Target gene activation by nuclear hormone receptors, including estrogen receptors (ERs), is thought to be mediated by a variety of interacting cofactors. Here we identify a number of nuclear extractderived proteins that interact with immobilized ER ligand binding domains in a 17␤-estradiol-dependent manner. The most prominent of these are components of the thyroid hormone receptor-associated protein (TRAP)͞Mediator coactivator complex, which interacts with ER␣ and ER␤ in both unfractionated nuclear extracts and purified form. Studies with extracts from TRAP220 ؊͞؊ fibroblasts reveal that these interactions depend on TRAP220, a TRAP͞Mediator subunit previously shown to interact with ER and other nuclear receptors in a ligand-dependent manner. The physiological relevance of the in vitro interaction is documented further by the isolation of an ER␣-TRAP͞Mediator complex from cultured cells expressing an epitopetagged ER␣. Finally, the complete TRAP͞Mediator complex is shown to enhance ER function directly in a highly purified cell-free transcription system. These studies firmly establish a direct role for TRAP͞ Mediator, through TRAP220, in ER function. N uclear hormone receptors comprise a superfamily of transcriptional activators that bind to and, in a ligand-dependent manner, activate target genes involved in diverse physiological processes (1). Conserved nuclear receptor domains include the central DNA binding domain and a C-terminal ligand binding domain (LBD) that contains the ligand-induced AF-2 activation domain. Many receptors also contain N-terminal AF-1 activation domains that are less conserved (2). The function of nuclear receptors on target genes involves a variety of commonly used coactivators that in many cases show ligand-dependent interactions (directly or indirectly) with the AF-2 domain (3-5). One prominent group includes the p160͞SRC family and the interacting p300͞CBP and PCAF proteins, which function at least in part through intrinsic histone acetyltransferase activities that modify chromatin structure to facilitate subsequent receptor͞coactivator-mediated recruitment and͞or function of the general transcription machinery (3-5).Another coactivator of increasing importance for nuclear receptors is the thyroid hormone receptor-associated protein (TRAP)͞ Mediator complex. Although now known to mediate the activity of a number of distinct activators through specific subunit interactions (refs. 6 and 7; reviewed in refs. 8 and 9), TRAP͞Mediator was identified first through a ligand-dependent interaction with thyroid hormone receptor (TR) and shown to be essential for TR function on DNA templates in a reconstituted cell-free system (10). The TRAP220 subunit was identified as the main anchor for TR on the basis of a selective ligand-dependent interaction of isolated TRAP220 with TR (6), and analyses with TRAP220 Ϫ͞Ϫ fibroblasts confirmed a receptor-selective function for TRAP220 (11,12). The early demonstration of ligand-dependent interactions of TRAP220 with a number of other nuclear receptors furth...

The biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor RNA can result in production of different protein isoforms with opposite biological activities. Therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. In this report, we demonstrate that individual steroid hormones had different and opposite effects on alternative splicing decisions, stimulating the production of different spliced variants produced from genes driven by steroid hormone-dependent promoters. Steroid hormone transcriptional effects are mediated by steroid hormone receptor coregulators that also modify alternative splicing decisions. Our data suggest that activated steroid hormone receptors recruit coregulators to the target promoter that participate in both the production and the splicing of the target gene transcripts. Because different coregulators activating transcription can have opposite effects on alternative splicing decisions, we conclude that the precise nature of the transcriptional coregulators recruited by activated steroid receptors, depending on the promoter and cellular contexts, may play a major role in regulating the nature of the spliced variants produced from certain target genes in response to steroid hormones. G ene expression regulation is a multistep process, including the synthesis of the pre-mRNA or transcription; the 5Ј and 3Ј end maturation of the transcript or capping and polyadenylation, respectively; the removal of the introns from the premRNA; and the export to the cytosol of the mRNA and its translation (1). Steroid hormones play a major role in the control of cellular fate and cellular homeostasis by modulating the expression of genes, the products of which are involved in cellular programs such as apoptosis, proliferation, differentiation, and in cellular metabolism (2, 3). Most of the studies of steroid hormone action focus on transcriptional effects that are mediated by the binding of steroid hormone receptors to hormone response elements localized in target gene promoters (4). Nevertheless, the biological consequences resulting from the modulation of the transcriptional activity of genes cannot be precisely predicted because of alternative splicing. Approximately 60% of human pre-mRNAs undergo an alternative splicing process that results in the synthesis from one gene of different mRNAs encoding different proteins having different biological actions (5). For instance, the products of many genes involved in apoptosis are alternatively spliced and this splicingcan result in the synthesis of isoforms that antagonize each other by having pro-vs. antiapoptosis effects (6). Therefore, the alternative splicing process can change considerably the biological consequences resulting from the transcriptional modulation of steroid hormone target genes and an important question to address is the mechanis...

