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. 2002 Mar 5;99(5):2642-7.
doi: 10.1073/pnas.261715899. Epub 2002 Feb 26.

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

Yun Kyoung Kang  1 , Mohamed Guermah, Chao-Xing Yuan, Robert G Roeder
Affiliations

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

Yun Kyoung Kang et al. Proc Natl Acad Sci U S A. .

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 extract-derived proteins that interact with immobilized ER ligand binding domains in a 17beta-estradiol-dependent manner. The most prominent of these are components of the thyroid hormone receptor-associated protein (TRAP)/Mediator coactivator complex, which interacts with ERalpha and ERbeta in both unfractionated nuclear extracts and purified form. Studies with extracts from TRAP220(-/-) fibroblasts reveal that these interactions depend on TRAP220, a TRAP/Mediator 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 ERalpha-TRAP/Mediator complex from cultured cells expressing an epitope-tagged ERalpha. Finally, the complete TRAP/Mediator complex is shown to enhance ER function directly in a highly purified cell-free transcription system. These studies firmly establish a direct role for TRAP/Mediator, through TRAP220, in ER function.

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Figures

Figure 1
Figure 1
E2-dependent interactions of HeLa nuclear extract proteins with ERα and ERβ LBDs. Immobilized GST (lanes 2 and 8), GST–ERαLBD (lanes 3 and 4) and GST–ERβLBD (lanes 6 and 7) proteins were incubated with HeLa nuclear extract in the absence (−) or presence (+) of 1 μM E2, and bound proteins were eluted and analyzed by SDS/PAGE and silver staining as described in Materials and Methods. Purified TRAP/Mediator complex from f:Nut2-expressing cells is analyzed in lane 5. Standard molecular mass markers (SM) with sizes in kDa indicated on the left were present in lane 1. Bands marked with an asterisk represent degradation products of GST–ER fusion proteins.
Figure 2
Figure 2
E2-dependent interactions of TRAP/Mediator with ERα and ERβ LBDs in nuclear extract. Immobilized GST (lane 2), GST–ERαAB (lane 3), GST–ERαLBD (lanes 4 and 5), GST–ERβAB (lane 6), and GST–ERβLBD (lanes 7 and 8) proteins were incubated with HeLa nuclear extract in the absence (−) or presence (+) of 1 μM E2, and bound proteins were eluted and analyzed by SDS/PAGE and Western blot (with antibodies to proteins indicated on the left) as described in Materials and Methods. A 1/10th equivalent of the input nuclear extract is shown in lane 1. TRAP/Mediator and TR–TRAP complexes immunopurified from cells expressing f:Nut2 and f:TR, respectively, are shown in lanes 9 and 10. The band observed in lane 6 with MED7 antibody is a nonspecific band that crossreacts with this antibody.
Figure 3
Figure 3
TRAP220-dependent interactions of TRAP/Mediator with ER LBDs in nuclear extract. Immobilized GST (lanes 2 and 11), GST–ERα LBD (lanes 3–6), and GST–ERβ LBD (lanes 7–10) were incubated with nuclear extracts from wild-type (WT, lanes 2, 3, 4, 7, and 8) and TRAP220−/− (KO, lanes 5, 6, 9, 10, and 11) MEFs in the absence (−) or presence (+) of 1 μM E2, and bound proteins were eluted and analyzed by SDS/PAGE and Western blot (with antibodies to proteins indicated on the left) as detailed in Materials and Methods. One-tenth equivalents of input nuclear extracts were analyzed in lanes 1 (WT) and 12 (KO).
Figure 4
Figure 4
Direct interactions of purified TRAP/Mediator with LBDs of ERα and ERβ. Immobilized GST (lanes 6 and 7), GST–ERα LBD (lanes 2 and 3), and GST–ERβ LBD (lanes 4 and 5) were incubated with immunopurified TRAP/Mediator complex from cells expressing f:CDK8/SRB10 in the absence (−) or presence (+) of 1 μM E2. After extensive washing, bound proteins were eluted and analyzed by SDS/PAGE and Western blot (with antibodies to proteins indicated on the left) as described in Materials and Methods. A 1/10th equivalent of the input TRAP/Mediator preparation was analyzed in lane 1.
Figure 5
Figure 5
Intracellular association of TRAP/Mediator with ERαΔAB. C8 cells that express FLAG-tagged ERα lacking the AB domain (f:hERαΔAB) were maintained in DMEM-PO4 medium supplemented with 10% calf serum. f:ERαΔAB and associated proteins were affinity-purified from C8-derived nuclear extract on M2 agarose. Proteins bound in the presence (+) or absence (−) of 1 μM E2 during purification were eluted with FLAG peptide and analyzed by SDS/PAGE and Western blot with antibodies to proteins indicated on the left. M2 agarose-bound proteins from HeLa extract were analyzed in lane 1, and a 1/10th equivalent of C8 nuclear extract input was analyzed in lane 4.
Figure 6
Figure 6
TRAP/Mediator interactions with full-length ERα and ERβ. (A) Ligand-dependent interactions between TRAP/Mediator and ERs in HeLa nuclear extract. M2 agarose-immobilized FLAG-ERα and FLAG-ERβ were incubated with HeLa nuclear extracts in the absence (−) or presence (+) of 1 μM E2, and bound proteins were eluted with FLAG peptide and analyzed by SDS/PAGE and Western blot (with antibodies to proteins indicated in the left) as described in Materials and Methods. As a control, HeLa nuclear extract proteins bound to M2 agarose alone were analyzed in lane 2. A 1/10th equivalent of input nuclear extract was analyzed in lane 1. (B) Purified f:TRAP220AB TRAP/Mediator complex. The complex was affinity-purified from cells expressing a FLAG-tagged TRAP220 lacking the C-terminal domain (f:TRAP220AB) and analyzed by SDS/PAGE and silver staining (lane 2). Lane 1 shows HeLa nuclear extract proteins bound nonspecifically to M2 agarose. (C) Direct interactions between purified TRAP/Mediator complex and full-length ERβ. M2 agarose-immobilized TRAP/Mediator (f:TRAP220AB) complex (lanes 3, 4, and 6) and M2 agarose alone (lanes 2 and 5) were incubated with 35S-labeled (in vitro translated) full-length ERβ in the presence (lane 3) or absence (lane 4) of 1 μM E2 or with control lysate (lanes 5 and 6). After washing, bound proteins were eluted with FLAG peptide and analyzed by autoradiography (Upper) or Western blot with antibodies to the indicated components of TRAP/Mediator complex (Lower). Lane 1 shows 1/20th of the 35S-labeled full-length ERβ input.
Figure 7
Figure 7
TRAP/Mediator complex directly mediates ER function in vitro. (A) Recombinant FLAG-tagged ER proteins. Affinity-purified ERs were analyzed by SDS/PAGE and either Coomassie brilliant blue R-250 staining (lanes 1 and 2) or Western blot with corresponding antibodies (lanes 3 and 4). (B) ER-dependent in vitro transcription. Reactions contained purified general TFs and cofactors, purified TRAP/Mediator complex, and ER proteins (20 nM) as indicated and both 4EREΔ53 G-less reporter and ML200 G-less control templates. TRAP/Mediator was purified from the cell line expressing f:TRAP220AB (Fig. 6B).
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