Identification of the Transactivation Domain of the Transcription Factor Sox-2 and an Associated Co-activator

Abstract

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.

Identification of Novel Domains within Sox-2 and Sox-11 Involved in Autoinhibition of DNA Binding and Partnership Specificity

“…Plasmids-Constructs pCATSO3, pCMVSox-2F 1-180 , pCMVSox-2, pCMVOct-3, pCMVOct-1, and CMV-␤-gal have been described previously (19). The promoter/reporter construct pCATDC3 was constructed similarly to pCATSO3 as follows.…”

“…Whole cell protein extracts were prepared as described previously (19). Nuclear extracts were prepared using the NE-PER kit (Pierce) following the manufacturer's protocol.…”

“…Cell Culture and Transient Transfection-HeLa cells were maintained and transfected using the calcium phosphate precipitation method as described previously (19). All transfections were performed in triplicate with representative experiments shown.…”

“…FGF-4 expression depends on a distal enhancer (13,14), which contains an essential binding site for Sox-2 (15)(16)(17). However, Sox-2 does not appear to be capable of substantial activation of this gene on its own, but rather requires cooperation with the POU transcription factor Oct-3 (18,19). The presence of Oct-3 at a site adjacent to Sox-2 promotes cooperative DNA binding, resulting in a synergistic increase in transactivation (18,19).…”

“…However, Sox-2 does not appear to be capable of substantial activation of this gene on its own, but rather requires cooperation with the POU transcription factor Oct-3 (18,19). The presence of Oct-3 at a site adjacent to Sox-2 promotes cooperative DNA binding, resulting in a synergistic increase in transactivation (18,19). The partnership of Sox-2 and Oct-3 is extremely specific for these two proteins because it requires stereospecific alignment and protein-protein interaction between the HMG and POU DNA binding domains when Sox-2 and Oct-3 are present at their adjacent binding sites (18).…”