The TRAP/Mediator coactivator complex interacts directly with estrogen receptors α and β through the TRAP220 subunit and directly enhances estrogen receptor function in vitro

Abstract

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...

An Extended LXXLL Motif Sequence Determines the Nuclear Receptor Binding Specificity of TRAP220

“…In addition, a recent report has shown that the TRAP complex can be purified from HeLa cell nuclear extracts using GST-ER␣ LBD in the presence of agonist ligand (50). In contrast to reports suggesting that TRAP170 interacts with steroid receptors, GST-ER␣ AF1 did not retain TRAP complex in HeLa cell nuclear extracts (50). Thus, recruitment of TRAP complexes to the promoters of estrogen-regulated genes may involve additional interactions with other components of this multiprotein complex.…”

“…Purified TRAP/DRIP/mediator complexes have been shown to enhance the activity of NRs in cell-free or purified in vitro transcription assays (17,50,54). In comparison, only very modest enhancement of TR␣/␤, VDR, and PPAR␥-mediated transcription has been observed due to ectopic expression of the NR binding subunit TRAP220/DRIP205/PBP in transiently transfected cells (17, 19 -21).…”

“…Nonetheless, chromatin immunoprecipitation experiments have suggested that TRAP/DRIP proteins are recruited to NR-regulated promoters, including ER␣-and AR-responsive promoters (51,55,56), and microinjection of HeLa cell nuclei with anti-TRAP220/PBP antibodies down-regulated ER␣ activation of a reporter gene (57). In addition, a recent report has shown that the TRAP complex can be purified from HeLa cell nuclear extracts using GST-ER␣ LBD in the presence of agonist ligand (50). In contrast to reports suggesting that TRAP170 interacts with steroid receptors, GST-ER␣ AF1 did not retain TRAP complex in HeLa cell nuclear extracts (50).…”

“…TRAP220 has also been reported to interact with ER␣, although this appears to be relatively weak (18,19,29,30). Recent reports have demonstrated that TRAP220 interacts more strongly with ER␤ (30,50). In addition, data from other studies have suggested that sequences flanking the core motif also influence NR binding specificity (30, 34 -39).…”

T:G Mismatch-specific Thymine-DNA Glycosylase Potentiates Transcription of Estrogen-regulated Genes through Direct Interaction with Estrogen Receptor α

“…Several coactivator proteins associate with ligand-bound NR and include the p160 proteins, SRC1, TIF2/ GRIP1, and pCIP/ACTR/AIB1/RAC3/TRAM-1 (13). The thyroid hormone receptor-associated protein complex (14), which is very similar to the vitamin D3 receptor-interacting protein complex (15), interacts with liganded NR and is required for thyroid hormone and vitamin D3 receptor as well as ER␣ activities in vitro (16). CBP and the highly related p300, which copurify with RNA polymerase II (17) and associate with p160 coactivators, can directly interact with NR (18 -20).…”