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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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Inhibition of TR3 agonist on p300 HAT activity. (A) Agonist up-regulates TR3 mRNA expression levels. HeLa cells were treated with different concentrations of agonist as indicated for 5 h, and total RNA was then prepared. The levels of TR3 mRNA were determined by RT-PCR. Representatives of at least two independent experiments with similar results are shown. The level of TR3 mRNA induced by agonist was determined by using densitometry. (B) Effect of TR3 agonist on p300 HAT activity. 293T cells transfected with HA-p300 (upper panel) or HeLa cells (lower panel) were treated with different concentrations of agonist (1–4 µg) as indicated for 5 h, then the p300 HAT activity was determined as described in Figure 3A. The expression levels of HA-p300, endogenous TR3 and histone H3 were determined by western blotting with anti-HA, -TR3 and -histone H3 antibody, respectively. The level of TR3 protein induced by agonist was determined by using densitometry. Right panels indicated IC50 values that were calculated by determining the concentration of agonist needed to promote 50% inhibition of agonist activity. (C) Effect of agonist on p300 HAT activity is attenuated by siRNA-TR3. siRNA-TR3 with p300 were transfected into 293T cells. Transfected cells were treated with agonist (4 µg) for 5 h. The HAT activity was determined as described in Figure 3A. (D) Effect of agonist on transcription induced by p300 HAT. Gal4-p300/HAT and a luciferase reporter gene pGAL4, with or without siRNA-TR3, was cotransfected into 293T cells. Transfected cells were treated with agonist (4 µg) for 5 h. Reporter gene activity was determined as described in Figure 1D. The bars represent the average ± mean from three independent experiments. (E) Effects of agonist and siRNA-TR3 on p300-induced transcriptional activity. p300 with different transcription factors and reporter genes was transfected into 293T cells, and cells were treated with agonist at different concentration (1, 2 and 5 µM). The transcriptional activity was determined as described in Figure 1D. In addition, siRNA-TR3 was introduced into cells to abolish agonist function as required. (F) TR3 agonist inhibits p300 and AR-mediated mitogenic activity. Different expression vectors, including HA-p300, GFP-AR and siRNA-TR3, were transfected into breast cancer MCF-7 cells as indicated, and the transfected cells were treated with or without agonist (4 µg) for 5 h. Cells were maintained in BrdU containing medium for 2 h and then identified by flow cytometry.
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Figure 5: Inhibition of TR3 agonist on p300 HAT activity. (A) Agonist up-regulates TR3 mRNA expression levels. HeLa cells were treated with different concentrations of agonist as indicated for 5 h, and total RNA was then prepared. The levels of TR3 mRNA were determined by RT-PCR. Representatives of at least two independent experiments with similar results are shown. The level of TR3 mRNA induced by agonist was determined by using densitometry. (B) Effect of TR3 agonist on p300 HAT activity. 293T cells transfected with HA-p300 (upper panel) or HeLa cells (lower panel) were treated with different concentrations of agonist (1–4 µg) as indicated for 5 h, then the p300 HAT activity was determined as described in Figure 3A. The expression levels of HA-p300, endogenous TR3 and histone H3 were determined by western blotting with anti-HA, -TR3 and -histone H3 antibody, respectively. The level of TR3 protein induced by agonist was determined by using densitometry. Right panels indicated IC50 values that were calculated by determining the concentration of agonist needed to promote 50% inhibition of agonist activity. (C) Effect of agonist on p300 HAT activity is attenuated by siRNA-TR3. siRNA-TR3 with p300 were transfected into 293T cells. Transfected cells were treated with agonist (4 µg) for 5 h. The HAT activity was determined as described in Figure 3A. (D) Effect of agonist on transcription induced by p300 HAT. Gal4-p300/HAT and a luciferase reporter gene pGAL4, with or without siRNA-TR3, was cotransfected into 293T cells. Transfected cells were treated with agonist (4 µg) for 5 h. Reporter gene activity was determined as described in Figure 1D. The bars represent the average ± mean from three independent experiments. (E) Effects of agonist and siRNA-TR3 on p300-induced transcriptional activity. p300 with different transcription factors and reporter genes was transfected into 293T cells, and cells were treated with agonist at different concentration (1, 2 and 5 µM). The transcriptional activity was determined as described in Figure 1D. In addition, siRNA-TR3 was introduced into cells to abolish agonist function as required. (F) TR3 agonist inhibits p300 and AR-mediated mitogenic activity. Different expression vectors, including HA-p300, GFP-AR and siRNA-TR3, were transfected into breast cancer MCF-7 cells as indicated, and the transfected cells were treated with or without agonist (4 µg) for 5 h. Cells were maintained in BrdU containing medium for 2 h and then identified by flow cytometry.

