10.1128/MCB.22.8.2842-2852.2002.

2002, 22(8):2842. DOI:Mol. Cell. Biol. Hwa Jin Baek, Sohail Malik, Jun Qin and Robert G. Roeder 

sIIConjunction with TFIID-Associated TAFActivator-Dependent Transcription inActivator-Independent and Requirement of TRAP/Mediator for Both

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MOLECULAR AND CELLULAR BIOLOGY, Apr. 2002, p. 2842–2852 Vol. 22, No. 80270-7306/02/$04.00�0 DOI: 10.1128/MCB.22.8.2842–2852.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Requirement of TRAP/Mediator for Both Activator-Independent andActivator-Dependent Transcription in Conjunction with

TFIID-Associated TAFIIsHwa Jin Baek,1 Sohail Malik,1 Jun Qin,2 and Robert G. Roeder1*

Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021,1 and Department ofBiochemistry and Department of Cell Biology, Baylor College of Medicine, Houston, Texas 770302

Received 15 October 2001/Returned for modification 26 November 2001/Accepted 8 January 2002

The multiprotein human TRAP/Mediator complex, which is phylogenetically related to the yeast SRB/Mediator coactivator, facilitates activation through a wide variety of transcriptional activators. However, itremains unclear how TRAP/Mediator functions in the context of other coactivators. Here we have identified apreviously uncharacterized integral subunit (TRAP25) of the complex that is apparently metazoan specific. Anantibody that is specific for TRAP25 allowed quantitative immunodepletion of essentially all TRAP/Mediatorcomponents from HeLa nuclear extract, without detectably affecting levels of RNA polymerase II and corre-sponding general transcription factors. Surprisingly, the TRAP/Mediator-depleted nuclear extract displayedseverely reduced levels of both basal and activator-dependent transcription from DNA templates. Both activ-ities were efficiently restored upon readdition of purified TRAP/Mediator. Moreover, restoration of basal andactivator-dependent transcription to extracts that were simultaneously depleted of TRAP/Mediator and TFIID(TBP plus the major TAFIIs) required addition of both TBP and associated TAFIIs, as well as TRAP/Mediator.These observations indicate that TAFIIs and Mediator are jointly required for both basal and activatedtranscription in the context of a more physiological complement of nuclear proteins. We propose a closemechanistic linkage between these components that most likely operates at the level of combined effects on thegeneral transcription machinery and, in addition, a direct role for Mediator in relaying activation signals tothis machinery.

Transcriptional regulation of eukaryotic protein-encodinggenes requires the concerted function of distinct classes offactors (reviewed in reference 40). These include (i) RNApolymerase II (Pol II) and its associated general transcriptionfactors (GTFs) (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, andTFIIH), which are sufficient for low levels of accurately initi-ated transcription from DNA templates in vitro (generallyreferred to as basal transcription), (ii) site-specific DNA-bind-ing regulatory factors, and (iii) transcriptional cofactors, whichmediate responsiveness of Pol II and GTFs to various activa-tors and repressors. Cofactors (coactivators or corepressors)have been operationally defined as factors that, while exhibit-ing no intrinsic site-specific DNA binding, preferentially influ-ence the function of DNA binding regulatory factors withoutaffecting basal (regulatory factor-independent) transcription.

Early biochemical studies employing metazoan systems re-constituted with partially purified factors pointed to TATAbox-binding protein (TBP)-associated factors (TAFIIs) inTFIID (7, 16, 37) and positive cofactors (PC1, PC2, PC3, andPC4) within the USA fraction (30) as coactivators required forthe function of diverse activators on DNA templates (reviewedin references 4, 40, and 45). Parallel studies with Saccharomy-ces cerevisiae revealed the existence of a coactivator or Medi-ator (8) that interacts with Pol II to form a Pol II holoenzyme

(19, 44) and that consists of genetically identified SRB andtranscriptional regulatory proteins, as well as novel Mediatorsubunits (reviewed in references 23 and 32). Subsequently, andunderscoring the evolutionary conservation of transcriptionalmechanisms, several metazoan Mediator-like multiproteincomplexes were identified (reviewed in reference 28). Theseinclude TRAP (9), SMCC (13), ARC (33), DRIP (38), NAT(43), murine Mediator (18), human Mediator (2), CRSP (41)and the USA-derived and TRAP-related complex PC2 (27).Except for minor differences in composition, the metazoancomplexes are rather similar and likely reflect either the samecellular entity (Mediator) or derived or modified forms (28).

The TRAP and SMCC complexes, for which equivalence hasbeen rigorously demonstrated (13, 17), consist of circa 25polypeptides that include the phylogenetically conserved sub-units RGR1, SRB10, MED7, MED6, SRB11, SRB7, SOH1,and NUT2, as well as subunits that are only found in themetazoan complex. The latter include, but are not limited to,the various TRAP polypeptides originally reported as part ofthe thyroid hormone receptor-associated TRAP complex (28).The identities of several additional subunits that appear to bespecifically associated with TRAP/Mediator, especially thoseclustered in the 20- to 25-kDa range, remain unknown.

The isolation of distinct coactivators has raised the issue ofwhether they act in a functionally redundant or in a synergisticmanner, or indeed whether they reflect alternative activationpathways. In the case of USA-derived positive cofactors, pre-vious studies demonstrated functional synergism between un-fractionated USA and TAFIIs (6), between PC4 and TAFIIs

* Corresponding author. Mailing address: Laboratory of Biochem-istry and Molecular Biology, The Rockefeller University, New York,New York 10021. Phone: (212) 327-7600. Fax: (212) 327-7949. E-mail:roeder@rockvax.rockefeller.edu.

