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


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.


RESULTS
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.


CONCLUSIONS
These results indicate that expression of FGF-2 in neonatal rat cardiomyocytes is associated with Sp1 binding to the FGF-2 promoter.
Tang
Wenjie
W
Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510080, China.
Pan
Qiuhui
Q
Sun
Fenyong
F
Ma
Ji
J
Tang
Shu
S
Le
Kang
K
Wan
Yang
Y
Chen
Qiongyu
Q
Liu
Peiqing
P
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D054730:Protein Interaction Domains and Motifs; D051381:Rats; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D016329:Sp1 Transcription Factor; D016222:Fibroblast Growth Factor 2