age and live a long time. (B) Rome. In Rome a lot of food is consumed and there are two consequences at the cellular level. First, high levels of NAD are reduced to make abundant NADH and second, high levels of ROS are produced. NAD levels are insufficient to maximize Sir2-dependent histone deacetylase activity (open green circle). Chromatin becomes less compact at specific genomic locations including telomeres and the mating locus because histone tails remain acetylated. Replication forks are not stabilized in rDNA allowing Rad52 epistasis group—dependent formation of ERCs. In addition, there are many DNA double-strand breaks generated in the general genomic DNA since there are high levels of ROS. The Rad52 epistasis group cannot repair all of these breaks with high fidelity such that mutations rapidly accumulate and Sir2 is translocated to these breaks leaving rDNA unprotected. Therefore, due to their excessive life style Roman yeast cells are gluttons that expend a lot of energy, reproduce quickly, look old for their age and live a short time.(Hasty, Mech Ageing Develop 2001;122:1651)
40 Histone methylationMethylation 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.Methylation occurs on the side-chain N of Lysine and Arginine by HMT (histone methyltransferases)HMTs transfer the methyl group from SAM, which is universal methyl donor for methyltransferases, to the lysine or arginine residues on histonesArginine can be either mono- or di-methylated and lysine can be mono-, di-, trimethylated.While DNA is only mono-methylated on cytosine base.This is distinct difference between DNA methylation and histone methylation.Methylation can result in either transcriptional activation or repression, depending on the residues and the pattern of other modifications.By contrast, LSD1 is highly selective for H3-K4 mono-and di-methylation but cannot demethylate H3-K4 tri-methylation
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)These are some of examples which show histone lysine methylation plays an important role in tumor suppressor gene silencing.These two studies have been done in colon cancer cells.In colon cancer cell lines, deacetylation and methylation at H3-K9 were related with promoter DNA methylation-associated MLH1 promoter.Kondo’s study in three different colon cancer cell lines, reduced H3-K4 methylation and increased H3-K9 play a critical role to maintain promoter methylation-associated silencing of p16, MLH1, MGMT.
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 demethylasePADI4 (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)
46 ATP-dependent chromatin remodeling complexes FIG. 1. Two-step model of SWI/SNF and RSC action in chromatin remodeling. The binding of the remodeling complex to chromatin is ATP independent (A). Upon ATP addition, the conformation of nucleosomes changes as a consequence of the alteration of histone-DNA interactions (B). This disruption results in remodeling of the chromatin (C), which might occur while the complex is still bound or might persist after it is released from the chromatin (indicated by the question mark). Remodeling that occurs may result in transfer of histone octamers to different DNA segments in trans or in sliding of the octamers in cis (i.e., to a different position in the same DNA molecule). The exact consequence of remodeling is likely dependent on the exact context of nucleosomes at a given promoter and can lead to either (i) activation of transcription or (ii) repression.
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
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
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
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 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
55 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;24:1269a rhizome is a horizontal stem of a plant that is usually found underground, often sending out roots and shoots from its nodes55
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;8:101HDAC 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;141:874In 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
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)
60 Evidence from animal studies-1 Animal models that develop tumors with diet alone:Methyl deficient dietDiets 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 dietWestern-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 dietMthfr+/+ and Mthfr+/- mice developed intestinal tumors after 1 year of low dietary folate.(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 Thymidylate Synthesis Methylation of DNA/histone one-carbon metabolismPurineSynthesisAdoMetMethionine*THFDHF10-formylTHFDimethylglycineSerine*SHMTB6MSB12Thymidylate SynthesisCholine*5,10-methenylTHFMethylation of DNA/histoneBetaine*glycineTSB2, B35-methylTHF5,10-methylene THFAdoHcyHomocysteineMTHFRCBSB6B6GlutathioneCystathionineCysteineTaurineMethylation pathwayNucleotide synthesis pathway*one-carbon donor nutrientsJ Nutr 2000;130:129
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 Age-adjusted CRC incidence from 1986 to 2002 in the United States (A) and Canada (B) based on nationally representative databases.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 (for Canada, the incidence is based on the average of the rates for men and women). ○, data points. A nonparametric loess smoother was fitted to the data and 95% confidence bands (gray areas) were drawn by using PROC LOESS of SAS for Windows, version 9.1.2, with its default settings.Mason J B et al. Cancer Epidemiol Biomarkers Prev
65 Animal Study Aim: Methods: To determine the effect of aging and dietary folate on DNA methylation status in the colonMethods: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 state2) 2 mg /kg: basal requirement of folate3) 8 mg /kg: folate-supplemented stateMice 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 Genomic DNA methylation in the young and old mice colon at 20 weeks p for trend=0.023****Genomic DNA methylation (%)Dietary folate groupsold vs young: * p<0.001, **p=0.032
67 p16 promoter methylation in the young and old mice colon at 20 weeks p for trend=0.009***p16 promoter methylation (%)Dietary folate groups*old vs young: p<0.001
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;49:4094During 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 dietPogribny, I. P. et al. J. Nutr. 2007;137:216S-222SAcid 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 Figure 2 Expression of Suv39h1, Suv4-20h2, and PRDM/Riz1 HMTs and HAT1 in liver of control rats and rats fed a methyl-deficient dietPogribny, I. P. et al. J. Nutr. 2007;137:216S-222SImmunoblotting 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 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
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 (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.MicroRNA biogenesisFigure 1 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. Reproduced with permission from Reference 24a.
