[Frontiers in Bioscience 12, 3899-3910, May 1, 2007]

Transcriptional activation of cartilage oligomeric matrix protein by Sox9, Sox5, and Sox6 transcription factors and CBP/p300 coactivators

Chuan-ju Liu1,2, Yan Zhang 1, Ke Xu 1, Deidre Parsons 1, Daniel Alfonso1, Paul E. Di Cesare 1,2

1 Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, New York, NY 10003, 2 Department of Cell Biology, New York University School of Medicine, New York, NY 10016

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Material and Methods
3.1. Preparation of nuclear extracts
3.2. Electrophoretic mobility shift assay (EMSA)
3.2.1. Labeling probe with digoxigenin
3.2.2. Binding reaction
3.2.3. Detection
3.3. Chromatin immunoprecipitation (ChIP)
3.4. Reporter gene assay
3.5. Generation and characterization of the constructs encoding small interfering RNA (siRNA) against Sox9 using pSUPER system
3.6. Real-time PCR assay
4. Results
4.1. Sox9 binds to the PRE in COMP promoter in vitro
4.2. Sox9 binds to the PRE in the COMP gene promoter in living cells
4.3. Identification of the Sox9-binding elements (SBEs) in the PRE
4.4. Sox9 activates COMP-specific reporter construct activities in a PRE-dependent manner
4.5. Suppression of Sox9 using the siRNA approach inhibits COMP gene expression
4.6. Full activation of COMP gene expression requires a combination of Sox9, Sox5, and Sox6
4.7. Transactivation of COMP gene by Sox9 is enhanced by coexpression of CBP/p300 histone acetylase
4.8. Sox9 overcomes LRF-mediated inhibition of the COMP-specific reporter construct
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

The gene for cartilage oligomeric matrix protein (COMP) encodes a noncollagenous matrix protein that is expressed predominantly in cartilage. COMP gene expression is deficient in the Sox9-null mouse, but the molecular mechanism remains unknown. We have previously delineated a 30-bp negative regulatory element (NRE) and a 51-bp positive regulatory element (PRE) in the regulatory region of the COMP gene. Subsequently we isolated LRF transcription repressor as an NRE-binding protein and established that LRF inhibits COMP gene expression via recruiting histone deacetylase 1 (HDAC1) to the COMP promoter. In this study we demonstrated that Sox9, an essential transcription factor of chondrogenesis, binds to the COMP promoter at the PRE in which 13 nucleotides (TGTTTACCTTGTG) are required for the binding of Sox9. Sox9 activates COMP gene expression and this activation is PRE-dependent. Sox9 is required for COMP gene expression during chondrogenesis, since repression of Sox9 expression using the small interfering RNA approach inhibited COMP gene expression. In addition, activation of COMP gene expression by Sox9 requires the participation of transcription factors Sox5 and Sox6 as well as the coactivators CBP and p300 histone acetylase. It appears that there exists a balance between LRF repressor and Sox9 activator in the control of COMP gene, since transactivation of COMP gene by Sox9 was abolished by the coexpression of LRF, and excess Sox9 overcame the LRF-mediated inhibition. This study provides the first evidence that Sox9 directly associates with COMP gene promoter and that mediation of COMP gene activation by Sox9 involves Sox5, Sox6, CBP, and p300 coactivators.