CpG methylation of gene promoter in the mouse liver by b8z9wwC6

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									Epigenetic regulation of liver specific
        genes in the mouse




      Laboratory Animal Research Center
       Ajou University School of Medicine
           Jin Bo Hwan, D.V.M, PhD
                  Epigenetics
• In Biology, the study of heritable changes in gene
  expression or cellular phenotype caused by
  mechanisms other than changes in the underlying
  DNA sequence

• Robin Holliday : "the study of the mechanisms of
  temporal and spatial control of gene activity during the
  development of complex organisms
                  Epigenetics
• Changes in gene expression caused by other
  mechanisms than changes in the underlying DNA
  sequence

• These changes may remain through cell divisions for
  the remainder of the cell's life and may also last for
  multiple generations.
                  Epigenetics
• Changes in gene expression caused by other
  mechanisms than changes in the underlying DNA
  sequence

• These changes may remain through cell divisions for
  the remainder of the cell's life and may also last for
  multiple generations.
       Epigenetic Mechanisms
• DNA methylation is the addition of methyl groups to
  the DNA, at CpG sites, to convert cytosine to 5-
  methylcytosine.

• Chromatin remodeling is post translational
  modification of the amino acids that make up histone
  proteins.

• The best example of epigenetic changes in eukaryotic
  biology is the process of cellular differentiation.
Epigenetic Mechanism
Source : NIH, 2005
(http://commonfund.nih.gov/epigenomics/figure.aspx)
        Epigenetic Mechanisms
•   Several layers of regulation of gene expression.

• One way that genes are regulated is through the
  remodeling of chromatin. Chromatin is the complex of
  DNA and the histone proteins with which it associates.

• Histone proteins are little spheres that DNA wraps
  around. If the way that DNA is wrapped around the
  histones changes, gene expression can change as
  well.
         Epigenetic Mechanisms
•    Chromatin remodeling is accomplished through two
    main mechanisms
     1. Post translational modification of the amino acids that
    make up histone proteins. Histone proteins are made up
    of long chains of amino acids. If the amino acids that are
    in the chain are changed, the shape of the histone
    sphere might be modified. DNA is not completely
    unwound during replication. It is possible, then, that the
    modified histones may be carried into each new copy of
    the DNA. Once there, these histones may act as
    templates, initiating the surrounding new histones to be
    shaped in the new manner.
     Epigenetic Mechanisms
2. The addition of methyl groups to the DNA, mostly at
CpG sites, to convert cytosine to 5-methylcytosine. 5-
Methylcytosine performs much like a regular cytosine,
pairing up with a guanine. However, some areas of the
genome are methylated more heavily than others, and
highly methylated areas tend to be less transcriptionally
active, through a mechanism not fully understood.
Methylation of cytosines can also persist from the germ
line of one of the parents into the zygote, marking the
chromosome as being inherited from this parent (genetic
imprinting).
Nucleosome structure.
 Source : By Richard Wheeler (Zephyris) 2005
 Epigenetics and environmental
             health
• Genomic imprinting and related disorders
  Some human disorders are associated with genomic
  imprinting, a phenomenon in mammals where the
  father and mother contribute different epigenetic
  patterns for specific genomic loci in their germ cells.
• Cancer
  A variety of compounds are considered as epigenetic
  carcinogens-they result in an increased incidence of
  tumors, but they do not show mutagen activity
  Examples include diethylstilbestrol, arsenite,
  hexachlorobenzene, and nickel compounds.

• Developmental abnormalities
  Many teratogens exert specific effects on the fetus by
  epigenetic mechanisms.
• Genomic imprinting
  A genetic phenomenon by which certain genes are
  expressed in a parent-of-origin-specific manner.
  An epigenetic process that involves methylation and
  histone modifications in order to achieve monoallelic
  gene expression without altering the genetic sequence.
Genetic imprinting.
 Source : The Australasian
Genetics Resource Book 2007
Genetic imprinting.
 Source : The Australasian
Genetics Resource Book 2007
             DNA Methylation
• DNA methylation is a type of
  chemical modification of DNA that
  can be inherited without changing
  the DNA sequence.

• DNA methylation involves the
  addition of a methyl group to
  DNA(the number 5’ carbon of the
  cytosine pyrimidine ring).
• In most vertebrates, 80% of cytosine residues within
  the dinucleotide sequence CpG are modified by
  methylation.


• DNA methylation is known to silence gene
  transcription either by preventing protein binding or by
  indirect mechanisms involving changes in chromatin
  structures.
• DNA methylation may impact the transcription of
  genes in two ways.

