HeLa细胞ezrin基因基本启动子区转录调控元件的鉴定

Identification of Transcriptional Regulatory Elements within Ezrin Gene Basal Promoter in HeLa Cells

目的鉴定HeLa细胞中调控ezrin基因基本启动子活性的顺式作用元件,探讨ezrin基因在HeLa细胞的表达调控机制。方法采用碱基定点突变实验和双荧光素酶报告基因分析系统,检测在HeLa细胞中Sp1结合位点(-75/-69,相对于转录起始位点)和AP-1结合位点(-64/-58)对人ezrin基因基本启动子活性的影响;采用电泳迁移率变动分析法(EMSA)实验,检测ezrin基因基本启动子区序列与HeLa细胞核蛋白提取物的特异性结合活性。结果在HeLa细胞中,单独删除和置换Sp1结合位点或AP-1结合位点,ezrin基因基本启动子活性降低50%左右;同时置换Sp1结合位点和AP-1结合位点,启动子活性几乎完全丧失。ezrin基因基本启动子序列能够与HeLa细胞核蛋白提取物相结合。结论HeLa细胞中,Sp1结合位点和AP-1结合位点为ezrin基因基本启动子区的重要转录调控元件,有可能存在某种转录因子与之结合,激活ezrin基因转录。

Objective To identify of the regulatory elements for human ezrin gene basal promoter activity in human cervical carcinoma cell line HeLa, and to elucidate the transcriptional regulatory mechanism of the ezrin gene in HeLa ceils. Methods Effect of Spl binding site （ - 75/- 69, relative to transcription start site） and AP-1 binding site （ - 64/- 58） on the human ezrin gene basal promoter activity in HeLa cells was performed using site-directed mutagenesis and dual-luciferase reporter assay system. Specific binding activity of nuclear extracts from HeLa cells to the human ezrin gene basal promoter region was detected by electrophoretic mobility shift assay. Results Deletion and replacement either the Spl site or the AP-1 site remained approximately 50% ezrin basal promoter activity in HeLa cells, while replace- ment both the Spl and AP-1 sites nearly abolished this activity. Nuclear extracts from HeLa cells could bind to the basal promoter region of human ezrin gene. Conclusion The Spl and AP-1 sites are two im- portant cis-elements for the human ezrin basal promoter activity, and some transcription factors possibly bind to the two elements to transactivate ezrin gene in HeLa cells.