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(11), and between the Mediator-like PC2 and PC4 and PC3(27). However, the relative contributions of Mediator compo-nents and TFIID-associated TAFIIs to activated transcriptionhave remained unclear. Consistent with earlier biochemicalstudies in yeast systems, where efficient Mediator-dependentactivation was observed in transcription assays that containedTBP in place of intact TFIID (19), genetic analysis revealedthat at least a subset of TFIID-specific TAFIIs were dispens-able, when individually depleted, for general activator functionin yeast cells (reviewed in reference 12). Furthermore, in sev-eral cases where a strong TAFII dependence was seen in thesegenetic studies, the TAFII requirement was found to be dic-tated by core promoter sequences (42). Consistent with theseobservations, TBP was found to support efficient function ofthe model activator Gal4-VP16 both in an immunodepletedHeLa nuclear extract lacking TFIID-specific TAFIIs (34) andin a purified cell-free system from HeLa cells (46), as well asthe function of the thyroid hormone receptor (TR)-TRAPcomplex in a purified cell-free system (10). In contrast, and inagreement with the earlier studies indicating TAFII require-ments for optimal function of various activators in partiallypurified metazoan cell-free systems (reviewed in reference 45),a recent study showed a strict TAFII requirement for the func-tion of a different activator (Sp1) in an assay system reconsti-tuted with purified human Pol II, GTFs, and Mediator-likeCRSP (41). Moreover, another recent study showed thatyTAFII40, a specific component of yeast TFIID, is essential formost transcription in vivo, which strongly suggests that someTAFIIs may be required as general cofactors and not just ascore promoter recognition factors (20).

As part of our ongoing structural and functional character-ization of TRAP/Mediator, we report here a novel bona fidesubunit of this complex (designated TRAP25). We have ex-ploited the ability of antibodies directed against this subunit toefficiently deplete TRAP/Mediator from nuclear extract to ex-plore broader TRAP/Mediator functions and to revisit issuesof coactivator redundancy versus coactivator synergy in thecontext of a more physiological complement of nuclear pro-teins. Our results indicate that in this system, which may alsocontain a number of negative cofactors (reviewed in reference25), TRAP/Mediator and TFIID-associated TAFIIs act syner-gistically to support the function of various activators via di-verse core promoter sequences. We further find, consistentwith a report that appeared while this article was in prepara-tion (31), that TRAP/Mediator is also needed for residuallevels of activator-independent (basal) transcription in this sys-tem.

MATERIALS AND METHODS

Cloning of TRAP25 cDNA and generation of specific antibody. Human ex-pressed sequence tag (EST) clone AI806623 was obtained from Research Ge-netics and completely sequenced. This EST clone contained the complete openreading frame for TRAP25. The full-length TRAP25 cDNA was inserted into6His-pET11d vector by PCR. Recombinant TRAP25 expressed in Escherichiacoli was insoluble. The protein was solubilized in denaturing buffer (6 M guani-dine HCl, 20 mM Tris [pH 7.9], and 500 mM NaCl) and purified by nickel-nitrilotriacetic acid-agarose column chromatography. The purified protein wasseparated by sodium dodecyl sulfate–15% polyacrylamide gel electrophoresis(SDS–15% PAGE) and visualized by using an ice-chilled 0.25 M KCl solution.The protein band was cut out for antibody production (Covance Research Prod-ucts Inc.).

TRAP/Mediator purification. Affinity purification of TRAP/Mediator fromnuclear extract was performed essentially as previously described (13). Briefly,5-ml aliquots of nuclear extracts prepared from cell lines expressing FLAG-tagged CDK8 (13), FLAG-tagged NUT2 (27), or FLAG-tagged TR (9) wereadjusted to 300 mM KCl and 0.5% NP-40 and incubated with 100 �l of M2-agarose beads (IBI/Kodak) at 4°C for 6 h with rotation. After four 5-ml washeswith BC300–0.1% NP-40, proteins were eluted from beads by incubation at 4°Cfor 30 min with 100 �l of BC100–0.05% NP-40 plus 0.2 mg of FLAG peptide/ml.

Highly purified TRAP/Mediator was obtained from nuclear extract preparedfrom cell lines expressing FLAG-tagged NUT2 (27). Briefly, after precipitationwith 33% ammonium sulfate, the resuspended protein was loaded on a phos-phocellulose P11 column at BC400. Protein fractions eluting between 0.5 and0.85 M KCl were collected and dialyzed to BC300. Finally, affinity purification ofTRAP/Mediator on M2 agarose was performed as described above.

In vitro transcription. Transcription reactions were carried out in a finalvolume of 25 �l and contained 50 ng of each supercoiled plasmid DNA template,2 or 5 �l of HeLa nuclear extract (at �10 mg/ml), 20 mM HEPES (pH 8.2), 11to 16% glycerol, 4 mM MgCl2, 60 mM KCl, 8 mM dithiothreitol, 0.5 mM ATPand CTP, 5 �M UTP, 0.1 mM 3�-O-methyl-GTP, 16 �Ci (0.6 MBq) of[�-32P]UTP, 0.4 mg of bovine serum albumin per ml, and 20 U of RNasin(Promega). Reaction mixtures were incubated at 30°C for 1 h, at which time theUTP concentration was increased to 25 �M and 15 U of RNase T1 was added.After 30 min of incubation at 30°C, the reactions were extracted with phenol-chloroform in the presence of 150 �l of stop solution (0.4 mM sodium acetate[pH 5.2], 13 mM EDTA, 0.33% SDS, and 0.67 mg of yeast tRNA per ml). Theaqueous layer was precipitated by ethanol and analyzed by gel electrophoresisfollowed by autoradiography. Correctly initiated transcripts were quantitated byPhosphorImager analysis with a STORM 840 system (Molecular Dynamics).

