| Gene transcription is a highly complex process that is strictly regulated to ensure proper spatial and temporal gene expression. To ensure proper expression, and prevent aberrant transcription, many, if not most, genes are regulated by enhancer elements. In this regard, transcription of the fibroblast growth factor-4 (FGF-4) gene during early development is controlled by a powerful distal enhancer located 3 kb downstream of the transcription start site within the 3′ untranslated region of the gene. Previous studies have shown that FGF-4 enhancer function is mediated by at least three critical positive cis-regulatory elements: an HMG, a POU, and a GT-box motif, which bind the transcription factors Sox-2, Oct-3, and Sp1/Sp3, respectively. In this dissertation, a second essential HMG motif is identified within the FGF-4 enhancer that binds the transcription factor Sox-2. Furthermore, it is shown using embryonic stem cells containing targeted disruptions of both Sp1 alleles, that the transcription factor Sp1 is not required for expression of FGF-4 transcripts in vivo. In addition, the transcription factor Sp3 is identified as a more potent mediator of the enhancer GT-box than Sp1, suggesting that Sp3 likely mediates that effects of the enhancer GT-box motif in vivo. Lastly, it is shown that a precise spatial arrangement of the new HMG motif, relative to the previously identified enhancer cis-regulatory elements, is required for optimal enhancer function. This is important, because enhancer mediated activation of many genes is accomplished by an intricate array of enhancer cis-regulatory elements that direct the assembly of a gene-specific activation complex known as an “enhanceosome.” Based on findings presented in this dissertation, and work published earlier demonstrating that the previously identified HMG and POU motifs of the FGF-4 enhancer function cooperatively and require a strict spatial arrangement, it is proposed that the FGF-4 enhancer functions, in part, as an enhanceosome. Thus, the work presented in this dissertation advances our understanding of FGF-4 gene regulation and the function of distal enhancer elements. |
