IDENTIFICATION OF NOVEL PAX-2 BINDING-SITES BY CHROMATIN PRECIPITATION

The Pax genes encode a family of developmental transcription factors t hat bind to specific DNA sequences via the paired domain and are neces sary for the morphogenesis of a variety of tissues, The murine Pax-2 g ene, through alternative splicing, encodes two nuclear proteins, Pax-2 A and Pax-2B, which are transiently expressed during the differentiati on of specific neural cell types and early kidney formation. In order to identify potential in vivo Pax-2 target sequences, chromatin from e mbryonic neural tube was immunoprecipitated with Pax-2 specific antibo dies and cloned. Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding as says using Pax-2 proteins produced in both Escherichia coil and eukary otic cells. In vitro DNA binding assays, using Pax-5 and Pax-8 DNA rec ognition sequences as well as the three immunopurified Pax-2 binding s ites, demonstrated that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain. The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequen ces for Pax-5 and Pax-2. The genomic clones can now be used as sequenc e tags to identify potential target loci.