【作者】 陈合格；

【导师】 张轩杰； 李万程；

【作者基本信息】 湖南师范大学 ， 发育生物学， 2008， 博士

【摘要】 蛋白磷酸酶PP2A是主要的丝氨酸/苏氨酸特异性磷酸酶,在真核生物中占到50%以上丝氨酸/苏氨酸磷酸酶活性。蛋白磷酸酶PP2A的全酶由3个亚基构成:结构亚基A,催化亚基C和调节亚基B。结构亚基以2种异构体形式存在即PP2A-Aα亚基和PP2A-Aβ亚基。结构亚基A为催化亚基C和调节亚基B提供了结合的支架和平台,因此在PP2A发挥活性时起着非常重要的作用。PP2A-Aα和PP2A-Aβ基因发生突变会导致人类的多种疾病,如癌症等。目前关于PP2A-Aα和PP2A-Aβ基因是如何被细胞内和细胞外因子调控的还知之甚少。在本研究中我们克隆了小鼠PP2A-Aα基因的启动子,构建了一系列从5′端删除部分序列的突变体和定点突变转录因子Ets-1,CREB,AP-2α和Sp1结合位点的突变体,并用这些质粒转染ARPE-19和FHL124细胞后用双荧光报告基因系统检测了其各自的相对荧光素酶的活性。我们的实验结果表明:(1)转录因子Ets-1,CREB,AP-2α和Sp1在ARPE-19和FHL124细胞系中都有表达;(2)小鼠PP2A-Aα基因的启动子无TATA框但有CAAT框,其活性区域主要在-625到+52区;(3)将与转录因子Ets-1所结合的序列突变后,其相对荧光素酶活性大大下降,约为野生型核心启动子A5荧光素酶活性的40%;(4)将与转录因子CREB所结合的序列突变后,其相对荧光素酶活性亦大大下降,约为野生型核心启动子A5荧光素酶活性的50%;(5)将与转录因子AP-2α所结合的2个位点的序列都突变后,其相对荧光素酶活性亦下降,约为野生型核心启动子A5荧光素酶活性的80%;(6)将与转录因子Sp1所结合的序列突变后,其相对荧光素酶活性增强,约为野生型核心启动子A5荧光素酶活性的120%。(7)转录因子Ets-1,CREB,AP-2α和Sp1的剂量依赖型荧光素酶的实验结果也显示:随着转入表达Ets-1,CREB,AP-2α蛋白的质粒的增加,PP2A-Aα核心启动子活性得到增强;随着转入表达Sp1蛋白的质粒的增加,小鼠PP2A-Aα核心启动子活性减弱。这进一步说明转录因子Ets-1,CREB和AP-2α能正向调节小鼠PP2A-Aα基因的表达,转录因子Sp1能负向调节PP2A-Aα基因的表达。(8)凝胶迁移滞后(EMSA)实验结果证实转录因子Ets-1,CREB,AP-2α和Sp1在体外都能结合到小鼠PP2A-Aα基因的启动子上的相应序列;(9)染色质免疫沉降(ChIP)的实验结果在活体细胞中证实了转录因子Ets-1和CREB能结合到小鼠PP2A-Aα基因的启动子上。总之,我们的研究结果显示多个转录因子共同调节着小鼠PP2A-Aα基因的表达。更多还原

【Abstract】 Protein phosphatases-2A(PP-2A)is a major serine/threonine phosphatase and accounts for more than 50%serine/threonine phosphatase activity in eukaryotes.The holoenzyme of PP-2A consists of three different subunits:the scaffold A subunit,the catalytic C subunit and the regulatory B subunit.The scaffold subunit appears in two isoforms,which are encoded by two genes:PP2A-Aa and PP2A-Aβ. The scaffold subunits provide a platform for both C and B subunits to bind,thus playing a crucial role in providing specific PP-2A activity. Mutation of both genes causes human diseases such as various types of cancers.Regulation of these genes by various factors,both extracellular and intracellular,remains largely unknown.In the present study,we have cloned the promoter of the mouse PP2A-Aa gene and made some deletion mutants from the promoter 5’ terminal and did in vitro mutagenesis on cis-elements for the binding of transcription factor Ets-1, CREB,AP-2a and Spl.We transfect ARPE-19 and FHL124 cell lines with these plasmids and then check their relative luciferase activity with dual-luciferase reporter assay system.Our results demonstrate that:(1).Transcription factor Ets-1,CREB,AP-2a and Spl are all present in ARPE-19 and FHL124 cell lines;(2).The core promoter of the mouse PP2A-Aa gene consists of 677 bp and contains numerous cis-elements for the binding of Ets-1,CREB, AP-2a and SP-1.The promoter region of mouse PP2A-Aa gene was found to lack TATA box but have a CAAT box and -625--+52 region provide the main activity to drive its expression;(3).When the binding site for transcription factor Ets-1 was mutated, compared with mouse wild type core promoter its relative luciferase activity was dramatically decreased to 40%;(4).When the binding site for transcription factor CREB was mutated,compared with mouse wild type core promoter its relative luciferase activity was dramatically decreased to 50%;(5).When all the two binding sites for transcription factor AP-2a was mutated,compared with mouse wild type core promoter its relative luciferase activity was dramatically decreased to 80%;(6).When the binding site for transcription factor Sp1 was mutated, compared with mouse wild type core promoter its relative luciferase activity was increased to 120%;(7).Transcription factor Ets-1,CREB,AP-2a and Sp1 dose-dependent luciferase assay reveal that transcription factor Ets-1, CREB and AP-2a all positively regulate the promoter of the PP2A-Aa gene while transcription factor SP-1 displays negative regulation;(8).Gel mobility shifting assays revealed that these four transcription factors can all bind to mouse PP2A-Aapromoter;(9).ChIP assay further confirms that both Ets-1 and CREB play important role in regulating mouse PP2A-Aa gene promoter.Taking it together,our results reveal that multiple transcription factors,such as Ets-1,CREB,AP-2a and Sp1,regulate the mouse PP2A-Aa gene.更多还原