Mediator and p300/CBP-steroid receptor coactivator complexes have distinct roles, but function synergistically, during estrogen receptor alpha-dependent transcription with chromatin templates

Abstract

Ligand-dependent transcriptional activation by nuclear receptors involves the recruitment of various coactivators to the promoters of hormone-regulated genes assembled into chromatin. Nuclear receptor coactivators include histone acetyltransferase complexes, such as p300/CBP-steroid receptor coactivator (SRC), as well as the multisubunit mediator complexes ("Mediator"), which may help recruit RNA polymerase II to the promoter. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the functional role for Mediator in the transcriptional activity of estrogen receptor alpha (ERalpha) with chromatin templates, as well as functional interplay between Mediator and p300/CBP during ERalpha-dependent transcription. Using three different approaches to functionally inactivate Mediator (immunoneutralization, immunodepletion, and inhibitory polypeptides), we find that Mediator is required for maximal transcriptional activation by ligand-activated ERalpha. In addition, we demonstrate synergism between Mediator and p300/CBP-SRC during ERalpha-dependent transcription with chromatin templates, but not with naked DNA. This synergism is important for promoting the formation of a stable transcription preinitiation complex leading to the initiation of transcription. Interestingly, we find that Mediator has an additional distinct role during ERalpha-dependent transcription not shared by p300/CBP-SRC: namely, to promote preinitiation complex formation for subsequent rounds of transcription reinitiation. These results suggest that one functional consequence of Mediator-ERalpha interactions is the stimulation of multiple cycles of transcription reinitiation. Collectively, our results indicate an important role for Mediator, as well as its functional interplay with p300/CBP-SRC, in the enhancement of ERalpha-dependent transcription with chromatin templates.

FIG. 1.

SDS-PAGE analysis of the purified recombinant proteins used in this study. Recombinant Flag-tagged, His-tagged, and GST-fused proteins were expressed in insect cells or in E. coli and were purified by using anti-Flag M2, nickel-NTA, and glutathione-agarose affinity chromatography, respectively, as described in Materials and Methods. Aliquots of the purified proteins were run on acrylamide-SDS gels with subsequent staining with Coomassie brilliant blue R-250. The sizes of molecular mass markers are shown.

FIG. 2.

Immunoneutralization of Mediator in HNEs inhibits ERα-dependent transcription. (A) Ligand- and p300-dependent transcription by ERα with chromatin templates in vitro. The plasmid template 2ERE-pS2 containing two EREs upstream of the human pS2 promoter (bottom, left) or the plasmid template pERE containing four EREs upstream of the adenovirus E4 promoter (bottom, right) was assembled into chromatin in the presence of recombinant ERα, 17β-estradiol (E₂), and p300, as indicated. The chromatin templates were transcribed with an HNE, and the resulting RNA products were analyzed by primer extension. Relative levels of transcription are indicated. (B) Effect of Mediator immunoneutralization on ERα-dependent transcription with chromatin templates. HNEs were incubated with anti-Med220 or anti-Med33 antibodies (40 ng/μl), a combination of both antibodies (20 ng/μl each), or a control antibody (40 ng/μl), as indicated. After the formation of immune complexes, the variously treated HNEs were tested for their ability to support ERα-dependent transcription with chromatin templates as described in panel A.

FIG. 3.

Immunodepletion of Mediator from HNEs. HNEs were immunodepleted with a mixture of antibodies to Med220 and Med33 (depleted) or mock depleted with control antibody (control). Aliquots of the original extract (input), Mediator-depleted extract, or control-depleted extract were analyzed by immunoblotting with an array of antibodies to Mediator subunits (Med220, Med150, Med130, Med78, Med34, Med33, and Cdk8) (A, left panel), components of the RNA pol II transcriptional machinery (the large subunit of RNA pol II, TBP, TFIIB, TFIIE p34, and TFIIF p74) (B), and coactivators and chromatin remodelers (p300, CBP, PCAF, SRC1, SRC2, SRC3, Brg1, and Brm) (C). The immunoprecipitated or coimmunoprecipitated material (IP or Co-IP, respectively) was also analyzed by immunoblotting for the presence of Mediator subunits (A, right), as well as RNA pol II, p300, CBP, and TBP (D). Note that the Mediator subunits are designated according to the unified nomenclature proposed by Rachez and Freedman (41).

