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Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5.

Hattori T, Coustry F, Stephens S, Eberspaecher H, Takigawa M, Yasuda H, de Crombrugghe B - Nucleic Acids Res. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmacy, 5-1 Shikata-cho, 2-chome, Okayama 700-8525, Japan. Email: hattorit@md.okayama-u.ac.jp

ABSTRACT
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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Tip60 and Sox5 associate with a Sox9-target locus in the Col2a1 enhancer. (A) ChIP analysis of human chondrocytic cell line HCS-2/8 (upper panel) and of primary mouse chondrocytes (lower panel) with antibodies against Tip60, Sox9, Sox5 demonstrates binding of all three factors to the Sox9 target. In human synovial cells, which do express Tip60 but not Sox9 (data not shown) the Col2a1 locus is not precipitated by these antibodies (middle panel). The enhancer region of Col2a1 intron1 was amplified by PCR. (B) CHIP of the β-actin gene of mouse primary chondrocytes with each antibody as a control confirms the absence of unspecific chromatin immunoprecipitates.
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Figure 7: Tip60 and Sox5 associate with a Sox9-target locus in the Col2a1 enhancer. (A) ChIP analysis of human chondrocytic cell line HCS-2/8 (upper panel) and of primary mouse chondrocytes (lower panel) with antibodies against Tip60, Sox9, Sox5 demonstrates binding of all three factors to the Sox9 target. In human synovial cells, which do express Tip60 but not Sox9 (data not shown) the Col2a1 locus is not precipitated by these antibodies (middle panel). The enhancer region of Col2a1 intron1 was amplified by PCR. (B) CHIP of the β-actin gene of mouse primary chondrocytes with each antibody as a control confirms the absence of unspecific chromatin immunoprecipitates.

Mentions: To further explore whether Tip60 associated with the same chromatin segment of the Col2a1 enhancer element of intron 1 which is the target of Sox9, we performed ChIP experiments using HCS-2/8 cells which express both Sox9 and Tip60, As shown in Figure 7, anti-Tip60 precipitated the same Col2a1 chromatin fragment in the Col2a1 intron 1 enhancer that was precipitated with the anti-Sox9 antibody; the coprecipitated Col2a1 fragment was identified by PCR (Figure 7A, upper photo). Interestingly, Sox5, which is known to bind to the same Col2a1 enhancer in EMSA assays, also interacted with this segment in the ChIP assay (Figure 7A). In human synovial cells, which express Tip60 but not Sox9, ChIP with anti Tip60 did not indicate any precipitation of the Col2a1 enhancer, suggesting that recruitment of Tip60 to the Col2a1 promoter requires Sox9. To confirm the association of Sox9, Tip60 and Sox5 with the Col2a1 enhancer, ChIP analysis was also performed with primary mouse chondrocytes. As in HCS-2/8cells, all three components were found to associate with the Col2a1 enhancer, while the β-actin gene which does not contain Sox9-binding elements [searched by TFsearch (http://www.cbrc.jp/research/db/TFSEARCH.html)] did not reveal any ChIP signals (Figure 7B).Figure 7.


Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5.

Hattori T, Coustry F, Stephens S, Eberspaecher H, Takigawa M, Yasuda H, de Crombrugghe B - Nucleic Acids Res. (2008)

Tip60 and Sox5 associate with a Sox9-target locus in the Col2a1 enhancer. (A) ChIP analysis of human chondrocytic cell line HCS-2/8 (upper panel) and of primary mouse chondrocytes (lower panel) with antibodies against Tip60, Sox9, Sox5 demonstrates binding of all three factors to the Sox9 target. In human synovial cells, which do express Tip60 but not Sox9 (data not shown) the Col2a1 locus is not precipitated by these antibodies (middle panel). The enhancer region of Col2a1 intron1 was amplified by PCR. (B) CHIP of the β-actin gene of mouse primary chondrocytes with each antibody as a control confirms the absence of unspecific chromatin immunoprecipitates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2396410&req=5

Figure 7: Tip60 and Sox5 associate with a Sox9-target locus in the Col2a1 enhancer. (A) ChIP analysis of human chondrocytic cell line HCS-2/8 (upper panel) and of primary mouse chondrocytes (lower panel) with antibodies against Tip60, Sox9, Sox5 demonstrates binding of all three factors to the Sox9 target. In human synovial cells, which do express Tip60 but not Sox9 (data not shown) the Col2a1 locus is not precipitated by these antibodies (middle panel). The enhancer region of Col2a1 intron1 was amplified by PCR. (B) CHIP of the β-actin gene of mouse primary chondrocytes with each antibody as a control confirms the absence of unspecific chromatin immunoprecipitates.
Mentions: To further explore whether Tip60 associated with the same chromatin segment of the Col2a1 enhancer element of intron 1 which is the target of Sox9, we performed ChIP experiments using HCS-2/8 cells which express both Sox9 and Tip60, As shown in Figure 7, anti-Tip60 precipitated the same Col2a1 chromatin fragment in the Col2a1 intron 1 enhancer that was precipitated with the anti-Sox9 antibody; the coprecipitated Col2a1 fragment was identified by PCR (Figure 7A, upper photo). Interestingly, Sox5, which is known to bind to the same Col2a1 enhancer in EMSA assays, also interacted with this segment in the ChIP assay (Figure 7A). In human synovial cells, which express Tip60 but not Sox9, ChIP with anti Tip60 did not indicate any precipitation of the Col2a1 enhancer, suggesting that recruitment of Tip60 to the Col2a1 promoter requires Sox9. To confirm the association of Sox9, Tip60 and Sox5 with the Col2a1 enhancer, ChIP analysis was also performed with primary mouse chondrocytes. As in HCS-2/8cells, all three components were found to associate with the Col2a1 enhancer, while the β-actin gene which does not contain Sox9-binding elements [searched by TFsearch (http://www.cbrc.jp/research/db/TFSEARCH.html)] did not reveal any ChIP signals (Figure 7B).Figure 7.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmacy, 5-1 Shikata-cho, 2-chome, Okayama 700-8525, Japan. Email: hattorit@md.okayama-u.ac.jp

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus