RAPID IDENTIFICATION OF HOMEODOMAIN BINDING-SITES IN THE WNT-5A GENE USING AN IMMUNOPRECIPITATION STRATEGY

Here we describe an immunoprecipitation approach for identifying homeo domain binding sites within uncharacterized genomic sequences of a put ative downstream target gene, Wnt-5a. Immunoprecipitation of Wnt-5a ge nomic fragments was performed using a purified Msx1 homeodomain polype ptide (Msx1) and its corresponding antisera (alpha-Msx1). This resulte d in isolation of three fragments containing multiple DNA binding site s for Msx1, as confirmed by DNA binding studies. The three fragments w ere 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 regulato ry element for Wnt-5a. We propose that the immunoprecipitation strateg y permits a rapid, initial approach for identifying functionally-relev ant homeodomain binding sites within target genes whose regulatory seq uences have not yet been previously elucidated. (C) 1996 Academic Pres s, Inc.