CpG methylation of gene promoter in the mouse liver

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1 Epigenetic regulation of liver specific genes in the mouse Gyeonggi Natural Products Research Institute BoHwan Jin, DVM, PhD

2 Epigenetics 인체를이루고있는다양한조직및장기의세포는동일한유전자정보를가지고있지만, 그기능과모습에서큰차이가있음. 이는각조직과장기의세포에서특정한유전자들만발현이되기때문임. 특이적인유전자발현은세포가분화하면서확립되는데, 조직특이적인 transcription factor 작용, DNA methylation, histone 의변경, 세포의시그널등이복합적으로작용하여이루어짐.

3 세포의분화는 DNA 염기서열의변화없이이루어지지만, 세포분화를통하여획득한형질적인특성은세포분열을통하여전달됨. 이와같이유전자염기서열과무관한게획득한특정형질이자손에게전달되는것을 Epigenetics 라함. Epigenetics 란, DNA 염기서열의변화, 즉유전자변이없이일어난유전자의기능변화를연구하는학문임.

4 Epigenetics 는발생과분화, X- 염색체의불활성화, 유전자각인 (Gene Imprinting) 에중요한역할을함. DNA methylation, Histone modification(methylation 과 deacetylation) 에의한유전자발현억제등이포함되어있음 DNA methylation 은대표적인 epigenetics 변화로서유전자코드의변화없이유전자의기능을직접변경시키고, 전달될수있으며, 유전자의발현억제및종양의발생과관련이있는것으로알려져있음

5 DNA methylation 유기화학에서 methylation : CH 3 그룹이첨가되는알킬화과정 DNA 구성 : Adenine(A), Guanine(G), Cytosine(C), Thymine(T) 1948 년 : 다섯번째염기인 5-methylcytosine(m 5 C) 발견

6 진핵생물에서 DNA methylation 은 DNA 합성후에일어나며, guanidine 앞의 cytosine pyrimidine ring 의 5 번탄소에 S-adenosyl-methionine 에의하여제공되는메틸그룹의첨가가 DNA methyltransferases(dnmts) 에의한촉매반응임.

8 인간에서약 1% 의 DNA 그리고, DNA 에존재하는모든 CpGs 의 60-70% 정도가 methytion 이되어있음. CG dinucleotide 는 CG basepair 와구분하기위하여 CpG 로표기, 비유전자에위치한 CpG 들은대부분 methylation 이되어있음 성인체세포에서는 CpG dinucleotide 에발생하고, embryonic stem cell 에서는 non-cpg methylation 이우세한것으로알려져있음

9 Methyl 그룹을 cytosine 링에전달하는효소 : cytosine 5-methyltransferase 또는 DNMTs 포유류의 DNMTs : Dnmt1, Dnmt3a, Dnmt3b

10 Dnmt1 : 포유류의주요 DNMTs 로서어디서나존재하며, maintenance methylation Dnmt3a, Dnmt3b : de novo methylation 기능, 초기배아, 발달하는생식세포에서고농도로발현되지만분화된체세포에서낮은농도로발현됨. 배아줄기세포나착상후배아에서일어나는 de novo methylation 에필수적이며, 생식세포에서 gene imprinting 의패턴유지

11 CpG islands 인간의유전체에서 CpG dinucleotide 의빈도는매우낮음.(CpG suppression) 특정부위에서는 CpG 의빈도가기대수치이상으로관찰됨, DNA 평균 100kb 당발생하고길이가 0.5-5kb 정도임 세포생존에필수적인단백질을 coding 하는 90% 이상의 house keeping genes 와특정조직에서만발현되는단백질을 coding 40% tissue-specific genes 의 promoter 에서 CpG Is 가발견

12 CpG Is 특징 : Unmethylated, High CG content, 개방된 chromtin 구조임. 즉, 이부위의 60-70% 의 CG content 를보이고 chromation 은 acetylation, histone 1 의결핍을나타냄 CpG Is 가아닌다른부위의 CpG 는대부분메틸화가되어있으며, methylcytosine 은자연적으로 deamination 에의해 thymine 으로변환되어 CpG 가 TpG 로전환, 전환된 TpG mismatch 가 DNA 복제가일어나기전에 DNA repair system 에의하여교정되지않으면, 자손세포로전달됨.

13 CpG Is 내의 methylation 은생리적으로 genomic imprintiong 과 X- 염색체의불활성화, 별리적으로발육성질환, 암발생, 줄기세포의성장과분화와관련이있는것으로알려져있음. 몇몇유전자에서의 methylation 은정상세포의전사억제와연과되지만, promoter CG Is 보다는 intragenic CpG Is 에서발생한다.

