Rapid identification of homeodomain binding sites in the Wnt-5a gene using an immunoprecipitation strategy

Here we describe an immunoprecipitation approach for identifying homeodomain binding sites within uncharacterized genomic sequences of a putative downstream target gene, Wnt-5a. Immunoprecipitation of Wnt-5a genomic fragments was performed using a purified Msx1 homeodomain polypeptide (Msx1) and its corresponding antisera (alpha-Msx1). This resulted in isolation of three fragments containing multiple DNA binding sites for Msx1, as confirmed by DNA binding studies. The three fragments were contiguous within a 3.4 kb intronic sequence of Wnt-5a. Moreover, at least one of the Msx1 sites has been conserved throughout evolution, suggesting that these sites may comprise or contribute to a regulatory element for Wnt-5a. We propose that the immunoprecipitation strategy permits a rapid, initial approach for identifying functionally-relevant homeodomain binding sites within target genes whose regulatory sequences have not yet been previously elucidated.