Immunodepletion from nuclear extract. Since recombinant His-taggedTRAP25 was totally insoluble in E. coli, the His-tagged TRAP25 was purifiedwith a nickel-nitrilotriacetic acid-agarose column under denaturing conditions (6M guanidine HCl, 0.05 M NaCl, 20 mM HEPES [pH 7.6], 100 mM EDTA). Thepurified His-tagged TRAP25 was cross-linked under the above conditions withCNBr-activated Sepharose 4B (Pharmacia) that had been pretreated with 1 mMHCl. After mixing for 2 h at room temperature, the cross-linking reaction wasblocked by adding Tris buffer (pH 8.0) to a final concentration of 0.1 M. Theresin was washed three times with 0.1 M sodium acetate (pH 4.0)–0.5 M NaCland three times with equilibration buffer (0.1 M Tris [pH 8.0], 0.5 M NaCl)before use.

For immunodepletion of TRAP/Mediator, anti-TRAP25 antisera were puri-fied by passage through a column containing His-tagged TRAP25 protein, whichwas covalently cross-linked to CNBr-activated Sepharose 4B (Pharmacia).Bound anti-TRAP25 antibodies were eluted with 200 mM glycine (pH 2.5). Theantibodies were further purified and concentrated by binding to protein A-Sepharose (Pharmacia) and then cross-linked to protein A with dimethylpimeli-date (Sigma). One milliliter of HeLa nuclear extract in BC100 was passed fourtimes over a 1-ml cross-linked anti-TRAP25–protein A–Sepharose column. Theflowthrough fractions were used for Western blotting and in vitro transcription.

TBP and TAFII depletions were carried out in BC500, and depleted extractswere dialyzed in BC100 essentially as described (34).

MS. Protein sequencing using mass spectrometry (MS) and tandem MS (MS/MS) was carried out as described previously (35), with the exception that 18O-labeled H2O was omitted. Briefly, the Coomassie blue-stained protein band wasin-gel digested with trypsin and the recovered peptides were analyzed using anelectrospray ion trap mass spectrometer (LCQ; Finnigan MAT, San Jose, Calif.)coupled on-line with a capillary high-pressure liquid chromatograph (LC) (C18

Magic MS column, Magic 2002; Michrom BioResources, Auburn, Calif.). Theflow was split with a Magic precolumn capillary splitter assembly (MichromBioResources). The LC/MS was programmed to run in a data-dependent fash-ion, in which the mass spectrometer switched to the MS/MS mode to acquirecollision-induced dissociation spectra, once an ion signal was detected to exceeda preset value in the MS mode during the entire LC run. Data derived from thecollision-induced dissociation spectrum were used to search a compiled proteindatabase that was composed of the nonredundant protein database and a six-reading-frame translated EST database to identify the protein.

RESULTS

Molecular cloning of a full-length cDNA encoding TRAP25.Our earlier analysis (27) of the polypeptide composition of theTRAP/Mediator complex revealed, in addition to the subunitswhose sequences were previously identified (13, 17), two or

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three polypeptides in the 20- to 25-kDa range that coelutedwith the complex regardless of whether it was purified from aFLAG-NUT2- or a FLAG-SRB10/CDK8-expressing cell line(13, 27). These bands were also consistently observed in PC2preparations that had been chromatographed over several col-umns (27), strongly suggesting that they represent authenticTRAP/Mediator subunits. To determine the identities of thesebands, we electrophoretically resolved a TRAP/Mediator prep-aration that was affinity purified from the FLAG-SRB10/CDK8-expressing cell line (13) (Fig. 1A). The bands (a to f)corresponding to these polypeptides were excised and sub-jected to mass-spectrometric sequence analysis.

Bands a and f appear, on the basis of derived sequences, tobe proteolytic degradation products of the previously reported

TRAP150�. Band d contained sequences from a polypeptidethat was reported both in PC2 (referred to as p37 in reference27) and in CRSP (referred to as CRSP34 in reference 41).Band b corresponded to immunoglobulin light chain derivedfrom the affinity resin. Additional sequences not previouslyfound in Mediator preparations were obtained from bands c, e,and f. The sequence YEYHWADGTNIK in band c identifiedESTs (AI393039 and AI557507) that encode the human or-tholog of yeast Mob1, which has been linked to the CCR4-NOT complex (21). However, several lines of evidence (datanot shown) suggest that Mob1 is not an authentic, stoichio-metric component of TRAP/Mediator. The sequence YLRP-PNTSLFVR in band e identified ESTs (AF047448 andAF067730) that encode TASR-2 (translocated in liposarcoma-

FIG. 1. Isolation and cDNA cloning of TRAP25. (A) Analysis of affinity-purified (M2-agarose) TRAP/Mediator from an f:CDK8 cell line.Samples were analyzed by SDS–11.5% PAGE and stained with silver. Bands (a to f) in the 25-kDa range were excised for mass-spectrometricsequence analysis. Peptide sequences thus obtained and corresponding proteins are indicated. (B) Amino acid sequence of TRAP25. Amino acidsequences that were identified by MS are underlined. An amino acid sequence comparison of human (TRAP25) and Drosophila TRAP25(dTRAP25) homologue is also shown. The alignment was performed with MacVector software, and identities (double dots) and similarities (singledots) are indicated. (C) Northern analysis of a multiple-tissue mRNA blot hybridized with a TRAP25 cDNA probe showing expression of TRAP25in human tissues.

