THAP11调控基因转录的分子机制研究

发布时间：2018-03-06 04:03

本文选题：THAP11　切入点：转录　出处：《安徽医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： THAP11属于新发现的THAP蛋白质家族成员。该蛋白家族是一类锌离子依赖的DNA结合蛋白质,广泛参与细胞增殖、凋亡调控,并可能与染色体修饰与重构相关。前期研究表明THAP11可通过直接结合c-Myc启动子区抑制c-Myc表达,进而抑制细胞增殖。此外,THAP11还可调控胚胎干细胞(ES)的自我更新和增殖。但目前对其作用机制尚无深入研究。本研究利用哺乳动物双杂交系统,发现THAP11同时具有转录激活和转录抑制活性,并通过系列缺失,确定了其功能结构域, N端82个氨基酸区具有转录抑制活性,C端185个氨基酸区具有转录激活活性。THAP11发挥转录抑制活性与染色体修饰相关因子关系。进一步利用酵母双杂交技术,从成人肝脏cDNA文库中筛选到CtBP1可与THAP11发生相互作用。利用免疫共沉淀技术,我们证实了THAP11与CtBP1存在相互作用,并确定了两种相互作用结构域。鉴于该蛋白可通过直接的蛋白质相互作用参与转录因子发挥转录抑制的过程,THAP11与CtBP1相互作用可能是THAP11发挥转录抑制活性的方式。为进一步探讨THAP11调控基因表达的分子机制,对全基因组进行THAP11潜在结合位点的检索,发现Oct4启动子含有THAP11潜在的结合位点。分离人Oct4启动子区,构建报告基因载体,发现THAP11可激活Oct4启动子转录,这种激活活性依赖于其C端转录激活区,而与其DNA结合能力无关。EMSA实验证实THAP11能够结合预测的结合位点结合,但系列缺失突变实验证实THAP11对Oct4启动子的激活并不依赖该结合位点,表明THAP11可能通过其它机制调控Oct4的表达。利用Real-time PCR实验表明,在PA-1细胞中过表达THAP11可明显上调内源Oct4的表达,表明Oct4是THAP11调控的靶基因。为进一步在ES细胞中检测THAP11功能及作用机制,构建了人THAP11的慢病毒表达载体,并对慢病毒进行了包装,获得了THAP11慢病毒颗粒,为后续的研究提供了有力的工具。
[Abstract]:THAP11 is a newly discovered member of THAP protein family, which is a zinc ion-dependent DNA binding protein, which is widely involved in cell proliferation and apoptosis regulation. Previous studies have shown that THAP11 can inhibit c-Myc expression by direct binding to c-Myc promoter region. In addition, THAP11 can also regulate the self-renewal and proliferation of embryonic stem cell (es). However, there is no in-depth study on the mechanism of its action. In this study, the mammalian two-hybrid system was used. We found that THAP11 has both transcriptional activation and transcriptional inhibitory activity, and through a series of deletions, Its functional domain was determined. The N-terminal 82 amino acid regions had transcriptional inhibitory activity and C-terminal 185 amino acid regions had transcriptional activation activity. THAP11 played a role in the relationship between transcription inhibitory activity and chromosome modification. Further more, yeast two-hybrid technique was used. CtBP1 could interact with THAP11 from adult liver cDNA library. We confirmed that there was interaction between THAP11 and CtBP1 by immunoprecipitation. Two kinds of interaction domains were identified. In view of the direct protein interaction this protein can participate in the process of transcription inhibition by transcription factors. The interaction between THAP11 and CtBP1 may be the way in which THAP11 exerts its transcription inhibitory activity. In order to further explore the molecular mechanism of gene expression regulated by THAP11, By searching the whole genome for the potential binding sites of THAP11, we found that the Oct4 promoter contains the potential binding site of THAP11. The reporter gene vector was constructed by isolating the promoter region of human Oct4. It was found that THAP11 could activate the transcription of Oct4 promoter. This activation activity is dependent on its C-terminal transcriptional activation region, but independent of its DNA binding ability. EMSA experiments confirm that THAP11 can bind to predicted binding sites. However, a series of deletion mutation experiments confirmed that the activation of Oct4 promoter by THAP11 does not depend on the binding site, suggesting that THAP11 may regulate the expression of Oct4 through other mechanisms. Overexpression of THAP11 in PA-1 cells significantly upregulated the expression of endogenous Oct4, suggesting that Oct4 was a target gene regulated by THAP11. In order to further detect the function and mechanism of THAP11 in es cells, the lentivirus expression vector of human THAP11 was constructed. The lentivirus was packaged and THAP11 lentivirus particles were obtained, which provided a powerful tool for further research.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

【相似文献】