吳成文臺灣大學：分子醫學研究所林佳慧Lin, Chia-HuiChia-HuiLin2007-11-262018-07-092007-11-262018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/51354C 型肝炎病毒感染在台灣已經成為慢性肝炎以及肝癌的主要致病原因，然而其致病機制並不清楚。核心蛋白為C 型肝炎病毒的結構蛋白，已被發現能調節許多細胞訊息傳遞路徑以及轉錄因子功能，並能於基因轉殖鼠內造成肝癌。因此，核心蛋白與細胞中分子的相互作用在致病機制中很可能扮演著重要的角色。在此篇論文中，我們探討了C型肝炎病毒核心蛋白對組蛋白甲基化酵素活性的調控。甲基化酵素為染色質修飾蛋白之一，而染色質修飾蛋白是由轉錄因子將其帶到基因的啟動子(promoter)對組蛋白進行修飾，使基因活化或抑制其表現。 利用組蛋白甲基化實驗(histone methyltransferase assay) ，我們發現C型肝炎病毒核心蛋白在試管內能抑制組蛋白甲基化酵素CARM1、PRMT1及SET9對組蛋白H3/H4的活性。核心蛋白本身亦能被CARM1及PRMT1進行甲基化，而SET9對於組蛋白H1的活性反而受核心蛋白影響而增加。利用核心蛋白的缺失突變，我們發現胺基酸51到101的片段對於核心蛋白抑制甲基化酵素是不可缺少的。目前已有許多報導指出C型肝炎病毒核心蛋白能調控細胞內許多轉錄因子如p53和NF-κB。我們的實驗發現核心蛋白會被帶到具有NF-κB結合位置的DNA上。利用luciferase assay，在HuH-7細胞中一些NF-κB下游基因Cox-2、IL-8、MCP-1 及 RANTES其啟動子的活性皆會被核心蛋白所抑制。而目前已知這三個甲基化酵素皆為p53的輔激活蛋白而CARM1在最近也被發現為NF-κB的輔激活蛋白。GST pull-down實驗的結果告訴我們全長的核心蛋白與細胞內的NF-κB組成分子p65、CARM1、PRMT1以及SET9有交互作用。此外，在HuH-7細胞中，核心蛋白的另一個目標基因—p53下游基因p21其啟動子活性也會受到核心蛋白所抑制。由chromatin immunoprecipitation實驗的結果，我們發現在HuH-7細胞中，在p21啟動子上，核心蛋白會減少CARM1以及PRMT1對其標的組蛋白精胺酸位置的甲基化。 總而言之，C型肝炎病毒核心蛋白抑制NF-κB及p53下游基因可能是經由抑制NF-κB及p53輔激活蛋白CARM1、PRMT1及SET9對組蛋白H3/H4的活性。我們的研究提供了一個C型肝炎病毒核心蛋白調控NF-κB和p53活性的新機制。其他受甲基化酵素調控的路徑亦可能會受到核心蛋白的影響。Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and hepatocellular carcinoma, especially in Taiwan. However, the detailed pathogenic mechanism remains unclear. It has been reported that core protein, a structure protein of HCV, regulates several cellular signaling pathways and transcriptional factors and induces hepatocellular carcinoma in transgenic mice. Thus, the interaction between core protein and its cellular targets may play important roles in the pathogenesis. Here, we characterize HCV core protein-mediated functional regulation of histone mehtyltransferases (HMTs). HMTs belong to members of chromatin modifiers that have to be recruited by specific transcription factors to the promoter region of genes to achieve transcriptional activation or repression. By performing in vitro HMT assays, we found that core protein represses the HMT activity of arginine-specific HMTs (R-HMTs) CARM1 and PRMT1 as well as lysine-specific HMT (K-HMT) SET9 on their target sites of histone H3 or H4. In contrast, core protein increases SET9 activity on histone H1. Interestingly, core protein itself can be methylated by CARM1 and PRMT1 in the presence of histones. By using deletion mutants in HMT assays, amino acids 51 to 101 of core protein seem to be the inhibitory domain on HMT. Core protein has been reported to regulate functions of cellular transcription factors, such as p53 and NF-κB. DNA affinity protein assays show that core protein is recruited to the NF-κB binding site, and the promoter activities of certain NF-κB target genes, Cox-2, IL-8, MCP-1 and RANTES, are reduced in the presence of core protein in HuH-7 cells. CARM1 has been reported as a novel coactivator of NF-κB. Our GST pull-down assay shows that full length core interacts with endogenous NF-κB p65 subunit, CARM1 and SET9. Moreover, CARM1, PRMT1 and SET9 have been reported to be coactivators of p53. The promoter activity of another core protein target, p53 downstream gene p21, is also repressed in the presence of core protein in HuH-7 cells, and the result of chromatin immunoprecipitation shows that core protein reduces CARM1-mediated histone H3 dimethylation at R2 and R17 and PRMT1-mediated histone H4 R3 dimethylation on p21 promoter in HuH-7 cells. Taken together, HCV core protein-mediated downregulation of NF-κB and p53 