The transcription factor B-Myb is a cell-cycle regulated phosphoprotein involved in cell cycle progression through the transcriptional regulation of many genes. In this study, we show that the promoter of the fibroblast growth factor-4 (FGF-4) gene is strongly activated by B-Myb in HeLa cells and it can serve as a novel diagnostic tool for assessing B-Myb activity. Specifically, B-Myb deletion mutants were examined and domains of B-Myb required for activation of the FGF-4 promoter were identified. Using phosphorylation-deficient mutant forms of B-Myb, we also show that phosphorylation is essential for B-Myb activity. Moreover, a mutant form of B-Myb, which lacks all identified phosphorylation sites and which has little activity, can function as a dominant-negative and suppress wild-type B-Myb activity. Acetylation is another post-translational modification known to affect the activity of other Myb family members. We show that B-Myb is acetylated by the coactivator p300. We also show that the bromo and histone acetyltransferase domains of p300 are sufficient to interact with and acetylate B-Myb. These data indicate that phosphorylation of B-Myb is an essential modification for activity and that acetylation of B-Myb may play a role in B-Myb activity.

The importance of interactions between Sox and POU transcription factors in the regulation of gene expression is becoming increasingly apparent. Recently, many examples of the involvement of Sox-POU partnerships in transcription have been discovered, including a partnership between Sox-2 and Oct-3. Little is known about the mechanisms by which these factors modulate transcription. To better understand the molecular interactions involved, we mapped the location of the transactivation domain of Sox-2. This was done in the context of its interaction with Oct-3, as well as its ability to transactivate as a fusion protein linked to the DNA-binding domain of Gal4. Both approaches demonstrated that Sox-2 contains a transactivation domain in its C-terminal half, containing a serine-rich region and the C terminus. We also determined that the viral oncoprotein E1a inhibits the ability of the Gal4/Sox-2 fusion protein to transactivate, as well as the transcriptional activation mediated by the combined action of Sox-2 and Oct-3. In contrast, a mutant form of E1a, unable to bind p300, lacks both of these effects. Importantly, we determined that p300 overcomes the inhibitory effects of E1a in both assays. Together, these findings suggest that Sox-2 mediates its effects, at least in part, through the co-activator p300.

Previous studies have shown that the transcription of the fibroblast growth factor-4 (FGF-4) gene is regulated by a powerful enhancer located approximately three kilobases downstream of the transcription start site. Several conserved cis-regulatory elements in the promoter and the enhancer have been identified, including two Sp1 motifs located in the promoter and one Sp1 motif located in the enhancer. Each of these Sp1 motifs has been shown previously to bind the transcription factors Sp1 and Sp3 in vitro. The main objective of this study was to examine the potential interaction of the FGF-4 promoter and enhancer Sp1 motifs. Using site-directed mutagenesis, we demonstrate that disruption of these sites, individually or in combination, reduce the expression of FGF-4 promoter/reporter gene constructs in embryonal carcinoma cells. Importantly, we demonstrate that disruption of the enhancer Sp1 motif exerts a more pronounced effect on the expression of these constructs than disruption of the promoter Sp1 motifs. We also demonstrate that changing the spacing and the stereo-alignment of the enhancer Sp1 motif, relative to the other cis-regulatory elements of the enhancer, has little effect on the ability of the enhancer to stimulate transcription. Furthermore, embryonic stem cells that contain two disrupted Sp1 alleles were used to demonstrate 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.

It has been shown previously that the FGF-4 gene is regulated by a powerful downstream enhancer in embryonal carcinoma (EC) cells. This enhancer contains an essential HMG motif; however, the transcription factor that binds to the HMG motif in EC cells has not been determined definitively. In earlier studies, this HMG motif was shown to bind a heat-stable, redox-insensitive factor expressed by F9 EC cells. Others have proposed that the transcription factor Sox-2 binds to the FGF-4 enhancer HMG motif. In this study, we demonstrate that the N-terminal half of Sox-2, which contains the DNA binding domain, binds to the FGF-4 enhancer HMG motif and we show that this binding is unaffected by heat and oxidation. In addition, we employed two experimental approaches to demonstrate that Sox-2 regulates the transcription of the FGF-4, gene in EC cells. As part of these studies, an expression plasmid that codes for a dominant-negative form of Sox-2 was used in transient expression assays. In other experiments, a Sox-2 antisense expression plasmid was used. When co-transfected into F9 EC cells along with an FGF-4 promoter/reporter gene construct, each expression plasmid caused a significant reduction in reporter activity. Our studies also demonstrate that Sox-2 affects the expression of the FGF-4 gene in the multipotent EC cell line, P19. Taken together, these studies argue strongly that Sox-2 plays an important role in the expression of the FGF-4 gene in vivo. Mol. Reprod. Dev. 50:377-386, 1998. (C) 1998 Wiley-Liss, Inc.

FGF-4 appears to be the first fibroblast growth factor (FGF) expressed during embryogenesis, and its expression is critical for early mammalian development. FGF-4 is expressed in the embryonic cell lines, F9, D3, and NT2/D1; but its expression in these cells is repressed upon differentiation. Transcription of the FGF-4 gene in embryonic cells is regulated by an enhancer in the third exon and by a positive regulatory region upstream of the transcription start site. A CCAAT box motif within the positive regulatory region has been shown to support FGF-4 expression, but the factor that binds to this site in vivo has not been identified. In this report, we demonstrate that the transcription factor complex NF-Y binds to the FGF-4 CCAAT box motif when nuclear extracts from each of the embryonic cell lines and their differentiated cells were examined by gel mobility shift analyses. Importantly, we demonstrate that expression of a dominant-negative NF-YA mutant protein reduces the expression of FGF-4 promoter/reporter gene constructs in F9 EC cells. Hence, we provide strong evidence that the transcription factor NF-Y is involved in the expression of the FGF-4 gene. Mol. Reprod. Dev. 48:301–309, 1997. © 1997 Wiley-Liss, Inc.