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.