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

賀博群(Bo-Qun He)

指導教授 ： 張大慈

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2007年)

摘要

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。

關鍵字

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

並列摘要

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.

並列關鍵字

EDN ； ECP

參考文獻

1. Sorrentino S, Libonati M: Structure-function relationships in human ribonucleases: main distinctive features of the major RNase types. FEBS Lett 1997, 404(1):1-5.

2. Nitto T, Lin C, Dyer KD, Wagner RA, Rosenberg HF: Characterization of a ribonuclease gene and encoded protein from the reptile, Iguana iguana. Gene 2005, 352:36-44.

3. Cho S, Beintema JJ, Zhang J: The ribonuclease A superfamily of mammals and birds: identifying new members and tracing evolutionary histories. Genomics 2005, 85(2):208-220.

4. Yang D, Chen Q, Rosenberg HF, Rybak SM, Newton DL, Wang ZY, Fu Q, Tchernev VT, Wang M, Schweitzer B et al: Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation. J Immunol 2004, 173(10):6134-6142.

5. Zhang J, Dyer KD, Rosenberg HF: Human RNase 7: a new cationic ribonuclease of the RNase A superfamily. Nucleic Acids Res 2003, 31(2):602-607.