FIG. 4.

Immunodepletion of Mediator from HNEs causes a modest reduction in basal transcription. Basal transcription with naked DNA and chromatin templates. The plasmid template pERE, either as naked DNA (left) or assembled into chromatin (right), was transcribed with control or Mediator-depleted HNEs, as indicated. The resulting RNA products were analyzed by primer extension. Note that the chromatin experiment represents a fourfold-longer exposure than the other chromatin transcriptions shown herein, so that the low levels of basal transcription with chromatin can be observed more readily.

FIG. 5.

ERα transcriptional activity with chromatin templates is impaired in Mediator-depleted nuclear extracts. (A) Analysis of ERα-dependent transcription with chromatin templates by using control and Mediator-depleted HNEs. The 2ERE-pS2 (pS2 promoter) and pERE (E4 promoter) plasmid templates were assembled into chromatin in the presence of purified recombinant ERα, E₂, and p300 as indicated. The chromatin samples were subjected to in vitro transcription analysis, and the resulting RNA products were analyzed by primer extension. The relative transcription for each promoter is indicated. (B) Summary of multiple experiments like those in panel A showing the effects of Mediator depletion on activated transcription by ERα with the human pS2 and adenovirus E4 promoters. The data are plotted as fold activation over basal to account for the modest effects that Mediator depletion has on basal transcription (Fig. 4). Each bar represents the mean + the standard error from three or more separate determinations.

FIG. 6.

NF-κB p65 transcriptional activity with chromatin templates is impaired in Mediator-depleted nuclear extracts. Analysis of NF-κB p65-dependent transcription was performed with chromatin templates by using control and Mediator-depleted HNEs. The MCP-1 promoter/enhancer construct pGLM-ENH was assembled into chromatin in the presence of purified recombinant NF-κB p65 as indicated. The chromatin samples were subjected to in vitro transcription analysis, and the resulting RNA products were analyzed by primer extension. The relative transcription for each condition is indicated.

FIG. 7.

Activity of a p300/CBP-dependent transcriptional activator is reduced in Mediator-depleted nuclear extracts. (A) Schematic representation of the mechanism of transcriptional activation by Gal4-SRC2(PID). Gal4-SRC2(PID) directly recruits endogenous p300/CBP in HNE to promoters assembled into chromatin via the PID of SRC2. (B) Analysis of transcriptional activation by Gal4-SRC2(PID) with control and Mediator-depleted HNEs. The 2Gal-pS2 (top panel) and 2Gal-E4 (bottom panel) plasmid templates containing two Gal4 UAS sites upstream of the pS2 and E4 promoters, respectively, were assembled into chromatin in the presence or absence of Gal4-SRC2(PID), as indicated. The chromatin samples were subjected to in vitro transcription analysis, and the resulting RNA products were analyzed by primer extension. The relative transcription for each promoter is indicated. (C) Summary of multiple experiments like those in panel B showing the effects of Mediator depletion on activated transcription by Gal4-SRC2(PID) with the human pS2 and adenovirus E4 promoters. The data are plotted as fold activation over basal to account for the modest effects that Mediator depletion has on basal transcription (Fig. 4). Each bar represents the mean + the standard error from three or more separate determinations.

FIG. 8.

Mediator and p300/CBP synergistically activate ERα-dependent transcription with chromatin templates. (A) Effect of p300/CBP inhibition on Mediator-stimulated ERα transcriptional activity with chromatin templates. ERα transcriptional activity with the pERE reporter template assembled into chromatin was assayed with control or Mediator-depleted HNEs in the presence or absence of the p300/CBP inhibitor GST-SRC2(PID) as indicated. The resulting RNA products were analyzed by primer extension. The relative transcription for each condition is indicated. GST-SRC2(PID), which blocks interactions between the endogenous p300/CBP and SRC proteins in the HNEs, was added at a 40-fold excess relative to the concentration of ERα. (B) Summary of selected conditions from experiments like those shown in panel A. The data are plotted as fold activation over basal and show the requirements for both Mediator and p300/CBP during ERα-activated transcription with chromatin templates. Each bar represents the mean + standard error from three or more separate determinations.

