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The mammalian Mediator complex and its role in transcriptional regulation
Review
TRENDS in Biochemical Sciences
Vol.30 No.5 May 2005
Mediator special issue
The mammalian Mediator complex and its role in transcriptional regulation Ronald C. Conaway1,2, Shigeo Sato1, Chieri Tomomori-Sato1, Tingting Yao1 and Joan W. Conaway1,2,3 1
Stowers Institute for Medical Research, Kansas City, MO 64110, USA Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, KS 66160, USA 3 Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA 2
Mediator is an essential component of the RNA polymerase II general transcriptional machinery and plays a crucial part in the activation and repression of eukaryotic mRNA synthesis. The Saccharomyces cerevisiae Mediator was the first to be defined and is a high molecular mass complex composed of O20 distinct subunits that performs multiple activities in transcription. Recent studies have defined the subunit composition and associated activities of mammalian Mediator, and revealed a striking evolutionary conservation of Mediator structure and function from yeast to man. Introduction The initiation stage of mRNA synthesis is a major site for the regulation of gene expression. In eukaryotes, mRNA synthesis is catalyzed by the multisubunit enzyme RNA polymerase II (pol II) and regulated by a host of DNAbinding transcription factors that activate or repress transcription in response to a myriad of signals emanating both from within the cell and from the cellular environment. Biochemical studies have shown that the initiation and regulation of eukaryotic mRNA synthesis requires a large collection of evolutionarily conserved ‘general’ transcription factors that seem to function at all, or most, genes. These general transcription factors include (i) the general initiation factors TFIIB, TFIID, TFIIE, TFIIF and TFIIH, which constitute the minimal set of auxiliary proteins necessary and sufficient for selective binding and accurate transcription initiation in vitro by pol II from the core regions of most promoters [1–4]; and (ii) the multiprotein Mediator complex, which functions, at least in part, as an adaptor that supports essential communication from transcription factors bound at upstream promoter elements and enhancers to pol II and the general initiation factors at the core promoter [5,6]. Although the compositions and many of the functions of the general initiation factors from yeast and higher eukaryotes were well established by the early 1990s, the structure and activities of the Mediator complex have only recently been illuminated. Mediator was first discovered and purified to near homogeneity from Saccharomyces Corresponding author: Conaway, J.W. ([email protected]). Available online 26 March 2005
cerevisiae by Kornberg and coworkers. They showed that it is required for transcription activation by the transcriptional activators Gcn4 or GAL4-VP16 in vitro using a reconstituted enzyme system composed of purified pol II and general initiation factors [7–9]. Yeast Mediator is composed of w20 subunits, which are present in three distinct Mediator subdomains referred to as the ‘head,’ ‘middle’ and ‘tail’ modules (reviewed in Ref. [10]) (Figure 1). An additional module, which includes a kinase–cyclin pair, is associated with a subset of yeast Mediator complexes and has, in yeast that is growing exponentially, been implicated in repression of a subset of genes [11–14]. Mammalian Mediator-like complexes were subsequently identified and characterized in several laboratories. However, whether the mammalian Mediator-like complexes isolated in different laboratories represented the same or different functional entities was unclear at first because they seemed to include distinct, but overlapping, sets of subunits. Furthermore, there was considerable controversy over the evolutionary relationship between yeast Mediator and mammalian Mediator-like complexes because there were obvious mammalian orthologs of only a subset of yeast Mediator subunits. As described in more detail later, recent efforts exploiting state-of-the-art proteomics methods have defined a set of consensus mammalian Mediator subunits, and improved bioinformatics approaches have revealed a striking evolutionary conservation of Mediator from yeast to man. Here, we discuss these recent developments in studies of the structure and function of mammalian Mediator. Isolation of mammalian Mediator-like complexes Mammalian Mediator-like complexes have been isolated by a variety of methods, including conventional and affinity chromatography. The first such complex was purified by Roeder and coworkers. It was designated the TRAP (thyroid hormone receptor-associated proteins) complex because it was isolated in association with the liganded thyroid hormone receptor (TR) [15,16]. Subsequently, related Mediator-like complexes were purified in several laboratories by conventional chromatography from mouse B cells [17], human HeLa cells [18–20], and rat liver [21] and designated mouse Mediator, CRSP
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TRENDS in Biochemical Sciences
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Figure 1. Comparison of mammalian Mediator-like complexes. Mediator subunits identified in different Mediator preparations are indicated with blue; Mediator subunits not identified are indicated with yellow ([29] and references therein). Subunits are grouped according to whether they are believed to be located in the head, middle or tail modules ([10] and references therein) or have not yet been assigned to a specific module. On the left portion of the table, the compositions of S. cerevisiae Mediator and various Mediator-like complexes from mammalian cells are summarized. The mammalian rat MED (rat mediator), TRAP, ARC, DRIP, mMED (mouse mediator), PC2, and CRSP Mediator-like complexes have been reported to fall into two size classes, large and small, and are grouped accordingly. The right portion summarizes the compositions determined by MudPIT of Mediator-like complexes purified via immunoaffinity chromatography of nuclear extracts prepared from extracts of HeLa cells expressing the indicated subunits with an N-terminal epitope tag with the amino sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (the FLAG epitope). f: denotes the FLAG epitope; proteins identified following immunoaffinity chromatography of extracts from parental HeLa or 293 cells are indicated.
(cofactor required for Sp1 activation) or PC2 (positive cofactor 2), and rat Mediator, respectively. In addition, Mediator-like complexes have been purified (i) in association with the liganded vitamin D receptor (VDR) from a HeLa cell line stably expressing FLAG-VDR and designated DRIP (vitamin D receptor-interacting proteins), (ii) by immunoaffinity chromatography from HeLa cell lines stably expressing epitope-tagged versions of mammalian orthologs of yeast Mediator subunits and designated SMCC [suppressor of RNA polymerase B (SRB)-mediatorcontaining cofactor] [22] or NAT (negative regulator of activated transcription) [23], and (iii) by affinity chromatography via interactions with immobilized transcriptional activation domains of the viral transactivators VP16 www.sciencedirect.com
and E1A, SREBP (sterol-response-element-binding protein), and the p65 subunit of NF-kB (nuclear factor-kB) and designated ARC (activator-recruited factor) or human Mediator [24,25]. The functional properties of these Mediator-like complexes have been characterized in vitro in transcription systems of varying purity and using both naked DNA and chromatin templates. Many of them, including the TRAP, SMCC, PC2, CRSP, human Mediator and DRIP complexes, have been reported to stimulate strongly activator-dependent transcription in vitro. In addition, the TRAP [22] and NAT [23] complexes have been reported to be capable of inhibiting activated transcription in vitro under some reaction conditions.