  1) the methylation of DNA may itself physically impede
  the binding of transcriptional proteins to the gene

  2) methylated DNA may be bound by proteins known
  as Methyl-CpG-binding domain proteins (MBDs).
          Bisulfite Sequencing
• Bisulfite sequencing : Treatment of DNA with bisulfite
  converts cytosine residues to uracil, but leaves 5-
  methylcytosine residues unaffected.
                   Acetylation
• A reaction that introduces an acetyl functional group
  into an organic compound. Deacetylation is the
  removal of the acetyl group.

• Moreover, it is that process of introducing an acetyl
  group (resulting in an acetoxy group) into a compound,
  specifically, the substitution of an acetyl group for an
  active hydrogen atom.
       N-α-terminal Acetylation
• Acetylation of the N-terminal α-amine of proteins is a
  widespread modification in eukaryotes. 40-50% of yeast
  proteins, and 80-90% of human proteins are modified in
  this manner, and the pattern of modification is found to
  be conserved throughout evolution.

• The modification is performed by N-α-acetyltransferases
  (NATs), which also include histone acetyl transferases.
        Lysine Acetylation and
            Deacetylation
• In histone acetylation and deacetylation, the histones
  are acetylated and deacetylated on lysine residues in
  the N-terminal tail as part of gene regulation.

• These reactions are catalyzed by enzymes with
  "histone acetyltransferase" (HAT) or "histone
  deacetylase" (HDAC) activity.
• Not only can the acetylation state of a protein modify
  its activity, there has been recent suggestion that this
  post-translational modification might crosstalk with
  phosphorylation, methylation, ubiquitination,
  sumoylation, and others for dynamic control of cellular
  signaling.
Epigenetic Regulation of CYP1A2 in
           Mouse Liver



   1. Jin B, Park DW, Nam KW, Oh GT, Lee YS, Ryu DY.
   CpG methylation of the mouse CYP1A2 promoter. Toxicol
   Lett. 2004 Aug 30;152(1):11-8.
   2. Jin B, Ryu DY. Regulation of CYP1A2 by histone
   deacetylase inhibitors in mouse hepatocytes. J Biochem
   Mol Toxicol. 2004;18(3):131-2.
                   Introduction
• Cytochrome P450 monooxygenases
  ► a major role in the oxidative activation
  ► inactivation of a wide range of xenobiotics.

• Cytochrome P450 1A2 (CYP1A2) is expressed
  preferentially in the liver of mammals.

• CYP1A2 is inducible after exposure to environmental
  compounds such as aromatic hydrocarbons and
  2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
• Cytochrome P450 1A2 promoter region
• 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
  The most potent compound of the series and became
  known as a contaminant in Agent Orange, a herbicide
  used in the Vietnam War, as well as the Seveso
  disaster.
  It is a persistent environmental contaminant usually
  present in a complex mixture of dioxin-like compounds.
• 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
  There is barely any organ without some effects by
  high doses of TCDD.
  In short-term toxicity studies in animals the typical
  effects are anorexia and wasting, and even after a
  huge dose animals die only 1 to 6 weeks after the
  TCDD administration.
  TCDD also affects the balance of several hormones.
  Taking into account the low doses of dioxins in the
  present human population, only two types of toxic
  effects have been considered to cause a relevant risk
  to humans: developmental effects and cancer.
                    Methods
• Animals
  C57BL/6 male mice, 4 – 5 weeks old.

• Isolation of primary hepatocytes
  Hepatocytes were isolated from the mouse liver using
  a modification of the C.A. McQueen’s method(Method
  in toxicology, vol1A, 1993).
• Cell culture and treatments of cells
  Hepa1c1c7 cell line was obtained from Korean Cell
  Line Bank (Seoul, Korea). Cells were treated with
  trichostatin A (TSA) and sodium butyrate (SB) for 24
  hours prior to harvesting.

