Negative Regulation Of Transcription Coactivator P300 By Orphan Receptor TR3

p300, a transcriptional co-activator, regulates the transcriptional activity of a variety of transcription factors by forming an activation complex and/or promoting histone acetylation. It plays a central role in co-ordinating and integrating multiple signal-dependent events with the transcription apparatus, allowing the appropriate level of gene activity to occur in response to diverse physiological cues that influence, for example, proliferation, differentiation and apoptosis.Nuclear orphan receptor TR3 belongs to the steroid/thyroid hormone receptor superfamily. TR3 is an immediate-early response gene whose expression is rapidly induced by a variety of growth stimuli, including serum, phorbol ester and growth factors etc. TR3 also exerts its biological functions in apoptosis, proliferation, differentiation and development.In our previous study, we found that p300-induced p53 acetylation can be dramatically suppressed by TR3 at the transcriptional level. By blocking the acetylation, TR3 down-regulates p53 transcriptional activity and lead to a decrease on the transcription level of MDM2. Similarly, TR3 also significantly inhibits the p300-induced RXRαacetylation, resulting in the disassociation of RXRαfrom DNA. These results strongly suggest that TR3 may also inhibit the activities of other p300-acetylated transcription factors.In the current study, 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 FLAT activity, as the p300-induced histone H3 acetylation and transcription were inhibited with the presence TR3. When TRAM was removed, TR3 still bound to p300 at the CH2 domain, but failed to inhibit the HAT activity. Therefore, TRAM is a necessary binding target for TR3 to inhibit p300 HAT activity. When the F and E in FLELFIL sequence were substituted with A simultaneously in full-length TR3 or TR3 deletion mutant, the effect of TR3 on HAT activity was attenuated. These results suggest that TR3 inhibition on p300 HAT activity may be dependent on an FLELFIL - TRAM interaction.In addition, we showed that TR3 agonist, a natural octaketide isolated from Dothiorella sp.HTF3 of endophytical fungus, possesses the ability to inhibit p300 HAT activity. More importantly, we found that TR3 could be the potential target of this agonist, as siRNA-TR3 attenuated agonist function on repression of p300 HAT activity and transcriptional activity of transcription factors. It has been reported that in breast cancer cells, p300 binding to AR alters expression of specific growth control genes and promotes aberrant cellular growth in vivo, which is closely associated with p300 acetylation for AR. In the present study, we found that when breast cancer cells were introduced with TR3 or treated with agonist, the cell proliferation mediated by AR via p300 acetylation was abolished through blockage of DNA synthesis in the cells. Taken together,our study not only reveals a novel function of TR3 to inhibit breast cancer cell growth through repression of p300 HAT activity, but also indicates that the TR3 agonist may be used as a lead compound for anticancer drug.