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The subunit composition of mammalian Mediator Analyses of proteins present in mammalian Mediator-like complexes from human, mouse and rat cells led to the identification of w30 potential mammalian Mediator subunits (reviewed in Ref. [5]). Only eight of these proteins could be unambiguously identified in pair-wise sequence alignments as orthologs of yeast Mediator subunits, which led to early suggestions that the mammalian Mediator-like complexes might be only distantly related in structure and function to the yeast Mediator. However, more sophisticated bioinformatic analyses exploiting multiple sequence alignments of Mediator subunits across many species have identified short regions of similarity between subunits of mammalian Mediatorlike complexes and all but three yeast Mediator subunits (MED2, MED3 and MED5) [10,21,26,27]. Further supporting the close evolutionary relationship between mammalian and yeast Mediator, pair-wise interactions between several of the mammalian Mediator proteins have been shown to recapitulate interactions between their presumptive yeast orthologs [26,27]. Recognition of these similarities motivated formulation of a unified nomenclature for Mediator subunits across species [28]; we use this new nomenclature to refer to specific Mediator subunits throughout this review. As noted, mammalian Mediator-like complexes purified in different laboratories using different purification procedures seemed to have distinct yet overlapping polypeptide compositions (Figure 1). These complexes fell into two classes: larger, 1–2 MDa complexes such as TRAP, DRIP and ARC, which were reported to include all or some combinations of the kinase module subunits MED12, MED13, Cyclin C, cyclin-dependent kinase CDK8; and smaller, 500–700 kDa complexes such as mouse Mediator, PC2 and CRSP, which lack kinase module subunits. Apparent differences in the subunit compositions of complexes that fell within these two classes were also notable. For example, the MED26 protein was initially identified only in the CRSP and DRIP complexes, the MED30 and MED31 proteins in the TRAP complexes, the MED8 protein only in the ARC and rat Mediator complexes, the MED25 protein only in the ARC, DRIP and CRSP complexes, the MED18 protein only in the mouse and rat Mediator complexes and the MED9, MED11, MED19, MED22, MED28 and MED29 proteins only in rat Mediator. As discussed in more detail later, some of the differences among mammalian Mediator-like complexes seem to reflect the existence of functionally distinct forms of Mediator in cells. Nevertheless, results of recent proteomic analyses suggest that at least some of the apparent differences between mammalian Mediator-like complexes might be due to use of insufficiently sensitive protein identification procedures and/or loss of some subunits during purification [29]. In these experiments, MudPIT (multidimensional protein identification technology), a sensitive proteomics method that links 2D chromatography and tandem mass spectrometry, was used to compare the subunit compositions of complexes purified by immunoaffinity chromatography from cultured HeLa or HEK293 cell lines stably expressing different epitopewww.sciencedirect.com
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tagged Mediator subunits. Complexes purified through any of four head-module subunits (MED8, MED19, MED28 or MED29) and two middle-module subunits (MED9 or MED10) included all 30 of the proteins previously identified as subunits of mammalian Mediator-like complexes (summarized in Figure 1). The only additional proteins associated with all of these complexes were pol II and alternative forms of the kinase module subunits MED12, MED13 and CDK8 (MED12L, MED13L and CDK11, respectively), raising the possibility that Mediator complexes containing different kinase modules might regulate different subsets of genes [29] (T. Yao, unpublished).
Role of mammalian Mediator in transcriptional regulation The mechanisms by which mammalian Mediator complexes control mRNA synthesis have not been firmly established. However, substantial evidence argues that Mediator activates transcription, at least in part, via direct interactions with DNA-binding transcriptional activators bound at upstream promoter elements and enhancers, with pol II and, most likely, with one or more of the general initiation factors bound at the core promoter. Notably, different Mediator subunits seem to be targets for interaction with the transcriptional activation domains (TADs) of different DNA-binding transcriptional activators. As a consequence, a major focus of current studies of the mechanism of action of mammalian Mediator complexes is to identify subunits that serve as contact sites for the large collection of DNA-binding transcriptional activators present in cells. The mammalian MED1 protein has an essential role in transcriptional activation by the large family of nuclear receptors. MED1 binds directly to liganded nuclear receptors via its L-eu-Xaa-Xaa-Leu-Leu (LxxLL) motifs, and depletion of MED1 specifically disrupts nuclearreceptor function both in cells and in vitro [30–35]. MED1 is also required for the activity of the arylhydrocarbon receptor and potentiates aryl-hydrocarbonreceptor-dependent transcription in cells in an LxxLLindependent manner [36]. Other Mediator subunits that serve as functional targets for TADs include: MED14, which interacts with STAT2 [37]; MED15, which interacts via its KIX (kinaseinducible interaction) domain with the SMAD2–SMAD4 and SMAD3–SMAD4 transcriptional activators in response to transforming growth factor-b signaling [38]; MED23, which interacts with the TADs of E1A, the etslike transcription factor Elk1, and CCAAT/enhancerbinding protein (C/EBPb) [25,28,39]; and MED25, which interacts with the potent VP16 TAD via its Von Willebrandassociated domain [40,41]. In addition, the mammalian Mediator subunit MED29 is the apparent ortholog of the Drosophila melanogaster Intersex protein, which interacts directly with, and functions as a transcriptional coactivator for, the DNA-binding transcription factor Doublesex [42,43]. Accordingly, it is likely that mammalian MED29 also serves as a target for one or more DNA-binding transcriptional activators. However, to date,
Review
TRENDS in Biochemical Sciences
mammalian MED29 has not been shown to function in the context of Mediator as a target for TADs. Results of biochemical experiments suggest that mammalian Mediator supports transcriptional activation, at least in part, by increasing the efficiency and/or rate of assembly of the pol II preinitiation complex. Consistent with this idea, the E1A and Elk1 transcriptional activation domains stimulate binding of Mediator subunits, pol II and general initiation factors to immobilized promoter DNA in extracts from wild-type cells but not in extracts from cells lacking MED23 [44], which is the target of the E1A and Elk1 TADs [25,28,39]. Mammalian Mediator complexes seem to affect several steps during assembly of the pol II preinitiation complex, including recruitment to the core promoter of TFIID (or its TATA-box binding subunit TBP) and of pol II and the other general initiation factors. Wu et al. [45] observed that Mediator complexes could enhance VP16 TAD-dependent recruitment to promoters of pol II – but not of the general initiation factors – in a highly purified, reconstituted transcription system when TFIID or TBP was present at saturating concentrations [45]. This suggests that Mediator can function at a step after the binding of TFIID (or TBP) to promoters. At low concentrations of TBP, however, Mediator complexes could also enhance activatordependent recruitment of TBP [45]. Carey and colleagues have shown that the model activator GAL4-VP16, Mediator and TFIID assemble onto promoters in a cooperative manner [46] and, furthermore, that – under appropriate reaction conditions – assembly of a complex that includes Mediator and either TFIID or TBP can bypass the requirement for a DNA-binding transcriptional activator for high levels of transcription in vitro [47]. Based on these observations, Johnson and Carey [47] proposed (i) that one function of activators might be to enhance the stability of a TFIID–Mediator complex at the promoter and (ii) that this TFIID–Mediator complex might serve as a stable platform for reiterative assembly of the pol II preinitiation complex. Evidence for multiple, functionally distinct forms of Mediator Although the results of proteomic studies suggest that many of the apparent differences in the compositions of Mediator purified by different laboratories or according to different procedures might be due to a lack of sensitivity in the protein identification methods used. Substantial evidence argues that some of these differences, indeed, reflect the existence of multiple forms of Mediator that either positively or negatively regulate transcription. In particular, several lines of evidence suggest that the kinase module can exert a repressive function when associated with the mammalian Mediator, whereas the MED26 protein is associated with an activating form of Mediator. First, it has been reported that Mediator complexes containing the kinase module have little or no effect on, or actually inhibit, in vitro transcription that is activated by the VP16, SREBP, Sp1 and other TADs [20,23,48,49]. By contrast, complexes that lack the kinase module [20,49] and, in at least some cases, include MED26 [49] support strong activation of transcription in vitro by these same TADs. Although several Mediator preparations www.sciencedirect.com
