Autoregulatory sequences are revealed by complex stability screening of the mouse brn-3.0 locus

The POU-IV or Brn-3 class of transcription factors exhibit conserved struct ure, DNA-binding properties, and expression in specific subclasses of neuro ns across widely diverged species. In the mouse CNS, Brn-3.0 expression cha racterizes specific neurons from neurogenesis through the life of the cell. This irreversible activation of expression suggests positive autoregulatio n. To search for cis-acting elements that could mediate autoregulation we u sed a novel method, complex stability screening, which we applied to rapidl y identify functional Brn-3.0 recognition sites within a large genomic regi on encompassing the mouse brn-3.0 locus. This method is based on the observ ation that the kinetic stability of Brn3.0 complexes with specific DNA sequ ences, as measured by their dissociation half-lives, is highly correlated w ith the ability of those sequences to mediate transcriptional activation by Brn-3.0. The principal Brn-3.0 autoregulatory region lies similar to 5 kb upstream from the Brn-3.0 transcription start site and contains multiple Br n-3.0-binding sites that strongly resemble the optimal binding site for thi s protein class. This region also mediates transactivation by the closely r elated protein Brn-3.2, suggesting a regulatory cascade of POU proteins in specific neurons in which Brn-3.2 expression precedes Brn-3.0.