A Helix‐Loop‐Helix Motif within a Genomic DNaseI Footprint of the Runx2 Gene Promoter is Essential for Basal Bone Tissue‐Specific Transcription

Abstract

Transcriptional mechanisms controlling Runx2 levels are critical for its osteogenic function during osteoblast differentiation. Here we examine the chromatin organization of the genomic Runx2 locus in intact cells. Chromatin immuno‐precipitations were performed with antibodies of modified histones and PCR probes spanning 120 kbp surrounding its bone‐specific (P1) promoter. The results show that a ~10 kbp genomic segment around the P1 promoter interacts with highly acetylated histones H3 and H4 reflecting an open chromatin conformation. Within this acetylated domain, we find a single major DNaseI hypersensitive (DHS) region that resolves into a cluster of smaller sites (−0.4 ~0.0 kbp) and coincides with the promoter segment that controls basal Runx2 transcription in osteoblasts. Genomic DNase I footprinting indicates that the DHS region contains several protein/DNA interaction sites (e.g., at AP1, HLH/E‐box and Runx motifs). HLH/E‐box occupancy of the DHS region persists in all mesenchymal cell types examined. Occupancy of the AP1 binding site occurs only during S phase when Runx2 expression is minimal. Point‐mutation of the HLH/E box causes a dramatic reduction of the P1 promoter activity. We propose that protein/DNA interactions at the AP1 and HLH/E box motifs may control Runx2 transcription when osteoblasts transit into a quiescent and differentiated state. Supported by NIH grant AR039588.