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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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Effects of different mutants of p300 and TR3 on regulation of HAT activity and transcriptional activity of transcription factors. (A and C) Different p300 or TR3 mutants impair TR3 inhibition on p300 HAT activity. Different Flag-p300 mutants (A) or Myc-TR3 mutants (C), together with full length of Myc-TR3 (A) or HA-p300 (C), were transfected into 293T cells. The HAT activity of p300 was determined as described above. The percentage of Ack-H3 inhibition was calculated by using densitometry. (B) Interaction of p300 with different TR3 mutants. HA-p300 and Myc-TR3 mutants were transfected into 293T cells. Cell lysates were prepared and then subjected to Co-IP/western blotting as described in Figure 2A. (D) Effect of different TR3 deletion mutants on transcriptional activity of various transcription factors. p300, together with different TR3 mutants, transcription factors and reporter genes, was cotransfected into 293T cells. The transcriptional activity was determined as described in Figure 1D.
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Figure 4: Effects of different mutants of p300 and TR3 on regulation of HAT activity and transcriptional activity of transcription factors. (A and C) Different p300 or TR3 mutants impair TR3 inhibition on p300 HAT activity. Different Flag-p300 mutants (A) or Myc-TR3 mutants (C), together with full length of Myc-TR3 (A) or HA-p300 (C), were transfected into 293T cells. The HAT activity of p300 was determined as described above. The percentage of Ack-H3 inhibition was calculated by using densitometry. (B) Interaction of p300 with different TR3 mutants. HA-p300 and Myc-TR3 mutants were transfected into 293T cells. Cell lysates were prepared and then subjected to Co-IP/western blotting as described in Figure 2A. (D) Effect of different TR3 deletion mutants on transcriptional activity of various transcription factors. p300, together with different TR3 mutants, transcription factors and reporter genes, was cotransfected into 293T cells. The transcriptional activity was determined as described in Figure 1D.

Mentions: Interestingly, when TR3 was cotransfected into 293T cells with either p300/M1 or p300/M2, both of which retain the p300 HAT activity fully, it only inhibited the HAT activity of p300/M2 with 78% approximately, but not that of p300/M1 (the approximate inhibition was 6%) (Figure 4A). Compared to p300/M1, p300/M2 contains extra sequences in which a motif (RKTNGGCPICKQ) called TRAM can be found (Figure 2D). TRAM has been shown to be conserved in all members of the CBP/p300 family of proteins and is responsible for specific recognition of target proteins (35). In p300, TRAM is located within the CH3 domain just after the HAT domain (36,37). Our result clearly demonstrated that TRAM plays an important role in mediating the inhibition of TR3 on p300 HAT activity.Figure 4.


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)

Effects of different mutants of p300 and TR3 on regulation of HAT activity and transcriptional activity of transcription factors. (A and C) Different p300 or TR3 mutants impair TR3 inhibition on p300 HAT activity. Different Flag-p300 mutants (A) or Myc-TR3 mutants (C), together with full length of Myc-TR3 (A) or HA-p300 (C), were transfected into 293T cells. The HAT activity of p300 was determined as described above. The percentage of Ack-H3 inhibition was calculated by using densitometry. (B) Interaction of p300 with different TR3 mutants. HA-p300 and Myc-TR3 mutants were transfected into 293T cells. Cell lysates were prepared and then subjected to Co-IP/western blotting as described in Figure 2A. (D) Effect of different TR3 deletion mutants on transcriptional activity of various transcription factors. p300, together with different TR3 mutants, transcription factors and reporter genes, was cotransfected into 293T cells. The transcriptional activity was determined as described in Figure 1D.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: Effects of different mutants of p300 and TR3 on regulation of HAT activity and transcriptional activity of transcription factors. (A and C) Different p300 or TR3 mutants impair TR3 inhibition on p300 HAT activity. Different Flag-p300 mutants (A) or Myc-TR3 mutants (C), together with full length of Myc-TR3 (A) or HA-p300 (C), were transfected into 293T cells. The HAT activity of p300 was determined as described above. The percentage of Ack-H3 inhibition was calculated by using densitometry. (B) Interaction of p300 with different TR3 mutants. HA-p300 and Myc-TR3 mutants were transfected into 293T cells. Cell lysates were prepared and then subjected to Co-IP/western blotting as described in Figure 2A. (D) Effect of different TR3 deletion mutants on transcriptional activity of various transcription factors. p300, together with different TR3 mutants, transcription factors and reporter genes, was cotransfected into 293T cells. The transcriptional activity was determined as described in Figure 1D.
Mentions: Interestingly, when TR3 was cotransfected into 293T cells with either p300/M1 or p300/M2, both of which retain the p300 HAT activity fully, it only inhibited the HAT activity of p300/M2 with 78% approximately, but not that of p300/M1 (the approximate inhibition was 6%) (Figure 4A). Compared to p300/M1, p300/M2 contains extra sequences in which a motif (RKTNGGCPICKQ) called TRAM can be found (Figure 2D). TRAM has been shown to be conserved in all members of the CBP/p300 family of proteins and is responsible for specific recognition of target proteins (35). In p300, TRAM is located within the CH3 domain just after the HAT domain (36,37). Our result clearly demonstrated that TRAM plays an important role in mediating the inhibition of TR3 on p300 HAT activity.Figure 4.

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