Title page
Acknowledgement
Contents
Abbreviations 10
CHAPTER 1. INTRODUCTION 11
1. Epigenetics 12
1-1. Chromatin remodeling 14
1-2. Histone Modification 15
1-3. DNA Methylation 16
1-3-1. DNA methylation in mammals 17
1-3-2. DNA methylation in humans 17
CHAPTER 2. Epigenetic Regulation of Viral Promoter for the Transcriptional Expression in Epstein-Barr Virus 21
2-1. Abstract 22
2-2. Introduction 23
2-3. Materials and Methods 25
2-3-1. Lymphoblastoid cell and the other cancer cell lines 25
2-3-2. DNA extraction and bisulfite treatment 25
2-3-3. PCR using universal biotinylated primer 26
2-3-4. Pyrosequencing 26
2-3-5. Real-time and Semi-quantitative RT-PCR Analysis 26
2-4. Results 30
2-4-1. Methylation status of the viral promoters (Cp, Qp and Wp) 30
2-4-2. Analysis of the methylation status of three promoters from the different passages of LCLs 33
2-4-3. Expression of four EBV Genes 35
2-5. Discussion 40
CHAPTER 3. Epigenetic Regulation of Cellular Promoter for the Transcriptional Expression in CYP17A1 gene 42
3-1. Abstract 43
3-2. Introduction 45
3-3. Materials and Methods 48
3-3-1. Patients and controls 48
3-3-2. Sequencing analysis of the human CYP17A1 genes 49
3-3-3. Genotyping of polymorphisms with fluorescence polarization detection 49
3-3-4. EPSTEIN-BARR VIRUS TRANSFORMATION OF B-CELLS 50
3-3-5. Preparation of CYP17A1 promoter reporter gene constructs 50
3-3-6. Transient transfection and assays for EGFP fluorescence 50
3-3-7. Methylation-specific PCR (MSP) 52
3-3-8. 5-AZA-dC TREATMENT 52
3-3-9. RNA ISOLATION AND QUANTITATIVE REAL-TIME PCR 52
3-3-10. Statistical analysis 53
3-4. Results 55
3-4-1. Resequencing of CYP17A1 genes and linkage analysis 55
3-4-2. Association analyses with male infertility risk and the level of hormones 55
3-4-3. Effect of the -600C and -600A alleles on activity of the CYP17A1 promoter 62
3-4-4. Methylation profiles -600C〉A polymorphic site 66
3-4-5. CYP17A1 EXPRESSION 66
3-5. Discussion 71
CHAPTER 4. CONCLUSION 74
REFERENCES 76
Table I-1. Specific epigenetic processes of interest 13
Table I-2. Characteristics of DNA Methyltransferase 20
Table II-1. Primers for pyrosequencing analysis of EBV three promoters (Cp, Wp and Qp) 27
Table II-2. Primers for realtime RT-PCR and semiquantitative RT-PCR 29
Table III-1. PCR primer sequences used in promoter construction, MSP and real-time PCR 51
Table III-2. Primer sequences for CYP17A1 sequence variants screening 58
Table III-3. Sequences of amplifying, Taqman probe for CYP17A1 polymorphisms genotyping 59
Table III-4. Frequencies of CYP17A1 polymorphisms (n=7) in male infertility patients and normal subjects 60
Table III-5. Genotype and allele frequencies for CYP17A1 polymorphisms in male infertility patients and normal subjects 61
Table III-6. Distribution of CYP17A1 -600 C/A genotype in infertile and fertile males 63
Table III-7. The frequency of CYP17A1 -600 C/A genotype according to the two subtype of infertile male group 64
Table III-8. The association of genotype of CYP17A1 gene with hormones (LH, FSH, Prolactin, Progesterone and Testosterone) in Ejaculate semen group 65
Figure I-1. Models for methylation effect on gene activity. 18
Figure II-1. Methylation status of CpG sites within the regulatory region of Wp and CP promoter. 31
Figure II-2. The methylation percentages EBV promoters from early (15), middle (55), late (110)-passage LCLs. 34
Figure II-3. Relative expression level of four genes (EBNA1, EBNA2, LMP1 and LMP2A) in early-passage LCLs (B95-A, B95-B, and B95-C) by Wp methylation. 36
Figure II-4. Relationship between Wp methylation and the expression pattern of four genes (EBNA2(■), LMP1(▤).EBNA1 U(□), EBNA1 Y3(■) and LMP2A(▥)) from three different passages. 39
Figure III-1. Androgen and estrogen metabolism. 46
Figure III-2. Gene maps, haplotypes, and linkage disequilibrium coefficients of CYP17A1. 56
Figure III-3. Comparison of CYP17A1 promoter activities for the -600C and -600A alleles of the -600C〉A SNP. 67
Figure III-4. Methylation patterns of -600C〉A polymorphic site in normal human tissue. 68
Figure III-5. CYP17A1 mRNA Expression pattern in 786-0 kidney cell by methylation inhibitor, 5-aza-2'-deoxycytidine. 70