Effects of three Sp1 motifs on the transcription of the FGF-4 gene

Previous studies have shown that the transcription of the fibroblast growth factor-4 (FGF-4) gene is regulated by a powerful enhancer located approxim ately three kilobases downstream of the transcription start site. Several c onserved cis-regulatory elements in the promoter and the enhancer have been identified, including two Sp1 motifs located in the promoter and one Sp1 m otif located in the enhancer. Each of these Sp1 motifs has been shown previ ously to bind the transcription factors Sp1 and Sp3 in vitro. The main obje ctive of this study was to examine the potential interaction of the FGF-4 p romoter and enhancer Sp1 motifs. Using site-directed mutagenesis, we demons trate that disruption of these sites, individually or in combination, reduc e the expression of FGF-4 promoter/reporter gene constructs in embryonal ca rcinoma cells. Importantly, we demonstrate that disruption of the enhancer Sp1 motif exerts a more pronounced effect on the expression of these constr ucts than disruption of the promoter Sp1 motifs. We also demonstrate that c hanging the spacing and the stereo-alignment of the enhancer Sp1 motif, rel ative to the other cis-regulatory elements of the enhancer, has little effe ct on the ability of the enhancer to stimulate transcription. Furthermore, embryonic stem cells that contain two disrupted Sp1 alleles were used to de monstrate that the transcription factor Sp1 is not necessary for expression of the endogenous FGF-4 gene. Finally, the significance of these findings relative to a looping model for the transcriptional activation of the FGF-4 gene is discussed. (C) 2000 Wiley-Liss, Inc.