14 Methylation 에의한유전자발현억제기전은명확하게밝혀지지않았지만, methyl-cytosine 에 MeCP2 와같은 metyl-dna 결합단백질이붙고, 여기에 histone deacethylase 등을내포한단백복합체가동원되어 histone deacetylation 을시켜, chromatin 을밀집화된폐쇄구조로변화시킴. 폐쇄적인구조를갖게된 promoter 에는전사인자가붙을수없고, 결과적으로유전자발현이억제됨.

15 Silenced promotor 의 Methylated cytosine 은 histone deacetylases(hdacs) 를포함하는복합형으로 methyl- CpG-binding-domain protein(mbds) 라는 Repressive protein 의특정그룹과결합함. HDACs 는 histone 의 N-terminal 에서아미노산으로부터아세틸그룹을제거할수있음. 이러한과정에서 heterochromatin 의 closed chromatin 구조가관여함. 반대로 unmethylated cytosine 을가진전사가활발한 promotor 는 histone acetyltransferase(hats) 에의하여 Atct

16 반대로 unmethylated cytosine 을가진전사가활발한 promotor 는 histone acetyltransferase(hats) 에의하여 Acetylation 되고, 이것은 transcription factor 와 coactivator protein 으로이루어진 transcription activator complex 를형성

18 Epigenetic Mechanism Source : NIH, 2005 (

19 Genomic Impriting Gametogenesis 과정중모성및부성유전체에조건을새기는것으로모성과부성대립유전자중한쪽대립유전자가배타적으로또는많이발현되는것. 발생기간중모성과부성기원대립유전자중어느한쪽만배타적으로발현되도록난자와정자에서 DNA methylation 에의한 epigenetics 기전으로각인

20 Genomic imprinting 기전으로는 DNA methylation 이유력함. DNA methylaion 은유전자를억제할수있고, epigenetic marker 로서자손세포에전달이가능하며, maintenance methyltransferase 가없는상태또는활동성 demethylase 의존재하에서 DNA 복제가이루어질경우삭제될수도있음. 많은 Imprinted genes 은 Allele-specific DNA methylation 을나타내고 maintenance methyltransferase 가없거나 methyltransferase inhibitor 가존재하는경우유전자각인이소실될수있음

21 Cancer 일반적으로암세포는 global genomic hypomethylation 와정상세포에서는 CpG Is 의 hypermethylation 의특징을나타냄 global genomic hypomethylation 는암세포에서흔히발견되는데주로반복염기서열 (repetitive sequence) 에서발생하지만, 특정유전자와연관되어있을수있음.

22 대장암에서 LINE1 repetitive sequence 의 global DNA hypmethylation 은정상상피세포가이형성됨. 갑상선암, 유방암, 전립선암, 방광암, 위암, 폐암, 식도암, 간암, 구강암등과 hypmethylation 의관련성이보고되고있음.

23 흔히, promotor hypomethylation 은전체적인 hypomethylation 과연관시키는데, promoter hypomethylation 은유전자의발현을증가시키고, proto-oncogenes 을활성화시킬수있음. 세포의기질과세포막의퇴화에중요한역할을하는 heparanase 는악성종양에서흔히증가됨. 방광암발생과정에서증가된 heparanase 는 early growth response gene-1(egr-1) 의 promotor CpG hypomethylation 때문임

25 Acetylation DNA 는 histone 중합체 (H2A, H2B, H3, H4) 염기성단백질에 2 번감겨있으면서좁은핵공간에압축되어있음. 이러한구조때문에전사인자와 RNA 중합효소가 DNA 에접근할수없음. 즉평소에는전사가일어날수없는형태로존재하고있으나, 전사가일어나기위해서는우선 histone 에아세틸기가붙어서 histone 의모양이바뀌고 DNA 구조가풀리게됨.

26 Acetylation

27 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.

28 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.

29 Bisulfite Sequencing Bisulfite sequencing : Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5- methylcytosine residues unaffected.

30 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 Aug 30;152(1): Jin B, Ryu DY. Regulation of CYP1A2 by histone deacetylase inhibitors in mouse hepatocytes. J Biochem Mol Toxicol. 2004;18(3):131-2.

31 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).

32 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

33 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.

34 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.

35 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).

36 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.

37 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. Results

38 Methylation profile of 18 CpG sites in the Cyp1a2 promoter in mouse tissues.

39 Developmental expression of CYP1A2 mrna and the methylation profile in C57BL/6 mouse liver

40 Methylation profile in the Cyp1a2 promoter in mouse hepatoma Hepa1c1c7 cell line.

41 CYP1A2 expression and the methylation of CpGs within the Cyp1a2 promoter in primary hepatocytes in culture.

42 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.

