Epigenetic connection

1
Epigenetic connection between nutrients and
cancer
IFCC Advanced Summer School in Biochemistry and
Molecular Cell Biology Epigenetics Molecular
Mechanisms, Biology and Human Diseases Shanghai,
China, July 14, 2010
2
La Cathédrale de Rouen, le portail et la tour
Saint-Romain Claude Monet
effet du matin
plein soleil, midi
après-midi
coucher du soleil
3
Epigenetics DNA isnt 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
Identical twins, Different Work-outs 
There are relatively few differences between the
twins when theyre 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 200510210604
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 20083191827

7
Agouti mouse model
Maternal methyl dietary contents affect the coat
color of the rodent offspring and alter the
susceptibility of the animal to certain chronic
diseases, obesity and cancer.
Nature Genetics 199923314
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 200644401-406

9
A bridge that connects environmental factors to
our genes and bring the phenotype into being.
10
Epigenetics

• The word epigenetics refers to genome
  information that is super-imposed on the DNA
  sequence.
• In the early 1940s Waddington described
  epigenetics as the interactions of genes with
  their environment, which bring the phenotype into
  being.
• Inheritable biological phenomena that modify DNA
  or chromatin structures, thereby affecting gene
  expression without altering base pairs.
• Epigenetic phenomena are critical for the
  embryonic development, aging, and the process of
  many diseases including cancers.
• Epigenetic phenomena are reversible and can be
  modulated by nutrients.

11
Epigenomics
Epigenome

• The epigenome is a catalog of the epigenetic
  modifications that occur in the genome.
• An epigenome can be described as the epigenomic
  profile of a specific cell or tissue type which
  reflects its biological condition or state and
  defines its transcriptional potential.

• Epigenomics is the study of epigenetic
  modification at a level much larger than a single
  gene

Human Molecular Genetics 200615(Review Issue
1)R95
12
What is nutritional epigenetics?
Genes
What your grandma didnt tell you about nutrition
13
What is Cancer?
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 points
Control of Apoptosis
15
Neoplasm
Cell Proliferation
Cell Death
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
Neoplasm
Tumor suppressor gene
Cell Proliferation
Oncogene gene
Cell Death
Tumor suppressor 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
overview

• Epigenetics and Nutrients
• DNA methylation
• Histone modifications
• Chromatin remodeling
• Nutrients, epigenetics and cancer
• Inflammation and histone modifications
• Folate, epigenetics and cancer
• Rodent hepatocellular carcinoma model of chronic
  dietary methyl deficiency
• Alcohol, epigeneticsand cancer
• Summary and future perspectives

20

• DNA methylation

21
DNA methylation

• DNA methylation is a unique modification of DNA
  and the most common epigenetic phenomenon in
  eukaryotic cells.
• DNA methyltransferases catalyze the transfer of
  methyl group from S-adenosylmethionine (AdoMet)
  to the carbon-5 position of cytosine in CpG
  dinucleotides.

22
DNA methylation

• 3-5 of cytosine residues in genomic DNA are
  modified to 5-methylcytosine in CpG
  dinucleotides.
• 70 of CpG dinucleotide sequences, which usually
  occur in CpG islands, contain 5-methylcytosine.
• DNA methylation is associated with gene
  expression and integrity.
• Aberrations in DNA methylation play a mechanistic
  role in carcinogenesis.
• (Cancer Res 2006668462)

unmethylated
methylated
23
Promoter DNA methylation
Wong, J J L et al. Gut 200756140-148
24
Progressive changes in promoter methylation at
CpG sites during cancer initiation and
progression
Nephew ,Huang, Cancer Lett. 2003190125
25
The possible role of DNA methylation in
carcinogenesis
Feinberg, Nat Rev 2006721
26
One-carbon metabolism and DNA methylation
Methionine
MAT
THF
AdoMet
Methylation of DNA/histone/ protein/RNA/ lipids/sm
all molecules
MTs
SHMT
MS
AdoHcy
methylTHF
methyleneTHF
MTHFR
SAHH
Homocysteine
CBS
AdoMet S-adenosylmethionine AdoHcy
S-adenosylhomoscysteine MTs methyltransferases CB
Scystathionine beta synthase MATmethionine
adenosyltransferase SAHH S-adenosylhomocysteine
hydrolase
Cystathionine
THF tetrahydrofolate MTHFR methylenetetrahydrofo
late 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.

C-rich
28
Nutrients that may affect DNA methylation
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
Lysine acetylation Arginine methylation Lysine
methylation Phosphorylation Ubiquitination
15 to 38 amino acids from each histone N
terminus for the histone tails, providing a
plat form for posttranslational modifications
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.

