- 學位論文
C型肝炎病毒核心蛋白調控組蛋白甲基化酵素活性之功能性研究
Functional Regulation of Histone Methyltransferase Activity by Hepatitis C Virus Core Protein
摘要
Table of Contents List of abbreviations…………………………………………...…………..1 中文摘要….………………………………………………..………………2 Abstract………………………………………...…………………………..4 Introduction………………………...………………………………………6 I. The contribution of HCV core protein to HCV pathogenesis………...6 I-1. Hepatitis C virus is an important human pathogen………………6 I-2. HCV core protein isoforms………………………………………7 I-3. HCV core protein interacts with cellular proteins……………….8 I-4. HCV core protein-mediated gene regulation………………….....9 II. Post-translational modification of histone proteins………………...10 II-1. Histone methylation and histone methyltransferases…………..11 II-1-1. Protein arginine methyltransferase 1 (PRMT1)…...………12 II-1-2. Coactivator-associated arginine methyltransferase 1 (CARM1)……………………………………..…………14 II-1-3. SET domain-containing lysine methyltransferase 9 (SET9)…………………………………………………...15 III. Histone modifiers are regulated by viral proteins………………….16 Preliminary Results……………………………………………………….18 Materials and Methods……………………………………………………22 Cell lines and DNA transfections……………………………………...22 Plasmids and antibodies………………………………………………..22 Reporter assay……………………………….…………………………24 Immunofluorescence assay……………………….……………………25 Immunoprecipitation/co-immunoprecipitation assay……………….…26 Western analysis….……………..……………………………………..27 IP-Mass spectrometry............………………………………………….28 In gel digestion…….............................................…………………...…28 Chromatin immunoprecipitation (ChIP)……………………………….29 Results…………………………………………………………...………..31 Interaction of HCV core protein and endogenous CARM1 and PRMT1 in vivo…………………………………………………………………..31 Nuclear localization of HCV core protein……………………………..31 Recruitment of HCV core protein to the p21 promoter did not impair p53 binding…………………………………………………...………..33 HCV core protein-mediated repression of p21 promoter activity.……34 Inhibition of CARM1- and PRMT1-induced p21 promoter activity and active histone modification by HCV core protein……………………35 HCV core protein-mediated inhibition of the promoter activitiy of NF-κB targeted genes……...………………………………..………..37 Methylation of HCV core protein……………………………………...38 Discussion…...……………………………………………………………40 References……...…………………………………………………………45 Figure 1. HCV core protein interacted with CARM1, PRMT1, but not SET9 in vivo………………………………………………………………55 Figure 2. Cellular distribution of HCV core protein……………………..56 Figure 3. Cellular distribution of CARM1, PRMT1 and SET9…………..57 Figure 4. Effects of HCV core protein on p21 promoter activity….……..58 Figure 5. HCV core protein repressed the CARM1- and PRMT1-enhanced p21 promoter activities…………………………………………………...59 Figure 6. HCV core protein inhibited the CARM1- and PRMT1-mediated active histone modification pattern on p21 promoter…………...………..61 Figure 7. HCV core protein reduced the promoter activities of NF-κB targeted genes….………………………………………………………….62 Figure 8. HCV core protein was methylated by R-HMTs………………..63 Table 1. Amino acid sequences of HCV core protein c173………………64 Table 2. The R17 residue of HCV core protein c173 was methylated.......64 Supplementary Results….………………………………………………...65 Acknowledgements……………………………………………………….68
並列摘要
Posttranslational modification of histone proteins constitutes the histone code which plays an important role in eukaryotic gene expression as well as viral pathogenesis. Previous studies have shown that viral proteins functionally interact with histone acetyltransferases/deacetylases. Whether cellular histone methyltransferases (HMTs) are also targeted by viral oncoproteins remains unclear. Our previous study indicated that the core protein of the hepatitis C virus (HCV) which causes chronic hepatitis, cirrhosis, fibrosis and hepatocellular carcinoma in infected individuals represses core histone methylation by coactivator-associated arginine methyltransferase 1 (CARM1), protein arginine methyltransferase 1 (PRMT1), and SET domain-containing lysine methyltransferase 9 (SET9) in in vitro HMT assays. It prompted us to investigate the relationship between HCV core protein and the three HMTs in vivo. The three HMTs (CARM1, PRMT1 and SET9) are the coactivators of p53, and p21 is the downstream of p53. We used human hepatoma cell line Huh 7 as our study model. We demonstrated that in reporter assays HCV core protein repressed CARM1- and PRMT1-enhanced p21 promoter activities as well as p21 synthesis. In addition, we showed the in vivo interactions between core protein and CARM1 and PRMT1. Using the chromatin-immunoprecipitation (ChIP) analysis, we further demonstrated that (1) core protein was recruited to the p21 promoter, (2) core protein did not affect p53 binding to the p21 promoter and (3) inhibited histone H3 acetylation and H3K4 mono-methylation, the active histone modification pattern. Furthermore, we unraveled that core protein 191 repressed promoter activities of four kinds of NF-κB target genes in reporter assay, and R17 site of core protein c173 was methylated. Taken together, our results indicate that HCV core protein inhibits HMT activities on core histones in vivo, which in turn resulted in the inhibition of the function of p53 and NF-κB.
參考文獻
延伸閱讀
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