AbstractEpigenetic mechanisms mediating expression of the Runt‐related transcription factor Runx2 are critical for controlling its osteogenic activity during skeletal development. Here, we characterized bona fide regulatory elements within 120 kbp of the endogenous bone‐related Runx2 promoter (P1) in osteoblasts by genomic DNase I footprinting and chromatin immuno‐precipitations (ChIPs). We identified a ∼10 kbp genomic domain spanning the P1 promoter that interacts with acetylated histones H3 and H4 reflecting an open chromatin conformation in MC3T3 osteoblasts. This large chromatin domain contains a single major DNaseI hypersensitive (DHS) region that defines a 0.4 kbp “basal core” promoter. This region encompasses two endogenous genomic protein/DNA interaction sites (i.e., footprints at Activating Protein 1 [AP1], E‐box and Runx motifs). Helix‐Loop‐Helix (HLH)/E‐box occupancy and presence of the DHS region persists in several mesenchymal cell types, but AP1 site occupancy occurs only during S phase when Runx2 expression is minimal. Point‐mutation of the HLH/E box dramatically reduces basal promoter activity. Our results indicate that the Runx2 P1 promoter utilizes two stable principal protein/DNA interaction domains associated with AP1 and HLH factors. These sites function together with dynamic and developmentally responsive sites in a major DHS region to support epigenetic control of bone‐specific transcription when osteoblasts transition into a quiescent or differentiated state. J. Cell. Physiol. 228: 313–321, 2013. © 2012 Wiley Periodicals, Inc.