target genes may likely occur through repression of activities of p53 and NF-κB coactivators, CARM1, PRMT1 and SET9 on histone H3/H4 methylation. Our study provides a novel pathway of core protein to modulate the activities of NF-κB and p53. And likely more HMT-regulated effects could be modulated by core protein.Abstract………………………………………………………………1 中文摘要………………………………………………………………3 Introduction…………………………………………………………5 Hepatitis C virus is an important human pathogen…………5 Core protein may contribute to HCV pathogenesis……………6 1. Core protein has three isoforms……………………………6 2. Core protein interacts with several cellular proteins…………7 3. Core protein regulates the activity of transcription factors……………8 Histones can be post-translationally modified………………9 1. Histone acetylation and methylation are keys to gene regulation……………10 2. Hsitone methyltransferases, CARM1, PRMT1 and SET9 are coactivators……………………………………………10 3. Histone modifiers are regulated by viral proteins……12 Study Purposes……………………………………………………14 Materials and Methods………………………………………………15 Cell culture and transfections…………………………………15 Plasmids and antibodies……………………………………………15 Purification of GST-fusion HCV core protein…………………17 Purification of His-tagged HPV E6 and E7 protein…………18 GST pull-down assay…………………………………………………19 In vitro histone methyltransferase assay……………………19 Preparation of cytoplasmic and nuclear fractions…………20 DNA affinity protein assay………………………………………21 Luciferase Assay……………………………………………………22 Chromatin immunoprecipitation……………………………………22 Results…………………………………………………………………24 GST-fusion HCV core protein and its deletion mutants are successfully purified………24 HCV core protein regulates CARM1 activity……………………24 HCV core protein regulates PRMT1 activity……………………26 HCV core protein regulates SET9 activity……………………26 Core protein regulates NF-κB activity possibly via HMT inhibition……………………28 Core protein reduces histone arginine dimethylation on p21 promoter in HuH-7 cells……………………………………………30 Discussion……………………………………………………………32 HCV core protein regulates HMT activity with specificity…………32 Histone H1 might be a novel target of SET9…………………33 Amino acids 51 to 101 of core inhibit CARM1-, PRMT1- and SET9-mediated H3/H4 methylation…………………………………34 The trnafected core protein locates in the nucleus………34 Core protein may downregulate NF-κB activity through HMT inhibition……………35 A novel pathway of p53 downregulation by core protein……37 References……………………………………………………………39 Figure 1. HCV Core protein and its deletion mutants are successfully purified……..49 Figure 2. HCV core protein inhibits the HMT activity of CARM1 on histone H3 and core itself is methylated by CARM1…………………………………………………50 Figure 3. HCV core protein inhibits the HMT activity of PRMT1 on histone H4 and is methylated by PRMT1……………………………………………………………….51 Figure 4. HCV core protein inhibits the HMT activity of SET9 on histone H3 and increases the HMT activity on Histone H1……………………………………………52 Figure 5. HPV type 11, 16 and 18 E6 protein regulate the activities of HMTs…………53 Figure 6. HPV type 16 and 18 E7 protein regulate the activities of HMTs………54 Figure 7. Regulation of NF-κB activity by HCV core protein…………………55 Figure 8. Regulation of p53 activity by HCV core protein………………………56 Table 1. Summary of the in vitro HMT assay…………………573500729 bytesapplication/pdfen-USC型肝炎核心蛋白組蛋白甲基化酵素hepatitis C virusHCVcore proteinhistone methyltransferase[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51354/1/ntu-95-R93448002-1.pdf