GATA4与STAT3协同激活EGF启动子活性

发布时间：2018-02-26 05:21

本文关键词： GATA4 STAT3 EGF 转录活性　出处：《大连理工大学》2011年硕士论文　论文类型：学位论文

【摘要】：心脏是胚胎发育过程中第一个形成的功能性器官,如果某一环节异常,则会引发先天性心脏病。GATA4是一个重要的调控心脏发育和分化的转录因子,GATA4基因的突变导致先天性心脏病的发生。表皮生长EGF促进胎儿心肌细胞增殖,抑制心肌细胞的分化,对心脏的正常发育和心功能的维持都起很重要的作用。但是目前心脏中EGF表达调节机制并不清楚。为了便于研究转录因子对EGF的转录调控,我们构建了含EGF启动子的报告基因。本研究旨在研究与GATA4相互作用的蛋白,并检测其对GATA4转录活性的影响。在本研究中,通过免疫共沉淀-免疫印迹实验,我们证实了在细胞内STAT3能与GATA4在细胞核中形成复合物。为了判断它们间的相互作用是直接还是间接,我们采用GST pull-down和免疫印迹实验验证了STAT3能直接与GATA4相互作用,并且通过缺失体实验发现GATA4与STAT3间的相互作用并不依赖GATA4的C-末端的锌指结构；STAT3的SH2结构域对其与GATA4间的相互作用是必需的。随后,我们从大鼠基因组中克隆了EGF启动子序列,并将其插入到无启动子的荧光素酶表达载体pGL3-basic上,构建含EGF启动子的荧光素酶报告基因。我们利用荧光素酶报告基因实验,验证了GATA4和STAT3都能激活EGF启动子活性,并且在细胞内具有协同性。综上所述,本研究中证实了在体内和体外,STAT3能和GATA4相互作用,并且STAT3与GATA4的相互作用是通过STAT3的SH2区域介导的,但是不依赖GATA4的C-末端锌指结构。我们构建了含有EGF启动子的报告基因,并且发现GATA4和STAT3能协同激活EGF启动子的转录活性。
[Abstract]:The heart is the first functional organ formed during embryonic development, and if a link is abnormal, GATA4 is an important transcription factor to regulate cardiac development and differentiation, which leads to the occurrence of congenital heart disease. Epidermal growth of EGF promotes the proliferation of fetal cardiomyocytes and inhibits the differentiation of cardiomyocytes. It plays an important role in the normal development of the heart and the maintenance of cardiac function. However, the regulation mechanism of EGF expression in the heart is not clear. In order to study the transcription regulation of EGF by transcription factors, We constructed a reporter gene containing EGF promoter. The aim of this study was to study the protein interacting with GATA4 and to detect its effect on the transcriptional activity of GATA4. In this study, we confirmed that STAT3 can form complex with GATA4 in the nucleus by immunoprecipitation-immunoblotting assay in order to determine whether the interaction between them is direct or indirect. We used GST pull-down and Western blot experiments to verify that STAT3 can interact with GATA4 directly. Furthermore, we found that the interaction between GATA4 and STAT3 was not dependent on the SH2 domain of GATA4's C-terminal zinc finger structure, STAT3. Subsequently, we cloned the EGF promoter sequence from the rat genome. The luciferase reporter gene containing EGF promoter was constructed by inserting it into the luciferase expression vector pGL3-basic without promoter. We have verified that both GATA4 and STAT3 can activate the activity of EGF promoter by luciferase reporter gene experiment. And there is synergy in the cells. In conclusion, it is confirmed that STAT3 can interact with GATA4 in vivo and in vitro, and that the interaction between STAT3 and GATA4 is mediated by the SH2 region of STAT3. We constructed a reporter gene containing EGF promoter and found that GATA4 and STAT3 can co-activate the transcriptional activity of EGF promoter.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

【相似文献】