臺灣博碩士論文加值系統

Epstein-Barr 病毒(EB 病毒)是一種γ皰疹病毒，超過90%人口都被傳染，而且EB病毒和許多淋巴性或皮膚性惡性腫瘤有密切的相關性。EB病毒從潛伏期轉活化的過程中，兩個即刻性早期的病毒促進者Zp和Rp，分別轉錄出Zta和Rta蛋白質，扮演重要的角色。先前的文獻指出，在潛伏期感染細胞中，EB病毒的基因是被高度甲基化的，且Zta蛋白質這個使EB病毒進入溶裂期的重要轉活化因子，會優先地結合到甲基化的病毒基因上進而活化此基因的表現。目前為止，在EB病毒的基因中已經發現很多Zta蛋白質的標地基因。而在EB病毒轉活化的過程中，宿主細胞中也發現到很多的基因表現量產生變化，但是否這些變化與Zta蛋白質調節的標地基因表現機制有關目前仍未知。先前報導中發現，宿主基因體中的一個轉譯因子，早期生長反應因子-1 (Egr-1)，可能是Zta的一個標地基因。Egr-1在受到化學藥物的刺激而走向轉活化的鼻咽癌惡性腫瘤細胞中，表現量會上升。且報導中也證實了Egr-1的啟動序列上有Zta蛋白質結合的位置 (ZRE)，因此，可以結合在Egr-1的啟動序列上，來調控Egr-1表現。由於Zta蛋白質結合的位置剛好座落在一段CpG群聚上，我們假設甲基化的修飾，在Zta大量表現的轉活化細胞中扮演一個重要的角色，極可能以改變Egr-1基因上啟動子區域的甲基化程度，來讓Zta蛋白質結合到此區域上，進而活化Egr-1表現。此次研究，我們利用Zta蛋白質大量表現的細胞模式來探討轉活化細胞中基因體甲基化改變的情形。為了檢驗宿主細胞中整體甲基化的狀態，我們分別利用人類雄性素接受器基因法去分析X染色體上不活化的情形，南方墨點轉漬法去分析基因體裡重複序列的甲基化程度，也利用即時定量PCR方法來分析控制印記基因IGF2/H19表現的區域上的甲基化情形，更利用高解析毛細電泳去定量出在基因體中有甲基的胞嘧啶的比例。在探討宿主基因體甲基化程度相關蛋白質的表現方面，我們利用了西方墨點漬法分析去氧核醣核酸甲基轉移酶的表現量，其中包含了有去氧核醣核酸甲基轉移酶1、3a及3b。此外，我們也以Egr-1這個Zta的標地基因去分析其啟動子上CpG群聚的甲基化情形。由結果發現，在Zta大量表現和沒有Zta表現的細胞中，不活化的X染色體似乎是選擇性的發生在特定的某一條X染色體上。但是在這兩株細胞中， X染色體上不活化的程度並沒有差異。在重複序列包括LINE-1、SINE-Alu和α-satellite，印記基因IGF2/H19上的甲基化程度，以及全基因體帶有甲基的胞嘧啶的比例，在Zta大量表現和沒有Zta表現的細胞中情形都是很相似的。除此之外，我們的結果也指出在Zta大量表現和沒有Zta表現的細胞裡， Egr-1啟動序列上的CpG群聚都沒有受到甲基化的保護。顯示Zta應是在大量表現後，藉著與Egr-1啟動子上的ZRE結合來調節Egr-1基因表現。由這些結果顯示在轉活化細胞中，Zta大量表現並不會改變宿主基因體甲基化的狀態。

Epstein-Barr virus, a γ herpesvirus, that infects more than 90% of the human population and closely associated with several lymphoid and epithelial malignancies. EBV reactivation from latent infection is initiated by activation of two immediate-early viral promoters, Zp and Rp, which encode BZLF1 (Zta) and BRLF1 (Rta) proteins, respectively. The EBV genome is highly methylated in latently infected cells. The Zta is a key lytic transactivator that preferentially binds to the methylated viral genome and activates lytic viral gene expression. While many targets of Zta have been identified in the EBV genome, expressions of a number of cellular genes were also shown to be regulated during EBV reactivation. But the underlying mechanism of Zta-induced transactivation on target genes expression are still unknown. One of these cellular genes, early growth response-1 (Egr-1), has been showed to be upregulated in EBV-infected nasopharyngeal carcinoma cell line that was treated by chemicals to induce reactivation. Further investigation identified Zta responsive element (ZRE) on Egr-1 promoter that are responsible for the induction of Egr-1 expression. We have found that ZRE on Egr-1 promoter was also located within a CpG island. We hypothesize that the methylation play a role in regulation of target gene expression in Zta overexpressed cells. The current study applied Zta-overexpressed cellular model to investigate the significance of altered genomic methylation upon EBV reactivation. We have employed several advanced technologies to assay the global methylation status in the host genome. These include the human androgen receptor (HUMARA) assay for X-chromosome inactivation, Southern blot analysis against genome repetitive sequences, and methylation-sensitive quantification real-time PCR to examine methylation status of imprinting control region (ICR) of the IGF2/H19 imprinting genes. In addition, we performed high performance capillary electrophoresis (HPCE) to measure the methyl-cytosine level in the cells. The expressions of methylation-related proteins were assayed by Western blot analysis using antibodies against human DNA methyltransferase 1 (DNMT1), DNMT3a, DNMT3b and LINE-1 ORFs. Finally, the Egr-1 promoter, as one example of Zta-targeted cellular gene, was also investigated by methylation-sensititive real-time PCR. Our data indicated in both Zta+ and Zta- cells, the patterns of inactivated X-chromosome were skewed X-chromosome inactivation. The methylation status of inactivated X-chromosome was not different in Zta+ and Zta- cells. The methylation level of the repetitive elements (LINE, SINE-Alu and α-satellite), methyl-cytosine level and imprinted gene, IGF2/H19, were similar between these two cells. Expressions of methylation-related proteins were also not changed by Zta expression. In addition, our data indicated that the CpG island of Egr-1 promoter is not protected by methylation in Zta+ and Zta- cells. Thus it suggests that Zta might bind to the ZRE on the Egr-1 promoter to regulate the gene expression in Zta-overexpressed cells. Theses results suggest the global methylation pattern in the host genome is not changed upon reactivation.

