Genomic occupancy of HLH, AP1 and Runx2 motifs within a nuclease sensitive site of the Runx2 gene.

Epigenetic 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 120kbp of the endogenous bone-related Runx2 promoter (P1) in osteoblasts by genomic DNase I footprinting and chromatin immuno-precipitations (ChIPs). We identified a similar to 10kbp 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 0.4kbp 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: 313321, 2013. (c) 2012 Wiley Periodicals, Inc.