Nucleic Acids Research Advance Access originally published online on April 4, 2008
Nucleic Acids Research 2008 36(9):3011-3024; doi:10.1093/nar/gkn150
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Nucleic Acids Research, 2008, Vol. 36, No. 9 3011-3024
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5
1Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmacy, 5-1 Shikata-cho, 2-chome, Okayama 700-8525, Japan, 2Department of Molecular Genetics, The University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 1006, Houston, TX 77030, USA and 3Bioscience Lab, Central Laboratory, Nippon Flour Mills Co., Ltd, Atsugi, Kanagawa 243-0041, Japan
*To whom correspondence should be addressed. Tel: +81 86 235 6646; Fax: +81 86 235 6649; Email: hattorit{at}md.okayama-u.ac.jp Correspondence may also be addressed to Benoit de Crombrugghe. Tel: +1 713 834 6376; Fax: +1 713 834 6396; Email: bdecromb{at}mdanderson.org
Received December 12, 2007. Revised March 9, 2008. Accepted March 17, 2008.
Sox9 is a transcription factor of the SRY family required for several steps of chondrogenesis. It activates the expression of various chondrocyte-specific genes, but the mechanisms and role of cofactors involved in Sox9-regulated gene transcription are not fully understood. Here, we report on the characterization of a Tat interactive protein-60 (Tip60) as Sox9-associated protein identified in a yeast two-hybrid screen. Both in vitro and in vivo assays confirmed the specificity of interactions between Sox9 and Tip60 including the existence of an endogenous complex containing both polypeptides in chondrocytes. Gel shift assays showed the presence of a complex containing Sox9, Tip60 and the DNA of an enhancer region of the Col2a1 promoter. Reporter assays using a Col2a1 promoter with multimerized Col2a1 Sox9-binding sites indicated that Tip60 enhanced the transcriptional activity of Sox9. A larger Col2a1 promoter showed that Tip60 increased the activity of this promoter in the presence of both Sox9 and Sox5. Ectopic expression of Sox9 and transient-cotransfection with Tip60 in COS7 cells showed a more diffuse subnuclear colocalization, suggesting changes in the chromatin structure. Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region. Consistent with a role of Tip60 in chondrogenesis, addition of Tip60 siRNA to limb-bud micromass cultures delayed chondrocyte differention. Tip60 enhances acetylation of Sox9 mainly through K61, 253, 398 residues; however, the K61/253/398A mutant of Sox9 still exhibited enhanced transcriptional activity by Tip60. Our results support the hypothesis that Tip60 is a coactivator of Sox9 in chondrocytes.
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