Laboratory Animal Research Center

Ajou University School of Medicine

Jin Bo Hwan, D.V.M, PhD

Epigenetic regulation of liver specific genes in the mouse

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.

• 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 Mechanisms

Epigenetic MechanismSource : NIH, 2005(http://commonfund.nih.gov/epigenomics/figure.aspx)

• 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).

Epigenetic Mechanisms

Nucleosome structure. Source : By Richard Wheeler (Zephyris) 2005

• 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.

Epigenetics and environmental health

• 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.

• 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.

N-α-terminal Acetylation

• 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.

Lysine Acetylation and Deacetylation

• 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.

• 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).

Methods

• 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

• 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.

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.

Results

　Gene

symbolID Gene Name

Fold increase

　 AzaC TCDDAzaC/

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

• List of genes up-regulated in the Hepa1c1c7 cell line following exposure to AzaC and TCDD

　Gene

symbolID Gene Name

Fold increase

　 AzaC TCDDAzaC/

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

Gene

symbolID Gene Name

Fold increase

AzaC TCDDAzaC/

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.