Analysis of the Mouse Splotch-Delayed Mutation Indicates that the Pax-3 Paired Domain can Influence Homeodomain DNA-Binding Activity

The murine Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain, and alterations in the Pax-3 gene are responsible for the neural tube defects observed in the Splotch (Sp) mouse mutant. Of five Sp alleles, Splotch-delayed (Spd) is the only one that encodes a full-length Pax-3 protein, containing a single glycine-to-arginine substitution within the paired domain. To better understand the consequence of this mutation on Pax-3 function, we have analyzed the DNA-binding properties of wild-type and SpdPax-3, using oligonucleotides that bind primarily to the paired domain (e5) or exclusively to the homeodomain (P2). Wild-type Pax-3 was found to bind e5in a specific manner. In contrast, the Spdmutation reduced binding of Pax-3 to e517-fold, revealing a defect in DNA binding by the paired domain. Surprisingly, the Spdmutation also drastically reduced the homeodomain-specific binding to P2 by 21-fold when compared with the wild-type protein. Interestingly, a deletion which removes the Spdmutation was found to restore P2-binding activity, suggesting that within the full-length Pax-3 protein, the paired domain and homeodomain may interact. We conclude, therefore, that the Spdmutation is phenotypically expressed in vitro by a defect in the DNA-binding properties of Pax-3. Furthermore, it is apparent that the paired domain and homeodomain of Pax-3 do not function as independent domains, since a mutation in the former impairs the DNA-binding activity of the latter.