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associated serine-arginine protein 2). TASR-2 may be involvedin RNA splicing via the Pol II C-terminal domain (15, 47).TASR-2 was not further characterized but, like Mob1, is un-likely to be an authentic TRAP/Mediator subunit. However, itremains possible that these proteins are representative of avariety of cellular components (other than the basic transcrip-tion apparatus) with which TRAP/Mediator normally inter-acts.

Band f yielded the additional sequence IGQETVQDIVYR,which identified an EST (AI806623) that encodes a hitherto-uncharacterized protein (termed TRAP25 based on evidencepresented below) with a predicted molecular weight of 20 kDa(Fig. 1B). A Drosophila TRAP25 ortholog (CG17183 geneproduct), which exhibited localized identity of 35% and simi-larity of 53% with human TRAP25 (Fig. 1B), was found in thedatabase. S. cerevisiae and Caenorhabditis elegans do not ap-pear to possess an equivalent protein, potentially implicatingthis subunit in signaling processes restricted to higher metazo-ans. A Northern blot analysis (Fig. 1C) indicated ubiquitousexpression of a 1-kb TRAP25 mRNA, although elevated levelswere seen in heart, placenta, and skeletal muscle. This patternis qualitatively similar to that previously reported for otherTRAP/Mediator subunits (17).

TRAP25 is a bona fide component of TRAP/Mediator. To-ward establishing that TRAP25 is a bona fide subunit ofTRAP/Mediator, we first expressed this protein in bacteria and

raised antisera against the purified recombinant protein. Im-munoblot analysis (Fig. 2A) with the TRAP25-specific anti-serum revealed the presence of TRAP25 in all preparations ofTRAP/Mediator regardless of how they were obtained, eitherthrough association with FLAG-TR or through intrinsicFLAG-tagged subunits (NUT2 or SRB10/CDK8). Further-more, the amount of TRAP25 relative to other authentic sub-units (TRAP80, MED6, and SRB7) was the same for thepurified complexes and for unfractionated nuclear extract,strongly suggesting that TRAP25 is a stoichiometric compo-nent of TRAP/Mediator. To further demonstrate that TRAP25 isstably associated with TRAP/Mediator complex, TRAP/Medi-ator purified from epitope-tagged SRB10/CDK8 cells was sub-jected to gel filtration chromatography on Superose 6 (Fig.2B). Consistent with previous reports (13), immunoblot anal-ysis of the resulting fractions with antibodies against represen-tative subunits revealed that the majority of the TRAP/Medi-ator eluted as a 1.8-MDa complex. Importantly, the bulk of thecomplex coeluted with TRAP25 (Fig. 2B, lanes 5 and 6). Thisresult provides additional strong evidence for the presence ofTRAP25 in the TRAP/Mediator complex.

In order to prove definitively that TRAP25 is an integralcomponent of TRAP/Mediator, we used our TRAP25 anti-serum to show that depletion of TRAP25 from nuclear extractsimultaneously resulted in depletion of other TRAP/Mediatorsubunits (Fig. 2C). As expected, immunoblot analysis of extract

FIG. 2. TRAP25 is a bona fide component of TRAP/Mediator. (A) Complexes from the indicated cell lines (f:TR, lane 2) were analyzed byimmunoblotting with anti-TRAP25 antibodies. Representative TRAP/Mediator subunits were also probed, as indicated. Total HeLa cell nuclearextract was also included (lane 1) as a control. (B) TRAP/Mediator purified from the f:CDK8 cell line was chromatographed (in 300 mM KCl)over a Superose 6 gel filtration column. Fractions were immunoblotted with the indicated antibodies. The elution range of molecular mass markersis indicated at the top. (C) An anti-TRAP25 antibody depletes TRAP/Mediator from HeLa nuclear extract. HeLa nuclear extract (input) waspassed over a control column containing immobilized preimmune antibodies (mock) or over a column containing anti-TRAP25. Unbound extractwas immunoblotted with the indicated antibodies.

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that had been passed through an anti-TRAP25 antibody col-umn revealed that TRAP25 was quantitatively removed by thistreatment (compare lanes 1 and 2 with lane 3). Furthermore,as predicted, the majority of TRAP/Mediator subunits werealso efficiently depleted. Exceptions included SRB11/cyclin C,which was only partially depleted; this likely reflects its pres-ence not only in TRAP/Mediator but also in a kinase-cyclinpair with SRB10/CDK8 that can exist in an unassociated form(13, 27). We therefore conclude that TRAP25 is a bona fideTRAP/Mediator subunit.

