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Regulatory Effects of Egr-1 on APP Gene Expression
Ma Lin1#, Zhou Yu2#, Li Yongling2, Chai Juan1, Jia Zhongfa2, Guo Ke2, Sun Tao1, Cui Jianqi1*
1Ningxia Key Laboratory of Cerebrocranial Diseases, the Incubation Base of National Key Laboratory, Yinchuan 750004, China;2School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, China
Abstract: The luciferase reporter plasmids which contain APP gene promoter and mutant APP gene promoter were constructed. The Egr-1 eukaryotic expression vector pCDNA3-Egr-1 and the constructed reporter plasmids were co-transfected into HEK293 and U87MG cells to analyze the effects of Egr-1 on APP promoter activities. The chromatin immunoprecipitation assay (ChIP) and electrophoretic mobility shift assay (EMSA) were performed to determine the binding sites of Egr-1 on 5′UTR in APP promoter. Cells were treated with histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and butyrate as well as Egr-1 inhibitor suramin and Western blot was performed to evaluate the changes of endogenous Egr-1 expression and the effects of Egr-1 on APP promoter activities were verified. The results of luciferase assay demonstrated that Egr-1 could up-regulate APP gene expression， and Egr-1 could bind to the specific binding sites in the 5′UTR of APP promoter. The up-regulatory effects of Egr-1 on APP promoter would be eliminated when these binding sites were deleted. ChIP and EMSA results confirmed that Egr-1 could bind to these sites. In addition， the effects of Egr-1 on APP promoter activities were dramatically influenced if the endogenous expression of Egr-1 was disturbed by HDAC inhibitor and suramin.
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