title: E1A-mediated repression of progesterone receptor-dependent transactivation involves inhibition of the assembly of a multisubunit coactivation complex.
abstract: The steroid hormone progesterone acts via high-affinity nuclear receptors that interact with specific DNA sequences located near the promoter of the hormone-responsive gene. Recent studies suggested that the hormone-occupied progesterone receptor (PR) mediates gene activation by recruiting a cellular coregulatory factor, termed coactivator, to the target promoter. The identity and mechanism of action of the coactivator(s) that regulates transcriptional activity of PR are currently under investigation. Here we provide evidence that the hormone-occupied PR forms a multisubunit receptor-coactivator complex containing two previously described coactivators, CREB-binding protein (CBP) and steroid receptor coactivator 1 (SRC-1, a member of the p160 family of coactivators), in nuclear extracts of human breast tumor T47D cells. The association of CBP and SRC-1/p160 with the receptor complex is entirely hormone dependent. Both CBP and SRC-1/p160 possess intrinsic histone acetyltransferase (HAT) activity, and it has been recently proposed that these coactivators function by modulating chromatin structure at the promoter of the target gene. Interestingly, addition of purified CBP to the nuclear extracts of T47D cells markedly stimulated progesterone- and PR-dependent transcription from a nucleosome-free, progesterone response element (PRE)-linked reporter DNA template. Furthermore, depletion of SRC-1/p160 by immunoprecipitation from these transcriptional extracts also significantly impaired PR-mediated RNA synthesis from a naked PRE-linked DNA template. These results strongly implied that CBP and SRC-1/p160 facilitate receptor-mediated transcription in these cell extracts through mechanisms other than chromatin remodeling. We also observed that the adenoviral oncoprotein E1A, which interacts directly with CBP, repressed PR-mediated transactivation when added to the nuclear extracts of T47D cells. Supplementation with purified CBP overcame this inhibition, indicating that the inhibitory effect of E1A is indeed due to a blockade of CBP function. Most importantly, we noted that binding of E1A to CBP prevented the assembly of a coactivation complex containing PR, CBP, and SRC-1/p160, presumably by disrupting the interaction between CBP and SRC-1/p160. These results strongly suggested that E1A repressed receptor-mediated transcription by blocking the formation or recruitment of coactivation complexes. Collectively, our results support the hypothesis that the assembly of a multisubunit coactivation complex containing PR, CBP, and SRC-1/p160 is a critical regulatory step during hormone-dependent gene activation by PR and that the fully assembled complex has the ability to control transcription through mechanisms that are independent of the histone-modifying activities of its component coactivators.
authors.affiliation: Population Council, The Rockefeller University, New York, New York 10021, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Xu
authors.forename: L
authors.initials: L
authors.lastname: Klein-Hitpass
authors.forename: M K
authors.initials: MK
authors.lastname: Bagchi
mesh_terms: D017500:Adenovirus E1A Proteins / Q000235:genetics*; D051548:Histone Acetyltransferases; D006801:Humans; D056920:Nuclear Receptor Coactivator 1; D011374:Progesterone / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011980:Receptors, Progesterone / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
chemical_list: D017500:Adenovirus E1A Proteins; D011980:Receptors, Progesterone; D014157:Transcription Factors; D011374:Progesterone; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1