Characterization of the upstream mouse Cbfa1/Runx2 promoter*

Abstract Cbfa1 (or Runx2/ AML‐3 /PEPB2α) 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 ∼0.8 kb 5′ upstream of the ATG encoding the MASN/p57 isoform and ∼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. J. Cell. Biochem. 82: 647–659, 2001. © 2001 Wiley‐Liss, Inc.