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that include the kinase module, such as TRAP, DRIP, human Mediator and ARC, have been shown to support activator-dependent transcription in vitro, results of fractionation studies suggest that these preparations contain mixtures of Mediator complexes that do and do not include MED12, MED13, CDK8 and Cyclin C [20,49,50]. Second, Leutz and coworkers have provided evidence that (i) the transcription factor C/EBPb represses transcription of target genes in cells by recruiting Mediator with the kinase module to promoters and (ii) RAS–MAPK-dependent C/EBPb phosphorylation might function as a switch that leads to release of the CDK8 module from Mediator and transcriptional activation [51]. How might association of the kinase module with Mediator complexes render them inactive or even inhibitory? The available evidence indicates several possibilities that are not mutually exclusive. First, the kinase module might interfere with the ability of Mediator complexes to bind pol II, either by phosphorylating polymerase and/or Mediator subunits or by simple steric hindrance. Results of one study indicate that mammalian Mediator complexes containing MED26 and lacking the kinase module bind to an immobilized GST (glutathione S transferase)fusion protein containing the C-terminal domain (CTD) of the largest pol II subunit, whereas complexes that include the kinase module do not [52]. In addition, MudPIT analyses of Mediator complexes immunopurified through CDK8 indicate that little or no pol II is associated with Mediator that contains stoichiometric amounts of kinase module (J.W. Conaway et al., unpublished results), whereas complexes purified through MED26, which contain only a small amount of kinase module, are associated with near stoichiometric amounts of polymerase [29]. Second, Mediator-associated kinase might phosphorylate and inactivate one or more of the general initiation factors. Consistent with this possibility, CDK8–Cyclin C can phosphorylate the Cyclin H subunit of mammalian TFIIH, blocking the activity of the TFIIH CTD kinase and inhibiting TFIIH activity in transcription [48]. Finally, as suggested by a recent study of the role of CDK8 in Notch-dependent regulation of the HES1 (hairyenhancer of split 1) promoter [53], recruitment of the kinase module to mammalian Mediator at promoters might target DNA-binding transcription factors for phosphorylation and, ultimately, for degradation via the ubiquitin-dependent proteolytic system, providing a mechanism to limit the length of time a given promoter is active. Upon activation of the Notch-signaling pathway, the Notch intracellular domain (ICD) translocates into the nucleus and forms a Notch enhancer complex with the DNA-binding transcription factor CBF1 and its coactivator Mastermind, leading to transient activation of Notch-responsive genes, including HES1. Fryer et al. [53] observed that CDK8, and presumably the entire kinase module, is recruited to the HES1 promoter by the Notch enhancer complex a short time after recruitment of other Mediator subunits and HES1 activation, coincident with the loss of Notch from the promoter and subsequent down-regulation of the gene. In further experiments, they showed that CDK8-dependent phosphorylation of the Notch ICD targets it for ubiquitination and degradation.
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Thus, transient activation of HES1 might be a result of initial recruitment of an activating form of Mediator to the promoter, followed shortly by recruitment of the Mediator kinase module and phosphorylation and destruction of the Notch ICD. Concluding remarks and future directions Significant progress in characterizing the structure and function of the mammalian Mediator complex has been achieved. A set of consensus mammalian Mediator subunits has been identified, and the striking evolutionary conservation of Mediator has been revealed. With the definition of what seems to be a complete set of ‘core’ mammalian Mediator subunits, an in-depth characterization of the roles these subunits have in reconstitution of the transcriptionally active Mediator complex is now possible. Major questions for future studies include: † How many functionally distinct forms of Mediator participate in pol II transcriptional regulation? As discussed, it is likely that Mediator assemblies both including and lacking the kinase module are present in mammalian cells and perform distinct functions in transcriptional regulation. The identification of isoforms of several subunits of the kinase module [29] raises the possibility that different kinase modules are operative at different promoters and/or in different cell types. Finally, Mediator complexes that lack subunits, such as MED1 and MED25 – which function as docking sites for specific TADs – have been described [40,54]. Although such complexes could simply result from dissociation of subunits during purification, it is also possible that alternative forms of Mediator that contain different repertoires of TAD-docking sites might function at different promoters in cells. † Which Mediator subunits orchestrate its interactions with the diverse collection of DNA-binding transcriptional activators present in mammalian cells? And does interaction of Mediator with TADs alter Mediator activities in ways dependent on the nature of the TAD? Notably, analyses of Mediator structure by electron microscopy and signal-particle reconstructions suggest that the binding of different activators to different subunits, or even the binding of different activators to the same subunit, causes Mediator complexes to adopt remarkably different structures [49,55]. These observations led to the proposal by Tjian and colleagues that Mediator complexes with the same polypeptide compositions could have quite different activities when brought to promoters via interactions with different TADs [56]. † How does the mammalian Mediator complex promote pol II transcriptional activation? Do Mediator and its subunits simply recruit polymerase to promoters or do they also directly regulate the activities of polymerase and the general initiation factors during transcription, promoter escape and perhaps transcription elongation. † What is the repertoire of enzymatic activities associated with Mediator, and what are their functions? It is likely that the mammalian Mediator-associated kinase CDK8 and CDK11 phosphorylate substrates in addition to those described here. Yeast CDK8, like its www.sciencedirect.com
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mammalian ortholog, is capable of phosphorylating DNA-binding transcriptional regulatory proteins, in some cases, targeting them for ubiquitination [57,58]. In addition, yeast CDK8 can phosphorylate other substrates, such as the Mediator subunit MED2 [59] and two of the TAF (TBP-associated transcription factor) subunits of TFIID [60]. Future experiments exploring the repertoire of substrates phosphorylated by mammalian Mediator-associated kinases are likely to provide new insights into their contributions to transcriptional regulation. In addition, evidence that the MED8 subunit of the mammalian Mediator complex is capable of assembling together with the Elongin B, Elongin C, Cul2 and Rbx1 proteins into a potential E3 ubiquitin ligase have raised the possibility that MED8 might facilitate recruitment of ubiquitin ligase activity directly to Mediator as part of its function in mRNA synthesis [21]. Clearly, many more studies addressing the functions of the intact mammalian Mediator complex in addition to its individual subunits will be necessary to provide a complete picture of the mechanism of Mediator action in pol II transcriptional regulation. Acknowledgements Work in the authors’ laboratory is supported, in part, by National Institutes of Health Grant R37 GM41628.