We also examined if the TRAP25 antibody depleted Pol IIand its associated GTFs. Immunoblotting analysis showed neg-ligible reductions in the amounts of Pol II, TFIIA, TFIIB,TFIID, TFIIH, TFIIE, and TFIIF in the supernatant (Fig. 2C),despite the fact that this depletion experiment was performedunder mild salt conditions (125 mM KCl). Therefore, a major-ity of TRAP/Mediator appears not to be tightly associated withPol II and other GTFs, consistent with previous results (13,43). However, as minor differences would not be evident fromthe present analysis, it remains possible that a subpopulation ofTRAP/Mediator complexes is associated with Pol II (S. Malikand R. G. Roeder, unpublished results).

Function of TRAP/Mediator in nuclear extract. Our previ-ous functional analyses of TRAP/Mediator (13, 17, 27) em-ployed an in vitro assay system that was reconstituted fromhighly purified Pol II, GTFs, and the USA-derived positivecofactor, PC4. The Mediator-depleted extract described abovenow provided an opportunity to assess the function of TRAP/Mediator in a less purified system that presumably contains amore normal complement of both positively and negativelyacting nuclear factors.

We thus tested the Mediator-depleted extract for its abilityto support activation by GAL4-p53 (Fig. 3A) and GAL4-VP16(Fig. 3B), which consist of the DNA binding domain of theyeast activator GAL4 fused, respectively, to the activation do-mains of the tumor suppressor p53 and the viral activatorVP16. Standard transcription assays contained two templates.The G5HML plasmid has five copies of a GAL4 binding siteupstream of a hybrid core promoter and permits us to assayGAL4-fused activation domains. The ML�53 plasmid containsonly the core promoter sequences (�53 to �10) from theadenovirus major late promoter and allows us to monitor basal(activator-independent) transcription. The G5HML is thus aweaker core promoter and yields lower levels (circa fourfold)of basal activity relative to the ML�53 promoter (Fig. 3, lanes1) (46). Under our conditions, GAL4-p53 (Fig. 3A, lanes 1 and2) activated transcription 72-fold and GAL-VP16 (Fig. 3B,lanes 1 and 2) activated transcription 13-fold in untreatedextracts. By contrast, in the Mediator-depleted extract(�MED) (Fig. 3) the absolute levels of activated transcriptionwere dramatically reduced for both GAL4-p53 (ca. 7.5-fold)(Fig. 3A, lane 2 versus lane 4) and GAL4-VP16 (ca. 15-fold)(Fig. 3B, lane 2 versus lane 4). In a control extract, which hadbeen passed over a column containing preimmune antiserum,the levels of activated transcription were essentially unaltered(Fig. 3, lanes 2 versus lanes 6). We conclude that depletion ofMediator from nuclear extract severely compromises its abilityto support high levels of activated transcription.

We also noted that the level of basal transcription, as mon-itored from both the ML�53 template and the G5HML tem-

plate in the absence of added activators, was reduced 6- to18-fold following depletion of Mediator (compare lanes 1, 3,and 5 in Fig. 3A and, especially, 3B). Thus, despite the lowabsolute levels, the level of activation appears to remain un-changed in the presence and absence of Mediator. While thisresidual activation might possibly be attributed to an alterna-tive mechanism (involving a distinct coactivator activity), theunderlying reason is currently unclear. It may arise as well fromtrace amounts of TRAP/Mediator that are not detected byimmunoblot analysis but whose effects would be evident in themore sensitive transcription assay. The slight reduction in tran-scription from the ML�53 template upon activator addition(e.g., lane 1 versus lane 2) is normal and has previously been

FIG. 3. Depletion of TRAP/Mediator from nuclear extract reducesbasal and activated transcription. (A) Transcription activation by Gal4-p53 is suppressed in the anti-TRAP25 antibody-depleted nuclear ex-tract. In vitro transcription reaction mixtures contained 50 �g of eachnuclear extract (lanes 1 and 2, untreated; lanes 3 and 4, anti-TRAP25antibody-depleted [�MED]; lanes 5 and 6, mock depleted) and 50 ngeach of pML�53 (�53) and pG5HML (G5HML) templates. Relativetranscription levels, determined by phosphorimager analysis, are indi-cated. (B) In vitro transcription reactions were performed as for panelA except that Gal4-VP16 was used as an activator.

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attributed to competition between the two templates for lim-iting factors.

We next asked if purified TRAP/Mediator could restore theability of Mediator-depleted extract to support activation byGAL4-p53 and GAL4-VP16. For this purpose, TRAP/Media-tor that had been affinity-purified from nuclear extract fromour epitope-tagged NUT2 cell-line, and which additionally hadbeen fractionated over phosphocellulose P11 (see Materialsand Methods), was added back to transcription reactions per-formed with the depleted extract (Fig. 4A and B). In thisexperiment, TRAP/Mediator was added over a concentrationrange that, as determined by immunoblot analysis (data notshown), either was just under (Fig. 4A and B, lanes 3 and 4) oractually approximated (lane 5) the concentration of Mediatorin standard nuclear extracts.

Upon addition of increasing amounts of TRAP/Mediator toMediator-depleted extract, in which activation by GAL-p53(Fig. 4A, lane 2) and GAL-VP16 (Fig. 4B, lane 2) was virtuallyundetectable, activated transcription was enhanced in an es-sentially linear dose-dependent fashion (lanes 3 to 5). Indeed,for both GAL4-p53 and GAL4-VP16, full recovery of activitywas observed when the normal concentration of Mediator innuclear extracts was reached (compare lanes 5 and 1 in Fig. 4Aand B). These results confirm that diminution of coactivatoractivity following depletion of Mediator is due solely to itsabsence and not to depletion of associated factors.

