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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 represses transcriptional activities of transcription factors through inhibiting p300-induced acetylation. (A) TR3 inhibits the acetylation of a broad spectrum of substrates. HA-p300, with increasing amount of Flag-TR3, was transfected into 293T cells. Lysates were prepared from transfected cells and subjected to western blotting. The acetylated proteins were detected by anti-acetylation-specific antibody. The levels of Flag-TR3 and HA-p300 were shown by anti-Flag and anti-HA antibodies, respectively. Tubulin was used to indicate the similar loading of proteins in each lane. (B) TR3 cannot be acetylated by p300. GFP-TR3 and HA-p300 were transfected into 293T cells. Cell lysates were prepared and GFP-TR3 was immunoprecipitated with anti-GFP antibody. The precipitated proteins were then subjected to western blotting with acetylation-specific antibody. p53, which is known to be acetylated by p300, was used as a positive control. GFP-TR3 and GFP-p53 were also detected by western blotting with anti-GFP antibody. (C) TR3 attenuates p300-induced acetylation of various transcription factors. HA-p300 and Flag-TR3 together with different tagged transcription factors as indicated were transfected into 293T cells. Acetylation for each transcription factor was determined as described above. The expression levels of various transcription factors were indicated by western blotting with antibodies corresponding to different tags. (D) TR3 represses p300-induced transcriptional activity of various transcription factors. Different transcription factors and their corresponding reporter genes, together with p300, β-gal expression vector and increasing amount of TR3, were transfected into 293T cells. Reporter gene activity was determined and normalized in relation to the cotransfected β-gal activity. The bars represent the average ± mean from three independent experiments.
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Figure 1: TR3 represses transcriptional activities of transcription factors through inhibiting p300-induced acetylation. (A) TR3 inhibits the acetylation of a broad spectrum of substrates. HA-p300, with increasing amount of Flag-TR3, was transfected into 293T cells. Lysates were prepared from transfected cells and subjected to western blotting. The acetylated proteins were detected by anti-acetylation-specific antibody. The levels of Flag-TR3 and HA-p300 were shown by anti-Flag and anti-HA antibodies, respectively. Tubulin was used to indicate the similar loading of proteins in each lane. (B) TR3 cannot be acetylated by p300. GFP-TR3 and HA-p300 were transfected into 293T cells. Cell lysates were prepared and GFP-TR3 was immunoprecipitated with anti-GFP antibody. The precipitated proteins were then subjected to western blotting with acetylation-specific antibody. p53, which is known to be acetylated by p300, was used as a positive control. GFP-TR3 and GFP-p53 were also detected by western blotting with anti-GFP antibody. (C) TR3 attenuates p300-induced acetylation of various transcription factors. HA-p300 and Flag-TR3 together with different tagged transcription factors as indicated were transfected into 293T cells. Acetylation for each transcription factor was determined as described above. The expression levels of various transcription factors were indicated by western blotting with antibodies corresponding to different tags. (D) TR3 represses p300-induced transcriptional activity of various transcription factors. Different transcription factors and their corresponding reporter genes, together with p300, β-gal expression vector and increasing amount of TR3, were transfected into 293T cells. Reporter gene activity was determined and normalized in relation to the cotransfected β-gal activity. The bars represent the average ± mean from three independent experiments.

Mentions: Recently, we found that TR3 negatively regulates the transcriptional activity of p53 (29) and retinoid X receptor (RXRα) (30) through the p300-dependent acetylation pathway. These results strongly suggest that TR3 may also inhibit the activity of other p300-acetylated transcription factors. To test this possibility, we first examined the effect of TR3 on the p300-induced acetylation of a broad spectrum of substrates. When whole-cell lysates were analyzed by western blotting with specific anti-acetyl-lysine antibody, a dramatic increase in total protein acetylation was observed upon the expression of p300 (Figure 1A, lane 2). As we expected, coexpression of TR3 with p300 led to a decrease in the total protein acetylation level in a dose-dependent manner (Figure 1A). We found that TR3 neither interfered with p300 expression (Figure 1A), nor was acetylated by p300 (Figure 1B). Clearly, TR3 has a unique characteristic in inhibiting p300-induced acetylation of numerous cellular proteins.Figure 1.


