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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors
Nature Communications Open Access 28 September 2022
Nature Communications Open Access 21 November 2018
Nature Communications Open Access 23 August 2018
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Roeder, R.G. Transcriptional regulation and the role of diverse coactivators in animal cells. FEBS Lett. 579, 909–915 (2005).
Malik, S. & Roeder, R.G. The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation. Nat. Rev. Genet. 11, 761–772 (2010).
Baek, H.J., Malik, S., Qin, J. & Roeder, R.G. Requirement of TRAP/mediator for both activator-independent and activator-dependent transcription in conjunction with TFIID-associated TAF(II)s. Mol. Cell. Biol. 22, 2842–2852 (2002).
Malik, S., Baek, H.J., Wu, W. & Roeder, R.G. Structural and functional characterization of PC2 and RNA polymerase II-associated subpopulations of metazoan Mediator. Mol. Cell. Biol. 25, 2117–2129 (2005).
Mittler, G., Kremmer, E., Timmers, H.T. & Meisterernst, M. Novel critical role of a human Mediator complex for basal RNA polymerase II transcription. EMBO Rep. 2, 808–813 (2001).
Poss, Z.C., Ebmeier, C.C. & Taatjes, D.J. The Mediator complex and transcription regulation. Crit. Rev. Biochem. Mol. Biol. 48, 575–608 (2013).
Black, J.C., Choi, J.E., Lombardo, S.R. & Carey, M. A mechanism for coordinating chromatin modification and preinitiation complex assembly. Mol. Cell 23, 809–818 (2006).
Wallberg, A.E., Yamamura, S., Malik, S., Spiegelman, B.M. & Roeder, R.G. Coordination of p300-mediated chromatin remodeling and TRAP/mediator function through coactivator PGC-1α. Mol. Cell 12, 1137–1149 (2003).
Lin, J.J. et al. Mediator coordinates PIC assembly with recruitment of CHD1. Genes Dev. 25, 2198–2209 (2011).
Malik, S., Barrero, M.J. & Jones, T. Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator. Proc. Natl. Acad. Sci. USA 104, 6182–6187 (2007).
Nock, A., Ascano, J.M., Barrero, M.J. & Malik, S. Mediator-regulated transcription through the +1 nucleosome. Mol. Cell 48, 837–848 (2012).
Takahashi, H. et al. Human mediator subunit MED26 functions as a docking site for transcription elongation factors. Cell 146, 92–104 (2011).
Lai, F. et al. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494, 497–501 (2013).
Kagey, M.H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010).
Eyboulet, F. et al. Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment. Genes Dev. 27, 2549–2562 (2013).
Schiano, C. et al. Involvement of Mediator complex in malignancy. Biochim. Biophys. Acta 1845, 66–83 (2014).
Spaeth, J.M., Kim, N.H. & Boyer, T.G. Mediator and human disease. Semin. Cell Dev. Biol. 22, 776–787 (2011).
Bourbon, H.M. Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex. Nucleic Acids Res. 36, 3993–4008 (2008).
Blazek, E., Mittler, G. & Meisterernst, M. The mediator of RNA polymerase II. Chromosoma 113, 399–408 (2005).
Baumli, S., Hoeppner, S. & Cramer, P. A conserved mediator hinge revealed in the structure of the MED7.MED21 (Med7.Srb7) heterodimer. J. Biol. Chem. 280, 18171–18178 (2005).
Koschubs, T. et al. Identification, structure, and functional requirement of the Mediator submodule Med7N/31. EMBO J. 28, 69–80 (2009).
Larivière, L. et al. Structure of the Mediator head module. Nature 492, 448–451 (2012).
Imasaki, T. et al. Architecture of the Mediator head module. Nature 475, 240–243 (2011).
Robinson, P.J., Bushnell, D.A., Trnka, M.J., Burlingame, A.L. & Kornberg, R.D. Structure of the mediator head module bound to the carboxy-terminal domain of RNA polymerase II. Proc. Natl. Acad. Sci. USA 109, 17931–17935 (2012).
Larivière, L. et al. Model of the Mediator middle module based on protein cross-linking. Nucleic Acids Res. 41, 9266–9273 (2013).
Guglielmi, B. et al. A high resolution protein interaction map of the yeast Mediator complex. Nucleic Acids Res. 32, 5379–5391 (2004).
Tsai, K.L. et al. Subunit architecture and functional modular rearrangements of the transcriptional mediator complex. Cell 157, 1430–1444 (2014).
Wang, X. et al. Redefining the modular organization of the core Mediator complex. Cell Res. 24, 796–808 (2014).
Malik, S., Gu, W., Wu, W., Qin, J. & Roeder, R.G. The USA-derived transcriptional coactivator PC2 is a submodule of TRAP/SMCC and acts synergistically with other PCs. Mol. Cell 5, 753–760 (2000).
Berger, I., Fitzgerald, D.J. & Richmond, T.J. Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol. 22, 1583–1587 (2004).
Ge, K. et al. Transcription coactivator TRAP220 is required for PPARã2-stimulated adipogenesis. Nature 417, 563–567 (2002).
Nonet, M.L. & Young, R.A. Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II. Genetics 123, 715–724 (1989).