References 1 Conaway, R.C. et al. (1991) Mechanism of promoter selection by RNA polymerase II: mammalian transcription factors a and bg promote entry of polymerase into the preinitiation complex. Proc. Natl. Acad. Sci. U. S. A. 88, 6205–6209 2 Conaway, J.W. and Conaway, R.C. (1997) General transcription factors for RNA polymerase II. Prog. Nucleic. Acids. Res. Mol. Biol. 56, 327–346 3 Orphanides, G. et al. (1996) The general transcription factors of RNA polymerase II. Genes Dev. 10, 2657–2683 4 Roeder, R.G. (1996) The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem. Sci. 21, 327–335 5 Myers, L.C. and Kornberg, R.D. (2000) Mediator of transcriptional regulation. Annu. Rev. Biochem. 69, 729–749 6 Koleske, A.J. and Young, R.A. (1995) The RNA polymerase II holoenzyme and its implications for gene regulation. Trends Biochem. Sci. 20, 113–116 7 Kim, Y.J. et al. (1994) A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77, 599–608 8 Li, Y. et al. (1995) Yeast global transcriptional regulators Sin4 and Rgr1 are components of mediator complex/RNA polymerase II holoenzyme. Proc. Natl. Acad. Sci. U. S. A. 92, 10864–10868 9 Myers, L.C. et al. (1998) The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain. Genes Dev. 12, 45–54 10 Boube, M. et al. (2002) Evidence for a Mediator of RNA polymerase II transcriptional regulation conserved from mammals to yeast. Cell 110, 143–151 11 Liao, S.M. et al. (1995) A kinase-cyclin pair in the RNA polymerase II holoenzyme. Nature 374, 193–196 12 Hengartner, C.J. et al. (1998) Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases. Mol. Cell 2, 43–53 13 Holstege, F.C.P. et al. (1998) Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717–728 14 Carlson, M. (1997) Genetics of transcriptional regulation in yeast: connections to the RNA polymerase II CTD. Annu. Rev. Cell Dev. Biol. 13, 1–23
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15 Fondell, J.D. et al. (1996) Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc. Natl. Acad. Sci. U. S. A. 93, 8329–8333 16 Fondell, J.D. et al. (1999) Thyroid hormone receptor-associated proteins and general positive cofactors mediate thyroid hormone receptor function in the absence of the TATA box-binding protein-associated factors of TFIID. Proc. Natl. Acad. Sci. U. S. A. 96, 1959–1964 17 Jiang, Y. et al. (1998) Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways. Proc. Natl. Acad. Sci. U. S. A. 95, 8538–8543 18 Ryu, S. et al. (1999) The transcriptional cofactor complex CRSP is required for activity of the enhancer binding protein Sp1. Nature 397, 446–450 19 Ryu, S. and Tjian, R. (1999) Purification of transcription cofactor complex CRSP. Proc. Natl. Acad. Sci. U. S. A. 96, 7137–7142 20 Malik, S. et al. (2000) The U. S. A.-derived transcriptional coactivator PC2 is a submodule of TRAP/SMCC and acts synergistically with other PC’s. Mol. Cell 5, 753–760 21 Brower, C.S. et al. (2002) Mammalian mediator subunit MED8 is an Elongin BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a ubiquitin ligase. Proc. Natl. Acad. Sci. U. S. A. 99, 10353–10358 22 Gu, W. et al. (1999) A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcriptional regulation. Mol. Cell 3, 97–108 23 Sun, X. et al. (1998) NAT, a human complex containing Srb polypeptides that functions as a negative regulator of activated transcription. Mol. Cell 2, 213–222 24 Naar, A.M. et al. (1999) Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828–832 25 Boyer, T.G. et al. (1999) Mammalian SRB/Mediator complex is targeted by adenovirus E1A protein. Nature 399, 276–279 26 Sato, S. et al. (2003) Identification of mammalian Mediator subunits with similarities to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3. J. Biol. Chem. 278, 15123–15127 27 Tomomori-Sato, C. et al. (2003) A mammalian mediator subunit that shares properties with Saccharomyces cerevisiae mediator subunit Cse2. J. Biol. Chem. 279, 5846–5851 28 Bourbon, H.M. et al. (2004) A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II. Mol. Cell 14, 553–557 29 Sato, S. et al. (2004) A set of consensus Mediator subunits identified by multidimensional protein identification technology. Mol. Cell 14, 685–691 30 Yuan, C.X. et al. (1998) 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. U. S. A. 95, 7939–7944 31 Ito, M. et al. (2000) Involvement of the TRAP220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action. Mol. Cell 5, 683–693 32 Ge, K. et al. (2002) Transcription coactivator TRAP220 is required for PPARg2-stimulated adipogenesis. Nature 417, 563–567 33 Kang, Y.K. et al. (2002) The TRAP/Mediator coactivator complex interacts directly with estrogen receptors a and b through the TRAP220 subunit and directly enhances estrogen receptor function in vitro. Proc. Natl. Acad. Sci. U. S. A. 99, 2642–2647 34 Malik, S. et al. (2002) 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 35 Malik, S. et al. (2002) 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
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36 Wang, S. et al. (2004) Role of Mediator in transcriptional activation by the aryl hydrocarbon receptor. J. Biol. Chem. 279, 13593–13600 37 Lau, J.F. et al. (2003) Role of metazoan Mediator proteins in interferon-responsive transcription. Mol. Cell. Biol. 23, 620–628 38 Kato, Y. et al. (2002) A component of the ARC/Mediator complex required for TGFb/Nodal signalling. Nature 418, 641–646 39 Stevens, J.L. et al. (2002) Transcription control by E1A and MAP kinase pathway via Sur2 mediator subunit. Science 296, 755–758 40 Mittler, G. et al. (2003) A novel docking site on Mediator is critical for activation by VP16 in mammalian cells. EMBO J. 22, 6494–6504 41 Yang, F. et al. (2004) The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator. Proc. Natl. Acad. Sci. U. S. A. 101, 2339–2344 42 Sato, S. et al. (2003) A mammalian homolog of Drosophila melanogaster transcriptional coactivator Intersex is a subunit of the mammalian Mediator complex. J. Biol. Chem. 278, 49671–49674 43 Garrett-Engele, C.M. et al. (2002) intersex, a gene required for femaile sexual development in Drosophila, is expressed in both sexes and functions together with doublesex to regulate terminal differentiation. Development 129, 4661–4675 44 Cantin, G.T. et al. (2003) Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA. Proc. Natl. Acad. Sci. U. S. A. 100, 12003–12008 45 Wu, S.Y. et al. (2003) Human mediator enhances activator-facilitated recruitment of RNA polymerase II and promoter recognition by TATAbinding protein (TBP) independently of TBP-associated factors. Mol. Cell. Biol. 23, 6229–6242 46 Johnson, K.M. et al. (2002) TFIID and human mediator coactivator complexes assemble cooperatively on promoter DNA. Genes Dev. 16, 1852–1863 47 Johnson, K.M. and Carey, M. (2003) Assembly of a Mediator/TFIID/ TFIIA complex bypasses the need for an activator. Curr. Biol. 13, 772–777 48 Akoulitchev, S. et al. (2000) TFIIH is negatively regulated by cdk8containing complexes. Nature 407, 102–106 49 Taatjes, D.J. et al. (2002) Structure, function, and activator-induced conformations of the CRSP coactivator. Science 295, 1058–1062 50 Wang, G. et al. (2001) Characterization of mediator complexes from HeLa cell nuclear extract. Mol. Cell. Biol. 21, 4604–4613 51 Mo, X. et al. (2004) Ras induces mediator complex exchange on C/EBPb. Mol. Cell 13, 241–250 52 Naar, A.M. et al. (2002) Human CRSP interacts with RNA polymerase II and adopts a specific conformation. Genes Dev. 16, 1339–1344 53 Fryer, C.J. et al. (2004) Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol. Cell 16, 509–520 54 Taatjes, D.J. and Tjian, R. (2004) Structure and function of CRSP/Med2: a promoter selective transcriptional coactivator complex. Mol. Cell 14, 675–683 55 Taatjes, D.J. et al. (2004) Distinct conformational states of nuclear receptor-bound CRSP-Med complexes. Nat. Struct. Mol. Biol. 11, 664–671 56 Taatjes, D.J. et al. (2004) Regulatory diversity amond metazoan coactivator complexes. Nat. Rev. Mol. Cell Biol. 5, 403–410 57 Chi, Y. et al. (2001) Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase. Genes Dev. 15, 1078–1092 58 Ansari, A.Z. et al. (2002) Transcriptional activating regions target a cyclin-dependent kinase. Proc. Natl. Acad. Sci. U. S. A. 99, 14706–14709 59 Hallberg, M. et al. (2004) Site-specific Srb10-dependent phosphorylation of the yeast Mediator subunit Med2 regulates gene expression from the 2-mm plasmid. Proc. Natl. Acad. Sci. U. S. A. 101, 3370–3375 60 Liu, Y. et al. (2004) Two cyclin-dependent kinases promote RNA polymerase II transcription and formation of the scaffold complex. Mol. Cell. Biol. 24, 1721–1735
