THE MOLECULAR-BASIS OF BOSTON-TYPE CRANIOSYNOSTOSIS - THE PRO148-]HISMUTATION IN THE N-TERMINAL ARM OF THE MSX2 HOMEODOMAIN STABILIZES DNA-BINDING WITHOUT ALTERING NUCLEOTIDE-SEQUENCE PREFERENCES

Craniosynostosis, Boston type is an autosomal dominant disorder that r esults in the premature fusion of calvarial bones and ensuing abnormal ities in skull shape, We showed previously that this disorder is tight ly linked to the Msx2 homeobox gene on the long arm of chromosome 5, a nd that affected individuals bear a mutated copy of Msx2. In addition, transgenic mice in which either mutant or wild-type mouse Msx2 is ove rexpressed in the developing skull also exhibit craniosynostosis, That both mutant and wild-type Msx2 elicit craniosynostosis in transgenic mice and that the Boston type mutation is dominant led us to hypothesi ze that the mutation might enhance the normal function of Msx2. The mu tation is located in position 7 of the N-terminal arm of the homeodoma in, a region implicated in both target sequence recognition and protei n-protein interactions. Here we test the hypothesis that the Pro148--> His mutation alters the DNA binding properties of Msx2. Using gel shif t and binding site selection analyses, we show that the mutation enhan ces the affinity of Msx2 for a set of known Msx2 target sequences but has little or no effect on the site specificity of Msx2 binding. The e nhancement of Msx2 binding is due largely if not entirely to an increa sed stability of the mutant Msx2-DNA complex. These data provide a mol ecular-level explanation of how the Pro148-->His mutation enhances Msx 2 function and thus leads to the dominant craniosynostosis phenotype.