Fig. 5.

The Brn-3.0 autoregulatory domains contain multiple stable Brn-3-binding sites. Complex stability EMSAs were performed as described in Materials and Methods. A andB show the interaction of a GST-Brn-3.0 POU-domain fusion protein with DNA fragments containing the distal (A) and proximal (B) autoregulatory domains from the Brn-3.0 locus. Lanes are designated by the time in minutes between the addition of a competitor oligonucleotide and the start of electrophoresis. The − lane represents electrophoresis of the complexes without competitor oligonucleotide. In the pre lane the competitor was added before the formation of the Brn-3.0 complex with the radiolabeled probe to demonstrate that the amount of competitor added is in large excess over Brn-3.0 protein. In C, the Brn-3.0 protein used was cleaved with thrombin to remove the GST-moiety, and complex stability EMSAs were conducted with the isolated Brn-3.0 POU-domain. As previously noted (Rhee et al., 1998), dissociation of the Brn-3.0–DNA complex is somewhat faster for the isolated POU domain than for the GST-fusion protein. The legend at rightrepresents the number of Brn-3.0 molecules contained in the Brn-3.0–DNA complexes, where zero denotes the free DNA probe. These results show that the proximal Brn-3.0 autoregulatory region can interact stably with at least six Brn-3.0 monomers, and that complexes containing an even number of Brn-3.0 molecules are highly favored, suggesting cooperative binding in pairs (Rhee et al., 1998). The lane designated −P contains the DNA probe alone.