Synergistic Activation of the Fibroblast Growth Factor 4 Enhancer by Sox2 and Oct-3 Depends on Protein-Protein Interactions Facilitated by a Specific Spatial Arrangement of Factor Binding Sites

Abstract

Octamer binding and Sox factors are thought to play important roles in development by potentiating the transcriptional activation of specific gene subsets. The proteins within these factor families are related by the presence of highly conserved DNA binding domains, the octamer binding protein POU domain or the Sox factors HMG domain. We have previously shown that fibroblast growth factor 4 (FGF-4) gene expression in embryonal carcinoma cells requires a synergistic interaction between Oct-3 and Sox2 on the FGF-4 enhancer. Sox2 and Oct-3 bind to adjacent sites within this enhancer to form a ternary protein-DNA complex (Oct-3*) whose assembly correlates with enhancer activity. We now demonstrate that increasing the distance between the octamer and Sox binding sites by base pair insertion results in a loss of enhancer function. Significantly, those enhancer "spacing mutants" which failed to activate transcription were also compromised in their ability to form the Oct* complexes even though they could still bind both Sox2 and the octamer binding proteins, suggesting that a direct interaction between Sox2 and Oct-3 is necessary for enhancer function. Consistent with this hypothesis, Oct-3 and Sox2 can participate in a direct protein-protein interaction in vitro in the absence of DNA, and both this interaction and assembly of the ternary Oct* complexes require only the octamer protein POU and Sox2 HMG domains. Assembly of the ternary complex by these two protein domains occurs in a cooperative manner on FGF-4 enhancer DNA, and the loss of this cooperative interaction contributes to the defect in Oct-3* formation observed for the enhancer spacing mutants. These observations indicate that Oct-3* assembly results from protein-protein interactions between the domains of Sox2 and Oct-3 that mediate their binding to DNA, but it also requires a specific arrangement of the binding sites within the FGF-4 enhancer DNA. Thus, these results define one parameter that is fundamental to synergistic activation by Sox2 and Oct-3 and further emphasize the critical role of enhancer DNA sequences in the proper assembly of functional activation complexes.

Rex-1, a Gene Encoding a Transcription Factor Expressed in the Early Embryo, Is Regulated via Oct-3/4 and Oct-6 Binding to an Octamer Site and a Novel Protein, Rox-1, Binding to an Adjacent Site

“…These vectors were previously described (35). The values for percent CAT conversion, presented as means Ϯ standard deviation, corresponding to p0.3Rex-CAT in the absence or presence of Oct-6, N157, N229, N2C52, and N229C52 are 18. regulating the expression of two additional cellular genes, encoding PDGF␣R and FGF-4, both of which are controlled by the Oct-3/4 gene product in EC cells (1,10,25,75). Transcriptional activation of the FGF-4 gene depends on a synergistic interaction between the Oct-3/4 and Sox2 (a member of the Sry-related Sox factors family) proteins, both binding sequences located very close to each other.…”

FGF4 dissociates anti-tumorigenic from differentiation signals of retinoic acid in human embryonal carcinomas

“…Of these diverse functions, FGF4 signals are especially interesting to examine in germ cell tumors since expression of FGF4 is tightly regulated during development and restricted to undi erentiated ECs (Miller and Rizzino, 1996;Miller et al, 1990;Mummery et al, 1993;Scho®eld et al, 1991;Strohmeyer et al, 1991;Tiesman and Rizzino, 1989;Yoshida et al, 1988a,b). Cell stage-speci®c expression depends on an enhancer element present 3' to FGF4 coding sequences, which contains an octamer binding site (Ambrosetti et al, 1997;Curatola and Basilico, 1990;Dailey et al, 1994;Yuan et al, 1995). RAtreatment represses FGF4 expression in di erentiation sensitive but not resistant human ECs (Miller and Rizzino, 1996;Miller et al, 1990Miller et al, , 1993Mummery et al, 1993;Scho®eld et al, 1991;Tiesman and Rizzino, 1989;Yoshida et al, 1988a,b).…”

Stem cell pluripotency and transcription factor Oct4

“…This enhancer contains an octamer motif adjacent to a binding site to which Oct4 and the high mobility group (HMG) transcription factor Sox-2 bind cooperatively to activate transcription synergistically [41]. This synergism is most likely mediated by protein-protein interactions [42]. In the absence of Sox-2, Oct4 is not sufficient for FGF4 enhancer activity, even in the presence of the bridging factor E1A [42].…”

“…This synergism is most likely mediated by protein-protein interactions [42]. In the absence of Sox-2, Oct4 is not sufficient for FGF4 enhancer activity, even in the presence of the bridging factor E1A [42]. In addition, the formation of Oct4/ Sox-2 complex also appears to be a reciprocal event, since the complex could unmask latent activation domains in both proteins, thus leading to transcriptional activation [43].…”