Regulation of the human Sox9 promoter by the CCAAT-binding factor.
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.
Colter
David C
DC
Department of Medicine, Division of Rheumatology, Thomas Jefferson University, Jefferson Medical College, 233 South 10th Street, Room 511 BLSB, Philadelphia, PA 19107, USA.
Piera-Velazquez
Sonsoles
S
Hawkins
David F
DF
Whitecavage
Mary Kate
MK
Jimenez
Sergio A
SA
Stokes
David G
DG
D000818:Animals; D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000502:physiology*; D002460:Cell Line; D017353:Gene Deletion; D006609:High Mobility Group Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000502:physiology*; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics*
D023081:CCAAT-Binding Factor; D006609:High Mobility Group Proteins; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C527933:Sox9 protein, mouse; D014157:Transcription Factors