• Bisulfite sequencing analysis
  2 mg of genomic DNA was diluted in a freshly
  prepared NaOH solution and denatured at 40°C for 30
  min. The denatured DNA was mixed in 2.5 M sodium
  bisulfite/20mM hydroquinone.
                          Results
• Regulation of CYP1A2 in
  hepatocytes exposed to
  TCDD and each histone
  deacetylase inhibitor.
  Hepatocytes isolated from
  C57Bl/6 male mouse liver
  were cultured up to 192 h,
  and were treated with TCDD
  (2nM) and either
  Trichostatin A (50nM) or
  Sodium butylate (50mM) for
  24 h before harvest.
• Methylation profile of 18 CpG sites in the Cyp1a2 promoter in mouse
  tissues.
• Developmental expression of CYP1A2 mRNA and the
  methylation profile in C57BL/6 mouse liver
• Methylation profile in the Cyp1a2 promoter in mouse hepatoma
  Hepa1c1c7 cell line.
• CYP1A2 expression and the methylation of CpGs within the Cyp1a2
  promoter in primary hepatocytes in culture.
                   Discussion
• CpG methylation is involved in the tissue-specific and
  developmetal regulation of CYP1A2 in the liver, and
  that the delayed de nove methylation of the CYP1A2
  promoter is a non-specific event that occurs after
  gene silencing in hepatocytes.

• Histone acetylation has a important role in the
  regulation of CYP1A2 in a pathway that is
  independent form, and possible conflicts with, AhR.
    Microarray analysis of gene
  regulation in the Hepa1c1c7 cell
line following exposure to the DNA
   methylation inhibitor 5-aza-2’-
     deoxycytidine and 2,3,7,8-
    tetrachlorodibenzo-p-dioxin

Jin B, Kim G, Park DW, Ryu DY. Microarray analysis of gene regulation
in the Hepa1c1c7 cell line following exposure to the DNA methylation
inhibitor 5-aza-2'-deoxycytidine and 2,3,7,8-tetrachlorodibenzo-p-
dioxin. Toxicol In Vitro. 2004 Oct;18(5):659-64.
                   Introduction
•   Principles of toxicogenomics are being applied to the
    prediction of toxic potential and the development of
    screening systems for untested chemicals which are
    based upon their capacity to alter transcriptional
    programs

•   cDNA microarray technology highthroughput
    measurement of transcriptional changes that occur
    as a consequence of xenobiotic exposure is
    facilitating the elucidation of toxicological
    mechanisms
                    Methods
• Microarray analysis
  Gene expression chips containing 7400 mouse genes
  were used to investigate global changes in gene
  expression.

  Fluorescent intensities of the printed cDNA targets
  were measured using a GenePix 4000 microarray
  scanner, and the log ratios of fluorescent intensities
  within each slide were adjusted for data normalization.
• Microarray analysis
  Gene expression chips containing 7400 mouse genes
  were used to investigate global changes in gene
  expression.

  Fluorescent intensities of the printed cDNA targets
  were measured using a GenePix 4000 microarray
  scanner, and the log ratios of fluorescent intensities
  within each slide were adjusted for data normalization.
Results
 • List of genes up-regulated in the Hepa1c1c7 cell
 line following exposure to AzaC and TCDD
                                                                                       Fold increase
           Gene
                     ID         Gene Name                                                              AzaC/
           symbol                                                                      AzaC TCDD
                                                                                                       TCDD

Group I    Atf2      AA445861   activating transcription factor 2                       0.84    4.34   3.69

           Cpo       AA259342   coproporphyrinogen oxidase                              1.01    2.80   2.32

           Fsp27     AA466094   fat specific gene 27                                    1.25    2.64   2.38

           Nqo1      AA068375   NAD(P)H dehydrogenase, quinone 1                        1.20    4.42   3.17

           Ramp3     AA387854   receptor (calcitonin) activity modifying protein 3      1.06    2.43   4.30

           Tm4sf3    AI892302   transmembrane 4 superfamily member 3                    1.28    2.48   2.92

           Xdh       AA472074   xanthine dehydrogenase                                  0.96    2.53   2.74

Group II   Arhgef6   AA140540   Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6    2.27    2.91   2.69

           Osmr      AA265259   oncostatin receptor                                     1.77    1.71   3.62

           Pdgfrl    AA030377   platelet-derived growth factor receptor-like            2.26    1.66   2.61

           Prkcd     AI893988   protein kinase C, delta                                 2.11    1.57   3.12

           Son       W97212     Son cell proliferation protein                          3.30    2.39   5.28

           Tnc       AA270625   tenascin C                                              2.34    1.68   2.66
                                                                                                              Fold increase
            Gene
                      ID         Gene Name                                                                                    AzaC/
            symbol                                                                                            AzaC TCDD
                                                                                                                              TCDD

Group III   Bhmt2     AA272010   betaine-homocysteine methyltransferase 2                                      4.77   0.84     4.99

            Ckmt2     AA038095   creatine kinase, mitochondrial 2                                              3.75   0.82     2.65

            Clcn2     AI430254   chloride channel 2                                                            3.09   0.78     2.43

