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Negative regulation of transcription coactivator p300 by orphan receptor TR3.

Li GD, Fang JX, Chen HZ, Luo J, Zheng ZH, Shen YM, Wu Q - Nucleic Acids Res. (2007)

Bottom Line: TR3 was found to interact with p300 and inhibited the acetylation of transcription factors induced by p300, resulting in the repression of their transcriptional activity.More importantly, this agonist could repress the transcriptional activity of transcription factors, and proliferation of cancer cells.Taken together, our results not only delineate a novel transcriptional repressor function for TR3, but also reveal its modulation on p300 HAT activity as the underlying mechanism.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China.

ABSTRACT
p300 regulates the transcriptional activity of a variety of transcription factors by forming an activation complex and/or promoting histone acetylation. Here, we show a unique characteristic of orphan receptor TR3 in negatively regulating the function of p300. TR3 was found to interact with p300 and inhibited the acetylation of transcription factors induced by p300, resulting in the repression of their transcriptional activity. Further analysis revealed that both a conserved transcriptional adapter motif (TRAM) in p300 and a specific sequence FLELFIL in TR3 were critical for their interaction. TR3 binding completely covered the histone acetyltransferase (HAT) domain of p300 and resulted in suppression of the HAT activity, as the p300-induced histone H3 acetylation and transcription were inhibited with the presence TR3. Furthermore, an agonist of TR3, a natural octaketide isolated from Dothiorella sp. HTF3 of an endophytical fungus, was shown to be a potent compound for inhibiting p300 HAT activity (IC(50) = 1.5 microg/ml) in vivo. More importantly, this agonist could repress the transcriptional activity of transcription factors, and proliferation of cancer cells. Taken together, our results not only delineate a novel transcriptional repressor function for TR3, but also reveal its modulation on p300 HAT activity as the underlying mechanism.

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Mapping of domains responsible for the interaction between TR3 and p300. (A) TR3 and p300 form a complex in 293T cells. Myc-TR3 and HA-p300 were transfected either alone or together into cells. Cell lysates were prepared and then subjected to immunoprecipitation, followed by western blotting analysis with anti-Myc or anti-HA antibodies as indicated. (B) Interaction of TR3 with p300 in the absence or presence of TR3 agonist in HeLa cells. Cell lysates were immunoprecipitated with anti-TR3 antibody and control IgG, respectively, followed by western blotting with anti-p300 antibody. (C) Pull-down of p300 by TR3. GST-TR3 was expressed in Escherichia coli cells and purified as described in Materials and Methods section. The beads-bound GST-TR3 was incubated with the lysate of 293T cells transfected with HA-p300. HA-p300 was indicated with anti-HA antibody. GST was used as a negative control. Lower panel indicated the amount of GST and GST-TR3 used in the assay. (D) Determination of TR3-binding sites in p300. Schematic diagrams depicting different p300 truncation mutants are shown in upper panel. Full-length Myc-TR3 and different HA-p300 mutants were transfected into 293T cells as indicated. Cell lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates and cell lysates were analyzed by western blotting with anti-HA and anti-Myc antibodies for p300 and TR3 proteins, respectively. (E) Determination of p300-binding sites in TR3. Schematic diagrams (upper panel) depict different truncation mutants of TR3. Full-length HA-p300 and different Flag-TR3 mutants were transfected into 293T cells as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates and cell lysates were analyzed by western blotting with anti-HA and anti-Myc antibodies for p300 and TR3 proteins, respectively.
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Figure 2: Mapping of domains responsible for the interaction between TR3 and p300. (A) TR3 and p300 form a complex in 293T cells. Myc-TR3 and HA-p300 were transfected either alone or together into cells. Cell lysates were prepared and then subjected to immunoprecipitation, followed by western blotting analysis with anti-Myc or anti-HA antibodies as indicated. (B) Interaction of TR3 with p300 in the absence or presence of TR3 agonist in HeLa cells. Cell lysates were immunoprecipitated with anti-TR3 antibody and control IgG, respectively, followed by western blotting with anti-p300 antibody. (C) Pull-down of p300 by TR3. GST-TR3 was expressed in Escherichia coli cells and purified as described in Materials and Methods section. The beads-bound GST-TR3 was incubated with the lysate of 293T cells transfected with HA-p300. HA-p300 was indicated with anti-HA antibody. GST was used as a negative control. Lower panel indicated the amount of GST and GST-TR3 used in the assay. (D) Determination of TR3-binding sites in p300. Schematic diagrams depicting different p300 truncation mutants are shown in upper panel. Full-length Myc-TR3 and different HA-p300 mutants were transfected into 293T cells as indicated. Cell lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates and cell lysates were analyzed by western blotting with anti-HA and anti-Myc antibodies for p300 and TR3 proteins, respectively. (E) Determination of p300-binding sites in TR3. Schematic diagrams (upper panel) depict different truncation mutants of TR3. Full-length HA-p300 and different Flag-TR3 mutants were transfected into 293T cells as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates and cell lysates were analyzed by western blotting with anti-HA and anti-Myc antibodies for p300 and TR3 proteins, respectively.