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Mediator special issue
The mammalian Mediator complex and its role in transcriptional regulation Ronald C. Conaway1,2, Shigeo Sato1, Chieri Tomomori-Sato1, Tingting Yao1 and Joan W. Conaway1,2,3 1
Stowers Institute for Medical Research, Kansas City, MO 64110, USA Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, KS 66160, USA 3 Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA 2
Mediator is an essential component of the RNA polymerase II general transcriptional machinery and plays a crucial part in the activation and repression of eukaryotic mRNA synthesis. The Saccharomyces cerevisiae Mediator was the first to be defined and is a high molecular mass complex composed of O20 distinct subunits that performs multiple activities in transcription. Recent studies have defined the subunit composition and associated activities of mammalian Mediator, and revealed a striking evolutionary conservation of Mediator structure and function from yeast to man. Introduction The initiation stage of mRNA synthesis is a major site for the regulation of gene expression. In eukaryotes, mRNA synthesis is catalyzed by the multisubunit enzyme RNA polymerase II (pol II) and regulated by a host of DNAbinding transcription factors that activate or repress transcription in response to a myriad of signals emanating both from within the cell and from the cellular environment. Biochemical studies have shown that the initiation and regulation of eukaryotic mRNA synthesis requires a large collection of evolutionarily conserved ‘general’ transcription factors that seem to function at all, or most, genes. These general transcription factors include (i) the general initiation factors TFIIB, TFIID, TFIIE, TFIIF and TFIIH, which constitute the minimal set of auxiliary proteins necessary and sufficient for selective binding and accurate transcription initiation in vitro by pol II from the core regions of most promoters [1–4]; and (ii) the multiprotein Mediator complex, which functions, at least in part, as an adaptor that supports essential communication from transcription factors bound at upstream promoter elements and enhancers to pol II and the general initiation factors at the core promoter [5,6]. Although the compositions and many of the functions of the general initiation factors from yeast and higher eukaryotes were well established by the early 1990s, the structure and activities of the Mediator complex have only recently been illuminated. Mediator was first discovered and purified to near homogeneity from Saccharomyces Corresponding author: Conaway, J.W. ([email protected]). Available online 26 March 2005
cerevisiae by Kornberg and coworkers. They showed that it is required for transcription activation by the transcriptional activators Gcn4 or GAL4-VP16 in vitro using a reconstituted enzyme system composed of purified pol II and general initiation factors [7–9]. Yeast Mediator is composed of w20 subunits, which are present in three distinct Mediator subdomains referred to as the ‘head,’ ‘middle’ and ‘tail’ modules (reviewed in Ref. [10]) (Figure 1). An additional module, which includes a kinase–cyclin pair, is associated with a subset of yeast Mediator complexes and has, in yeast that is growing exponentially, been implicated in repression of a subset of genes [11–14]. Mammalian Mediator-like complexes were subsequently identified and characterized in several laboratories. However, whether the mammalian Mediator-like complexes isolated in different laboratories represented the same or different functional entities was unclear at first because they seemed to include distinct, but overlapping, sets of subunits. Furthermore, there was considerable controversy over the evolutionary relationship between yeast Mediator and mammalian Mediator-like complexes because there were obvious mammalian orthologs of only a subset of yeast Mediator subunits. As described in more detail later, recent efforts exploiting state-of-the-art proteomics methods have defined a set of consensus mammalian Mediator subunits, and improved bioinformatics approaches have revealed a striking evolutionary conservation of Mediator from yeast to man. Here, we discuss these recent developments in studies of the structure and function of mammalian Mediator. Isolation of mammalian Mediator-like complexes Mammalian Mediator-like complexes have been isolated by a variety of methods, including conventional and affinity chromatography. The first such complex was purified by Roeder and coworkers. It was designated the TRAP (thyroid hormone receptor-associated proteins) complex because it was isolated in association with the liganded thyroid hormone receptor (TR) [15,16]. Subsequently, related Mediator-like complexes were purified in several laboratories by conventional chromatography from mouse B cells [17], human HeLa cells [18–20], and rat liver [21] and designated mouse Mediator, CRSP
www.sciencedirect.com 0968-0004/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2005.03.002
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Figure 1. Comparison of mammalian Mediator-like complexes. Mediator subunits identified in different Mediator preparations are indicated with blue; Mediator subunits not identified are indicated with yellow ([29] and references therein). Subunits are grouped according to whether they are believed to be located in the head, middle or tail modules ([10] and references therein) or have not yet been assigned to a specific module. On the left portion of the table, the compositions of S. cerevisiae Mediator and various Mediator-like complexes from mammalian cells are summarized. The mammalian rat MED (rat mediator), TRAP, ARC, DRIP, mMED (mouse mediator), PC2, and CRSP Mediator-like complexes have been reported to fall into two size classes, large and small, and are grouped accordingly. The right portion summarizes the compositions determined by MudPIT of Mediator-like complexes purified via immunoaffinity chromatography of nuclear extracts prepared from extracts of HeLa cells expressing the indicated subunits with an N-terminal epitope tag with the amino sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (the FLAG epitope). f: denotes the FLAG epitope; proteins identified following immunoaffinity chromatography of extracts from parental HeLa or 293 cells are indicated.
(cofactor required for Sp1 activation) or PC2 (positive cofactor 2), and rat Mediator, respectively. In addition, Mediator-like complexes have been purified (i) in association with the liganded vitamin D receptor (VDR) from a HeLa cell line stably expressing FLAG-VDR and designated DRIP (vitamin D receptor-interacting proteins), (ii) by immunoaffinity chromatography from HeLa cell lines stably expressing epitope-tagged versions of mammalian orthologs of yeast Mediator subunits and designated SMCC [suppressor of RNA polymerase B (SRB)-mediatorcontaining cofactor] [22] or NAT (negative regulator of activated transcription) [23], and (iii) by affinity chromatography via interactions with immobilized transcriptional activation domains of the viral transactivators VP16 www.sciencedirect.com
and E1A, SREBP (sterol-response-element-binding protein), and the p65 subunit of NF-kB (nuclear factor-kB) and designated ARC (activator-recruited factor) or human Mediator [24,25]. The functional properties of these Mediator-like complexes have been characterized in vitro in transcription systems of varying purity and using both naked DNA and chromatin templates. Many of them, including the TRAP, SMCC, PC2, CRSP, human Mediator and DRIP complexes, have been reported to stimulate strongly activator-dependent transcription in vitro. In addition, the TRAP [22] and NAT [23] complexes have been reported to be capable of inhibiting activated transcription in vitro under some reaction conditions.