(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 197451786)
• 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 200537391)

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 200523635)
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 200627344)

37
Examples of dietary compounds able to modulate
HAT or HDAC activities
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 20011221651)
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 2001152343)
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 2002627213)
• 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
  200323206)

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 20022469, Nature Reviews
Genetics 200910295)
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 200835551)

45

• Chromatin remodeling

46
ATP-dependent chromatin remodeling complexes
47
(No Transcript)
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 2001841372 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 2007252191

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

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 2004170141, PNAS
  2002998921

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
NF?B transcriptional activity and
pro-inflammatory gene expression. In contrast,
glucocorticoids and natural chromatin-modifying
agents trigger HDAC recruitment and HAT
inhibition, which results in NF?B 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 2003425191, Am J Physiol Lung
  Cell Mol Physiol 2007292L567

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 20062169, J Biol Chem 20032782758 ,
  J Clin Immunol 2007 2719, Carcinogenesis
  2003241269

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 20058101
• 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 2004141874
• 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 20072719

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
  1992525002)
• 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 2000605434)

60
Evidence from animal studies-1

• Animal models that develop tumors with diet
  alone
• Methyl deficient diet
• Diets that are deficient in methionine, choline,
  folate and B-12 lead to spontaneous development
  of liver cancer with hepatic DNA hypomethyation.
  
• (Cancer Res 1989494094)
• 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. 2001221871)
• Folate deficient diet
• Mthfr/ and Mthfr/- mice developed intestinal
  tumors after 1 year of low dietary folate.
• (Knock, Cancer Res 20066610349)

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 200027529318)

62
one-carbon metabolism
Purine Synthesis
Methionine
THF
DHF
AdoMet
10-formyl THF
Dimethyl glycine
Serine
SHMT

B6
Thymidylate Synthesis
B12
MS
5,10- methenyl THF
Choline
Methylation of DNA/histone
glycine
Betaine
TS
B2, B3
5-methyl THF
5,10-methylene THF
AdoHcy
Homocysteine
MTHFR
B6
CBS
Glutathione
B6
Cysteine
Cystathionine
Taurine
Methylation pathway
Nucleotide synthesis pathway
one-carbon donor nutrients
J Nutr 2000130129
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.
Mason J B et al. Cancer Epidemiol Biomarkers Prev

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, n32) and 18 month old
  (n34) 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 1371713)
66
Genomic DNA methylation in the young and old mice
colon at 20 weeks
p for trend0.023



Genomic DNA methylation ()
Dietary folate groups
old vs young plt0.001, p0.032
67
p16 promoter methylation in the young and old
mice colon at 20 weeks
p for trend0.009



p16 promoter methylation ()
Dietary folate groups
old vs young plt0.001
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 1989494094
• 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 199711531

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
Figure 1 Western blot analysis of histone H3 and
H4 modifications in liver of control rats and
rats fed methyl-deficient diet
Pogribny, I. P. et al. J. Nutr. 2007137216S-222S
Acid extracts of total histones were separated by
SDS-PAGE and subjected to immunoblotting using
primary antibodies against H3K9me3, H3K9me2,
H3K9me1, H3K9 ac, and H3S10ph (A) and H4k20me3,
H4K20me2, H4K20me1, and H4K16 ac (B),
respectively. Results are presented as change
relative to age-matched control rats.
Significantly different from control at the same
(n 5, means SEM).
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 diet
Pogribny, I. P. et al. J. Nutr. 2007137216S-222S
Immunoblotting using primary antibodies against
Suv420h2, Suv39h1, PRDM/Riz1, and HAT1. The
lower part of the figure shows a quantitative
evaluation of the Suv420h2, 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 (n5, meansSEM).
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 200848479
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 20073021, Cell Cycle
  20087602, Mol Carcinog 200948479

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
1924-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 biogenesis
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
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 20072746317, Hepatology
199318984, Alcohol Clin Exp Res 2005292188,
Biochem Pharmacol 1994 471561, Nutrition
200016296, JCI 197453693, Alcohol 199815305)
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 methyltransferase
• This results in a drastic waste of betaine as
  well as increased AdoHcy and homocysteine.
• Hepatology 199318984

83
Effect of total folate intake and alcohol on the
relative risk of breast cancer in the Nurses'
Health Study
Bailey, L. B. J. Nutr. 20031333748S-3753S
84
Relative risk of colon cancer in participants in
the Physician's Health Study
Bailey, L. B. J. Nutr. 20031333748S-3753S
85
Alcohol and one-carbon metabolism
MAT
Methionine
AdoMet
THF
?
Thymidylate and purine synthesis
Dimethyl glycine
MTs
Serine
SHMT

