Model for the transcriptional activation of RAR/RXR by MED25. For details, see the Discussion section.

Biological role of MED25 in RA signaling. MCF-7 cells stably transfected with empty, MED25 sense (S), or MED25 antisense (AS) expression vector were selected by G-418 treatment. (A) Cellular levels of MED25 were monitored by WB, using polyclonal anti-MED25 antibody. (B) RARβ2 expression was monitored by RT–PCR as described. (C, D) MTT assays (Cho et al, 2006) were performed to monitor cell growth in the absence (C) and presence of 5 μM AtRA (D).

The MED25 domain responsible for transcriptional activation of RAR. (A) Mapping of the domain required for autonomous transcriptional activity. Gal4-DBD full-length mouse MED25 and -deletion mutant expression vectors (0.25 μg each) were transfected into HeLa cells together with a Gal4-responsive 17-mer tk-luciferase reporter. A schematic representation of the MED25 fragments. Relative luciferase activity was determined by luciferase assay after normalizing to the observed β-galactosidase activity. (B–D) Dominant-negative effect of the MED25 mutants. HeLa cells were transfected with expression vectors for RARα and MED25 (0.4 μg) with RARE-tk-luciferase in the presence of AtRA. The cells were further transfected with increasing amounts (0, 0.1, 0.2, 0.3, and 0.4 μg) of each MED25 deletion mutant. The residues included in each fragment are indicated: (1–290), containing the VWA domain; (546–745), the NR box; (1–545), the VWA and AD, as defined above. Luciferase activity was determined as described above. Each graph includes the data obtained from three independent experiments.

Role of Mediator binding in MED25 function. (A) HeLa cells were transfected with the expression vectors for full-length MED25 (FL) or a MED25 fragment (residues 395–745 or 1–394) with RARE-tk-luciferase in the absence or presence of ligand (1 μM). The fold activation is a comparison between the luciferase activity in the presence and absence of ligand. (B) Flag-MED25 FL or a MED25 fragment (residues 395–745 or 1–394) was expressed in HeLa cells. The interaction was determined by IP using anti-Flag antibody and WB using anti-CBP, anti-TRAP220, and anti-MED6 antibodies. (C) After the transfections described above, crosslinked chromatin was prepared and immunoprecipitated with the antibodies indicated on the right. The precipitates were subjected to PCR analysis using primer pairs spanning the human RARβ2 promoter. (D) HeLa cells were treated with MED25 siRNA (−, untreated; C, control siRNA) and the extracts were subjected to WB with the indicated antibodies. The interaction was determined by IP followed by WB using the indicated antibodies.

MED25 interaction with RAR in vitro and in mammalian cells. (A) In vitro translated [³⁵S]-labeled NR (hRAR and mRXR) was incubated with 2 μg of GST-fused MED25 (residues 564–747) in the presence of the cognate ligand (2 μM). Bound proteins were visualized by SDS–PAGE and autoradiography. (B) NIH3T3 cells were cotransfected with 2 μg of GFP-NR (hRAR and mRXR) and 2 μg of the Flag-mMED25 (mouse, full length) expression vectors. IP was carried out using anti-Flag antibody. The precipitated proteins were visualized by WB using anti-GFP antibody. (C) HeLa cells were cotransfected with 2 μg each of the GFP-MED25 and Flag-RXRα expression vectors in the presence of the RXR-specific agonist methoprene acid. IP was carried out using anti-Flag antibody. The precipitated proteins were visualized by WB using anti-GFP antibody. (D) Interaction between endogenous proteins. HeLa cells were cultured in the absence and presence of AtRA. Cell extracts were subjected to IP with preimmune serum or anti-RAR antibody. Interactions were detected by WB using polyclonal anti-MED25 antibody raised against residues 48–53 of human MED25 in rabbit. The inputs were 10% of the IP samples, and are shown by WB using anti-RAR and -MED25 antibodies. (E) Deletion of the NR box abrogates the interaction. Flag-mMED25 or Flag-mMED25 ΔNR (NR box deletion) was expressed in HEK293 cells in the presence of AtRA. The interaction was determined by IP using anti-RARα antibody followed by WB using anti-Flag antibody. The inputs were 10% of the IP samples.

Cooperation of MED25 with CBP. (A) The interaction between MED25 and CBP in yeast. Yeast two-hybrid assays were performed using LexA DBD fused to CBP deletions and Gal4 AD fused to MED25. Interactions were monitored by β-galactosidase assay. (B) In vitro interactions between MED25 and CBP. [³⁵S]-labeled MED25 (residues 395–545) or MED25 (residues 546–745) was mixed with GST, GST-CBP (residues 1–460), or GST-CBP (residues 602–1096). The GST pull-down results were visualized by autoradiography. (C) Interaction between MED25 and CBP in mammalian cells. HA-tagged CBP and Flag-tagged MED25 expression vectors were transfected alone or together into NIH3T3 cells. IP of the cellular extracts with anti-CBP antiserum was followed by WB with anti-Flag antibody. (D) Mapping of the MED25 domain needed to bind CBP. NIH3T3 cells were transfected with HA-tagged CBP and Flag-tagged MED25 (or its deletions). WB using anti-Flag antibody was performed using precipitates obtained from cellular extracts and anti-HA antibody. The locations of the IgG heavy and light chains are indicated. (E) Effect of CBP on the intrinsic transcriptional activity of MED25. NIH3T3 cells were transfected with Gal4 DBD-responsive luciferase reporter, 0.1 μg of Gal4 DBD-fused MED25 (or its deletions), and increasing amounts (0, 0.01, 0.05, 0.1, and 0.2 μg) of CBP expression vector. (F) Effect of CBP on MED25-mediated transcriptional enhancement of RAR. NIH3T3 cells were transfected with RARE-tk-luciferase reporter, RARα, and increasing amounts (0, 0.1, 0.2, and 0.4 μg) of CBP expression vector in the absence or presence of MED25. Extracts were subjected to luciferase assays as described (E, F).