We also observed that basal transcription from the ML�53template was fully restored upon complementation of the de-pleted extract with TRAP/Mediator. This is especially evidentin Fig. 4B (compare lane 5 and lane 1). Nonetheless, to ensurethat these Mediator effects on basal transcription are entirelyindependent of activators, we repeated the depletion-and-complementation experiment in the absence of any exogenousactivators (Fig. 4C). Once again, basal transcription activityfrom both G5HML and ML�53 templates was both severelyreduced in Mediator-depleted extract (Fig. 4C, lane 2) andfully restored by complementation with purified TRAP/Medi-ator (lane 5 versus lane 1). Together, these results demonstratethat, in nuclear extract, Mediator plays a dominant role inregulating both activator-dependent and activator-indepen-dent transcription.

Joint requirement of TRAP/Mediator and TAFIIs in TFIIDfor activated transcription. We also made use of our ability todeplete Mediator from nuclear extracts to address the out-standing question of whether the coactivator activities of Me-diator and TAFIIs within TFIID are functionally redundant orsynergistic. We therefore prepared HeLa nuclear extract lack-ing both TFIID and Mediator by sequential passages overcolumns containing immobilized antibodies against TBP andTAFII100 (to deplete TFIID) (34) and against TRAP25 (todeplete Mediator). As judged by immunoblot analysis of rep-resentative TFIID subunits (TBP, TAFII135, and TAFII100),and consistent with previous results (34), the extract (�MED/�IID) was efficiently depleted of TFIID (Fig. 5A, lane 2 versuslane 1). Similarly, the extract was simultaneously depleted ofMediator. Importantly, none of the other GTFs tested showedany significant decrease. Our strategy was to supplement theseextracts with Mediator and either TBP or intact TFIID, eithersingly or in combination. Thus, together with an extract(�MED) that was selectively depleted of only Mediator (see

FIG. 4. Restoration of activated and basal transcription by exog-enously added purified TRAP/Mediator. (A) Increasing amounts ofpurified TRAP/Mediator (lanes 3 to 5) were added to anti-TRAP25antibody-depleted nuclear extract (�MED) in in vitro transcriptionreactions. Reaction mixtures were as for Fig. 3 except that they con-tained 20 �g of each nuclear extract. As determined by immunoblot-ting, the amount of TRAP/Mediator added to the reactions corre-sponded to approximately 20% (lane 3), 70% (lane 4), or 100% of theTRAP/Mediator concentration in untreated nuclear extract. GAL4-p53 was used as the activator in all reactions. Lane 1 shows the productof a control reaction with untreated extract. (B and C) In vitro tran-scription reactions were performed as for panel A except that GAL4-VP16 was used as the activator (B) or no activator was added (C).

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above) (Fig. 5A, lane 3), this provided a system for assessingthe relative contribution of TFIID-associated TAFIIs andTRAP/Mediator in transcription. These assays were per-formed both with the above-described G5HML template (Fig.5B and D) and with a template (G5E1b) in which the GAL4sites are located upstream of the adenovirus E1b core pro-moter, which contains a relatively weak TATA box (Fig. 5Cand E).

As expected, �MED/�IID extract failed to support bothbasal and activated transcription by GAL-p53 and GAL-VP16(Fig. 5B to E, lanes 7 and 8 versus lanes 1 and 2). Furthermore,whereas the activated transcription in �MED extracts could bereadily recovered upon supplementation with purified TRAP/Mediator in this experiment (Fig. 5B to E, lanes 6 versus lanes2 and 4), consistent with earlier data (Fig. 4A and B), this wasnot sufficient to restore activity to the �MED/�IID extract

FIG. 5. Synergistic effect of TRAP/Mediator and TFIID-associated TAFIIs on transcription activation in vitro. (A) Immunoblot analysis ofTFIID- and TRAP/Mediator-depleted HeLa nuclear extract (Material and Methods) with the indicated antibodies. Lane 1, mock-depleted extract;lane 2, nuclear extract depleted with anti-TBP, anti-TAFII100, and anti-TRAP25 antibodies (�MED/�IID); lane 3, anti-TRAP25-depleted nuclearextract (�MED). Each lane was loaded with 20 �g of each nuclear extract and analyzed on a 4-to-20% gradient SDS-PAGE gel, followed byimmunoblotting. (B and C) In vitro transcription reaction mixtures contained the indicated nuclear extract plus 25 ng each of pML�53 and eitherpG5HML (B) or pG5E1b (C) in a 12.5-�l reaction volume. GAL4-p53 (50 ng), recombinant TBP (rTBP, 3 ng), affinity-purified TFIID containingan equivalent molar amount of TBP (as determined by immunoblotting), and an amount of TRAP/Mediator corresponding to that in standardnuclear extract were added where indicated. (D and E) In vitro transcription reactions were performed as for panels B and C, respectively, exceptthat Gal4-VP16 (50 ng) was the activator.

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(Fig. 5B to E, lanes 14 versus lanes 2 and 8). We also addedaffinity-purified TFIID to the �MED/�IID extract in amountsthat were empirically determined to support baseline levels (inthe absence of exogenous Mediator) of transcription equiva-lent to those seen with the �MED extract (Fig. 5B to E,compare lanes 12 with lanes 4). This resulted in the addition ofan amount of TFIID whose TBP content was only about a fifthof that in standard extract, which is to be expected in view ofthe fact that TBP is shared among several nuclear multiproteincomplexes. Under these conditions, addition of an equivalentmolar amount of TBP had negligible effects (Fig. 5B to E,compare lanes 9 and 10 with lanes 7 and 8). When the �MED/�IID extract was supplemented with both TFIID and TRAP/Mediator, the resulting transcription levels for both GAL-p53

(Fig. 5B and C) and GAL-VP16 (Fig. 5D and E) were virtuallyindistinguishable from those observed with control extract(lanes 18 versus lanes 2).

