AbstractThe molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are poorly understood. The cartilage oligomeric matrix protein gene (COMP) encodes a noncollagenous extracellular matrix protein whose expression pattern correlates with chondrocyte differentiation and arthritis. We have used the COMP promoter as a model to identify regulatory sequences necessary for chondrocyte‐specific expression and to identify cell type‐specific proteins that bind these sequences. We have previously cloned 1.9 kilobases of the 5′ flanking promoter sequence of the murine COMP gene and by deletion analysis have identified two spatially distant chondrocyte‐specific regulatory regions. One element is situated proximally (−125 to −75), and a second region is located distally (−1925 to −592) relative to the transcription start site. In the present study, we performed a finer deletion analysis of the region of the COMP promoter from −1925 to −592 and identified a silencer region situated between −1775 and −1725. This silencer binds sequence‐specific protein complexes; the intensity of these complexes is greater in two different fibroblast cell lines (NIH3T3 and 10T1/2) than in chondrocytic RCS cells. Competition experiments localized the binding site of these protein complexes from −1775 to −1746; deletion of this 30‐bp site results in a selective increase in COMP promoter activity in fibroblasts. Four tandem repeats of this 30‐bp site are sufficient to confer negative transcriptional regulation on a heterologous promoter (SV40) in NIH3T3 fibroblasts. These results suggest that negative regulation of transcription is an important mechanism for chondrocyte‐specific expression of the COMP gene. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.