80 Many facets of alcoholIn 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 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)
82 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 methyltransferaseThis results in a drastic waste of betaine as well as increased AdoHcy and homocysteine.Hepatology 1993;18:984
83 Effect of total folate intake and alcohol on the relative risk of breast cancer in the Nurses' Health StudyFIGURE 1 Effect of total folate intake and alcohol on the relative risk of breast cancer in the Nurses' Health Study (26). The two categories for alcohol consumption were 15 g/d and <15 g/d, the approximate quantity of alcohol in one drink of any kind. In women who consumed 15 g/d, the breast cancer risk was significantly elevated except in the highest folate intake group ( 600 µg/d), which included diet plus supplements. Folate intake was not associated with breast cancer risk for women who consumed <15 g/d. Reproduced from Zhang et al. (26) with permission.Bailey, L. B. J. Nutr. 2003;133:3748S-3753S
84 Relative risk of colon cancer in participants in the Physician's Health Study FIGURE 2 Relative risk of colon cancer in participants in the Physician's Health Study (19). Diets were classified as high methyl [high folate (750 µg/d), high methionine (2.6 g/d), low alcohol (<5 g/d)]; intermediate methyl [intermediate folate (440 µg/d), intermediate methionine (1.8 g/d), intermediate alcohol (10 g/d)]; and low methyl [low folate (200 µg/d), low methionine (1.5 g/d), high alcohol (>20 g/d)]. In men consuming the low methyl diets, colon cancer risk was increased greater than sevenfold compared with the high methyl diet.Bailey, L. B. J. Nutr. 2003;133:3748S-3753S
85 Thymidylate and purine synthesis Methylation of DNA/protein Alcohol and one-carbon metabolismMATAdoMetMethionineTHF?Thymidylate and purine synthesisDimethylglycineMTsSerineSHMTMethylation of DNA/protein(histone)/RNA/lipidsMSB12B6CholineglycineBetaine5-methylTHF5,10-methylene THFAdoHcyHomocysteineMTHFRCBSB6B6CystathionineCysteineGlutathioneMethylation pathwayNucleotide synthesis pathway(J Nutr 2000;130:129, Biochem Pharm 1994;47:1561)
86 Hypothesis Alcohol disturbs methyl transfer in one-carbon metabolism Aberrations in DNA methylation and histone modificationsAlters carcinogenesis
87 Chronic alcohol consumption induces genomic DNA hypomethylation in the rat colon. Animal StudyTo determine the effect of chronic alcohol consumption on DNA methylation in the colonTwenty 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 PlasmaGroupsAlcohol-fed ratsControl ratsHomocysteine (µmol/L)17.23 ± 4.63*10.73 ± 2.76Folate (nmol/L)250.8 ± 61.5295.1 ± 38.4PLP (nmol/L)± 85.15± 89.09Vitamin B-12 (pmol/L)62.91 ± 21.4676.58 ± 12.54LiverGroupsAlcohol-fed ratsControl ratsAdoMet (nmol/g liver)34.1 ± 5.6*48.8 ± 13.9AdoHcy (nmol/g liver)14.8 ± 1.7*10.4 ± 2.7Folate (nmol/g liver)29.3 ± 17.035.6 ± 12.3All values are mean ± SD, n=10, *Significantly different from control rats, p<0.05Alcohol Clin Res Exp, 2000;24:259
89 Genomic DNA methylation in the colonic DNA from alcohol-fed rats *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
90 Mice studyYoung 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.OldYoungDiet5 week10 weekControl4.58 ± 0.064.37 ± 0.08*4.37 ± 0.154.75 ± 0.0818% EtOH4.52 ± 0.064.60 ± 0.064.67 ± 0.074.44 ± 0.10#18% EtOH and low folate4.22 ± 0.12†4.31 ± 0.114.61 ± 0.094.63 ± 0.07DNA 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 1. Alcohol dose-dependent and time-dependent acetylation of histone H3 lysine 9 (H3-K9) acetylation in rat hepatic stellate cells(Alcohol Alcohol :367)Figure 2. H3-K9 acetylation in the rat liver after binge drinking(Alcohol Alcohol 