In sharp contrast, when the �MED/�IID extract was sup-plemented with TBP and TRAP/Mediator, the absolute tran-scription levels remained low (less than 5% of that with intactTFIID) (Fig. 5B to E, lanes 16 versus lanes 18). Similarly,although TBP could support low-level (circa three- to sixfoldactivation) activated transcription (Fig. 5B to E, lanes 15 and16 versus lanes 17 and 18) in the presence of TRAP/Mediator,the results clearly show that TAFIIs contribute an additionalpotential that allows a further 5-fold enhancement (8- to 28-fold activation in the presence of TFIID and TRAP/Mediator).At the same time, because basal levels are also elevated sig-

FIG. 5—Continued.

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nificantly as a result of the combined effects of TFIID andTRAP/Mediator (Fig. 5B to E, lanes 17 versus lanes 7) butmuch less so with TBP and TRAP/Mediator (lanes 15 versuslanes 7), the final extent of transcription is significantly higherin the former situation. We note, however, that the TBP- andTFIID-nucleated basal transcription in this system is criticallydependent on the amount of exogenously added factor. Thus,the levels of basal transcription reconstituted with f:TFIID andTRAP/Mediator were similar to those with rTBP and TRAP/Mediator when a fivefold excess of exogenous f:TFIID andrTBP was added to the reaction mixtures (data not shown).

As indicated above, these assays were performed with tem-plates containing either combined TATA and initiator ele-ments (G5HML) or a relatively weak TATA (G5E1b) elementwithin the core promoter. The fact that the two templatesshowed qualitatively similar results indicates that, at least withrespect to the limited number of core promoters tested, theobserved synergy between TFIID-specific TAFIIs and Media-tor is not a function of core promoter sequences.

DISCUSSION

The main conclusions of this work are (i) that TRAP25 is anovel, integral subunit of human Mediator, (ii) that Mediatoris required for both basal (activator-independent) and acti-vated transcription in the context of nuclear extract, and (iii)that Mediator and TAFIIs within TFIID function synergisti-cally in both basal and activated transcription in this system.

Role of metazoan Mediator in basal transcription. Initialcharacterization of the yeast Mediator complex had pointed tothree distinct associated biochemical activities. It was found toplay a critical role as a coactivator (the function most closelyidentified with the Mediator) and to stimulate basal transcrip-tion activity in reaction mixtures reconstituted with Pol II andGTFs, leading to the hypothesis that the two activities may beinterdependent (19). Functions in both basal and activatedtranscription were also evident from analyses of extracts fromyeast with a deletion mutation in the SRB5 subunit of Medi-ator (44). In vitro studies also showed that Mediator can stim-ulate the Pol II carboxy-terminal repeat domain kinase activityof TFIIH (19). Although our earlier functional analysis of thevarious human Mediator complexes (13, 17) clearly revealedtheir coactivator potential for a wide range of activators (in-cluding the thyroid hormone receptor in conjunction with theretinoid X receptor, GAL4-p53, and GAL4-VP16), the effectson activator-independent transcription were not as marked.Our previous reconstituted transcription system relied on anadditional coactivator, PC4, that, in addition to promotingactivated transcription, strongly suppresses basal transcription(11, 26). Similarly, in experiments in which ARC (33) andDRIP (38) coactivator effects were seen only in the context ofchromatin templates, basal transcription may have been sup-pressed (either by histones or by negative cofactors in thecrude chromatin assembly extracts) to such an extent that itwas refractory to stimulation by Mediator alone. By contrast,the unfractionated nuclear extract-based assays employed hereand by Mittler et al. (31) have helped uncover a prominentbasal function of human Mediator. By thus extending our ini-tial characterization, these findings now suggest that, consis-tent with the phylogenetic conservation of some subunits, the

fundamental mechanisms by which the Mediator functionsmay also be conserved from yeast to humans.

It is likely that basal effects of Mediator are readily observedin an unfractionated nuclear extract because transcription insuch a system is subject to a much broader range of bothpositively and negatively acting cofactors than exist in the pu-rified reconstituted systems. Thus, it may be that net transcrip-tion, both basal and activated, in less purified systems entails amajor antirepression component and that one function of Me-diator is to help overcome restrictions imposed by negativelyacting cofactors. These cofactors are presumably distinct fromhistones and PC4 but could include cofactors such as NC2(reviewed in reference 25). It perhaps is relevant that in puresystems, basal transcription (in the absence of PC4) is rela-tively high, providing another possible reason why basal effectsof Mediator have been undetected (26).

