Requirement of TRAP/mediator for both activator-independent and activator-dependent transcription in conjunction with TFIID-associated TAF(II)s

Abstract

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, it remains unclear how TRAP/Mediator functions in the context of other coactivators. Here we have identified a previously uncharacterized integral subunit (TRAP25) of the complex that is apparently metazoan specific. An antibody that is specific for TRAP25 allowed quantitative immunodepletion of essentially all TRAP/Mediator components from HeLa nuclear extract, without detectably affecting levels of RNA polymerase II and corresponding general transcription factors. Surprisingly, the TRAP/Mediator-depleted nuclear extract displayed severely reduced levels of both basal and activator-dependent transcription from DNA templates. Both activities were efficiently restored upon readdition of purified TRAP/Mediator. Moreover, restoration of basal and activator-dependent transcription to extracts that were simultaneously depleted of TRAP/Mediator and TFIID (TBP plus the major TAF(II)s) required addition of both TBP and associated TAF(II)s, as well as TRAP/Mediator. These observations indicate that TAF(II)s and Mediator are jointly required for both basal and activated transcription in the context of a more physiological complement of nuclear proteins. We propose a close mechanistic linkage between these components that most likely operates at the level of combined effects on the general transcription machinery and, in addition, a direct role for Mediator in relaying activation signals to this machinery.

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-spectrometric sequence analysis. Peptide sequences thus obtained and corresponding proteins are indicated. (B) Amino acid sequence of TRAP25. Amino acid sequences 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 (single dots) are indicated. (C) Northern analysis of a multiple-tissue mRNA blot hybridized with a TRAP25 cDNA probe showing expression of TRAP25 in human tissues.

FIG. 2.

TRAP25 is a bona fide component of TRAP/Mediator. (A) Complexes from the indicated cell lines (f:TR, lane 2) were analyzed by immunoblotting with anti-TRAP25 antibodies. Representative TRAP/Mediator subunits were also probed, as indicated. Total HeLa cell nuclear extract 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 markers is indicated at the top. (C) An anti-TRAP25 antibody depletes TRAP/Mediator from HeLa nuclear extract. HeLa nuclear extract (input) was passed over a control column containing immobilized preimmune antibodies (mock) or over a column containing anti-TRAP25. Unbound extract was immunoblotted with the indicated antibodies.

FIG. 3.

Depletion of TRAP/Mediator from nuclear extract reduces basal and activated transcription. (A) Transcription activation by Gal4-p53 is suppressed in the anti-TRAP25 antibody-depleted nuclear extract. In vitro transcription reaction mixtures contained 50 μg of each nuclear extract (lanes 1 and 2, untreated; lanes 3 and 4, anti-TRAP25 antibody-depleted [ΔMED]; lanes 5 and 6, mock depleted) and 50 ng each of pMLΔ53 (Δ53) and pG5HML (G5HML) templates. Relative transcription levels, determined by phosphorimager analysis, are indicated. (B) In vitro transcription reactions were performed as for panel A except that Gal4-VP16 was used as an activator.

FIG. 4.

Restoration of activated and basal transcription by exogenously added purified TRAP/Mediator. (A) Increasing amounts of purified TRAP/Mediator (lanes 3 to 5) were added to anti-TRAP25 antibody-depleted nuclear extract (ΔMED) in in vitro transcription reactions. Reaction mixtures were as for Fig. 3 except that they contained 20 μg of each nuclear extract. As determined by immunoblotting, the amount of TRAP/Mediator added to the reactions corresponded to approximately 20% (lane 3), 70% (lane 4), or 100% of the TRAP/Mediator concentration in untreated nuclear extract. GAL4-p53 was used as the activator in all reactions. Lane 1 shows the product of a control reaction with untreated extract. (B and C) In vitro transcription reactions were performed as for panel A except that GAL4-VP16 was used as the activator (B) or no activator was added (C).

FIG. 5.

Synergistic effect of TRAP/Mediator and TFIID-associated TAF_IIs on transcription activation in vitro. (A) Immunoblot analysis of TFIID- 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-TAF_II100, and anti-TRAP25 antibodies (ΔMED/ΔIID); lane 3, anti-TRAP25-depleted nuclear extract (ΔMED). Each lane was loaded with 20 μg of each nuclear extract and analyzed on a 4-to-20% gradient SDS-PAGE gel, followed by immunoblotting. (B and C) In vitro transcription reaction mixtures contained the indicated nuclear extract plus 25 ng each of pMLΔ53 and either pG5HML (B) or pG5E1b (C) in a 12.5-μl reaction volume. GAL4-p53 (50 ng), recombinant TBP (rTBP, 3 ng), affinity-purified TFIID containing an equivalent molar amount of TBP (as determined by immunoblotting), and an amount of TRAP/Mediator corresponding to that in standard nuclear extract were added where indicated. (D and E) In vitro transcription reactions were performed as for panels B and C, respectively, except that Gal4-VP16 (50 ng) was the activator.

FIG. 5.

Synergistic effect of TRAP/Mediator and TFIID-associated TAF_IIs on transcription activation in vitro. (A) Immunoblot analysis of TFIID- 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-TAF_II100, and anti-TRAP25 antibodies (ΔMED/ΔIID); lane 3, anti-TRAP25-depleted nuclear extract (ΔMED). Each lane was loaded with 20 μg of each nuclear extract and analyzed on a 4-to-20% gradient SDS-PAGE gel, followed by immunoblotting. (B and C) In vitro transcription reaction mixtures contained the indicated nuclear extract plus 25 ng each of pMLΔ53 and either pG5HML (B) or pG5E1b (C) in a 12.5-μl reaction volume. GAL4-p53 (50 ng), recombinant TBP (rTBP, 3 ng), affinity-purified TFIID containing an equivalent molar amount of TBP (as determined by immunoblotting), and an amount of TRAP/Mediator corresponding to that in standard nuclear extract were added where indicated. (D and E) In vitro transcription reactions were performed as for panels B and C, respectively, except that Gal4-VP16 (50 ng) was the activator.

FIG. 5.

Synergistic effect of TRAP/Mediator and TFIID-associated TAF_IIs on transcription activation in vitro. (A) Immunoblot analysis of TFIID- 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-TAF_II100, and anti-TRAP25 antibodies (ΔMED/ΔIID); lane 3, anti-TRAP25-depleted nuclear extract (ΔMED). Each lane was loaded with 20 μg of each nuclear extract and analyzed on a 4-to-20% gradient SDS-PAGE gel, followed by immunoblotting. (B and C) In vitro transcription reaction mixtures contained the indicated nuclear extract plus 25 ng each of pMLΔ53 and either pG5HML (B) or pG5E1b (C) in a 12.5-μl reaction volume. GAL4-p53 (50 ng), recombinant TBP (rTBP, 3 ng), affinity-purified TFIID containing an equivalent molar amount of TBP (as determined by immunoblotting), and an amount of TRAP/Mediator corresponding to that in standard nuclear extract were added where indicated. (D and E) In vitro transcription reactions were performed as for panels B and C, respectively, except that Gal4-VP16 (50 ng) was the activator.