肝細胞核因子4對人類嗜酸性白血球神經毒性蛋白的表現調節之影響

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研究生：	賀博群
論文名稱：	肝細胞核因子4對人類嗜酸性白血球神經毒性蛋白的表現調節之影響 Role of liver transcription factor HNF4 on regulation of human ribonuclease2
指導教授：	張大慈 Margaret Dah-Tsyr Chang
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2007
畢業學年度：	95
語文別：	中文
論文頁數：	68
中文關鍵詞：	人類嗜酸性白血球神經毒性蛋白、人類嗜酸性白血球陽離子蛋白
外文關鍵詞：	EDN, ECP
相關次數：	點閱：2 下載：0
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本論文係探討人類嗜酸性白血球神經毒性蛋白（核醣核酸水解酵素2）與人類嗜酸性白血球陽離子蛋白（核醣核酸水解酵素3）之不同生物演化功能，研究的重點為二者啟動子活性及轉錄因子的差異。人類嗜酸性白血球神經毒性蛋白與人類嗜酸性白血球陽離子蛋白為核醣核酸水解酵素家族的成員，二者的基因均位於第14號染色體長臂第2區第4條帶和長臂第3區第4條帶之間。比對其密碼區上游一千個鹼基對的啟動子序列顯示二者具92%的相似度，但主要的差異為嗜酸性白血球神經毒性蛋白啟動子-81/-48位置含有一段34個鹼基對的區域，而人類嗜酸性白血球陽離子蛋白則無；以及六個鹼基對序列差異較高的次要區域。由啟動子分析的實驗得知，在人類肝癌細胞株中將人類嗜酸性白血球神經毒性蛋白啟動子的第二個次要差異區域突變成人類嗜酸性白血球陽離子蛋白者，明顯降低人類嗜酸性白血球神經毒性蛋白之轉錄能力。膠體電泳流動轉移試驗(EMSA)以及抗體膠位移試驗(supershift assay)則發現轉錄因子中肝細胞核因子4(HNF4)會結合在人類嗜酸性白血球神經毒性蛋白的第二個次要差異區域，繼而達到調控其轉錄能力的效果。若將此區域從人類嗜酸性白血球神經毒性蛋白啟動子序列中剔除，則將造成其轉錄能力急劇下降，證明本區域對人類嗜酸性白血球神經毒性蛋白的轉錄調控扮演重要的角色。本實驗室已知轉錄因子Sp1和MAZ能與含34個鹼基對區域結合以調控人類嗜酸性白血球神經毒性蛋白的表達能力，結合本論文研究之新穎發現，具體證明HNF4、Sp1、和MAZ共同調節機制，使得人類嗜酸性白血球神經毒性蛋白與人類嗜酸性白血球陽離子蛋白之啟動子活性在肝癌細胞中顯現差異。

Human eosinophil-derived neurotoxin (EDN, RNase2) and human eosinophil cationic protein (ECP, RNase3) belong to the ribonuclease A (RNase A) superfamily, and the duplicated edn and ecp genes are located in the q24-q31 region of chromosome 14. Multiple sequence alignment of upstream 1 kb region of human edn and ecp showed 92% identity, but a major difference was a 34-nucleotide (34-nt) element (-81/-48) only appeared in the edn promoter. However, some minor differences between edn and ecp promoters mainly located in six different regions might also be involved. In this study, luciferase reporter assay was employed to find out which minor region also plays the role to regulate the promoter activity, and a mount of decrease in edn promoter activity was detected when region 2 sequence mutated to the same with ecp. Electrophoretic mobility shift assays (EMSA) and supershift results show that the transcription factor HNF4 could bind to region 2 of edn promoter. Greatly decrease of promoter activity was detected in deletion of region 2 in edn promoter results, so this region might play important role with edn gene. In previous research, edn could be regulated by Sp1 and MAZ depending on that binding to 34nt. Combining this study, edn gene expression might be co-regulated by interaction between its promoter and transcription factors HNF4, Sp1 and MAZ.