Thompson, C.M., Koleske, A.J., Chao, D.M. & Young, R.A. A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast. Cell 73, 1361–1375 (1993).
Malik, S. & Roeder, R.G. Isolation and functional characterization of the TRAP/mediator complex. Methods Enzymol. 364, 257–284 (2003).
Dotson, M.R. et al. Structural organization of yeast and mammalian mediator complexes. Proc. Natl. Acad. Sci. USA 97, 14307–14310 (2000).
Sato, S. et al. A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology. Mol. Cell 14, 685–691 (2004).
Conaway, R.C. & Conaway, J.W. The Mediator complex and transcription elongation. Biochim. Biophys. Acta 1829, 69–75 (2013).
Ito, M. et al. Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear receptors and diverse mammalian activators. Mol. Cell 3, 361–370 (1999).
Yuan, C.X., Ito, M., Fondell, J.D., Fu, Z.Y. & Roeder, R.G. The TRAP220 component of a thyroid hormone receptor-associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion. Proc. Natl. Acad. Sci. USA 95, 7939–7944 (1998).
Larivière, L. et al. Structure and TBP binding of the Mediator head subcomplex Med8–Med18–Med20. Nat. Struct. Mol. Biol. 13, 895–901 (2006).
Cai, G. et al. Interaction of the mediator head module with RNA polymerase II. Structure 20, 899–910 (2012).
Baek, H.J., Kang, Y.K. & Roeder, R.G. Human Mediator enhances basal transcription by facilitating recruitment of transcription factor IIB during preinitiation complex assembly. J. Biol. Chem. 281, 15172–15181 (2006).
Leitner, A. et al. Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics. Mol. Cell. Proteomics 9, 1634–1649 (2010).
Soutourina, J., Wydau, S., Ambroise, Y., Boschiero, C. & Werner, M. Direct interaction of RNA polymerase II and mediator required for transcription in vivo. Science 331, 1451–1454 (2011).
Tsai, K.L. et al. A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction. Nat. Struct. Mol. Biol. 20, 611–619 (2013).
Näär, A.M., Taatjes, D.J., Zhai, W., Nogales, E. & Tjian, R. Human CRSP interacts with RNA polymerase II CTD and adopts a specific CTD-bound conformation. Genes Dev. 16, 1339–1344 (2002).
Jishage, M. et al. Transcriptional regulation by Pol II(G) involving mediator and competitive interactions of Gdown1 and TFIIF with Pol II. Mol. Cell 45, 51–63 (2012).
Lemaire, M., Xie, J., Meisterernst, M. & Collart, M.A. The NC2 repressor is dispensable in yeast mutated for the Sin4p component of the holoenzyme and plays roles similar to Mot1p in vivo. Mol. Microbiol. 36, 163–173 (2000).
Marr, S.K., Lis, J.T., Treisman, J.E. & Marr, M.T. II The metazoan-specific Mediator Subunit 26 (Med26) is essential for viability and is found at both active genes and pericentric heterochromatin in Drosophila melanogaster. Mol. Cell. Biol. 34, 2710–2720 (2014).
Gu, W. et al. A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation. Mol. Cell 3, 97–108 (1999).
Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).
Malik, S., Wallberg, A.E., Kang, Y.K. & Roeder, R.G. TRAP/SMCC/mediator-dependent transcriptional activation from DNA and chromatin templates by orphan nuclear receptor hepatocyte nuclear factor 4. Mol. Cell. Biol. 22, 5626–5637 (2002).
Shi, Y. et al. Structural characterization by cross-linking reveals the detailed architecture of a coatomer-related heptameric module from the nuclear pore complex. Mol. Cell. Proteomics 10.1074/mcp.M114.041673 (26 August 2014).
Algret, R. et al. Molecular architecture and function of the SEA complex, a modulator of the TORC1 pathway. Mol. Cell. Proteomics 10.1074/mcp.M114.039388 (29 July 2014).
Leitner, A. et al. Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography. Mol. Cell. Proteomics 11 M111.014126 (2012).
Yang, B. et al. Identification of cross-linked peptides from complex samples. Nat. Methods 9, 904–906 (2012).
Qin, J. & Chait, B.T. Matrix-assisted laser desorption ion trap mass spectrometry: efficient isolation and effective fragmentation of peptide ions. Anal. Chem. 68, 2108–2112 (1996).
Michalski, A., Neuhauser, N., Cox, J. & Mann, M. A systematic investigation into the nature of tryptic HCD spectra. J. Proteome Res. 11, 5479–5491 (2012).
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.
The authors declare no competing financial interests.
Integrated supplementary information
(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.
(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.
(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.
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 Figures 1–6 (PDF 2899 kb)
DSS cross-link dataset of the reconstituted Mediator complex (PDF 364 kb)
Annotated HCD MS/MS spectra of the cross-linked peptides identified from the reconstituted Mediator complex (PDF 9896 kb)
Uncropped gels from Figs. 1 and 2 (PDF 7543 kb)
Uncropped gels from Figs. 3 and 4 (PDF 6334 kb)
About this article
Cite this article
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
This article is cited by
Nature Reviews Molecular Cell Biology (2022)
The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors
Nature Communications (2022)
Nature Genetics (2020)