活化转录因子ATF1、ATF2对USP22基因启动子活性的调节  

Regulation of USP22 Promoter Transcription Activity by ATF1 and ATF2 Genes

作　　者：刘建云[1] 周小鸥[2] 吴萍[1] 熊建军[2] 

机构地区：[1]九江学院基础医学院江西省系统生物医学重点实验室,江西九江332000 [2]九江学院基础医学院药理教研室,江西九江332000

出　　处：《南昌大学学报（医学版）》2016年第2期9-13,52,共6页Journal of Nanchang University：Medical Sciences

基　　金：国家自然科学基金(31000581;81460172)

摘　　要：目的克隆人活化转录因子ATF1基因和ATF2基因蛋白编码序列,分别构建真核细胞表达载体,进而研究高表达ATF1和ATF2对泛素水解酶22(ubiquitin-specific processing enzyme 22,USP22)基因启动子转录活性的影响。方法 RT-PCR扩增ATF1基因和ATF2基因蛋白编码序列,分别与pVAX1真核表达载体连接;测序、酶切鉴定重组载体;Western Blot检测外源性ATF1和ATF2在靶细胞Hela细胞中的表达;pVAX1-ATF1、pVAX1-ATF2分别与含野生型USP22启动子的萤光素酶报告质粒或其CREB位点突变体质粒共转染,双萤光素酶报告系统检测萤光素酶活性的表达。结果重组质粒pVAX1-ATF1、pVAX1-ATF2构建成功,外源性ATF1、ATF2在Hela细胞内有效表达;转染pVAX-ATF1促进USP22启动子活性升高(83%±11%),转染pVAX-ATF2促进USP22启动子活性升高约(52%±8%);而突变CREB位点均可导致上升作用的消失。结论成功构建了ATF1、ATF2真核表达质粒,高表达外源性ATF1和ATF2均可通过CREB位点激活USP22启动子的转录活性。Objective To construct the eukaryotic expression vector carrying ATF1 or ATF2gene,and to investigate the effect of exogenous ATF1 or ATF2on USP22 promoter transcription activity.Methods Human ATF1 or ATF2cDNA was amplified by RT-PCR and was cloned into the eukaryotic expression vector pVAX1.The recombinant plasmid was identified by sequencing and restriction endonuclease digestion.The exogenous expression of ATF1 or ATF2in Hela cells was detected by Western blot.The pVAX1-ATF1 or pVAX1-ATF2 was co-transfected with luciferase reporter plasmids containing USP22 promoter or its mutant isoform on CREB binding site.The promoter activity was evaluated by luciferase reporter system.Results The recombinant plasmids pVAX1-ATF1 and pVAX1-ATF2 were constructed successfully,and the exogenous ATF1 and ATF2were expressed in Hela cells.The USP22 promoter transcription activity was increased by（83% ±11%）and（52% ±8%）after transfection of pVAX1-ATF1 and pVAX1-ATF2,respectively.However,the mutation of CREB binding site abrogated the effects of exogenous ATF1 and ATF2.Conclusion The eukaryotic expression plasmid carrying ATF1 or ATF2gene has been constructed successfully,and the overexpression of ATF1 or ATF2can enhance USP22 promoter transcription activity via CREB binding site.

关 键 词：ATF1 ATF2 USP22基因 启动子 萤光素酶 

分 类 号：Q78[生物学—分子生物学]