            Cml1      AA124476   camello-like 1                                                                2.90   0.87     2.41

            Eral1     AA261675   Era (G-protein)-like 1 (E. coli)                                              2.00   0.81     2.94

            Flt4      AA086840   FMS-like tyrosine kinase 4                                                    3.01   1.14     1.42

            Frat1     AA200201   frequently rearranged in advanced T-cell lymphomas                            2.58   0.69     1.45

            Gys2      AA537291   glycogen synthase 2                                                           2.93   0.91     2.29

            Hdgfrp2   AI425908   Hepatoma-derivedgrowthfactor,relatedprotein2                                  2.77   0.72     2.86

            Hs3st1    W62484     heparan sulfate (glucosamine) 3-O-sulfotransferase 1                          2.64   1.33     1.97

            Lrrc2     AA416235   leucine-rich repeat-containing 2                                              4.04   0.82     2.79

            Mthfd1    AI322702   methylenetetrahydrofolate dehydrogenase (NADP+ dependent)                     2.87   0.98     3.03

            Pdgfa     AA242310   platelet derived growth factor, alpha                                         2.80   1.23     2.18

            Pitrm1    AA003165   pitrilysin metalloprotease 1                                                  2.75   0.83     2.27

            Ptges     AA178132   prostagladin E synthase                                                       1.75   0.99     3.48

            Slc1a2    AA238533   solute carrier family 1, member 2                                             2.25   0.97     2.59

            Smarca5   AA415240   SWI/SNF related, matrix associated, actin dependent regulator of chromatin    2.79   1.02     2.02

            Sp1       AA183548   trans-acting transcription factor 1                                           2.79   0.89     2.69

            Sprr1a    AA230988   small proline-rich protein 1A                                                 5.35   1.24     6.03

            Sprr2a    AA497620   small proline-rich protein 2A                                                16.39   0.86    15.04
• List of genes down-regulated in the Hepa1c1c7 cell
line following exposure to AzaC and TCDD
                                                                                               Fold increase
      Gene
                   ID                                        Gene Name                                     AzaC/
      symbol                                                                               AzaC    TCDD
                                                                                                           TCDD

Ak4            W82313     adenylate kinase 4                                               -2.59   -3.17   -3.15

Alox12b        W36511     arachidonate 12-lipoxygenase, 12R type                           -2.40   -2.42   -3.32

Bnip3          AA105295   BCL2/adenovirus E1B 19kDa-interacting protein 1                  -3.73   -3.74   -4.03

Dnajc3         AA277325   Dnajc3 DnaJ (Hsp40) homolog, subfamily C, member 3               -3.40   -2.46   -3.55

Gabarapl1      AA276338   gamma-aminobutyric acid receptor-associated protein-like 1       -3.35   -2.80   -3.11

Gnb2-rs1       AA048915   guanine nucleotide binding protein, beta 2                       -0.34   -2.86   -3.55

H47            AA237415   H47 histocompatibility 47                                        -2.65   -2.69   -3.06

Myd116         AA050417   myeloid differentiation primary response                         -2.91   -2.89   -3.59

Risc-pending   W10703     Risc-pending retinoid-inducible serine caroboxypetidase          -1.66   -2.02   -3.14

Sel1h          W74907     Sel1 (suppressor of lin-12) 1 homolog (C. elegans)               -3.38   -2.91   -3.49

Smpd2          AA028477   sphingomyelin phosphodiesterase 2                                -3.49   -3.86   -4.21

Stard5         AA275581   Stard5 StAR-related lipid transfer (START) domain containing 5   -2.72   -2.06   -3.20

Tyrobp         AA242315   TYRO protein tyrosine kinase binding protein                     -3.10   -3.08   -3.25
Conclusions
• CpG methylation is involved in the tissue-specific and
  developmental regulation of CYP1A2 in the liver, and
  that the delayed de novo methylation of the CYP1A2
  promoter is a non-specific event that occurs after gene
  silencing in hepatocytes.

• DNA methylation has an important role in the tissue-
  specific expression of GLUT2 in the mouse and that it is
  also involved in gene regulation in mouse liver-derived
  cells.

• Using cDNA microarray technology, mRNA expression
  analyzes to investigate methylation-dependent genes
  that are susceptible to induction by xenobiotic exposure .
• In summary, these results demonstrated a correlation
  between the expression and the promoter methylation of
  specific gene in the mouse liver and liver-derived cells.

• Further studies are required to elucidate the role of
  epigenetic modifications in liver-specific gene regulation.

								
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