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The subunit composition of mammalian Mediator Analyses of proteins present in mammalian Mediator-like complexes from human, mouse and rat cells led to the identification of w30 potential mammalian Mediator subunits (reviewed in Ref. [5]). Only eight of these proteins could be unambiguously identified in pair-wise sequence alignments as orthologs of yeast Mediator subunits, which led to early suggestions that the mammalian Mediator-like complexes might be only distantly related in structure and function to the yeast Mediator. However, more sophisticated bioinformatic analyses exploiting multiple sequence alignments of Mediator subunits across many species have identified short regions of similarity between subunits of mammalian Mediatorlike complexes and all but three yeast Mediator subunits (MED2, MED3 and MED5) [10,21,26,27]. Further supporting the close evolutionary relationship between mammalian and yeast Mediator, pair-wise interactions between several of the mammalian Mediator proteins have been shown to recapitulate interactions between their presumptive yeast orthologs [26,27]. Recognition of these similarities motivated formulation of a unified nomenclature for Mediator subunits across species [28]; we use this new nomenclature to refer to specific Mediator subunits throughout this review. As noted, mammalian Mediator-like complexes purified in different laboratories using different purification procedures seemed to have distinct yet overlapping polypeptide compositions (Figure 1). These complexes fell into two classes: larger, 1–2 MDa complexes such as TRAP, DRIP and ARC, which were reported to include all or some combinations of the kinase module subunits MED12, MED13, Cyclin C, cyclin-dependent kinase CDK8; and smaller, 500–700 kDa complexes such as mouse Mediator, PC2 and CRSP, which lack kinase module subunits. Apparent differences in the subunit compositions of complexes that fell within these two classes were also notable. For example, the MED26 protein was initially identified only in the CRSP and DRIP complexes, the MED30 and MED31 proteins in the TRAP complexes, the MED8 protein only in the ARC and rat Mediator complexes, the MED25 protein only in the ARC, DRIP and CRSP complexes, the MED18 protein only in the mouse and rat Mediator complexes and the MED9, MED11, MED19, MED22, MED28 and MED29 proteins only in rat Mediator. As discussed in more detail later, some of the differences among mammalian Mediator-like complexes seem to reflect the existence of functionally distinct forms of Mediator in cells. Nevertheless, results of recent proteomic analyses suggest that at least some of the apparent differences between mammalian Mediator-like complexes might be due to use of insufficiently sensitive protein identification procedures and/or loss of some subunits during purification [29]. In these experiments, MudPIT (multidimensional protein identification technology), a sensitive proteomics method that links 2D chromatography and tandem mass spectrometry, was used to compare the subunit compositions of complexes purified by immunoaffinity chromatography from cultured HeLa or HEK293 cell lines stably expressing different epitopewww.sciencedirect.com
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tagged Mediator subunits. Complexes purified through any of four head-module subunits (MED8, MED19, MED28 or MED29) and two middle-module subunits (MED9 or MED10) included all 30 of the proteins previously identified as subunits of mammalian Mediator-like complexes (summarized in Figure 1). The only additional proteins associated with all of these complexes were pol II and alternative forms of the kinase module subunits MED12, MED13 and CDK8 (MED12L, MED13L and CDK11, respectively), raising the possibility that Mediator complexes containing different kinase modules might regulate different subsets of genes [29] (T. Yao, unpublished).
Role of mammalian Mediator in transcriptional regulation The mechanisms by which mammalian Mediator complexes control mRNA synthesis have not been firmly established. However, substantial evidence argues that Mediator activates transcription, at least in part, via direct interactions with DNA-binding transcriptional activators bound at upstream promoter elements and enhancers, with pol II and, most likely, with one or more of the general initiation factors bound at the core promoter. Notably, different Mediator subunits seem to be targets for interaction with the transcriptional activation domains (TADs) of different DNA-binding transcriptional activators. As a consequence, a major focus of current studies of the mechanism of action of mammalian Mediator complexes is to identify subunits that serve as contact sites for the large collection of DNA-binding transcriptional activators present in cells. The mammalian MED1 protein has an essential role in transcriptional activation by the large family of nuclear receptors. MED1 binds directly to liganded nuclear receptors via its L-eu-Xaa-Xaa-Leu-Leu (LxxLL) motifs, and depletion of MED1 specifically disrupts nuclearreceptor function both in cells and in vitro [30–35]. MED1 is also required for the activity of the arylhydrocarbon receptor and potentiates aryl-hydrocarbonreceptor-dependent transcription in cells in an LxxLLindependent manner [36]. Other Mediator subunits that serve as functional targets for TADs include: MED14, which interacts with STAT2 [37]; MED15, which interacts via its KIX (kinaseinducible interaction) domain with the SMAD2–SMAD4 and SMAD3–SMAD4 transcriptional activators in response to transforming growth factor-b signaling [38]; MED23, which interacts with the TADs of E1A, the etslike transcription factor Elk1, and CCAAT/enhancerbinding protein (C/EBPb) [25,28,39]; and MED25, which interacts with the potent VP16 TAD via its Von Willebrandassociated domain [40,41]. In addition, the mammalian Mediator subunit MED29 is the apparent ortholog of the Drosophila melanogaster Intersex protein, which interacts directly with, and functions as a transcriptional coactivator for, the DNA-binding transcription factor Doublesex [42,43]. Accordingly, it is likely that mammalian MED29 also serves as a target for one or more DNA-binding transcriptional activators. However, to date,
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mammalian MED29 has not been shown to function in the context of Mediator as a target for TADs. Results of biochemical experiments suggest that mammalian Mediator supports transcriptional activation, at least in part, by increasing the efficiency and/or rate of assembly of the pol II preinitiation complex. Consistent with this idea, the E1A and Elk1 transcriptional activation domains stimulate binding of Mediator subunits, pol II and general initiation factors to immobilized promoter DNA in extracts from wild-type cells but not in extracts from cells lacking MED23 [44], which is the target of the E1A and Elk1 TADs [25,28,39]. Mammalian Mediator complexes seem to affect several steps during assembly of the pol II preinitiation complex, including recruitment to the core promoter of TFIID (or its TATA-box binding subunit TBP) and of pol II and the other general initiation factors. Wu et al. [45] observed that Mediator complexes could enhance VP16 TAD-dependent recruitment to promoters of pol II – but not of the general initiation factors – in a highly purified, reconstituted transcription system when TFIID or TBP was present at saturating concentrations [45]. This suggests that Mediator can function at a step after the binding of TFIID (or TBP) to promoters. At low concentrations of TBP, however, Mediator complexes could also enhance activatordependent recruitment of TBP [45]. Carey and colleagues have shown that the model activator GAL4-VP16, Mediator and TFIID assemble onto promoters in a cooperative manner [46] and, furthermore, that – under appropriate reaction conditions – assembly of a complex that includes Mediator and either TFIID or TBP can bypass the requirement for a DNA-binding transcriptional activator for high levels of transcription in vitro [47]. Based on these observations, Johnson and Carey [47] proposed (i) that one function of activators might be to enhance the stability of a TFIID–Mediator complex at the promoter and (ii) that this TFIID–Mediator complex might serve as a stable platform for reiterative assembly of the pol II preinitiation complex. Evidence for multiple, functionally distinct forms of Mediator Although the results of proteomic studies suggest that many of the apparent differences in the compositions of Mediator purified by different laboratories or according to different procedures might be due to a lack of sensitivity in the protein identification methods used. Substantial evidence argues that some of these differences, indeed, reflect the existence of multiple forms of Mediator that either positively or negatively regulate transcription. In particular, several lines of evidence suggest that the kinase module can exert a repressive function when associated with the mammalian Mediator, whereas the MED26 protein is associated with an activating form of Mediator. First, it has been reported that Mediator complexes containing the kinase module have little or no effect on, or actually inhibit, in vitro transcription that is activated by the VP16, SREBP, Sp1 and other TADs [20,23,48,49]. By contrast, complexes that lack the kinase module [20,49] and, in at least some cases, include MED26 [49] support strong activation of transcription in vitro by these same TADs. Although several Mediator preparations www.sciencedirect.com
