HDAC3和NCoR介导了Ataxin-3缺失诱导的转录异常

发布时间：2018-02-21 07:27

本文关键词： ataxin-3 基因表达芯片逆转录实时定量PCR 小鼠胚胎成纤维细胞 EphA3基因启动子荧光素酶报告基因载体转录活性 Ataxin-3 EphA3 组蛋白乙酰化组蛋白去乙酰化酶3 核受体共抑制因子组蛋白去乙酰化酶抑制剂　出处：《华中科技大学》2012年博士论文　论文类型：学位论文

【摘要】：第一部分：Ataxin-3敲除导致基因表达谱改变目的：采用基因表达芯片技术(Microarray)观察ataxin-3敲除对基因表达变化的影响。方法：选取ataxin-3敲除的小鼠胚胎成纤维细胞(ataxin-3 KO MEF)和正常小鼠胚胎成纤维细胞(ataxin-3 WT MEF)为研究对象,提取RNA并用基因表达谱芯片技术观察ataxin-3敲除对基因表达的影响,所得实验结果数据经ThomsonReuters/GeneGo公司的生物信息学软件进行分析,并采用逆转录实时定量PCR技术证实基因表达芯片结果的可靠性。结果：与正常细胞相比,ataxin-3敲除导致410个基因表达明显异常(基因表达信号上调或下调超过2倍及以上),其中260个基因表达上调,而150个基因表达下调。GeneGo软件分析这410个基因的生物学功能,并对其功能进行分类。发现ataxin-3敲除导致多个细胞通路转录异常,尤其是炎症免疫和细胞粘附相关基因。结论：Ataxin-3分子丢失导致多种细胞通路相关基因转录异常。第二部分：EphA3基因启动子区域定位表达及活性分析目的：构建含有不同长度EphA3基因启动子片段的报告基因载体,研究其在293T细胞和MEF细胞中的转录活性。方法：以Balb/C小鼠基因组DNA为模板,扩增不同长度的EphA3基因启动子片段,并克隆进入荧光素酶报告基因质粒pGL3-Basic真核表达载体内。酶切鉴定及基因测序无误后,将重组质粒和pRL-CMV内对照质粒共转染293T或MEF细胞,分析不同长度的EphA3基因启动子片段的转录活性。结果：酶切鉴定和基因测序结果提示表达载体构建成功,EphA3基因的核心启动子区域位于-279～+110bp之间,在293T细胞和MEF细胞中启动子片段的转录活性相似。结论：成功构建了荧光素酶报告基因重组质粒,并确定了EphA3基因的核心启动子区域。第三部分：HDAC3和NCoR介导了ataxin-3对EphA3基因的转录调控脊髓小脑共济失调3型(SCA3)是多聚谷氨酸诱导神经退行性疾病的一种,其疾病基因编码一个小分子蛋白ataxin-3。Ataxin-3蛋白外显子区域CAG异常拷贝导致疾病的发生。研究发现Ataxin-3分子是一个去泛素化酶,调节某些蛋白的活性和稳定性,是泛素-蛋白酶体的重要成分。同时,ataxin-3分子也参与细胞转录调控,病理性ataxin-3导致多种细胞通路基因转录异常,加重疾病进程。为探讨正常Ataxin-3分子转录调控的机制,我们选用ataxin-3 KO MEF和ataxin-3 WT MEF细胞为研究对象。基因表达芯片、逆转录实时定量PCR和免疫印迹结果均显示ataxin-3敲除导致EphA3基因表达明显上调。因此我们构建了EphA3基因的启动子片段,双荧光素酶活性测定结果发现ataxin-3缺失导致EphA3基因启动子转录活性增强,而瞬时表达人源ataxin-3分子进入ataxin-3 KO MEF细胞内可抑制EphA3基因启动子的转录活性。为阐明ataxin-3分子对EphA3基因启动子调控的机制,我们采用染色体免疫共沉淀技术,结果发现虽然ataxin-3分子不能直接结合到EphA3启动子区域,但可以调节EphA3启动子区域组蛋白H3和H4乙酰化的水平。进一步免疫印迹结果证实Ataxin-3丢失导致总的组蛋白H3和H4乙酰化水平增加,而调节组蛋白H3和H4乙酰化的酶组蛋白去乙酰化酶3(HDAC3)和核受体共抑制因子(NCoR)的水平减少。在正常MEF细胞内给予HDAC3特异的阻断剂可以模拟ataxin-3丢失的情况。综上所述,我们的研究首次发现,ataxin-3敲除可导致多种基因转录异常。Ataxin-3通过调节组蛋白去乙酰化酶HDAC3和NCoR的水平来调控基因启动子区域组蛋白的乙酰化,进而调控基因的转录。Ataxin-3功能缺失导致的转录异常可能也贡献于SCA3疾病病理,探索Ataxin-3分子的转录调控机制,有助于寻找合适的治疗靶点。
[Abstract]:Part I : Ataxin - 3 Knockout Causes Gene Expression Profile Changes Objective : To observe the effect of attemperance - 3 knock on gene expression by gene expression chip technique . Methods : The experimental results were analyzed by the bioinformatics software of Thomson Reuters / GeneGo , and the reliability of gene expression chip was confirmed by reverse transcription - real - time quantitative PCR . Results : Compared with normal cells , at least one hundred and forty - four genes were differentially expressed ( up - regulated or down - regulated by more than 2 - fold or more ) , and the expression of 260 genes was up - regulated and 150 genes were down - regulated . GeneGo software was used to analyze the biological functions of the 410 genes and classify their functions . Conclusion : The loss of Ataxin - 3 molecules leads to abnormal gene transcription in various cell pathways . Second part : Expression and activity analysis of EphA3 gene promoter region Objective : To construct a reporter gene vector containing EphA3 gene promoter fragment with different length , and to study the transcriptional activity of EphA3 gene promoter . Methods : Using Balb / C mouse genomic DNA as a template , the EphA3 gene promoter fragment of different length was amplified and cloned into the luciferase reporter plasmid pGL3 - Basic eukaryotic expression vector . After the enzyme digestion and the gene sequencing were correct , the recombinant plasmid and the control plasmid in pRL - CMV were co - transfected into 293 cells , and the transcription activity of EphA3 gene promoter fragment of different length was analyzed . Results : The results of enzyme digestion and gene sequencing showed that the expression vector was constructed successfully . The core promoter region of EphA3 gene was located between -279 and + 110bp , and the transcriptional activity of the promoter fragment was similar to that of the promoter fragment . Conclusion : The recombinant plasmid of luciferase reporter gene was successfully constructed and the core promoter region of EphA3 gene was determined . The third part : HDAC3 and NCoR mediate the transcriptional regulation of EphA3 gene . In this paper , we have constructed the promoter fragment of EphA3 gene . The results showed that the expression of Ataxin - 3 could not bind directly to the promoter region of EphA3 , but it could regulate the transcription of histone H3 and H4 .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R346

【共引文献】