Skeletal malformations are a significant proportion of birth defects which occur in 3% of newborns. They are often associated with syndromic and non-syndromic disorders of the craniofacies and limb, and arise from either cell autonomous or cell non-autonomous mechanisms. One consequence of dysregulation of osteoblast differentiation is the abnormal skeletogenesis found in cleidocranial dysplasia (CCD). This condition is caused by mutations in a transcriptional determinant of osteoblast cell fate, core binding factor alpha 1 (CBFA1). By delineating its mechanism of action, we learn how dysregulation of osteoblast differentiation cause skeletal malformations. CBFA1 acts in vertebrate acts in vertebrate development in a context-dependent manner which is specified by interaction of CBFA1 domains with other cellular proteins. Two components which may be integral in this process is a newly isolated CBFA1 interacting protein (CIP) and a unique polyglutamine/polyanine domain (Q/A) found in CBFA1, but not in the other mammalian runt domain proteins CBFA2 and CBFA3. Biochemical and genetic data in humans suggest that CIP interacts with CBFA1 to modulate its action, perhaps in a repressive manner. While a transactivation function has been attributed to the region overlapping the Q/A domain, in vitro studies have focused on the polyalanine stretch. In addition, the presence of the Q/A domain may prevent interaction of CBFA1 with CBFbeta, a normally required interacting protein CBFA2 function during hematopoiesis. This study will determine the function of CIP and the CBFA1 Q/A domain by both in vitro studies and in vivo studies using null and over-expression mouse models. Ultimately, understanding how dysregulation of osteoblast differentiation translate into a clinical phenotype will be critical for correlating CBFA1 structure with function, and for diagnosis and prevention of these diseases. By generating a large allelic series of CBFA1 mutation and correlating their functional consequences in vitro with a well characterized CCD patients, we will be able to determine the effects of hypomorphic mutations. The sensitivity of such correlations will be improved by application of new quantitative measures of craniofacial development such as 3D laser surface scanning. These studies will elucidate basic mechanisms governing osteoblast differentiation and show how dysregulation of CBFA1 function translates into the craniofacial and skeletal abnormalities in CCD. Moreover, they will establish a model for understanding how other genes may interact in this pathway and contribute to skeletal malformations, while also developing new technology which will aid in the characterization and diagnosis of other congenital malformations involving dysmorphic facies.
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