FIG. 9.

Inhibition of ERα-dependent transcription by GST-SRC2(PID) does not occur with naked DNA templates and is relieved by the addition of purified recombinant p300. (A) Effect of p300/CBP inhibition on Mediator-stimulated ERα transcriptional activity with naked DNA templates. ERα transcriptional activity with the pERE reporter template as naked DNA was assayed with HNE in the presence or absence of the p300/CBP inhibitor GST-SRC2(PID) as indicated. The resulting RNA products were analyzed by primer extension. The relative transcription for each condition is indicated. GST-SRC2(PID), which blocks interactions between the endogenous p300/CBP and SRC proteins in the HNEs, was added at a 40-fold excess relative to the concentration of ERα (compare to Fig. 8A). (B) Relief of GST-SRC2(PID)-mediated inhibition of ERα transcriptional activity with chromatin templates by exogenously added p300. ERα transcriptional activity with the pERE reporter template assembled into chromatin was assayed with HNE in the presence or absence of GST-SRC2(PID) and purified recombinant p300, as indicated. The resulting RNA products were analyzed by primer extension. The relative transcription for each condition is indicated.

FIG. 10.

Both Mediator and p300/CBP-SRC are required for the efficient stimulation of preinitiation complex formation by liganded ERα. (A) Effect of p300/CBP inhibition at various points in the process of ERα-dependent transcription in the presence of Mediator. (Top) The pERE (E4 promoter) template was assembled into chromatin in the presence of purified recombinant ERα and 17β-estradiol (E₂) as indicated. The p300/CBP inhibitor GST-SRC2(PID) was added at the time points shown in the schematic diagram of the experimental setup (bottom). The samples were then subjected to in vitro transcription analysis, with the addition of the detergent Sarkosyl (0.2% [wt/vol]) after the initiation of transcription (i.e., 10 s after the addition of rNTPs). Under these conditions, Sarkosyl inhibits transcription reinitiation, but not elongation, and thus a single round of transcription is obtained. The resulting RNA products were analyzed by primer extension. Note that transcription initiates primarily from the most 3′ start site of the E4 promoter in experiments in which Sarkosyl is added (26). (Bottom) Experimental setup. GST-SRC2(PID) was added as follows: [1] to the chromatin, before the addition of the HNE; [2] simultaneously with the HNE, [3] after preinitiation complex formation, just prior to the addition of rNTPs; or [4] after transcription initiation. RT, room temperature. (B) Analysis of ERα-dependent transcription with chromatin in a single round by using control and Mediator-depleted HNEs. The 2ERE-pS2 (pS2 promoter) and pERE (E4 promoter) templates were assembled into chromatin in the presence of purified recombinant ERα and E₂ as indicated. The chromatin samples were then subjected to in vitro transcription analysis, with the addition of Sarkosyl after the initiation of transcription as described in panel A. The resulting RNA products were analyzed by primer extension.

FIG. 11.

Mediator and p300/CBP-SRC have distinct roles in the ERα-dependent transcription process. The effect of blocking Mediator-ERα and p300/CBP-SRC-ERα interactions at different times during the transcription cycle is shown. The pERE (E4 promoter) template was assembled into chromatin in the presence of purified recombinant ERα and E₂ as indicated. The GST-Med220(RID) or GST-SRC2(RID) inhibitors were added at a 50-fold molar excess relative to ERα at the time points shown in the schematic diagram of the experimental setup (bottom) as follows: [1] to the chromatin, before the addition of the HNE and [2] after preinitiation complex formation, just prior to the addition of rNTPs. The samples were then subjected to in vitro transcription analysis with or without the addition of Sarkosyl (0.2% [wt/vol]) after the initiation of transcription (i.e., 10 s after the addition of rNTPs) as described in the legend to Fig. 10. The resulting RNA products were analyzed by primer extension. Each bar represents the mean + standard error from three or more separate determinations.