Chromatin association of MED25 with RAR, CBP, and TRAP220. (A) Binding of MED25 to endogenous RAR, CBP, and TRAP220. HEK293 cells were transfected with Flag-MED25 expression vector in the absence or presence of 2 μM AtRA. RAR and the indicated cofactors were immunoprecipitated from whole extracts of transfected cells using the indicated antibodies. MED25 was detected in precipitates by WB with anti-Flag antibody. (B) AtRA-dependent recruitment of MED25 and other RAR cofactors. HEK293 cells were transfected with Flag-MED25 or MED25 ΔNR expression vector or MED25 siRNA and then treated with or without 2 μM AtRA for 2 h. Crosslinked chromatin was prepared and immunoprecipitated with the antibodies indicated on the right. The precipitates were subjected to PCR analysis using primer pairs spanning the human RARβ2 promoter. The control represents the PCR product obtained before IP. (C) Sequential recruitment of MED25 and other RAR cofactors. ChIP assays were performed as in Figure 7B, except that HeLa cells were treated with AtRA for varying lengths of time (shown above the lanes) and another RA-responsive promoter was included (p21WAF1). The primers used for the p21WAF1 promoter were obtained from Zeng et al (2002). The antibodies used are indicated on the right. (D) Real-time quantitative PCR was performed using the double-stranded DNA binding dye SYBR Green in an iCycler (Bio-Rad). All of the data (the average of three independent amplifications) represent the recovery of each DNA fragment relative to the total input DNA.

Characterization of the interaction between MED25 and RAR in yeast. (A) Structural features of MED25 (human and mouse homologs) and PTOV1 (human). Notable domains include a von Willebrand factor type A (VWA) domain, a synapsin I domain 1 (SD1), a PTOV (a conserved region found in prostate tumor overexpressed protein 1) domain, a VP16 activator-interacting domain (ACID), an SD2 domain, and an NR box (LRSLL). (B) Isolation of MED25, which interacts with RAR DEF in the presence of retinoid, by yeast two-hybrid screening. Interactions between other known RAR cofactors (SRC-1 and TRAP220) and RAR are shown for comparison. (C) Interaction of MED25 with other nuclear receptors. The assays were performed using the indicated NRs in the presence of their cognate ligands. (D) The AF-2 AD core of RAR/RXR is critical for its ligand-dependent interaction with MED25. The location of the AF-2 AD core is shown (Edn, E region with deleted AF-2 AD, residues 212–403 of hRARα; D3Edn, D3E region with deleted AF-2 AD, residues 237–448 of mRXRα), and the point mutations are indicated. (E) Mapping of the MED25 domain responsible for the interaction with RAR. Additional deletions were constructed using the original human MED25 (residues 564–747) clone isolated by yeast-two hybrid screening. For full-length MED25, the mouse homolog was used. Schematic representations of full-length MED25 and its deletion mutants. (F) The NR box (LRSLL) of MED25 is critical for the interaction. Four NR box mutants (L → A) were designed on the basis of an alignment of MED25 from various mammalian species, as indicated. In all experiments, yeast two-hybrid and β-galactosidase assays were used to determine the interaction between LexA-fused nuclear receptors, including RAR, and Gal4 AD-fused RAR cofactors, including MED25 (and its mutants). Fold activity indicates the value relative to that of the Gal4 AD empty control.

MED25 is a coactivator of RAR as determined using overexpression (A–D) and reduced expression (E–H) conditions. (A, B) Effect of MED25 on the transcriptional activity of RAR and RXR. NIH3T3 cells were transfected with expression vectors for RAR (or RXR) and MED25 (or ΔNR), with RARE- or RXRE-tk-luciferase, in the absence or presence of 1 μM ligand (AtRA for RAR; 9-cis RA for RXR). Relative luciferase activity was determined by luciferase assay after normalizing to the observed β-galactosidase activity. The fold activation is a comparison between the luciferase activity in the presence and absence of ligand. The data shown are averages from three independent experiments. (C, D) Effect of MED25 on endogenous RARβ2 expression. (C) HeLa cells were transiently transfected with a MED25 sense or antisense expression vector and treated with MED25 siRNA. (D) MCF-7 cells stably expressing MED25 were selected by G-418 treatment. Total RNA was extracted from each cell and subjected to RT–PCR using primer pairs specific for the RARβ2 coding sequence. GAPDH was used as an internal control. (E–H) Downregulation of MED25 resulted in a significant and specific reduction in RAR activity. (E) Increasing amounts (0, 80, 160, and 240 pmol) of MED25 or TRAP220 siRNA were added to HeLa cells. The extent of knockdown was monitored by WB. β-Actin was used as an internal control. (F) HeLa cells were transfected with control, MED25, or TRAP220 siRNA (0, 40, 80, and 120 pmol) and the RARE-tk-luciferase reporter in the presence of 1 μM of AtRA. The level of endogenous RARα was determined by WB (inset). Luciferase activity was determined as described above. (G) The DR4-tk-luciferase reporter was used instead, and the level of TR is shown (inset). Each graph shows the average data obtained from three independent experiments. (H) The knockdown effect was analyzed by RT–PCR using primer pairs specific for the RARβ2 coding sequence.