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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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TR3 inhibits p300 HAT activity. (A) Effect of TR3 on p300 HAT activity. HA-p300 and increasing amount of Falg-TR3 were transfected into 293T cells. Cell lysates were prepared and HA-p300 was immunoprecipitated with anti-HA antibody. The precipitates were then incubated with histone (10 µg) and Lys-CoA (10 µM) at 30°C for 30 min. The reaction products were subjected to western blotting with anti-Ack-H3 antibody to show the HAT activity. The levels of TR3, p300 and histone H3 were indicated by western blotting with anti-Flag, -HA and -histone antibodies. The percentage of Ack-H3 inhibition was calculated by determining the Ack-H3 protein amount of individual bands using densitometry. (B) Different TR3 mutants impair p300 HAT activity. Flag-TR3 mutants together with full-length HA-p300, were transfected into 293T cells. The HAT activity of p300 was determined as described above. Ack-H3 inhibition was determined by using densitometry. (C and D) TR3 inhibits p300 HAT-mediated transcription. Gal4-p300/HAT, together with a luciferase reporter gene pGAL4 and increasing amount of TR3 (C) or TR3 deletion mutants (D), was cotransfected into 293T cells. Reporter gene activity was determined as described in Figure 1D. The bars represent the average ± mean from three independent experiments.
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Figure 3: TR3 inhibits p300 HAT activity. (A) Effect of TR3 on p300 HAT activity. HA-p300 and increasing amount of Falg-TR3 were transfected into 293T cells. Cell lysates were prepared and HA-p300 was immunoprecipitated with anti-HA antibody. The precipitates were then incubated with histone (10 µg) and Lys-CoA (10 µM) at 30°C for 30 min. The reaction products were subjected to western blotting with anti-Ack-H3 antibody to show the HAT activity. The levels of TR3, p300 and histone H3 were indicated by western blotting with anti-Flag, -HA and -histone antibodies. The percentage of Ack-H3 inhibition was calculated by determining the Ack-H3 protein amount of individual bands using densitometry. (B) Different TR3 mutants impair p300 HAT activity. Flag-TR3 mutants together with full-length HA-p300, were transfected into 293T cells. The HAT activity of p300 was determined as described above. Ack-H3 inhibition was determined by using densitometry. (C and D) TR3 inhibits p300 HAT-mediated transcription. Gal4-p300/HAT, together with a luciferase reporter gene pGAL4 and increasing amount of TR3 (C) or TR3 deletion mutants (D), was cotransfected into 293T cells. Reporter gene activity was determined as described in Figure 1D. The bars represent the average ± mean from three independent experiments.

Mentions: The HAT domain of p300 is located at the middle region of the protein that shows a physical interaction with TR3 (Figure 2D), indicating that the HAT activity of p300 may also be impaired by TR3 binding. We therefore assayed the p300 HAT activity by utilizing previously described systems (28,32). We first examined the acetylation level of histone H3, as Histone H3 acetylation is usually used to reflect p300 HAT activity (25,32). In 293T cells that were cotransfected with TR3 and p300, TR3 indeed exhibited a strong inhibition on p300 HAT activity, as ∼74% of such activity had lost when 2 µg of TR3 was introduced (Figure 3A). We further examined the effect of different TR3 deletion mutants on the HAT activity of p300. Similar to full-length TR3, TR3/D1, TR3/D4 and TR3/D6 could efficiently inhibit p300 HAT activity, reaching to 60% inhibition (Figure 3B). A common feature for these deletion mutants is that they all contain the region of aa 439–521 that interacts with p300 (Figure 2E). In contrast, TR3/D2, TR3/D3 and TR3/D5 showed no effect on p300 HAT activity (the activity was remained at almost 100% in all the tested mutants except TR3/D5) (Figure 3B), even though TR3/D5 could interact with p300 via the DBD (Figure 2E). Thus, these results suggest that the region of aa 439–521, rather than the DBD, is essential for TR3 to inhibit p300 HAT activity.Figure 3.