43 Tissue-specific and de novo promoter methylation of the mouse glucose transporter 2 Jin B, Seong JK, Ryu DY. Tissue-specific and de novo promoter methylation of the mouse glucose transporter 2. Biol Pharm Bull Nov;28(11):

44 Introduction GLUT2 : glucose homeostasis in glucose uptake from the intestine, reabsorption by the kidney, sensing in the pancreatic beta cells, and uptake and release by the liver. Glucose transporter 2 (GLUT2) is tissue-specifically expressed in liver and kidney.

45 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).

46 Cell culture and treatments of cells Hepa1c1c7 cell line was obtained from Korean Cell Line Bank. Cultured cells were treated with 5-aza-2 - deoxycytidine (AzaC), up to 50 μm, for 72 h 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.

47 Results

50 Discussion A correlation between the expression and the promoter methylation of GLUT2 in mouse tissue and liver-derived cells. The de novo promoter methylation of GLUT2 happened independently of its expression in isolated hepatocytes, suggesting that de novo methylation of GLUT2 is not casually related to its expression.

51 However, the down-regulation of GLUT2 expression could be rescued by demethylation in isolated hepatocytes and hepatoma cells, suggesting that methylation is responsible for gene regulation in liver-derived cells.

52 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-pdioxin. Toxicol In Vitro Oct;18(5):

53 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

54 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.

55 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.

56 Results

57 List of genes up-regulated in the Hepa1c1c7 cell line following exposure to AzaC and TCDD Gene symbol ID Gene Name Fold increase AzaC TCDD AzaC/ TCDD Group I Atf2 AA activating transcription factor Cpo AA coproporphyrinogen oxidase Fsp27 AA fat specific gene Nqo1 AA NAD(P)H dehydrogenase, quinone Ramp3 AA receptor (calcitonin) activity modifying protein Tm4sf3 AI transmembrane 4 superfamily member Xdh AA xanthine dehydrogenase Group II Arhgef6 AA Rac/Cdc42 guanine nucleotide exchange factor (GEF) Osmr AA oncostatin receptor Pdgfrl AA platelet-derived growth factor receptor-like Prkcd AI protein kinase C, delta Son W97212 Son cell proliferation protein Tnc AA tenascin C

58 Gene symbol ID Gene Name Fold increase AzaC TCDD AzaC/ TCDD Group III Bhmt2 AA betaine-homocysteine methyltransferase Ckmt2 AA creatine kinase, mitochondrial Clcn2 AI chloride channel Cml1 AA camello-like Eral1 AA Era (G-protein)-like 1 (E. coli) Flt4 AA FMS-like tyrosine kinase Frat1 AA frequently rearranged in advanced T-cell lymphomas Gys2 AA glycogen synthase Hdgfrp2 AI Hepatoma-derivedgrowthfactor,relatedprotein Hs3st1 W62484 heparan sulfate (glucosamine) 3-O-sulfotransferase Lrrc2 AA leucine-rich repeat-containing Mthfd1 AI methylenetetrahydrofolate dehydrogenase (NADP+ dependent) Pdgfa AA platelet derived growth factor, alpha Pitrm1 AA pitrilysin metalloprotease Ptges AA prostagladin E synthase Slc1a2 AA solute carrier family 1, member Smarca5 AA SWI/SNF related, matrix associated, actin dependent regulator of chromatin Sp1 AA trans-acting transcription factor Sprr1a AA small proline-rich protein 1A Sprr2a AA small proline-rich protein 2A

59 List of genes down-regulated in the Hepa1c1c7 cell line following exposure to AzaC and TCDD Gene symbol ID Gene Name Fold increase AzaC/ AzaC TCDD TCDD Ak4 W82313 adenylate kinase Alox12b W36511 arachidonate 12-lipoxygenase, 12R type Bnip3 AA BCL2/adenovirus E1B 19kDa-interacting protein Dnajc3 AA Dnajc3 DnaJ (Hsp40) homolog, subfamily C, member Gabarapl1 AA gamma-aminobutyric acid receptor-associated protein-like Gnb2-rs1 AA guanine nucleotide binding protein, beta H47 AA H47 histocompatibility Myd116 AA myeloid differentiation primary response Risc-pending W10703 Risc-pending retinoid-inducible serine caroboxypetidase Sel1h W74907 Sel1 (suppressor of lin-12) 1 homolog (C. elegans) Smpd2 AA sphingomyelin phosphodiesterase Stard5 AA Stard5 StAR-related lipid transfer (START) domain containing Tyrobp AA TYRO protein tyrosine kinase binding protein

61 Conclusions

62 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 tissuespecific 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.

63 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.

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

65 참고문헌 오정환외 : 후생유전학 (Epigenetics) 과 DNA methylation 의이해 김도엽 : Epigenetics 와유전자조절에관하여 Partha M. Das 외 : DNA Methylation and Cancer

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract