hsp90β基因在Jurkat细胞中表达调控机理的研究

【作者】 刘秉胜；

【导师】 沈珝琲； 吴宁华；

【作者基本信息】 中国协和医科大学 ， 分子生物学及生物化学， 2000， 博士

【摘要】 热休克蛋白90(HSP90)在正常生长条件下细胞内就有较高水平的组成性表达，并可被热和丝裂原双重诱导。这提示HSP90在细胞内具有重要的功能。事实证明，作为一种特异的分子伴侣，HSP90除在细胞正常生长和应急保护中具有重要生理功能外，还与信号传导、细胞周期调控、激素应答等细胞内重要生理过程有关。因此研究HSP90蛋白基因的表达调控机制具有重要的生物学意义。本论文首先研究了两个重要顺式元件CRE和AP-1位点及相关的反式作用因子在hsp90β基因表达调控中的作用；另外，从染色质水平对hsp90β基因组在组成性、热诱导和PHA激活表达条件下DNaseI高敏位点进行了分析。 一、CRE和AP-1位点及相关反式作用因子在hsp90β基因表达调控中的作用 1．首先构建了CRE和AP-1位点系列缺失的CAT和Luc报告基因质粒。转染Jurkat细胞并分析报告基因的活性。结果发现，在组成性表达中，单独删除CRE或单独删除AP-1位点，报告基因的活性变化不显著，但同时删除这两个元件时，报告基因的活性降低了40％。这表明CRE和AP-1位点共同对hsp90β基因的高组成性表达起重要作用；在热诱导表达中，缺失CRE、AP-1位点和两者同时缺失，报告基因热诱导倍数分别降低67％、50％、67％，表明CRE和AP-1位点参与了hsp90β基因的热诱导表达。在PHA诱导表达中，保留CRE和AP-1位点中的任何一个PHA均能激活报告基因的表达，但同时删除这两个元件时，PHA不能激活报告基因的表达。这表明CRE和AP-1位点介导了PHA激活hsp90β基因的表达。 2．我们随后分析了CRE相关的反式作用因子对hsp90β基因表达的影响。利用EMSA实验，发现热休克处理的Jurkat细胞核抽提物能与hsp90β基因5’上游含有CRE的-173／-91DNA片段形成三条结合带，野生型冷探针可特异竞争这三条结合带而CRE共有系列的核心碱基突变的寡聚核苷酸片段却不能竞争，表明这三条结合更多还原

【Abstract】 Human hsp90 genes are usually constitutively expressed in most mammalian cell types and can be further induced by heat shock and mitogen. This suggests that HSP90 proteins are critical to the cells’ normal growth. In fact, as a specific molecular chaperone, HSP90 proteins are possibly involved in many important physiological processes such as signal transduction, cell cycle regulation and hormone response. So it is very meanful for us to study the regulatory mechanisms of expression of human hsp90 genes. Here, we first present the evidence of the role of CRE, AP-1 site and their own related transactivators in the regulation of human hsp90p gene; then we identify the DNase I hypersensitive sites in human hsp90p gene in Jurkat cells.— The role of CRE, AP-1 site and the related trans-activators in the expression of human hsp90p gene1. We first constructed the CAT and Luc reporter plasmid deleting CRE or AP-1 site or both. These deleting mutants were then transfected into Jurkat cells and the reporter activities were detected. The results show that CRE together with AP-1 site are critical for the high constitutive expression of human hsp90p gene; CRE and AP-1 site also participate in heat inducible expression of human hsp90p gene. We also found that the full-length promoter, only CRE deleted or only AP-1 site deleted mutants presented inducible reporter gene activity after PHA treatment; however, deletion mutant lacking both CRE and AP-1 site showed no inducible reporter gene activity after PHA treatment. These results indicate that CRE and AP-1 site mediate the PHA activated expression of human hsp90p gene.2. We then analyzed the roles of CRE related transactivators in the expression of hsp90p gene. By EMSA, we found that heat shock nuclear extracts and the labeled probe of -173/-91 fragment containing CRE can form three bands. The addition of the unlabeled wide type probe can efficiently compete three specific bands while the更多还原