The long-term goal of this study is to determine how distal enhancers regulate gene transcription. Since the 1990s, the FGF-4 gene, which is repressed when embryonal carcinoma (EC) cells differentiate, has emerged as a powerful model to study how differentiation regulates gene transcription and how distal enhancers function. The FGF-4 enhancer contains at least three critical cis-regulatory elements: an HMG motif, which binds the transcription factor Sox-2; an octamer binding motif, which binds the transcription factor Oct-3; and a GT/CACC box, which binds the transcription factors Spi and Sp3. Although a great deal is now known about the FGF-4 enhancer, many important questions remain unanswered, in particular: How does the enhancer of the FGF-4 gene, which is located three kilobases from the transcription start site, mediate its effects? Recent studies argue that Sox-2 and Oct-3 can mediate their effects through the co-activators p300 and CBP (p300/CBP). On the basis 01 these and other findings, it is hypothesized that p300/CBP are required for the action of the FGF-4 enhancer. This hypothesis will be tested in Specific Aim 1. The studies proposed in Specific Aim 2 seek to understand mechanistically how p300/CBP mediate the effects of Sox-2 and Oct-3. Other studies focus on the function of the GT/CACC box in the FGF-4 enhancer. Although this cis-regulatory element can bind two related transcription factors, Sp 1 and Sp3, in vitro, recent studies have shown that Sp 1 is not essential for transcription of the FGF-4 gene. Hence, it has been hypothesized that Sp3 mediates the effect 01 the FGF-4 enhancer GT/CACC box. Alternatively, it is possible that Sp3 and Spi are redundant and both are able to stimulate transcription of the FGF-4 gene. The goal of Specific Aim 3 is to test the hypothesis regarding Sp3. Recent studies have shown that the FGF-4 enhancer is influenced by at least one other essential cis-regulatory element that has been localized to a small region of the FGF-4 enhancer. The studies proposed in Specific Aim 4 seek to map the precise location of this cis-regulatory element(s) and to determine whether the factor(s) that binds to this site is regulated by the differentiation of EC cells. Together, the studies proposed in this application are significant for at least four reasons: 1)They focus on a gene that codes for an essential embryonic growth factor, which is expressed in many tumors. 2)They will be performed in a well characterized model system in which the FGF-4 gene is turned off at the transcriptional level when the cells differentiate. 3)They will provide critical information regarding the mechanisms used by one of the most powerful distal enhancers described to date. 4) They will provide a new understanding of the mechanisms by which the transcription factors, Sox-2 and Oct-3, utilize p300/CBP to stimulate transcription. The last studies are highly significant in their own right, because they will provide novel insights into the mechanisms used by members of two large transcription factor families to stimulate transcription.
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