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that include the kinase module, such as TRAP, DRIP, human Mediator and ARC, have been shown to support activator-dependent transcription in vitro, results of fractionation studies suggest that these preparations contain mixtures of Mediator complexes that do and do not include MED12, MED13, CDK8 and Cyclin C [20,49,50]. Second, Leutz and coworkers have provided evidence that (i) the transcription factor C/EBPb represses transcription of target genes in cells by recruiting Mediator with the kinase module to promoters and (ii) RAS–MAPK-dependent C/EBPb phosphorylation might function as a switch that leads to release of the CDK8 module from Mediator and transcriptional activation [51]. How might association of the kinase module with Mediator complexes render them inactive or even inhibitory? The available evidence indicates several possibilities that are not mutually exclusive. First, the kinase module might interfere with the ability of Mediator complexes to bind pol II, either by phosphorylating polymerase and/or Mediator subunits or by simple steric hindrance. Results of one study indicate that mammalian Mediator complexes containing MED26 and lacking the kinase module bind to an immobilized GST (glutathione S transferase)fusion protein containing the C-terminal domain (CTD) of the largest pol II subunit, whereas complexes that include the kinase module do not [52]. In addition, MudPIT analyses of Mediator complexes immunopurified through CDK8 indicate that little or no pol II is associated with Mediator that contains stoichiometric amounts of kinase module (J.W. Conaway et al., unpublished results), whereas complexes purified through MED26, which contain only a small amount of kinase module, are associated with near stoichiometric amounts of polymerase [29]. Second, Mediator-associated kinase might phosphorylate and inactivate one or more of the general initiation factors. Consistent with this possibility, CDK8–Cyclin C can phosphorylate the Cyclin H subunit of mammalian TFIIH, blocking the activity of the TFIIH CTD kinase and inhibiting TFIIH activity in transcription [48]. Finally, as suggested by a recent study of the role of CDK8 in Notch-dependent regulation of the HES1 (hairyenhancer of split 1) promoter [53], recruitment of the kinase module to mammalian Mediator at promoters might target DNA-binding transcription factors for phosphorylation and, ultimately, for degradation via the ubiquitin-dependent proteolytic system, providing a mechanism to limit the length of time a given promoter is active. Upon activation of the Notch-signaling pathway, the Notch intracellular domain (ICD) translocates into the nucleus and forms a Notch enhancer complex with the DNA-binding transcription factor CBF1 and its coactivator Mastermind, leading to transient activation of Notch-responsive genes, including HES1. Fryer et al. [53] observed that CDK8, and presumably the entire kinase module, is recruited to the HES1 promoter by the Notch enhancer complex a short time after recruitment of other Mediator subunits and HES1 activation, coincident with the loss of Notch from the promoter and subsequent down-regulation of the gene. In further experiments, they showed that CDK8-dependent phosphorylation of the Notch ICD targets it for ubiquitination and degradation.
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Thus, transient activation of HES1 might be a result of initial recruitment of an activating form of Mediator to the promoter, followed shortly by recruitment of the Mediator kinase module and phosphorylation and destruction of the Notch ICD. Concluding remarks and future directions Significant progress in characterizing the structure and function of the mammalian Mediator complex has been achieved. A set of consensus mammalian Mediator subunits has been identified, and the striking evolutionary conservation of Mediator has been revealed. With the definition of what seems to be a complete set of ‘core’ mammalian Mediator subunits, an in-depth characterization of the roles these subunits have in reconstitution of the transcriptionally active Mediator complex is now possible. Major questions for future studies include: † How many functionally distinct forms of Mediator participate in pol II transcriptional regulation? As discussed, it is likely that Mediator assemblies both including and lacking the kinase module are present in mammalian cells and perform distinct functions in transcriptional regulation. The identification of isoforms of several subunits of the kinase module [29] raises the possibility that different kinase modules are operative at different promoters and/or in different cell types. Finally, Mediator complexes that lack subunits, such as MED1 and MED25 – which function as docking sites for specific TADs – have been described [40,54]. Although such complexes could simply result from dissociation of subunits during purification, it is also possible that alternative forms of Mediator that contain different repertoires of TAD-docking sites might function at different promoters in cells. † Which Mediator subunits orchestrate its interactions with the diverse collection of DNA-binding transcriptional activators present in mammalian cells? And does interaction of Mediator with TADs alter Mediator activities in ways dependent on the nature of the TAD? Notably, analyses of Mediator structure by electron microscopy and signal-particle reconstructions suggest that the binding of different activators to different subunits, or even the binding of different activators to the same subunit, causes Mediator complexes to adopt remarkably different structures [49,55]. These observations led to the proposal by Tjian and colleagues that Mediator complexes with the same polypeptide compositions could have quite different activities when brought to promoters via interactions with different TADs [56]. † How does the mammalian Mediator complex promote pol II transcriptional activation? Do Mediator and its subunits simply recruit polymerase to promoters or do they also directly regulate the activities of polymerase and the general initiation factors during transcription, promoter escape and perhaps transcription elongation. † What is the repertoire of enzymatic activities associated with Mediator, and what are their functions? It is likely that the mammalian Mediator-associated kinase CDK8 and CDK11 phosphorylate substrates in addition to those described here. Yeast CDK8, like its www.sciencedirect.com
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mammalian ortholog, is capable of phosphorylating DNA-binding transcriptional regulatory proteins, in some cases, targeting them for ubiquitination [57,58]. In addition, yeast CDK8 can phosphorylate other substrates, such as the Mediator subunit MED2 [59] and two of the TAF (TBP-associated transcription factor) subunits of TFIID [60]. Future experiments exploring the repertoire of substrates phosphorylated by mammalian Mediator-associated kinases are likely to provide new insights into their contributions to transcriptional regulation. In addition, evidence that the MED8 subunit of the mammalian Mediator complex is capable of assembling together with the Elongin B, Elongin C, Cul2 and Rbx1 proteins into a potential E3 ubiquitin ligase have raised the possibility that MED8 might facilitate recruitment of ubiquitin ligase activity directly to Mediator as part of its function in mRNA synthesis [21]. Clearly, many more studies addressing the functions of the intact mammalian Mediator complex in addition to its individual subunits will be necessary to provide a complete picture of the mechanism of Mediator action in pol II transcriptional regulation. Acknowledgements Work in the authors’ laboratory is supported, in part, by National Institutes of Health Grant R37 GM41628.