ABSTRACT IN CHINESE ……………………………………....Ι
ABSTRACT IN ENGLISH …………………………………….IV
ACKNOWLEDGEMENT ……………………………………ⅤI
TABLE OF CONTENTS …………………………………..VIII
LIST OF TABLES …………………………………………...XII
LIST OF FIGURES …………………………………………..XIII

1 INTRODUCTION ………………………………………………..1
1.1 EPIGENTIC MODIFICATION …………………………………..1
1.1.1 DNA methylation ……………………………………………1
1.1.2 Histone modification ……………………………………….4
1.1.3 RNA-mediated regulation …………………………………..6
1.2 THE EPSTEIN-BARR VIRUS …………………………………..7
1.2.1 The EBV genome ……………………………………………8
1.2.2 Life cycle of EBV infection …………………………………..8
1.2.3 EBV reactivation …………………………………………..10
1.2.4 EBV hypermethylated genome to escape host immune system ……………………………………………………………..11
1.3 EBV TRANSACTIVATOR, ZTA …………………………………12
1.3.1 Molecular properties of Zta protein ………………………...12
1.3.2 Zta overexpression …………………………………………..14
1.4 EGR-1 FUNCTION UNDER EBV INFECTION ……………….15
1.5 RESEARCH OBJECTIVES OF THIS STUDY ……………………15
2 MATERIAL AND METHODS …………………………………17
2.1 Cell line ……………………………………………………………..17
2.2 DNA extraction ……………………………………………………17
2.3 Human androgen receptor assay (HUMARA) ……………………18
2.3.1 Subjects and Sample Preparations ………………………...19
2.3.1.1 Genomic DNA digestion ……………………………..19
2.3.1.2 Purification from DNA digestion ……………………19
2.3.2 Polymerase Chain Reaction …………………………………20
2.4 Southern Blotting ……………………………………………….20
2.4.1 Genomic DNA digestion …………………………………20
2.4.2 Gel electrophoresis of the restricted DNA ……………….21
2.4.3 Transfer ……………………………………………………21
2.4.4 Probe preparation …………………………………………..22
2.4.5 Hybridization ……………………………………………….23
2.4.6 Detection ……………………………………………………24
2.5 Preparation of nuclear extracts …………………………………24
2.6 Protein concentration determination ……………………………..25
2.7 Western blotting ……………………………………………………26
2.7.1 Gel electrophoresis …………………………………………..26
2.7.2 Transfer ……………………………………………………26
2.7.3 Hybridization ……………………………………………….27
2.7.3.1 Hybridization against DNMT1 and DNMT3a ……...27
2.7.3.2 Hybridization against DNMT3b ……………………27
2.7.3.3 Hybridization against LINE-1 ORF1 ……………….28
2.7.3.4 Hybridization against PCNA ……………………28
2.7.3.5 Hybridization against Pan-actin ……………………28
2.7.4 Detection ……………………………………………………29
2.8 Methylation-sensitive quantitative real-time PCR ……………….29
2.8.1 Subjects and sample preparations ………………………...29
2.8.1.1 Genomic DNA digestion ………………………...29
2.8.1.2 Purification from DNA digestion ……………………29
2.8.2 Quantitative real-time PCR …………………………………30
2.8.3 Relative quantitation method ……………………………..31
2.8.4 Validating relative efficiency of target and reference ……...32
2.9 High performance capillary electrophoresis (HPCE) …………..32
2.9.1 Subjects and sample preparation ……………………………..32
2.9.1.1 Standards …………………………………………..32
2.9.1.2 Genomic DNA hydrolysis ………………………...33
2.9.2 High performance capillary electrophoresis ……………….33
2.10 Statistical analysis …………………………………………..33
3 RESULT ……………………………………………………………..35
3.1 The global methylation level in Zta overexpressed cells …………..35
3.1.1 The DNA methylation status of inactivative X chromosome ……………………………………………………………..35
3.1.2 The global DNA methylation level of repetitive elements in Zta overexpressed cells …………………………………………..36
3.1.3 The DNA methylation level of imprinted genes …………..37
3.1.4 The level of methyl-cytosine ……………………………..38
3.2 The expression level of methyltion-related proteins …………..39
3.2.1 The expression level of DNA methyltransferases …………..39
3.2.2 The expression of LINE-1 ORFs ………………………...40
3.3 The methylation status of Egr-1 promoter region ……………….40
4 DISCUSSION ………………………………………………………...42
4.1 The Zta-responsive cellular genes in the host cells ……………….42
4.2 The pattern of X chromosome inactivation in human genome …43
4.3 The methylation status of the imprinted gene in host genome ....45
4.4 The limitation of methylation-sensitive real-time PCR …………..46
4.5 The expression of LINE-1 ORFs in Zta+ and Zta- cells …………..46
4.6 The possible mechanism of Zta induce Egr-1 expression ……...47
4.7 Conclusion ………………………………………………………. 49

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