中文摘要    I
Abstract    II
Table of Contents    III
Abbreviations    VII

1.    Introduction    1
2.    Materials and methods    3
2.1 Microbial strains and plasmids    3
2.2 Competent cell preparation    3
2.3 Culture media    4
2.4 Polymerase chain reaction (PCR)    4
2.5 Site-directed mutagenesis    4
2.6 Restriction enzyme digestion and DNA fragment recovery    5
2.7 Ligation    5
2.8 Construction of plasmid    6
2.9 Plasmid mini-preparation and Transformation    6
2.10 In situ PCR    6
2.11 DNA Sequencing    7
2.12 DNA sequence analysis    8
2.13 Cell culture    8
2.14 Transient transfection    8
2.15 Promoter activity assay    9
2.16 Preparation of nuclear extract    9
2.17 Determination of protein concentration    10
2.18 Electrophoretic mobility shift assays (EMSA)    10
2.19 Expression of fusion protein by E. coli    11
2.20 Purification of His-tag fusion protein    11
2.21 Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis    12
2.22 Western blotting    13
2.23 DNA affinity precipitation assay (DAPA)    13
2.24 Chromatin immunoprecipitation (ChIP)    14
3.    Results    16
3.1 Promoter activity of human edn is higher than that of ecp with insertion of 34-nt segment    16
3.2 Six regions with minor segment variations exist in human edn and ecp promoters    16
3.3 Replacement of region 2 segment of edn with that of ecp decreases promoter activity    16
3.4 Region 2 segment in edn promoter is crucial for transcriptional regulation    17
3.5 Transcription factors are associated with the region 2 segments of edn and ecp promoters    18
3.6 ConSite predicts transcription factor binding sites in ednR2 and ecpR2 segments    19
3.7 PROMO predicts transcription factor binding sites in ednR2 and ecpR2 segments    19
3.8 Potential transcription factor(s) bound to ednR2 are identified by EMSA    20
3.9 COUP-TF, HNF-4 and LEF-1 may bind to ednR2    20
3.10 Supershift assay demonstrates specific binding between HNF4 and ednR2    21
3.11 HNF4 binding site is located at -342/-335 in edn promoter    21
3.12 Recombinant and dominant negative HNF4 are expressed in E. coli    22
3.13 Recombinant HNF4 binds to ednR2    23
3.14 HNF4 is associated with edn promoter in vivo    23
3.15 HNF4 regulates edn promoter activity in vivo    23
3.16 HNF-4 interacts with Sp1 in vivo    24
3.17 HNF4 binding to the ednR2 is associated with Sp1    24
4.    Discussion    26
5.    References    30
Table 1  Primer sequences for site-directed mutagenesis    36
Table 2  Oligonucleotide sequences for EMSA screening    37
Table 3  HNF4 cloning primers    38
Table 4  ChIP primers    38
Table 5  Variable regions 1 to 6 in edn and ecp promoters    38
Figure 1  Promoter activity of edn and ecp    39
Figure 2  Alignment of edn and ecp promoters by ClustalW    40
Figure 3  Transcription activity of wild type and mutant edn promoters in HepG2    41
Figure 4  Transcription activity of wild type and mutant ecp promoters in HepG2    42
Figure 5  The role of region 2 in edn promoter    43
Figure 6  Transcription factors binding with ednR2 and ecpR2    44
Figure 7  Putative transcription factor binding sites in region 2 segment of edn and ecp promoters predicted by ConSite    45
Figure 8  Putative transcription factor binding sites in region 2 segments of edn and ecp promoters predicted by PROMO    46
Figure 9  Scanning of transcription elements in ednR2 by EMSA    47
Figure 10  Specific binding between ednR2 and transcription factors    48
Figure 11  Identification of HNF4 in the ednR2-containing complex    49
Figure 12  Localization of HNF4 binding site in edn promoter by EMSA mutagenesis scanning    50
Figure 13  Purification of HNF4 fusion proteins    51
Figure 14  Identification of recombinant wild type and mutant HNF4    52
Figure 15  Characterization of specific HNF4 binding to ednR2    53
Figure 16  In vivo binding of HNF4 to edn promoter    54
Figure 17  Repression of edn promoter activity by HNF4    55
Figure 18  Co-immunoprecipitation of HNF4 and Sp1 from HepG2 cell lysates      56
Figure 19  Interaction of HNF4 bound to ednR2 with Sp1    57
Figure 20  Alignment of region 2 segments in primate edn and ecp promoters    58
Figure 21  Schematic representation of the binding of transcription factors to edn and ecp promoter    59

                                

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