Involvement of Sp1 binding sequences in basal transcription of the rat fibroblast growth factor-2 gene in neonatal cardiomyocytes

Abstract

Aims The identification of transcription factors that regulate the transcription of the fibroblast growth factor-2 (FGF-2) gene has facilitated the understanding of the etiology of cardiovascular diseases. The purpose of this study was to determine the molecular mechanism underlying the activation of FGF-2 gene transcription in cardiomyocytes from neonatal rats. Main methods To identify the factors involved in cardiac expression of FGF-2, we used transient transfections in neonatal rat cardiomyocytes coupled with electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) analyses. Key findings Deletion analyses showed that the region between − 16 and + 59 was essential for maximal FGF-2 promoter activity. Three putative stimulating protein 1 (Sp1) regulatory sites located at positions − 3, + 14, and + 27 were predicted within this region by computer analysis. EMSA showed the existence of two atypical G-rich Sp1-binding elements located at positions − 3 and + 14. Mutation of these two sites resulted in a significant decline in FGF-2 promoter activity compared to wild type promoter activity. Combinatorial mutation of these sites reduced the promoter activity to background levels. Mutation of the Sp1 motif at + 27 did not affect promoter activity. Lastly, ChIP analyses revealed that Sp1 binds to the FGF-2 promoter region in vivo. Significance These results indicate that expression of FGF-2 in neonatal rat cardiomyocytes is associated with Sp1 binding to the FGF-2 promoter.