Characterization of the upstream mouse Cbfa1/Runx2 promoter

Cbfa1 (or Runx2/AML-3/PEPB2alpha) is a transcriptional activator of osteoblastic differentiation. To investigate the regulation of Cbfa1 expression, we isolated and characterized a portion of the 5'-flanking region of the Cbfa1 gene containing its "bone-related" or P1 promoter and exon 1. We identified additional coding sequence in exon 1 and splice donor sites that potentially give rise to a novel Cbfa1 isoform containing an 18 amino acid insert. In addition, primer extension mapping identified in the Cbfa1 promoter a minor mRNA start site located approximately 0.8 kb 5' upstream of the ATG encoding the MASN/p57 isoform and approximately 0.4 kb upstream of the previously reported start site. A luciferase reporter construct containing 1.4 kb of the mouse Cbfa1 promoter was analyzed in Ros 17/2.8 and MC3T3-E1 osteoblast cell lines that express high levels of Cbfa1 transcripts. The activity of this construct was also examined in non-osteoblastic Cos-7 and NIH3T3 cells that do not express Cbfa1 and mesenchymal-derived cell lines, including CH3T101/2, C2C12, and L929 cells, that express low levels of mature Cbfa1 transcripts. The 1.4 kb 5' flanking sequence of the Cbfa1 gene directed high levels of transcriptional activity in Ros 17/2.8 and MC3T3-E1 osteoblasts compared to non-osteoblasts Cos-7 cells, but this construct also exhibited high levels of expression in C310T1/2, L929, and C2C12 cells as well as NIH3T3 cells. In addition, Cbfa1 mRNA expression, but not the activity of the Cbfa1 promoter, was upregulated in a dose-dependent manner in pluripotent mesenchymal C2C12 by bone morphogenetic protein-2 (BMP-2). These data indicate that Cbfa1 is expressed in osteogenic as well as non-osteogenic cells and that the regulation of Cbfa1 expression is complex, possibly involving both transcriptional and post-transcriptional mechanisms. Additional studies are needed to further characterize important regulatory elements and to identify additional regions of the promoter and/or post-transcriptional events responsible for the cell-type restricted regulation of Cbfa1 expression.