Sp1 and finb interact with BETA2/NeuroD to enhance its transactivation of the secretin promoter

Abstract

of full length fragment which covers all three gastrin responsive elements were cloned for yeast one hybrid screening. Electrophoretic mobility shift assays (EMSAs) were then used to confirm the positives. To determine the functional importance of the target factor(s), transient transfection and reporter assays were performed by overexpression of the target nuclear factor(s) in the presence of the HDC promoter construct which contains 125bp upstream sequence and 126bp downstream sequence followed by the luciferase gene. Results: Yeast one hybrid screening indicated that gut-enriched Kruppel like factor (GKLF) binds downstream gastrin responsive elements of the HDC promoter. Electrophoretic mobility shift assays confirmed the binding of GKLF with all three downstream gastrin responsive elements. EMSA competition assay also showed the binding of Spl with all three gastrin responsive elements. Furthermore, EMSAs indicated that GKLF binds Spl bound GC-box fragment which is located upstream of the HDC promoter. Overexpression of GKLF and Spl transactivated HDC promoter activity, which was further supported by the repressive effect o f their dominant negative plasmids. Conclusion: GKLF and Sp 1 synergistically regulate HDC promoter activity through binding both upstream GC box and downstream gastrin responsive elements.