Alternative, but not mutually exclusive, mechanisms bywhich Mediator could promote basal transcription involve di-rect interactions with the basal transcription machinery. In-deed, in yeast, genetic interactions of SOH1 with Pol II andTFIIB have been described (reviewed in reference 5). How-ever, it should be noted that while SOH1 is an integral subunitof the human Mediator (13; Malik and Roeder, unpublished),it has not been documented as a yeast Mediator subunit. Fur-thermore, in vitro recruitment experiments have revealed thatthe yeast holoenzyme (consisting minimally of Pol II and Me-diator) is dependent on both TBP and TFIIB for incorporationinto the preinitiation complex formation (39). It is conceivable,therefore, that the Mediator could directly stabilize the preini-tiation complex. A more dynamic effect of the Mediator onbasal transcription also might be exerted via its ability to mod-ulate TFIIH activities (19). Although it has been proposed thatthe SRB10/CDK8 kinase subunit of the human Mediatordown-regulates TFIIH activity by phosphorylating its cyclin Hsubunit (1), the yeast Mediator preparation that was shown tostimulate TFIIH carboxy-terminal repeat domain kinase activ-ity lacked the SRB10 kinase (19).

Synergistic transcriptional activation by Mediator andTAFIIs. Our results also have revealed that, in unfractionatedsystems, Mediator and TAFIIs (in TFIID) functionally syner-gize to give high levels of activated transcription that result,albeit only in part, from favorable TAFII- and Mediator-de-pendent basal effects discussed above. Although at apparentvariance (but see below) with earlier results (34) indicatingthat the TBP subunit of TFIID could support activated tran-scription in a similar unfractionated system in the absence ofassociated TAFIIs, the present experiments have clearly servedto define experimental conditions in which functional synergybetween Mediator and TFIID-specific TAFIIs can be readilyobserved. Our use of near-physiological concentrations of ex-ogenously added factors in depleted extracts may account inpart for the present results. However, it also remains possiblethat our particular protocol (depletion of both TFIID andMediator and subsequent complementation with highly puri-fied preparations of Mediator and TFIID) results in conditionsthat favor multiple rounds of transcription and thus render thesystem TAFII dependent (34). The combined depletion ofTFIID and Mediator may also result in depletion of compo-nents that facilitate TAFII-independent transcription and thatare not present in the highly purified TFIID and Mediator

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preparations. Nonetheless, the synergistic function of TFIIDand Mediator is in accord with the coactivator properties as-cribed to each (9, 13, 45) and with recently published datashowing synergistic effects of TFIID and Mediator in highlypurified reconstituted systems (14, 27, 41).

While the role of TFIID-specific TAFIIs as conventionalcoactivators remains somewhat controversial, a consensus isemerging that they may play major roles in core promoterselectivity both in yeast and in metazoans (reviewed in refer-ence 12). This is especially evident from studies showing anabsolute requirement for metazoan TAFIIs in basal transcrip-tion from promoters that contain divergent core promoterelements, including initiators and downstream promoter ele-ments (DPEs), but often no TATA element (3, 22, 29). TAFIIsare also required for functional synergy between TATA andinitiators or DPEs, indicating promoter-selective functions oncomposite core promoters (29). Preliminary indications arethat, mechanistically, this may involve direct interactions withcore promoter sequences, especially in the case of promotersmade up of divergent (non-TATA) promoter elements (3).The recently published results of Park et al. (36), who exam-ined the relative contribution of TAFIIs and Mediator in acti-vation of several Drosophila promoters, are also in agreementwith the hypothesis that TFIID-associated TAFIIs are primar-ily involved in core promoter effects. These authors observedthat a TATA-box-containing promoter, Adh, could be acti-vated by GAL4-VP16 (through upstream GAL4 sites) withonly TBP (in the absence of TAFIIs) and Mediator. However,activation through the engrailed promoter, which lacks aTATA box but contains a DPE, required intact TFIID andMediator.

In view of the above results and given that this study, incontrast to an earlier report (34), utilized relatively weak corepromoters (the adenovirus E1b promoter and the syntheticHML promoter that contains the TATA box from the humanimmunodeficiency virus promoter and the initiator elementfrom adenovirus ML promoter), it is likely that the TAFII

dependence observed here has its basis in how the transcrip-tion apparatus senses the promoter sequences. Together withthe newly uncovered basal transcription effect of the Mediatorreported here and by Mittler et al. (31), it appears that theabilities of human TFIID and Mediator complexes, originallythought only to mediate activator signals to the basal appara-tus, to directly modulate the basal machinery and to processactivation signals are inextricably conjoined. This implies thatone mechanism by which these coactivators function is to“present” the basal machinery in a form that is now responsiveto activators. For TFIID (including TBP and TAFIIs) this maytake the form of appropriately positioning Pol II and GTFs(i.e., in promoter recognition); for Mediator, this could entailany of the mechanisms discussed above. Interestingly, by thisview, and on the basis of the results presented here, Mediatorwould appear to be functionally indistinguishable from any ofthe GTFs. However, because it is likely the key component thatcontacts activators (2, 9, 17, 33, 38) and because the level ofactivation in the presence of Mediator is significantly higherthan what would be expected if it were simply contributing asa basal factor, Mediator is also clearly functioning as a coac-tivator in the conventional sense. Therefore, the activationprocess must additionally involve a Mediator-dependent signal

transduction event. This notion is also supported by studiesindicating that mutations in yeast Mediator subunits may alterfunction without noticeably affecting interactions of Mediatorwith activator and Pol II (24). Clearly, future studies on a widevariety of naturally occurring combinations of core promotersand upstream activating sequences, together with detailed mu-tagenesis of the Mediator components, will be required tofurther substantiate and elaborate these concepts.

ACKNOWLEDGMENTS

We thank Y. K. Kang for a purified TRAP (Flag-NUT2) prepara-tion.

This work was supported by NIH grant CA42567 to R.G.R.

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