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. 2007;35(18):6161-9.
doi: 10.1093/nar/gkm661. Epub 2007 Sep 7.

The Mediator subunit MED1/TRAP220 is required for optimal glucocorticoid receptor-mediated transcription activation

Wei Chen  1 , Robert G Roeder
Affiliations

The Mediator subunit MED1/TRAP220 is required for optimal glucocorticoid receptor-mediated transcription activation

Wei Chen et al. Nucleic Acids Res. 2007.

Abstract

The MED1/TRAP220 subunit of the Mediator plays a key role in facilitating ligand-dependent interactions of this multisubunit coactivator complex with nuclear receptors through their ligand binding domains. The isolated MED1/TRAP220 protein previously was shown to interact with glucocorticoid receptor (GR) in a ligand-dependent manner. However, the functional role of MED1/TRAP220, within the context of the entire Mediator, is not well studied in GR-mediated transcription. In this study, we show that GR binds directly to the Mediator complex and that both LXXLL motifs of MED1/TRAP220 contribute to its binding to GR. Furthermore, using a Med1/Trap220-/- mouse embryonic fibroblast (MEF) line that lacks entirely MED1/TRAP220, we show that MED1/TRAP220 enhances GR-mediated transcription from an MMTV promoter based-reporter gene and that mutations in the MED1/TRAP220 LXXLL motifs reduce, but do not eliminate, GR-dependent transcription. An analysis of endogenous genes in Med1/Trap220-/- cells has confirmed a variable MED1/TRAP220 requirement for different GR target genes. Taken together, these findings support the idea that Mediator, at least in part through MED1/TRAP220, plays a coregulatory role in ligand-dependent GR-mediated gene expression.

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Figures

Figure 1.
Figure 1.
GR interacts with the Mediator complex. (A) Binding of Mediator in nuclear extract. (B) Binding of purified Mediator. Beads containing bound GST or GST-GR-LBD were incubated with HeLa nuclear extract (A) or purified Mediator complex (B) in the presence of 10 µM Dex and processed as described in Materials and Methods. Associated components were identified by immunoblot with antibodies (all generated in this lab) to MED1/TRAP220, MED14/TRAP170, MED17/TRAP80 and MED20/TRFP.
Figure 2.
Figure 2.
The NR boxes of MED1/TRAP220 interact with GR-LBD in a ligand-dependent manner. (A) Schematic representation of GR. (B) Schematic representation of MED1/TRAP220 and MED1/TRAP220 mutant proteins fused with GST. Numbers indicate positions of amino acid residues, black bars represent NR (LXXLL) boxes, shaded bars represent NR mutants (LXXAA). (C) Comassie staining of purified GST-MED1/TRAP220 fusion proteins. (D) Binding of MED1/TRAP220 NR to GR domains. Glutathione-Sepharose beads containing immobilized GST or GST-MED1/TRAP220NR (amino acids 580–701) proteins were incubated with in vitro translated, 35S-labelled GR-AF1 or GR-LBD proteins (shown in A) as indicated and bound proteins were resolved by SDS-PAGE and visualized by autoradiography.
Figure 3.
Figure 3.
Both NR boxes contribute to the interaction between MED1/TRAP220 and GR. (A) Binding assays with the indicated fusion proteins (Figure 2B) and 35S-labeled GR-LBD were performed as described in Figure 2D. (B) Quantitation of the binding results in A. Values are from the representative experiment shown in A. Similar results were obtained from an independent experiment.
Figure 4.
Figure 4.
MED1/TRAP220 and GR co-localize in the nucleus after ligand treatment. Nuclear (NE) and cytoplasmic (S100) fractions were prepared from HeLa S cells grown in the absence or presence of Dex. MED1/TRAP220 and GR were detected by immunoblot with corresponding anti-MED1/TRAP220 and anti-GR antibodies.
Figure 5.
Figure 5.
MED1/TRAP220 enhances GR-mediated transcription. (A) MED1/TRAP220 requirement for optimum GR-mediated transcription activation. Med1/Trap220−/− and WT MEFs were co-transfected with MMTV-luc reporter (150 ng) and vectors expressing GR (25 ng) or MED1/TRAP220 as indicated and cultured in the presence or absence of Dex. Conditions were as described in Materials and Methods. (B) Effect of GR dosage on MED1/TRAP220 enhanced transcription. Luciferase activities are shown as relative luciferase units (RLU) after normalization by β-gal activity. Values are the mean and SD of triple (A) or duplicate (B) samples in a representative experiment of at least three. * indicates p < 0.05. ** indicates p < 0.01 with unpaired Student's t-test.
Figure 6.
Figure 6.
Effect of MED1/TRAP220 mutations on enhancement of GR-mediated transcription. (A) Functional analysis. Med1/Trap220−/− MEFs were transfected with MMTV-luc reporter (150 ng) and vectors expressing GR (10 ng) or MED1/TRAP220 (300 ng) mutants (described in C) as indicated, grown in the presence or absence of Dex as indicated, and processed as described in Materials and Methods. Luciferase activities are shown as relative luciferase units (RLU) after normalization to β-gal activity. Values are the mean and SD of duplicate samples in a representative experiment of at least three. Lanes 3-8 were compared with lane 2, lanes 4–8 were compared with lane 3. * indicates p < 0.05, ** indicates p < 0.01 with unpaired Student's t-test. (B) Mutation of the two LXXLL motives does not affect the function of the N-terminal domain of TRAP220 in enhancing GR-mediated transcription. The experiments were performed and analyzed as described in (A). Lanes 3–5 were compared to lane 2. * indicates p < 0.05. (C) Schematic representation of MED1/TRAP220 and MED1/TRAP220 mutants.
Figure 7.
Figure 7.
Expression profile of endogenous GR target genes in wild-type and Med1/TRAP220−/− MEFs. Cells were treated as described in the text, and real-time PCR was performed as described in Materials and Methods. (A) GILZ is induced to similar levels in wild-type and Med1/TRAP−/− MEFs. (B) Induction of IRF8 is reduced in Med1/TRAP220−/− MEFs relative to wild-type MEFs. * indicates p < 0.05.
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