Mentions: Recently, we screened a bank of the natural products from fungi and found an agonist of TR3. This agonist specifically activated TR3 transactivation through targeting to the TR3 ligand-binding domain (Zhan et al., unpublished data), and promoted the expression of TR3 mRNA and protein (Figure 5A–C). Co-IP assay showed that agonist could enhance the TR3–p300 interaction at endogenous level (Figure 2B). If TR3 is a genuine factor for inhibition of p300 HAT activity, it is expected that this agonist would also have such an inhibitory effect on p300 HAT activity as well as the transcriptional activity of p300-induced transcription factors. We first analyzed the role of TR3 agonist in p300-dependent HAT activity. As shown in Figure 5B, agonist did not impair the expression levels of p300 and Histone H3, but indeed down-regulated p300 HAT activity in a concentration-dependent manner; meanwhile, the expression levels of endogenous TR3 were up-regulated with increase of agonist dose (Figure 5B, left panels). The IC50 (inhibitory concentration of agonist for 50% reduction of the HAT activity) was 2.2 µg/ml in the in vitro assay and 1.5 µg/ml in the in vivo assay (Figure 5B, right panels). To confirm that such inhibition was directly mediated by TR3, a siRNA against TR3 was introduced into cells to inhibit endogenous TR3 expression. When endogenous TR3 was inhibited by its RNA interfere, the inhibitory effect of agonist on p300 HAT activity was not detected (Figure 5C). TR3 agonist also repressed p300-activated transcription of GAL4 response elements linked luciferase gene, and such repression could be diminished by siRNA-TR3 (Figure 5D). In addition, while introduction of p300 into 293T cells activated the expression of luciferase reporter gene for a series of transcription factors (Figure 5E, lane 2), agonist exerted an obvious inhibition on reporter gene activities with a dose-dependent fashion (Figure 5E), but failed to do so in the presence of siRNA-TR3 (Figure 5E, lane 6). Clearly, the repression of p300 HAT activity and activities of reporter genes by agonist is mediated by TR3.Figure 5.


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)

Inhibition of TR3 agonist on p300 HAT activity. (A) Agonist up-regulates TR3 mRNA expression levels. HeLa cells were treated with different concentrations of agonist as indicated for 5 h, and total RNA was then prepared. The levels of TR3 mRNA were determined by RT-PCR. Representatives of at least two independent experiments with similar results are shown. The level of TR3 mRNA induced by agonist was determined by using densitometry. (B) Effect of TR3 agonist on p300 HAT activity. 293T cells transfected with HA-p300 (upper panel) or HeLa cells (lower panel) were treated with different concentrations of agonist (1–4 µg) as indicated for 5 h, then the p300 HAT activity was determined as described in Figure 3A. The expression levels of HA-p300, endogenous TR3 and histone H3 were determined by western blotting with anti-HA, -TR3 and -histone H3 antibody, respectively. The level of TR3 protein induced by agonist was determined by using densitometry. Right panels indicated IC50 values that were calculated by determining the concentration of agonist needed to promote 50% inhibition of agonist activity. (C) Effect of agonist on p300 HAT activity is attenuated by siRNA-TR3. siRNA-TR3 with p300 were transfected into 293T cells. Transfected cells were treated with agonist (4 µg) for 5 h. The HAT activity was determined as described in Figure 3A. (D) Effect of agonist on transcription induced by p300 HAT. Gal4-p300/HAT and a luciferase reporter gene pGAL4, with or without siRNA-TR3, was cotransfected into 293T cells. Transfected cells were treated with agonist (4 µg) for 5 h. Reporter gene activity was determined as described in Figure 1D. The bars represent the average ± mean from three independent experiments. (E) Effects of agonist and siRNA-TR3 on p300-induced transcriptional activity. p300 with different transcription factors and reporter genes was transfected into 293T cells, and cells were treated with agonist at different concentration (1, 2 and 5 µM). The transcriptional activity was determined as described in Figure 1D. In addition, siRNA-TR3 was introduced into cells to abolish agonist function as required. (F) TR3 agonist inhibits p300 and AR-mediated mitogenic activity. Different expression vectors, including HA-p300, GFP-AR and siRNA-TR3, were transfected into breast cancer MCF-7 cells as indicated, and the transfected cells were treated with or without agonist (4 µg) for 5 h. Cells were maintained in BrdU containing medium for 2 h and then identified by flow cytometry.
