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Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit

Abstract

The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular complex. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator complex.

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Figure 1: Reconstitution of human Mediator subcomplexes.
Figure 2: Critical roles of MED14 and MED26 in Mediator-stimulated basal transcription.
Figure 3: Critical roles of MED14 and MED26 in Mediator coactivator function.
Figure 4: MED14-dependent Mediator–Pol II interaction and MED26-dependent Pol II recruitment in nuclear extract.
Figure 5: Molecular architecture of the reconstituted Mediator complex revealed by chemical cross-linking and MS (CX-MS).
Figure 6: Schematic representation of subunit interactions in the human core Mediator complex, based on composite data from CX-MS and biochemical approaches.

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Acknowledgements

We thank T. Richmond (Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Zurich) for the MultiBac baculovirus system, J. Fernandez-Martinez and M.P. Rout (Rockefeller University, Laboratory of Cellular and Structural Biology) for assistance with the offline Agilent HPLC system and M. Guermah (Rockefeller University, Laboratory of Biochemistry and Molecular Biology) for discussion. Funding for this work was provided by US Department of Defense grant W81XWH-13-1-0172 (R.G.R.) and by US National Institute of Health grants CA129325 (R.G.R.), GM090929 (R.G.R. and S.M.), GM103511 (B.T.C.), GM109824 (B.T.C.) and GM103314 (B.T.C.). M.A.C. was supported by an American Cancer Society Eastern Division–New York Cancer Research Fund Postdoctoral Fellowship.

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Contributions

M.A.C., S.M., R.G.R., Y.S. and B.T.C. designed the experiments and wrote the manuscript. M.A.C. carried out biochemical experiments including cDNA preparations, reconstitutions, in vitro transcriptions and coimmunoprecipitation experiments. D.L. helped M.A.C. in the generation of partial head-module complexes (in Supplementary Fig. 5d). Y.S. carried out the CX-MS experiments.

Corresponding author

Correspondence to Robert G Roeder.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Isolation of the recombinant middle and head + middle modules.

(a) Scheme illustrating the multi-step purification protocol for the reconstituted middle module. Extract from infected cells co-expressing middle module subunits was incubated with nickel-NTA agarose for purification via His-MED10, followed by M2 agarose for selection via f-MED7. Further purification was achieved through SP-Sepharose chromatography. (b) SDS-PAGE analysis (Coomassie staining) of middle module preparations after each of the three purification steps. (c) Scheme illustrating the multi-step purification protocol for the reconstituted H+M complex. Extract from infected cells co-expressing middle and head subunits was purified over M2 agarose for selection via f-MED17 followed by chromatography on an anti-HA resin for selection via HA-MED7. Further purification was achieved through AKTA Superose 6 gel filtration chromatography. (d) SDS-PAGE analysis (Coomassie staining) of H+M preparations after each of the three purification steps. (e) Superose 6 gel filtration profile of the reconstituted H+M complex. Column fractions were analyzed by immunoblotting with the indicated Mediator antibodies. Fractions at which the various molecular mass standards elute are identified.

Supplementary Figure 2 MED26 is part of the middle module.

(a) Extract from infected cells co-expressing MED26 plus all middle module subunits was subjected to purification over an anti-HA resin (through HA-MED7). Coomassie staining shows strong enrichment of MED26. (b) Extract from infected cells co-expressing f-MED26 plus all middle module subunits together with all head module subunits except MED17 (which precludes H+M formation) was purified over M2 agarose and probed for representative head and middle subunits. Middle module subunits were selectively enriched in the eluates. (c) SDS-PAGE analysis of a reconstituted H+M+26 complex following M2 agarose chromatography.

Supplementary Figure 3 Purification of reconstituted head + middle + MED14 + MED26 complex.

Recombinant H+M+14+26 complex was purified according to the three-step scheme in Supplementary Fig. 1c-e. (a) SDS-PAGE analysis. (b) Superose 6 elution profile (immunoblotting of fractions) as in figure S2c.

Supplementary Figure 4 Cross-linking efficiency of lysine residues in CX-MS.