Mentions: The finding that TR3 regulates the transcriptional activity of p300-acetylated transcription factors promoted us to determine whether TR3 physically interacts with p300 in cells. To test this possibility, Myc-TR3 and HA-p300 were coexpressed in 293T cells, followed by reciprocal immunoprecipitation with anti-Myc for TR3 and anti-HA for p300. Western blotting showed that TR3 was readily detected when p300 was immunoprecipitated (upper panel); similarly, p300 was also detected in the immunoprecipitates of TR3 (lower panel), suggesting TR3 interaction with p300 at exogenous level. We carried out another parallel experiment to test whether endogenous TR3 and p300 could interact with each other using lysates of untransfected HeLa cells. The results showed that p300 could be detected in the immunoprecipitates of TR3 (Figure 2B), indicating the association of TR3 with p300 at the endogenous level. GST pull-down experiment further demonstrated that GST-TR3 can bind HA-p300 that was transfected into 293T cells and immunoblotted by HA antibody (Figure 2C). Together, these results suggest that TR3 has a physical interaction with p300 in the cells.Figure 2.


Negative regulation of transcription coactivator p300 by orphan receptor TR3.

Li GD, Fang JX, Chen HZ, Luo J, Zheng ZH, Shen YM, Wu Q - Nucleic Acids Res. (2007)