2006;41:126)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 hepatocytesIn the northern blot assay Alcohol dehydrogennase-1 (Adh-1) and GlutathionS-transferase Yc2 (GST-Yc2)] wereupregulated and l-serine dehydratase (Lsdh) and Cytochrome P450 2C11 (CYP2C11) were down regulated by ethanol.CHIP assay demonstrating association of histone H3me2K9 and H3me2K4 methylation at the promoter of the genes up- and downregulated by ethanol in hepatocytes.Treatment of hepatocytes with ethanol reduces H3 Lysine-9 methylation with subsequent increase of H3 Lysine-4 methylation in the regulatory region of the upregulatory genes. Whereas, in down regulatory genes, the dimethyl Lysine-9 accumulated at the promoter with negligible trace of Lysine-4 methylated histone H3Figure 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 Reduced methyl availability and inhibition of one-carbon metabolism by alcohol NCM460, Human colonic epithelial cell lineDMEM + 10% fetal bovine serumAdded 100mmol/L EthanolCultured cells for 0, 24, 48, 72h
96 Time-dependent trimethylation of histone H3 at Lys4 (H3K4me3) by ethanol ControlEthanol for 24hEthanol for 48hEthanol for 72hTSA for 24h
97 Time-dependent acetylation of histone H3 at Lys9 (H3K9ac) by ethanol 55.54p<=0.0001H3K9acH4ControlEthanol for 24hEthanol for 48hEthanol for 72hTSA for 24h4.5
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.
105 ChIP assayChromatin 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 Chromatin immunoprecipitations (ChIPs) How do we measure distribution of histone modifications at specific loci?Chromatin immunoprecipitations(ChIPs)Formaldehyde treat live cells to crosslink proteins and DNAShear chromatin into small fragments (~ 500bp)Prepare cell extractImmunoprecipitate wi Ab of interestReverse cross-linksAnalyze immunoprecipitated DNA by quantitative PCR
107 Immuno-preciptation with specific antibody Chromatin purification Schematic of ChIP500 bpSonicationwith formaldehydeCrosslinkingImmuno-preciptation with specific antibodyChromatin purificationK562InputNo ABUSF IITF II INF-E2PCR of genomic fragmentsYReverse of CrosslinkingDNA purification
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 ( bp).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 p16 gene specific histone modifications (an example for ChiP assay) No Ab controltriM3H3K4triMeH3K9AcH3K9Input DNACONTROL100uM Adox for 24hr100uM Adox for 48hr100uM Adox for 72hr5uM ADC for 72hr
110 0.20.40.60.81H3K4me3H3K9me3CONTROL100uM Adox for24hr48hr72hrratio (precipitated DNA/input DNA)Input DNANo Ab control234Figure 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 promoterH3K9 methylation of p16 promoterH3K4 methylation of p16 promoterFIG. 1. Examples of histone H3 lysine 9 CHIP assays. (A) Schema of the P16, MLH1, and MGMT promoter regions. The distribution of CpG sites is represented by vertical bars. Two Alu sequences are upstream of the P16 promoter region; arrows indicate transcription initiation sites. Lines shown below each promoter indicate regions amplified by each set of PCR primers. To the right of each gene, methylation status is indicated by filled circles (fully methylated), partially filled circles (partially methylated), and unfilled circles (unmethylated) in the three cell lines studied. (B) Examples of CHIP assays using anti-acetylated histone H3 Lys-9 antibody. (C) Examples of CHIP assays using anti-methylated histone H3 Lys-9 antibody. (D) Examples of CHIP assays using anti-methylated histone H3 Lys-4 antibody. In these assays, DNA combined with acetylated or methylated histone H3 Lys-9 antibody is immunoprecipitated and detected by PCR amplification. In panel B, note that the histone deacetylase