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)

TR3 inhibits p300 HAT activity. (A) Effect of TR3 on p300 HAT activity. HA-p300 and increasing amount of Falg-TR3 were transfected into 293T cells. Cell lysates were prepared and HA-p300 was immunoprecipitated with anti-HA antibody. The precipitates were then incubated with histone (10 µg) and Lys-CoA (10 µM) at 30°C for 30 min. The reaction products were subjected to western blotting with anti-Ack-H3 antibody to show the HAT activity. The levels of TR3, p300 and histone H3 were indicated by western blotting with anti-Flag, -HA and -histone antibodies. The percentage of Ack-H3 inhibition was calculated by determining the Ack-H3 protein amount of individual bands using densitometry. (B) Different TR3 mutants impair p300 HAT activity. Flag-TR3 mutants together with full-length HA-p300, were transfected into 293T cells. The HAT activity of p300 was determined as described above. Ack-H3 inhibition was determined by using densitometry. (C and D) TR3 inhibits p300 HAT-mediated transcription. Gal4-p300/HAT, together with a luciferase reporter gene pGAL4 and increasing amount of TR3 (C) or TR3 deletion mutants (D), was cotransfected into 293T cells. Reporter gene activity was determined as described in Figure 1D. The bars represent the average ± mean from three independent experiments.
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Figure 3: TR3 inhibits p300 HAT activity. (A) Effect of TR3 on p300 HAT activity. HA-p300 and increasing amount of Falg-TR3 were transfected into 293T cells. Cell lysates were prepared and HA-p300 was immunoprecipitated with anti-HA antibody. The precipitates were then incubated with histone (10 µg) and Lys-CoA (10 µM) at 30°C for 30 min. The reaction products were subjected to western blotting with anti-Ack-H3 antibody to show the HAT activity. The levels of TR3, p300 and histone H3 were indicated by western blotting with anti-Flag, -HA and -histone antibodies. The percentage of Ack-H3 inhibition was calculated by determining the Ack-H3 protein amount of individual bands using densitometry. (B) Different TR3 mutants impair p300 HAT activity. Flag-TR3 mutants together with full-length HA-p300, were transfected into 293T cells. The HAT activity of p300 was determined as described above. Ack-H3 inhibition was determined by using densitometry. (C and D) TR3 inhibits p300 HAT-mediated transcription. Gal4-p300/HAT, together with a luciferase reporter gene pGAL4 and increasing amount of TR3 (C) or TR3 deletion mutants (D), was cotransfected into 293T cells. Reporter gene activity was determined as described in Figure 1D. The bars represent the average ± mean from three independent experiments.
Mentions: The HAT domain of p300 is located at the middle region of the protein that shows a physical interaction with TR3 (Figure 2D), indicating that the HAT activity of p300 may also be impaired by TR3 binding. We therefore assayed the p300 HAT activity by utilizing previously described systems (28,32). We first examined the acetylation level of histone H3, as Histone H3 acetylation is usually used to reflect p300 HAT activity (25,32). In 293T cells that were cotransfected with TR3 and p300, TR3 indeed exhibited a strong inhibition on p300 HAT activity, as ∼74% of such activity had lost when 2 µg of TR3 was introduced (Figure 3A). We further examined the effect of different TR3 deletion mutants on the HAT activity of p300. Similar to full-length TR3, TR3/D1, TR3/D4 and TR3/D6 could efficiently inhibit p300 HAT activity, reaching to 60% inhibition (Figure 3B). A common feature for these deletion mutants is that they all contain the region of aa 439–521 that interacts with p300 (Figure 2E). In contrast, TR3/D2, TR3/D3 and TR3/D5 showed no effect on p300 HAT activity (the activity was remained at almost 100% in all the tested mutants except TR3/D5) (Figure 3B), even though TR3/D5 could interact with p300 via the DBD (Figure 2E). Thus, these results suggest that the region of aa 439–521, rather than the DBD, is essential for TR3 to inhibit p300 HAT activity.Figure 3.

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