References 1 Conaway, R.C. et al. (1991) Mechanism of promoter selection by RNA polymerase II: mammalian transcription factors a and bg promote entry of polymerase into the preinitiation complex. Proc. Natl. Acad. Sci. U. S. A. 88, 6205–6209 2 Conaway, J.W. and Conaway, R.C. (1997) General transcription factors for RNA polymerase II. Prog. Nucleic. Acids. Res. Mol. Biol. 56, 327–346 3 Orphanides, G. et al. (1996) The general transcription factors of RNA polymerase II. Genes Dev. 10, 2657–2683 4 Roeder, R.G. (1996) The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem. Sci. 21, 327–335 5 Myers, L.C. and Kornberg, R.D. (2000) Mediator of transcriptional regulation. Annu. Rev. Biochem. 69, 729–749 6 Koleske, A.J. and Young, R.A. (1995) The RNA polymerase II holoenzyme and its implications for gene regulation. Trends Biochem. Sci. 20, 113–116 7 Kim, Y.J. et al. (1994) A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77, 599–608 8 Li, Y. et al. (1995) Yeast global transcriptional regulators Sin4 and Rgr1 are components of mediator complex/RNA polymerase II holoenzyme. Proc. Natl. Acad. Sci. U. S. A. 92, 10864–10868 9 Myers, L.C. et al. (1998) The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain. Genes Dev. 12, 45–54 10 Boube, M. et al. (2002) Evidence for a Mediator of RNA polymerase II transcriptional regulation conserved from mammals to yeast. Cell 110, 143–151 11 Liao, S.M. et al. (1995) A kinase-cyclin pair in the RNA polymerase II holoenzyme. Nature 374, 193–196 12 Hengartner, C.J. et al. (1998) Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases. Mol. Cell 2, 43–53 13 Holstege, F.C.P. et al. (1998) Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717–728 14 Carlson, M. (1997) Genetics of transcriptional regulation in yeast: connections to the RNA polymerase II CTD. Annu. Rev. Cell Dev. Biol. 13, 1–23
Review
TRENDS in Biochemical Sciences
15 Fondell, J.D. et al. (1996) Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc. Natl. Acad. Sci. U. S. A. 93, 8329–8333 16 Fondell, J.D. et al. (1999) Thyroid hormone receptor-associated proteins and general positive cofactors mediate thyroid hormone receptor function in the absence of the TATA box-binding protein-associated factors of TFIID. Proc. Natl. Acad. Sci. U. S. A. 96, 1959–1964 17 Jiang, Y. et al. (1998) Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways. Proc. Natl. Acad. Sci. U. S. A. 95, 8538–8543 18 Ryu, S. et al. (1999) The transcriptional cofactor complex CRSP is required for activity of the enhancer binding protein Sp1. Nature 397, 446–450 19 Ryu, S. and Tjian, R. (1999) Purification of transcription cofactor complex CRSP. Proc. Natl. Acad. Sci. U. S. A. 96, 7137–7142 20 Malik, S. et al. (2000) The U. S. A.-derived transcriptional coactivator PC2 is a submodule of TRAP/SMCC and acts synergistically with other PC’s. Mol. Cell 5, 753–760 21 Brower, C.S. et al. (2002) Mammalian mediator subunit MED8 is an Elongin BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a ubiquitin ligase. Proc. Natl. Acad. Sci. U. S. A. 99, 10353–10358 22 Gu, W. et al. (1999) A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcriptional regulation. Mol. Cell 3, 97–108 23 Sun, X. et al. (1998) NAT, a human complex containing Srb polypeptides that functions as a negative regulator of activated transcription. Mol. Cell 2, 213–222 24 Naar, A.M. et al. (1999) Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828–832 25 Boyer, T.G. et al. (1999) Mammalian SRB/Mediator complex is targeted by adenovirus E1A protein. Nature 399, 276–279 26 Sato, S. et al. (2003) Identification of mammalian Mediator subunits with similarities to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3. J. Biol. Chem. 278, 15123–15127 27 Tomomori-Sato, C. et al. (2003) A mammalian mediator subunit that shares properties with Saccharomyces cerevisiae mediator subunit Cse2. J. Biol. Chem. 279, 5846–5851 28 Bourbon, H.M. et al. (2004) A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II. Mol. Cell 14, 553–557 29 Sato, S. et al. (2004) A set of consensus Mediator subunits identified by multidimensional protein identification technology. Mol. Cell 14, 685–691 30 Yuan, C.X. et al. (1998) 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. U. S. A. 95, 7939–7944 31 Ito, M. et al. (2000) Involvement of the TRAP220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action. Mol. Cell 5, 683–693 32 Ge, K. et al. (2002) Transcription coactivator TRAP220 is required for PPARg2-stimulated adipogenesis. Nature 417, 563–567 33 Kang, Y.K. et al. (2002) The TRAP/Mediator coactivator complex interacts directly with estrogen receptors a and b through the TRAP220 subunit and directly enhances estrogen receptor function in vitro. Proc. Natl. Acad. Sci. U. S. A. 99, 2642–2647 34 Malik, S. et al. (2002) 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 35 Malik, S. et al. (2002) 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
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36 Wang, S. et al. (2004) Role of Mediator in transcriptional activation by the aryl hydrocarbon receptor. J. Biol. Chem. 279, 13593–13600 37 Lau, J.F. et al. (2003) Role of metazoan Mediator proteins in interferon-responsive transcription. Mol. Cell. Biol. 23, 620–628 38 Kato, Y. et al. (2002) A component of the ARC/Mediator complex required for TGFb/Nodal signalling. Nature 418, 641–646 39 Stevens, J.L. et al. (2002) Transcription control by E1A and MAP kinase pathway via Sur2 mediator subunit. Science 296, 755–758 40 Mittler, G. et al. (2003) A novel docking site on Mediator is critical for activation by VP16 in mammalian cells. EMBO J. 22, 6494–6504 41 Yang, F. et al. (2004) The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator. Proc. Natl. Acad. Sci. U. S. A. 101, 2339–2344 42 Sato, S. et al. (2003) A mammalian homolog of Drosophila melanogaster transcriptional coactivator Intersex is a subunit of the mammalian Mediator complex. J. Biol. Chem. 278, 49671–49674 43 Garrett-Engele, C.M. et al. (2002) intersex, a gene required for femaile sexual development in Drosophila, is expressed in both sexes and functions together with doublesex to regulate terminal differentiation. Development 129, 4661–4675 44 Cantin, G.T. et al. (2003) Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA. Proc. Natl. Acad. Sci. U. S. A. 100, 12003–12008 45 Wu, S.Y. et al. (2003) Human mediator enhances activator-facilitated recruitment of RNA polymerase II and promoter recognition by TATAbinding protein (TBP) independently of TBP-associated factors. Mol. Cell. Biol. 23, 6229–6242 46 Johnson, K.M. et al. (2002) TFIID and human mediator coactivator complexes assemble cooperatively on promoter DNA. Genes Dev. 16, 1852–1863 47 Johnson, K.M. and Carey, M. (2003) Assembly of a Mediator/TFIID/ TFIIA complex bypasses the need for an activator. Curr. Biol. 13, 772–777 48 Akoulitchev, S. et al. (2000) TFIIH is negatively regulated by cdk8containing complexes. Nature 407, 102–106 49 Taatjes, D.J. et al. (2002) Structure, function, and activator-induced conformations of the CRSP coactivator. Science 295, 1058–1062 50 Wang, G. et al. (2001) Characterization of mediator complexes from HeLa cell nuclear extract. Mol. Cell. Biol. 21, 4604–4613 51 Mo, X. et al. (2004) Ras induces mediator complex exchange on C/EBPb. Mol. Cell 13, 241–250 52 Naar, A.M. et al. (2002) Human CRSP interacts with RNA polymerase II and adopts a specific conformation. Genes Dev. 16, 1339–1344 53 Fryer, C.J. et al. (2004) Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol. Cell 16, 509–520 54 Taatjes, D.J. and Tjian, R. (2004) Structure and function of CRSP/Med2: a promoter selective transcriptional coactivator complex. Mol. Cell 14, 675–683 55 Taatjes, D.J. et al. (2004) Distinct conformational states of nuclear receptor-bound CRSP-Med complexes. Nat. Struct. Mol. Biol. 11, 664–671 56 Taatjes, D.J. et al. (2004) Regulatory diversity amond metazoan coactivator complexes. Nat. Rev. Mol. Cell Biol. 5, 403–410 57 Chi, Y. et al. (2001) Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase. Genes Dev. 15, 1078–1092 58 Ansari, A.Z. et al. (2002) Transcriptional activating regions target a cyclin-dependent kinase. Proc. Natl. Acad. Sci. U. S. A. 99, 14706–14709 59 Hallberg, M. et al. (2004) Site-specific Srb10-dependent phosphorylation of the yeast Mediator subunit Med2 regulates gene expression from the 2-mm plasmid. Proc. Natl. Acad. Sci. U. S. A. 101, 3370–3375 60 Liu, Y. et al. (2004) Two cyclin-dependent kinases promote RNA polymerase II transcription and formation of the scaffold complex. Mol. Cell. Biol. 24, 1721–1735