(a) Reconstituted complexes were treated with various concentrations of DSS crosslinker prior to SDS-PAGE and silver staining. A DSS concentration of 1 mM (20 min at 4OC) was chosen for scaled-up CX-MS experiments. (b) Graph showing lysine prevalence within the cross-linked Mediator subunits. (c) Graph showing percentage of cross-linked lysines within the scored Mediator subunits.

Supplementary Figure 5 Subunit organization of the middle and head modules, based on immunoprecipitations and partial reconstitutions.

(a) MED21 interacts with all subunits of the middle module. Extracts from insect cells co-expressing f-MED21 and either MED10, MED31, MED7 or MED4 were incubated with M2 agarose and the eluates were analyzed by Western blot. (b) MED7 co-purifies with all tested middle module subunits. f-MED7 was co-expressed with: (i) MED4, MED31, and MED10 (lane 1); (ii) MED21 and MED10 (lane 2); and (iii) MED4 and MED10 (lane 5). Infected cell extracts were purified over M2 agarose and characterized by SDS-PAGE/Coomassie stain (lanes 1 and 2) or by Western blot (lane 5). (c) Schematic representation of the interaction pattern of the middle module subunits based on the results from panels a and b. Pairwise interactions established in these assays are shown by solid lines; interactions implied, but not established, are shown by broken lines. (d) Co-purification of MED17 with the majority of the subunits of the head module and heterodimer formation by MED11 and MED22 and by MED18 and MED20. Complexes were isolated from cell extracts co-expressing the following combinations: all head subunits (lane 1); all head subunits except MED6 and MED20 (-6, -20; lane 2); f-MED17, MED18, and MED8 (lane 3); f-MED17, MED18, and MED6 (lane 4); f-MED17 and MED8 (lane 5); f-MED22, MED11, and MED17 (lane 6); f-MED22 and MED11 (lane 7); and f-MED20 and MED18 (lane 8). In purifications (M2 agarose followed by SDS-PAGE of eluates) shown in lanes 1-5, complexes were selected through f-MED17. In lanes 6-8, selections were through f-MED22 (lanes 6 and 7) or f-MED20 (lane 8). MED18 fails to interact with MED17 in the absence of MED8 (compare lane 3 [MED18 in the presence of MED8] vs. lane 4 [MED18 in the absence of MED8]). However, MED18 and MED20 form a strong heterodimer. Further, leaving out MED20 does not affect MED18 incorporation into the complex (lane 2), which copurifies with MED17 and MED8 (lane 3). Thus, MED20 is anchored to MED17 via MED8. (e) Schematic representation of the interaction pattern of the head module subunits based on the results from panel d. Pairwise interactions established in these assays are shown by solid lines; interactions implied, but not established, are shown by broken lines. (f) MED17 co-purifies with middle module. Extracts with co-expressed f-MED17 and all middle subunits were purified over M2 agarose and eluates were characterized by Western blot. (g) MED17 interacts with MED7: Extract from Hi5 cells co-expressing f-MED17 and HA-MED7 subunits was incubated with M2 agarose and the eluate characterized by SDS-PAGE with Coomassie staining.

Supplementary Figure 6 Copurification of MED14 with all three major Mediator modules.

Extracts from infected cells co-expressing f-MED14 and either the complete middle module (a), the complete head (b), or the tail subunits MED16, MED23, and MED24 that are known to form a sub-module (c) were affinity purified over M2 agarose and analyzed by SDS-PAGE and Coomassie staining.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 2899 kb)

Supplementary Table 1

DSS cross-link dataset of the reconstituted Mediator complex (PDF 364 kb)

Supplementary Table 2

Annotated HCD MS/MS spectra of the cross-linked peptides identified from the reconstituted Mediator complex (PDF 9896 kb)

Supplementary Data Set 1

Uncropped gels from Figs. 1 and 2 (PDF 7543 kb)

Supplementary Data Set 2

Uncropped gels from Figs. 3 and 4 (PDF 6334 kb)

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Cevher, M., Shi, Y., Li, D. et al. Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit. Nat Struct Mol Biol 21, 1028–1034 (2014). https://doi.org/10.1038/nsmb.2914

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