Methylation of DNA/protein (histone)/RNA/ lipids
B12
MS
B6
Choline
glycine
Betaine
5-methyl THF
5,10-methylene THF
Homocysteine
AdoHcy
MTHFR
B6
CBS
B6
Glutathione
Cysteine
Cystathionine
Methylation pathway
Nucleotide synthesis pathway
(J Nutr 2000130129, Biochem Pharm 1994471561)
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 1291945
88
Effect of chronic alcohol consumption on
one-carbon metabolism in rats
Plasma
Liver
All values are mean SD, n10, Significantly
different from control rats, plt0.05
Alcohol Clin Res Exp, 200024259
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 (plt0.05).
J Nutr 19991291945
90
Mice study

• Young and old C57B6 mice (n10 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 EtOHlow
folate level for 5 and 10 weeks.
DNA methylation is significantly lower in overall
old mice compared to the all young mice
(4.430.04 vs 4.580.04, plt0.02).
Significantly different from corresponding young
mice (plt0.02) Different tendency from
corresponding young mice (p0.08) Different
tendency from young control mice (p0.08) 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 EtOHlow folate for 5 and 10 weeks.
Values are meansSEM (p old vs. young
lt0.001).  
93
Effect of alcohol on histone acetylation
Figure 2. H3-K9 acetylation in the rat liver
after binge drinking (Alcohol Alcohol
200641126)
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 40367)
Alcohol modulates H3K9 acetylation via increasing
HAT activity. (Alcohol Clin Exp Res
2008321)
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. 200781979)
95
Reduced methyl availability and inhibition of
one-carbon metabolism by alcohol

• NCM460, Human colonic epithelial cell line
• DMEM 10 fetal bovine serum
• Added 100mmol/L Ethanol
• Cultured cells for 0, 24, 48, 72h

96
Time-dependent trimethylation of histone H3 at
Lys4 (H3K4me3) by ethanol
97
Time-dependent acetylation of histone H3 at Lys9
(H3K9ac) by ethanol
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.

100
Cheeeeeeeeers !!!!!
sang.choi_at_tufts.edu
101
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102
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103
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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
PCR of genomic fragments
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
p16 gene specific histone modifications (an
example for ChiP assay)
No Ab control
triM3H3K4
triMeH3K9
AcH3K9
Input DNA
CONTROL
100uM Adox for 24hr
100uM Adox for 48hr
100uM Adox for 72hr
5uM ADC for 72hr
110
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
H3K4 methylation of p16 promoter
(Kondo, Mol Cell Biol 200323206)
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 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 4C, 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
Pogribny, Carcinogenesis 2006271180
115
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116
A method to assess genomic DNA methylation using
HPLC/ESI/MS
HPLC
DNA
LC/MS interface
hydrolysis
CH3
ESI-SOURCE
CH3
ION TRAP MS
CH3
m/z112
m/z126
H
H
(Friso, Analyt Chem 2002744526)
117
Four ion peaks
118
Genomic DNA methylation in the young and old mice
colon at 20 weeks
p for trend0.04


plt0.05 old vs young
Keyes et al. J Nutr in press
119
Plt0.042
Genomic DNA methylation (mCyt ng /µg DNA)
(n13)
(n13)
Friso et al. Br J Nutr 200797617
120
Question?
A
400
300
m/z 112.1
Abundance (x103)
200
100
m/z 114.9
0
0
2
4
8
10

6
12
B
400
300
Abundance (x103)
m/z 126.1
200
100
m/z 130.1
0
0
2
4
8
10
Time (min)
6
C
D
NH2
NH2
H3C
N
N
m/z 126.1
m/z 112.1
O
O
N
N
H
H
Choi et al. Mol Biochem Parasitology
2006150350
121
no peak at 126
122
Gene-specific DNA methylation
123
Principle of bisulfite modification
Unmethylated DNA
ggg gcg gac cgc
bisulfite modification
ggg gug gau ugu
Methylated DNA
ggg gcmg gacm cmgcm
bisulfite modification
ggg gcmg gacm cmgcm
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 M



p16 promoter methylation in old mice colon after
20 weeks of folate deplete diet partially
methylated


U M U M U M


.


p16 promoter methylation in young mice colon
after 20 weeks of folate deplete diet
unmethylated

126
p16 promoter methylation in the young and old
mice colon at 20 weeks
p for trend0.04
p0.03
p0.05



plt0.05 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 control
triM3H3K4
triMeH3K9
AcH3K9
Input DNA
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