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 represses transcriptional activities of transcription factors through inhibiting p300-induced acetylation. (A) TR3 inhibits the acetylation of a broad spectrum of substrates. HA-p300, with increasing amount of Flag-TR3, was transfected into 293T cells. Lysates were prepared from transfected cells and subjected to western blotting. The acetylated proteins were detected by anti-acetylation-specific antibody. The levels of Flag-TR3 and HA-p300 were shown by anti-Flag and anti-HA antibodies, respectively. Tubulin was used to indicate the similar loading of proteins in each lane. (B) TR3 cannot be acetylated by p300. GFP-TR3 and HA-p300 were transfected into 293T cells. Cell lysates were prepared and GFP-TR3 was immunoprecipitated with anti-GFP antibody. The precipitated proteins were then subjected to western blotting with acetylation-specific antibody. p53, which is known to be acetylated by p300, was used as a positive control. GFP-TR3 and GFP-p53 were also detected by western blotting with anti-GFP antibody. (C) TR3 attenuates p300-induced acetylation of various transcription factors. HA-p300 and Flag-TR3 together with different tagged transcription factors as indicated were transfected into 293T cells. Acetylation for each transcription factor was determined as described above. The expression levels of various transcription factors were indicated by western blotting with antibodies corresponding to different tags. (D) TR3 represses p300-induced transcriptional activity of various transcription factors. Different transcription factors and their corresponding reporter genes, together with p300, β-gal expression vector and increasing amount of TR3, were transfected into 293T cells. Reporter gene activity was determined and normalized in relation to the cotransfected β-gal activity. The bars represent the average ± mean from three independent experiments.
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Figure 1: TR3 represses transcriptional activities of transcription factors through inhibiting p300-induced acetylation. (A) TR3 inhibits the acetylation of a broad spectrum of substrates. HA-p300, with increasing amount of Flag-TR3, was transfected into 293T cells. Lysates were prepared from transfected cells and subjected to western blotting. The acetylated proteins were detected by anti-acetylation-specific antibody. The levels of Flag-TR3 and HA-p300 were shown by anti-Flag and anti-HA antibodies, respectively. Tubulin was used to indicate the similar loading of proteins in each lane. (B) TR3 cannot be acetylated by p300. GFP-TR3 and HA-p300 were transfected into 293T cells. Cell lysates were prepared and GFP-TR3 was immunoprecipitated with anti-GFP antibody. The precipitated proteins were then subjected to western blotting with acetylation-specific antibody. p53, which is known to be acetylated by p300, was used as a positive control. GFP-TR3 and GFP-p53 were also detected by western blotting with anti-GFP antibody. (C) TR3 attenuates p300-induced acetylation of various transcription factors. HA-p300 and Flag-TR3 together with different tagged transcription factors as indicated were transfected into 293T cells. Acetylation for each transcription factor was determined as described above. The expression levels of various transcription factors were indicated by western blotting with antibodies corresponding to different tags. (D) TR3 represses p300-induced transcriptional activity of various transcription factors. Different transcription factors and their corresponding reporter genes, together with p300, β-gal expression vector and increasing amount of TR3, were transfected into 293T cells. Reporter gene activity was determined and normalized in relation to the cotransfected β-gal activity. The bars represent the average ± mean from three independent experiments.
Mentions: Recently, we found that TR3 negatively regulates the transcriptional activity of p53 (29) and retinoid X receptor (RXRα) (30) through the p300-dependent acetylation pathway. These results strongly suggest that TR3 may also inhibit the activity of other p300-acetylated transcription factors. To test this possibility, we first examined the effect of TR3 on the p300-induced acetylation of a broad spectrum of substrates. When whole-cell lysates were analyzed by western blotting with specific anti-acetyl-lysine antibody, a dramatic increase in total protein acetylation was observed upon the expression of p300 (Figure 1A, lane 2). As we expected, coexpression of TR3 with p300 led to a decrease in the total protein acetylation level in a dose-dependent manner (Figure 1A). We found that TR3 neither interfered with p300 expression (Figure 1A), nor was acetylated by p300 (Figure 1B). Clearly, TR3 has a unique characteristic in inhibiting p300-induced acetylation of numerous cellular proteins.Figure 1.

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