Mapping of domains responsible for the interaction between TR3 and p300. (A) TR3 and p300 form a complex in 293T cells. Myc-TR3 and HA-p300 were transfected either alone or together into cells. Cell lysates were prepared and then subjected to immunoprecipitation, followed by western blotting analysis with anti-Myc or anti-HA antibodies as indicated. (B) Interaction of TR3 with p300 in the absence or presence of TR3 agonist in HeLa cells. Cell lysates were immunoprecipitated with anti-TR3 antibody and control IgG, respectively, followed by western blotting with anti-p300 antibody. (C) Pull-down of p300 by TR3. GST-TR3 was expressed in Escherichia coli cells and purified as described in Materials and Methods section. The beads-bound GST-TR3 was incubated with the lysate of 293T cells transfected with HA-p300. HA-p300 was indicated with anti-HA antibody. GST was used as a negative control. Lower panel indicated the amount of GST and GST-TR3 used in the assay. (D) Determination of TR3-binding sites in p300. Schematic diagrams depicting different p300 truncation mutants are shown in upper panel. Full-length Myc-TR3 and different HA-p300 mutants were transfected into 293T cells as indicated. Cell lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates and cell lysates were analyzed by western blotting with anti-HA and anti-Myc antibodies for p300 and TR3 proteins, respectively. (E) Determination of p300-binding sites in TR3. Schematic diagrams (upper panel) depict different truncation mutants of TR3. Full-length HA-p300 and different Flag-TR3 mutants were transfected into 293T cells as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates and cell lysates were analyzed by western blotting with anti-HA and anti-Myc antibodies for p300 and TR3 proteins, respectively.
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Figure 2: Mapping of domains responsible for the interaction between TR3 and p300. (A) TR3 and p300 form a complex in 293T cells. Myc-TR3 and HA-p300 were transfected either alone or together into cells. Cell lysates were prepared and then subjected to immunoprecipitation, followed by western blotting analysis with anti-Myc or anti-HA antibodies as indicated. (B) Interaction of TR3 with p300 in the absence or presence of TR3 agonist in HeLa cells. Cell lysates were immunoprecipitated with anti-TR3 antibody and control IgG, respectively, followed by western blotting with anti-p300 antibody. (C) Pull-down of p300 by TR3. GST-TR3 was expressed in Escherichia coli cells and purified as described in Materials and Methods section. The beads-bound GST-TR3 was incubated with the lysate of 293T cells transfected with HA-p300. HA-p300 was indicated with anti-HA antibody. GST was used as a negative control. Lower panel indicated the amount of GST and GST-TR3 used in the assay. (D) Determination of TR3-binding sites in p300. Schematic diagrams depicting different p300 truncation mutants are shown in upper panel. Full-length Myc-TR3 and different HA-p300 mutants were transfected into 293T cells as indicated. Cell lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates and cell lysates were analyzed by western blotting with anti-HA and anti-Myc antibodies for p300 and TR3 proteins, respectively. (E) Determination of p300-binding sites in TR3. Schematic diagrams (upper panel) depict different truncation mutants of TR3. Full-length HA-p300 and different Flag-TR3 mutants were transfected into 293T cells as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates and cell lysates were analyzed by western blotting with anti-HA and anti-Myc antibodies for p300 and TR3 proteins, respectively.
Mentions: The finding that TR3 regulates the transcriptional activity of p300-acetylated transcription factors promoted us to determine whether TR3 physically interacts with p300 in cells. To test this possibility, Myc-TR3 and HA-p300 were coexpressed in 293T cells, followed by reciprocal immunoprecipitation with anti-Myc for TR3 and anti-HA for p300. Western blotting showed that TR3 was readily detected when p300 was immunoprecipitated (upper panel); similarly, p300 was also detected in the immunoprecipitates of TR3 (lower panel), suggesting TR3 interaction with p300 at exogenous level. We carried out another parallel experiment to test whether endogenous TR3 and p300 could interact with each other using lysates of untransfected HeLa cells. The results showed that p300 could be detected in the immunoprecipitates of TR3 (Figure 2B), indicating the association of TR3 with p300 at the endogenous level. GST pull-down experiment further demonstrated that GST-TR3 can bind HA-p300 that was transfected into 293T cells and immunoblotted by HA antibody (Figure 2C). Together, these results suggest that TR3 has a physical interaction with p300 in the cells.Figure 2.

Bottom Line: TR3 was found to interact with p300 and inhibited the acetylation of transcription factors induced by p300, resulting in the repression of their transcriptional activity.More importantly, this agonist could repress the transcriptional activity of transcription factors, and proliferation of cancer cells.Taken together, our results not only delineate a novel transcriptional repressor function for TR3, but also reveal its modulation on p300 HAT activity as the underlying mechanism.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China.

ABSTRACT
p300 regulates the transcriptional activity of a variety of transcription factors by forming an activation complex and/or promoting histone acetylation. Here, we show a unique characteristic of orphan receptor TR3 in negatively regulating the function of p300. TR3 was found to interact with p300 and inhibited the acetylation of transcription factors induced by p300, resulting in the repression of their transcriptional activity. Further analysis revealed that both a conserved transcriptional adapter motif (TRAM) in p300 and a specific sequence FLELFIL in TR3 were critical for their interaction. TR3 binding completely covered the histone acetyltransferase (HAT) domain of p300 and resulted in suppression of the HAT activity, as the p300-induced histone H3 acetylation and transcription were inhibited with the presence TR3. Furthermore, an agonist of TR3, a natural octaketide isolated from Dothiorella sp. HTF3 of an endophytical fungus, was shown to be a potent compound for inhibiting p300 HAT activity (IC(50) = 1.5 microg/ml) in vivo. More importantly, this agonist could repress the transcriptional activity of transcription factors, and proliferation of cancer cells. Taken together, our results not only delineate a novel transcriptional repressor function for TR3, but also reveal its modulation on p300 HAT activity as the underlying mechanism.

Show MeSH
Related in: MedlinePlus