Fig. 4. Mutagenesis of the autoregulatory region of the Brn3a enhancer. (A) Shows the location of the Brn3a autoregulatory region, including four consensus Brn3a-binding domains (red) and one octamer binding site (blue). Two rounds of mutagenesis were required to eliminate Brn3a binding completely, finally incorporating 19 nucleotide changes (mut2). (B) The stoichiometry and stability of Brn3a/DNA complexes were assessed in complex-stability EMSA assays. In these assays, a competitor oligonucleotide was added at the stated time prior to the start of electrophoresis. The wild-type enhancer forms a stable complex with multiple Brn3a molecules, while the Mut1 enhancer retains a single stable binding site and binding is eliminated in Mut2. NP, no protein; NC, no competitor. (C) Luciferase reporter constructs containing wild-type or mutant enhancer domains, or a minimal promoter (-36prl) were co-transfected into CV1 epithelial cells with a Brn3a expression plasmid or vector alone (pcDNA). Elimination of the identified Brn3a-binding sites in the mutant enhancer construct eliminated transactivation by Brn3a. Bars represent the mean of triplicate assays, and similar results were observed in three experiments.