Altered pharmacology and distinct coactivator usage for estrogen receptor-dependent transcription through activating protein-1.

Estrogen signaling occurs through at least two distinct molecular pathways: (i) direct binding of liganded estrogen receptors (ERs) to estrogen-responsive DNA elements (EREs) (the "ER/ERE pathway") and (ii) indirect recruitment of liganded ERs to activating protein-1 (AP-1)-responsive DNA elements via heterodimers of Fos and Jun (the "ER/AP-1 pathway"). We have developed a biochemical assay for examining ligand-regulated transcription by ERs in the ER/AP-1 pathway. This assay recapitulates the altered (i.e., agonistic) pharmacology of selective estrogen receptor modulator drugs in this pathway reported previously by using various cell-based assays. We used our biochemical assay to examine the detailed mechanisms of ER/AP-1-dependent transcription. Our studies indicate that (i) ERalpha/AP-1 complexes play a critical role in promoting the formation of stable RNA polymerase II preinitiation complexes leading to transcription initiation, (ii) chromatin is a key determinant of estrogen and selective estrogen receptor modulator signaling in the ERalpha/AP-1 pathway, (iii) distinct domains of ERalpha are required for recruitment to DNA-bound Fos/Jun heterodimers and transcriptional activation at AP-1 sites, and (iv) different enhancer/activator combinations in the ERalpha and AP-1 pathways use coactivators in distinct ways. These studies have increased our understanding of the molecular mechanisms underlying ligand-dependent signaling in the ER/AP-1 pathway and demonstrate the usefulness of this biochemical approach.

Transcriptional activation by nuclear receptors.

Transcriptional activation by nuclear receptors (NRs) involves the recruitment of distinct classes of co-activators and other transcription-related factors to target promoters in the chromatin environment of the nucleus. Chromatin has a general repressive effect on transcription, but also provides opportunities for NRs to regulate transcription by directing specific patterns of chromatin remodelling and histone modification. Ultimately, the transcription of hormone-regulated genes by NRs is critically dependent on co-ordinated physical and functional interactions among the receptors, chromatin, co-activators with chromatin-, histone- and factor-modifying activities, and the RNA polymerase II transcriptional machinery. In addition, several mechanisms exist to terminate or attenuate NR-dependent signalling, including modification, recycling, subcellular redistribution and degradation of the receptors or their associated cofactors. The complexity of NR-dependent transcription provides multiple targets for regulatory inputs, thus allowing each hormone-responsive cell to direct its transcriptional output in a physiologically appropriate manner.

Selective recognition of distinct classes of coactivators by a ligand-inducible activation domain.

How nuclear receptors (NRs) coordinate the sequential, ligand-dependent recruitment of multiple coactivator complexes (e.g., SRC complexes and Mediator) that share similar receptor binding determinants is unclear. We show that although the receptor binding subunits of these complexes (i.e., SRCs and Med220, respectively) share overlapping binding sites on estrogen receptor alpha (ERalpha), information contained in the receptor-coactivator interface allows the receptor to distinguish between them. In support of this conclusion, we have identified an ERalpha AF-2 point mutant (L540Q) that selectively binds and recruits Med220, but not SRCs, both in vitro and in vivo. In cells expressing this mutant, the recruitment of Med220 to the pS2 promoter is delayed, and the expression of the vast majority of estrogen target genes is impaired, suggesting a nearly global functional interdependence of these coactivators. Collectively, our results suggest that "facilitated recruitment," rather than competition, drives the sequential recruitment of SRC complexes and Mediator by NRs.

Mediator and p300/CBP-steroid receptor coactivator complexes have distinct roles, but function synergistically, during estrogen receptor alpha-dependent transcription with chromatin templates.

Ligand-dependent transcriptional activation by nuclear receptors involves the recruitment of various coactivators to the promoters of hormone-regulated genes assembled into chromatin. Nuclear receptor coactivators include histone acetyltransferase complexes, such as p300/CBP-steroid receptor coactivator (SRC), as well as the multisubunit mediator complexes ("Mediator"), which may help recruit RNA polymerase II to the promoter. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the functional role for Mediator in the transcriptional activity of estrogen receptor alpha (ERalpha) with chromatin templates, as well as functional interplay between Mediator and p300/CBP during ERalpha-dependent transcription. Using three different approaches to functionally inactivate Mediator (immunoneutralization, immunodepletion, and inhibitory polypeptides), we find that Mediator is required for maximal transcriptional activation by ligand-activated ERalpha. In addition, we demonstrate synergism between Mediator and p300/CBP-SRC during ERalpha-dependent transcription with chromatin templates, but not with naked DNA. This synergism is important for promoting the formation of a stable transcription preinitiation complex leading to the initiation of transcription. Interestingly, we find that Mediator has an additional distinct role during ERalpha-dependent transcription not shared by p300/CBP-SRC: namely, to promote preinitiation complex formation for subsequent rounds of transcription reinitiation. These results suggest that one functional consequence of Mediator-ERalpha interactions is the stimulation of multiple cycles of transcription reinitiation. Collectively, our results indicate an important role for Mediator, as well as its functional interplay with p300/CBP-SRC, in the enhancement of ERalpha-dependent transcription with chromatin templates.