inhibitor TSA induces moderate increased Lys-9 acetylation while the combination of the DNA methyltransferase inhibitor 5Aza-dC and TSA induces remarkable Lys-9 hyperacetylation. In panel C, note that 5Aza-dC and a combination of 5Aza-dC and TSA remarkably decrease Lys-9 methylation. In panel D, note that 5Aza-dC and a combination of 5Aza-dC and TSA remarkably increase Lys-4 methylation. IN, input DNA from whole-cell lysate. The intensities of the bands of PCR products were quantitated by densitometry or using the Agilent 2100 Bioanalyzer.DNA methylation of p16 promoter(Kondo, Mol Cell Biol 2003;23:206)
112 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
113 Histone extractionThe 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.
114 Western blot analysis of methylation status of histone H3-Lys9 and histone H4-Lys20 Fig. 3. Western blot analysis of methylation status of histone H3-Lys9 and histone H4-Lys20. Acid extracts of total histones were separated by SDS-PAGE and subjected to immunoblotting using specific antibodies against trimethyl histone H3-Lys9 and trimethyl histone H4-Lys20. Results are presented as change in methylation relative to control animals and shown as the mean ± SD. Gray bars—trimethyl histone H3-Lys9; black bars-trimethyl histone H4-Lys20. Equal sample loading was confirmed by immunostaining against histone H3 and histone H4. These results were reproduced in two independent experiments. Representative immunoblot images are shown. *Significantly different from control.Pogribny, Carcinogenesis 2006;27:1180
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.
125 p16 promoter methylation in mice colon U M U M U Mp16 promoter methylation in old mice colon after 20 weeks of folate deplete diet: partially methylatedU M U M U M.p16 promoter methylation in young mice colon after 20 weeks of folate deplete diet: unmethylated
126 p16 promoter methylation in the young and old mice colon at 20 weeks p for trend=0.04p=0.03p=0.05**** p<0.05 old vs young
127 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)
128 p16 gene specific histone modifications (an example for ChiP assay) No Ab controltriM3H3K4triMeH3K9AcH3K9Input DNACONTROL100uM Adox for 24hr100uM Adox for 48hr100uM Adox for 72hr5uM ADC for 72hr
129 ChIP-chip Protocol 1. Sample cross-linking and fragmentation 2. Immunoprecipitation3. Enrichment4. Amplification5. Labeling6. Hybridization7. Data AnalysisChIP-chip ProtocolSample Cross-linking and Fragmentation: Chromatin preparations are crosslinked to preserve protein/DNA interactions, and then fragmented by sonication to produce low molecular weight fragments ( bp).Immunoprecipitation: Remove control input DNA sample aliquot (save for step 4) and immunoprecipitate remaining chromatin sample with a ChIP-grade antibody directed against target protein.Enrichment: Antibody-chromatin complexes are affinity enriched and unbound chromatin fragments are washed away.Amplification: Immunoaffinity-enriched DNA (IP) fragments and the input sample are purified and amplified using whole-genome amplification (WGA)* or ligation mediated-PCR (LM-PCR) to obtain adequate amounts of DNA for labeling.Labeling: IP and input samples are labeled in separate reactions with Cy5 and Cy3, respectively.Hybridization: IP and input samples are pooled and co-hybridized to a NimbleGen ChIP-chip array.Data Analysis: Data is extracted using NimbleScan software, the ratio of IP versus input sample is calculated and peaks are called from ratio data and mapped to the transcription start site of a gene.
130 DNA methylation microarray 1. Sample fragmentation 2. DNA denaturation3. Immunoprecipitation4. Enrichment5. Amplification6. Labeling7. Hybridization