ABSTRACT
The transcription factor SOX9 is essential for multiple steps during skeletal development, including mesenchymal cell chondrogenesis and endochondral bone formation. We recently reported that the human SOX9 proximal promoter region is regulated by the CCAAT-binding factor through two CCAAT boxes located within 100 bp of the transcriptional start site. Here we report that the human SOX9 proximal promoter is also regulated by the cyclic-AMP response element binding protein (CREB) and Sp1. We show by DNaseI protection and EMSA analysis that CREB and Sp1 interact with specific sites within the SOX9 proximal promoter region. By transient transfection analysis we also demonstrate that mutations of the CREB and Sp1 binding sites result in a profound reduction of SOX9 promoter activity. Chromatin immunoprecipitation (ChIP) assay demonstrated that both Sp1 and CREB interact with the SOX9 promoter in vivo. Finally, we demonstrate that IL-1beta treatment of chondrocytes isolated from human normal and osteoarthritic (OA) cartilage down-regulates SOX9 promoter activity, an effect accompanied by a reduction of Sp1 binding to the SOX9 proximal promoter.
Subject(s)
Cyclic AMP Response Element-Binding Protein/genetics , Gene Expression Regulation , High Mobility Group Proteins/genetics , Promoter Regions, Genetic , Sp1 Transcription Factor/genetics , Transcription Factors/genetics , Animals , Base Sequence , Cell Line , Chondrogenesis , Cyclic AMP Response Element-Binding Protein/metabolism , Deoxyribonuclease I/genetics , Electrophoretic Mobility Shift Assay , High Mobility Group Proteins/metabolism , Humans , Interleukin-1beta/pharmacology , Mice , Molecular Sequence Data , Protein Binding , Response Elements , SOX9 Transcription Factor , Transcription Factors/metabolism , TransfectionABSTRACT
Sox9 is an essential transcriptional regulator of chondrogenesis and chondrocyte-specific gene expression; however, the identity and function of transcription factors that regulate Sox9 gene expression are not well understood. Here, we have undertaken an analysis of the human Sox9 proximal promoter region in an effort to elucidate the function and identity of transcriptional regulators that are important for controlling Sox9 gene transcription. By transfection analysis, we show that elements residing between -256 bp and +67 bp are important for the overall level of Sox9 promoter activity. Previously, two CCAAT boxes were identified in the Sox9 mouse and human promoters (position -60 bp and -100 bp) by sequence analysis (Kanai, Y., Koopman, P., 1999. Structural and functional characterization of the mouse Sox9 promoter: implications for campomelic dysplasia. Hum. Mol. Genet., 8: 691-696). We demonstrate by electrophoretic mobility shift (EMSA) competition and supershift assays that the CCAAT-binding factor (CBF) can form a complex with both Sox9 CCAAT boxes in nuclear extracts from multiple cell lines. Transfection of human Sox9 promoter-luciferase constructs containing mutated or deleted CCAAT boxes demonstrated that both CCAAT boxes are important for Sox9 promoter activity in chondrogenic cell lines and primary chondrocytes. Chromatin immunoprecipitation (ChIP) experiments demonstrated that CBF interacts with the Sox9 promoter in vivo. Together, these studies show that the Sox9 promoter is regulated by CBF through its interaction with two functional CCAAT boxes.
