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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: Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region.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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Subnuclear co-localization of Sox9 with Tip60 in the nucleus. (A) Sox9 protein and Tip60 protein in HCS-2/8 cells were stained with anti-Sox9 (green) and Tip60 (red) antibodies, respectively. The subnuclear localization of proteins was detected by indirect immunofluorescence and analyzed by deconvolution microscopy. The subnuclear distribution of endogenous Sox9 and Tip60 in HCS-2/8 cells overlapped in a diffuse staining pattern. (B–D) COS7 cells were transfected with expression vectors encoding HA3-Sox9 (B), or GFP-Tip60 (C) or both (D). Sox9 was stained with anti-Sox9 antibody and detected by indirect immunofluorescence, and Sox9 and Tip60 were analyzed by deconvolution microscopy. Sox9 alone displayed a punctuated distribution in the nucleus (B), while Tip60 alone showed a somewhat patchy distribution in the nucleus (C), but the Tip60 distribution became more diffused in the presence of Sox9 (D); about 50% of the nuclear area shows codistribution of Sox9 and Tip60.
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Figure 5: Subnuclear co-localization of Sox9 with Tip60 in the nucleus. (A) Sox9 protein and Tip60 protein in HCS-2/8 cells were stained with anti-Sox9 (green) and Tip60 (red) antibodies, respectively. The subnuclear localization of proteins was detected by indirect immunofluorescence and analyzed by deconvolution microscopy. The subnuclear distribution of endogenous Sox9 and Tip60 in HCS-2/8 cells overlapped in a diffuse staining pattern. (B–D) COS7 cells were transfected with expression vectors encoding HA3-Sox9 (B), or GFP-Tip60 (C) or both (D). Sox9 was stained with anti-Sox9 antibody and detected by indirect immunofluorescence, and Sox9 and Tip60 were analyzed by deconvolution microscopy. Sox9 alone displayed a punctuated distribution in the nucleus (B), while Tip60 alone showed a somewhat patchy distribution in the nucleus (C), but the Tip60 distribution became more diffused in the presence of Sox9 (D); about 50% of the nuclear area shows codistribution of Sox9 and Tip60.

Mentions: Because Tip60 is reported to act as a HAT, we evaluated potential effects of Tip60 on the subnuclear localization of Sox9. In the chondrocyte cell line HCS-2/8 expressing both endogenous Tip60 and Sox9, both proteins showed colocalization in the nucleus in a diffuse pattern (Figure 5A). Cos7 cells in which HA-tagged Sox9 was overexpressed revealed a granular subnuclear localization, whereas Cos7 cells expressing a green fluorescent protein (GFP)-Tip60 fusion polypeptide showed a punctated subnuclear localization when each of these was overexpressed separately (Figure 5B). When HA-Sox9 and GFP-Tip60 were co-overexpressed in COS7 cells, Sox9 showed a slightly more diffuse localization, whereas the Tip60 distribution changed from a punctuated to a diffuse localization, largely overlapping with Sox9. A proportion of Sox9 and Tip60 staining remained separate (Figure 5B).Figure 5.


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)

Subnuclear co-localization of Sox9 with Tip60 in the nucleus. (A) Sox9 protein and Tip60 protein in HCS-2/8 cells were stained with anti-Sox9 (green) and Tip60 (red) antibodies, respectively. The subnuclear localization of proteins was detected by indirect immunofluorescence and analyzed by deconvolution microscopy. The subnuclear distribution of endogenous Sox9 and Tip60 in HCS-2/8 cells overlapped in a diffuse staining pattern. (B–D) COS7 cells were transfected with expression vectors encoding HA3-Sox9 (B), or GFP-Tip60 (C) or both (D). Sox9 was stained with anti-Sox9 antibody and detected by indirect immunofluorescence, and Sox9 and Tip60 were analyzed by deconvolution microscopy. Sox9 alone displayed a punctuated distribution in the nucleus (B), while Tip60 alone showed a somewhat patchy distribution in the nucleus (C), but the Tip60 distribution became more diffused in the presence of Sox9 (D); about 50% of the nuclear area shows codistribution of Sox9 and Tip60.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Subnuclear co-localization of Sox9 with Tip60 in the nucleus. (A) Sox9 protein and Tip60 protein in HCS-2/8 cells were stained with anti-Sox9 (green) and Tip60 (red) antibodies, respectively. The subnuclear localization of proteins was detected by indirect immunofluorescence and analyzed by deconvolution microscopy. The subnuclear distribution of endogenous Sox9 and Tip60 in HCS-2/8 cells overlapped in a diffuse staining pattern. (B–D) COS7 cells were transfected with expression vectors encoding HA3-Sox9 (B), or GFP-Tip60 (C) or both (D). Sox9 was stained with anti-Sox9 antibody and detected by indirect immunofluorescence, and Sox9 and Tip60 were analyzed by deconvolution microscopy. Sox9 alone displayed a punctuated distribution in the nucleus (B), while Tip60 alone showed a somewhat patchy distribution in the nucleus (C), but the Tip60 distribution became more diffused in the presence of Sox9 (D); about 50% of the nuclear area shows codistribution of Sox9 and Tip60.
Mentions: Because Tip60 is reported to act as a HAT, we evaluated potential effects of Tip60 on the subnuclear localization of Sox9. In the chondrocyte cell line HCS-2/8 expressing both endogenous Tip60 and Sox9, both proteins showed colocalization in the nucleus in a diffuse pattern (Figure 5A). Cos7 cells in which HA-tagged Sox9 was overexpressed revealed a granular subnuclear localization, whereas Cos7 cells expressing a green fluorescent protein (GFP)-Tip60 fusion polypeptide showed a punctated subnuclear localization when each of these was overexpressed separately (Figure 5B). When HA-Sox9 and GFP-Tip60 were co-overexpressed in COS7 cells, Sox9 showed a slightly more diffuse localization, whereas the Tip60 distribution changed from a punctuated to a diffuse localization, largely overlapping with Sox9. A proportion of Sox9 and Tip60 staining remained separate (Figure 5B).Figure 5.

Bottom Line: Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region.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