An Osteoblast-Specific Enhancer and Subenhancer Cooperatively Regulate Runx2 Expression in Chondrocytes

Runx2 is an essential transcription factor for osteoblast differentiation and chondrocyte maturation. The spatiotemporal expression of Runx2 is regulated by enhancers. We previously identified a 1.3 kb osteoblast-specific enhancer; however, mice with this deletion showed no phenotypes. A 0.8 kb conserved region detected near the 1.3 kb enhancer did not exhibit enhancer activity in reporter assays, whereas four tandem repeats of 452 bp (452 × 4) containing the most conserved region of 0.8 kb induced strong reporter activity in chondrocyte cell lines. However, chondrocytes of enhanced green fluorescent protein (EGFP) reporter mice using 452 × 4 did not express EGFP. When 452 × 4 was combined with the 1.3 kb enhancer, hypertrophic chondrocytes highly expressed EGFP. Moreover, the 0.8 kb region combined with the 1.3 kb enhancer induced EGFP expression in prehypertrophic and hypertrophic chondrocytes. The deletion of both the 1.3 kb enhancer and the 0.8 kb conserved region slightly reduced Runx2 expression in the limbs. However, neither homozygous nor heterozygous deletions in the Runx2^+/- background showed phenotypes. The 0.8 kb conserved region itself lacked enhancer activity, but when combined with the 1.3 kb enhancer, EGFP expression was induced in chondrocytes with a similar expression pattern to Runx2. Therefore, the 0.8 kb conserved region has a novel function as a subenhancer.