Spatial expression of Cbfa1/Runx2 isoforms in teeth and characterization of binding sites in the DSPP gene

Abstract

Cbfa1/Runx2 is an essential transcription factor for osteoblast and odontoblast differentiation. Heterogeneous mutations of Cbfa1 gene result in cleidocranial dysplasia, an autosomal dominant syndrome, characterized by abnormal skeletal genesis and dental disorders. Recently three Cbfa1/Runx isoforms (Pebp2 alpha A/type I, til-1/type II, and Osf2/type III) have been identified that differ in their amino-terminal sequences. The precise roles of Cbfa1/Runx2 isoforms in odontoblast development are not known. The purpose of this study was to determine and compare expression patterns of the three Cbfa1/Runx2 isoforms in newborn tooth organs. Toward this aim, we developed three probes: type I and type II, which specifically hybridize with Pebp2 alpha A and til-1, respectively, and type II/III, which hybridizes with osf2 and partially with til-1. In addition, Cbfa1/Runx2 binding sites were identified in the regulatory elements of mouse dentin sialophosphoprotein (mDSPP) gene, which encodes a matrix protein expressed during odontogenesis. In situ hybridization performed with the specific Cbfa1/Runx2 isoform probes demonstrated that all isoforms are expressed in teeth and bone. The type I isoform was expressed at higher levels than isoforms type II and type II/III in developing newborn mouse incisors. Genomic mDSPP clones were isolated and characterized containing approximately 2.6 kb of the promoter region. Computer analysis of the promoter segment and intron 1 revealed a number of potential transcriptional factor binding sites including five Cbfa1/Runx2 binding sites, three in the promoter region and two within intron 1. DNA-protein assay and antibody supershift experiments showed that these binding sites interact with nuclear extracts isolated from the mouse odontoblast cell line MO6-G3. Further characterization of the functional role of Cbfa1/Runx2 in the regulation of the mDSPP gene expression is being investigated.