The transcription of eukaryotic genes is mediated by functional interactions of the general transcription machinery with common core promoter (e.g. TATA) elements and further regulated by gene-specific factors bound to distal regulatory elements (1). Studies with purified factors have demonstrated that pol II 4 and cognate initiation factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (general transcription machinery) are necessary and sufficient for robust basal (activator-independent) transcription from TATA-containing DNA templates and that the formation of a functional PIC involves the ordered stepwise assembly of these factors (2, 3). In contrast, activator-dependent transcription from DNA templates was found to require additional cofactors, the most prominent of which is the multisubunit Mediator complex, which is conserved from yeast to human (4, 5). Early demonstrations of its interaction with pol II (6, 7), along with later demonstrations of direct interactions with activators (4, 8), led to the notion that Mediator serves as a bridge between DNA-binding regulatory factors and the general transcription machinery to facilitate formation and/or function of the PIC (4, 5). Various biochemical and genetic studies have substantiated this view.Despite its paramount role in activator-dependent transcription, Mediator also has been implicated in basal transcription events by both biochemical and genetic assays. Thus, consistent with the ability of yeast Mediator to enhance basal transcription in a purified system (7), mutations in Mediator subunits were shown to eliminate or reduce basal transcription in crude nuclear extracts (9, 10). This is consistent with genetic studies implicating yeast Mediator in transcription of essentially all genes in yeast (11). Similarly, human Mediator has also been shown to be essential for basal transcription in nuclear extracts (12, 13). As nuclear extracts contain a more natural complement of cellular factors, these assays likely give a more accurate reflection of Mediator requirements in living cells and support the emerging view that Mediator functions as a general transcription factor under more physiological assay conditions. The mechanism(s) by which Mediator facilitates basal transcription remain to be fully understood and could include direct or indirect functions at any of the steps (PIC formation, promoter melting, initiation, promoter clearance, etc.) leading to productive transcription (3). The isolation of stable Mediator-pol II (holoenzyme) complexes, initially in yeast (6, 7) and later in mammalian cells (4, 14 -16), suggested the possibility of Mediator recruitment to, and possible stabilization of, the PIC through Mediator-pol II interactions. A role for Mediator in basal PIC formation or function was also suggested by the ability of yeast Mediator to enhance TFIIH-mediated phosphorylation of the pol II CTD (7) and an associated CTD requirement both for basal transcription in yeast and mammalian nuclear extracts (17) and for formation (activator-dependent) of ...

In contrast to previous findings in cell-free systems reconstituted with partially purified metazoan factors, we demonstrate dramatic activation of transcription in a TBP-dependent but TAFII-independent manner in HeLa nuclear extracts immunodepleted of TBP and major TAFIIs. Single-round transcription assays reveal that TAFII-independent activation is manifested at the level of productive preinitiation complex formation and that TAFIIs actually impair functional preinitiation complex assembly in a core promoter-specific manner. Furthermore, TAFIIs appear to elevate absolute levels of transcription under multiple-round transcription conditions, presumably by facilitating secondary initiation events. Finally, human coactivator activities related to those in yeast RNA polymerase II/mediator complexes appear to function in unfractionated HeLa nuclear extracts.

The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liverspecific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regulatory module of the transcription apparatus. Using a system that has been reconstituted from purified transcription factors, as well as one consisting of unfractionated nuclear extract from which TRAP/SMCC/Mediator has been depleted by specific antibodies, we demonstrate a strong dependence of HNF-4 function on this coactivator. Importantly, we further show a TRAP/SMCC/Mediator-dependence for HNF-4 transcriptional activation from chromatin templates. The latter involves cooperation with the histone acetyltransferase-containing coactivator p300, in accord with a synergistic mode of action of the two divergent coactivators. We also show that HNF-4 and TRAP/SMCC/Mediator can interact physically. This interaction likely involves primary HNF-4 activation function 2 (AF-2)-dependent interactions with the TRAP220 subunit of TRAP/SMCC/Mediator and secondary (AF-2-independent) interactions with TRAP170/RGR1. Finally, recruitment experiments using immobilized templates strongly suggest that the functional consequences of the physical interaction probably are manifested at a postrecruitment step in the activation pathway.