Sp1 is required for transcriptional activation of the fibroblast growth factor receptor 1 gene in neonatal cardiomyocytes.
Fibroblast growth factor receptor 1 (FGFR1) is the predominant FGFR in cardiac tissue and regulates proliferation, differentiation, and maintenance of normal myocardium. During development of cardiac tissue, FGFR1 gene expression regulates cardiomyocyte proliferation. The focus of this study was to determine the molecular mechanism of transcriptional activation of the FGFR1 gene in proliferating neonatal cardiomyocytes. Analysis of DNA sequence of the FGFR1 gene identified three potential Sp factor binding sites located at 49 bp, 68 bp, and 100 bp upstream from the 3' end of the promoter segment. Mutation of each of these sites resulted in a significant decline in FGFR1 promoter activity compared to wild type promoter activity, and combinatorial mutation of all three sites completely abrogated promoter activity to background levels. In addition, overexpression of Sp1 in neonatal cardiomyocytes resulted in a dose-dependent increase in wild type FGFR1 promoter activity. However, Sp1-mediated up-regulation of promoter activity was abrogated when all three Sp interacting sites were mutated. Chromatin immunoprecipitation (ChIP) assays were used to demonstrate direct interactions of Sp1 with the proximal promoter region of the FGFR1 gene in neonatal cardiomyocytes. ChIP assays using Drosophila Schneider Line 2 (SL2) cells transiently transfected with wild type or mutant FGFR1 promoter constructs verified the direct interaction between Sp1 and the three Sp1 interacting sites of the promoter. Western blot analyses indicated that Sp1 was present in cytoplasmic and nuclear extracts of neonatal myocardium. These results indicate that Sp1 is a necessary positive regulator of FGFR1 gene transcription in neonatal cardiomyocytes.
Seyed
Mahdie
M
Rosalind Franklin University of Medicine and Science, The Chicago Medical School, Department of Cell Biology and Anatomy, 3333 Green Bay Road, North Chicago, Illinois 60064, USA.
Dimario
Joseph X
JX
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D009154:Mutation; D032383:Myocytes, Cardiac; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection
D016329:Sp1 Transcription Factor; D051496:Receptor, Fibroblast Growth Factor, Type 1