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Figure 5: Inhibition of TR3 agonist on p300 HAT activity. (A) Agonist up-regulates TR3 mRNA expression levels. HeLa cells were treated with different concentrations of agonist as indicated for 5 h, and total RNA was then prepared. The levels of TR3 mRNA were determined by RT-PCR. Representatives of at least two independent experiments with similar results are shown. The level of TR3 mRNA induced by agonist was determined by using densitometry. (B) Effect of TR3 agonist on p300 HAT activity. 293T cells transfected with HA-p300 (upper panel) or HeLa cells (lower panel) were treated with different concentrations of agonist (1–4 µg) as indicated for 5 h, then the p300 HAT activity was determined as described in Figure 3A. The expression levels of HA-p300, endogenous TR3 and histone H3 were determined by western blotting with anti-HA, -TR3 and -histone H3 antibody, respectively. The level of TR3 protein induced by agonist was determined by using densitometry. Right panels indicated IC50 values that were calculated by determining the concentration of agonist needed to promote 50% inhibition of agonist activity. (C) Effect of agonist on p300 HAT activity is attenuated by siRNA-TR3. siRNA-TR3 with p300 were transfected into 293T cells. Transfected cells were treated with agonist (4 µg) for 5 h. The HAT activity was determined as described in Figure 3A. (D) Effect of agonist on transcription induced by p300 HAT. Gal4-p300/HAT and a luciferase reporter gene pGAL4, with or without siRNA-TR3, was cotransfected into 293T cells. Transfected cells were treated with agonist (4 µg) for 5 h. Reporter gene activity was determined as described in Figure 1D. The bars represent the average ± mean from three independent experiments. (E) Effects of agonist and siRNA-TR3 on p300-induced transcriptional activity. p300 with different transcription factors and reporter genes was transfected into 293T cells, and cells were treated with agonist at different concentration (1, 2 and 5 µM). The transcriptional activity was determined as described in Figure 1D. In addition, siRNA-TR3 was introduced into cells to abolish agonist function as required. (F) TR3 agonist inhibits p300 and AR-mediated mitogenic activity. Different expression vectors, including HA-p300, GFP-AR and siRNA-TR3, were transfected into breast cancer MCF-7 cells as indicated, and the transfected cells were treated with or without agonist (4 µg) for 5 h. Cells were maintained in BrdU containing medium for 2 h and then identified by flow cytometry.
Mentions: Recently, we screened a bank of the natural products from fungi and found an agonist of TR3. This agonist specifically activated TR3 transactivation through targeting to the TR3 ligand-binding domain (Zhan et al., unpublished data), and promoted the expression of TR3 mRNA and protein (Figure 5A–C). Co-IP assay showed that agonist could enhance the TR3–p300 interaction at endogenous level (Figure 2B). If TR3 is a genuine factor for inhibition of p300 HAT activity, it is expected that this agonist would also have such an inhibitory effect on p300 HAT activity as well as the transcriptional activity of p300-induced transcription factors. We first analyzed the role of TR3 agonist in p300-dependent HAT activity. As shown in Figure 5B, agonist did not impair the expression levels of p300 and Histone H3, but indeed down-regulated p300 HAT activity in a concentration-dependent manner; meanwhile, the expression levels of endogenous TR3 were up-regulated with increase of agonist dose (Figure 5B, left panels). The IC50 (inhibitory concentration of agonist for 50% reduction of the HAT activity) was 2.2 µg/ml in the in vitro assay and 1.5 µg/ml in the in vivo assay (Figure 5B, right panels). To confirm that such inhibition was directly mediated by TR3, a siRNA against TR3 was introduced into cells to inhibit endogenous TR3 expression. When endogenous TR3 was inhibited by its RNA interfere, the inhibitory effect of agonist on p300 HAT activity was not detected (Figure 5C). TR3 agonist also repressed p300-activated transcription of GAL4 response elements linked luciferase gene, and such repression could be diminished by siRNA-TR3 (Figure 5D). In addition, while introduction of p300 into 293T cells activated the expression of luciferase reporter gene for a series of transcription factors (Figure 5E, lane 2), agonist exerted an obvious inhibition on reporter gene activities with a dose-dependent fashion (Figure 5E), but failed to do so in the presence of siRNA-TR3 (Figure 5E, lane 6). Clearly, the repression of p300 HAT activity and activities of reporter genes by agonist is mediated by TR3.Figure 5.

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