Synergistic regulation of the Runx2 P1 promoter in mesenchymal cells by a conserved HLH box and purine‐rich elements (GAY motifs)

Abstract

Transcriptional control of Runx2 gene expression through two alternative promoters (P1 and P2) is critical for the execution of its function as an osteogenic cell fate determining factor. Using genomic DNaseI footprinting and in vivo DNA binding assays, we identified a helix‐loop‐helix (HLH) motif that overlaps with a Runx motif in the P1 promoter. The HLH mutation decreases P1 transcription 10‐fold in mesenchymal cells, while mutation of the Runx site enhances transcription 2 fold. Although the HLH element is not cell‐type specific, its function may depend on promoter context. Promoter deletion analysis shows that a GA‐rich region (−200 to −128) positively regulates Runx2 P1 transcription and synergizes with the HLH motif. Examination of the −200/−128 region reveals two tandemly repeated GA‐rich elements (GAY motifs) that each contain recognition motifs for purine‐rich DNA binding factors (i.e., SP1 and KLF10/TGFâ Inducible Factor [TIEG]). Forced expression of SP1 or TIEG modulates Runx2 P1 promoter activity through the GAY motifs. Taken together, our data suggest that the Runx2 gene transcription is regulated by synergistic interactions at multiple conserved composite promoter elements to control Runx2 expression and skeletal development.