TIBS 25 – JUNE 2000 REVIEWS termed Mediator, that was necessary to support activated transcription in vitro in conjunction with general initiation Transcriptional regulation factors8, but apparently distinct from the TAF (Ref. 5) and USA (Ref. 7) coacti- through Mediator-like vators identified in metazoans (see below). Concurrent genetic analysis also implicated two broad groups of pro- coactivators in yeast and teins in transcriptional regulation in vivo. The SRB (suppressor of RNA metazoan cells polymerase B) proteins first emerged from genetic screens for suppressors of partial truncations in the C-terminal domain (CTD) of the largest subunit of Sohail Malik and Robert G. Roeder Pol II (Ref. 9). Other gene products, including GAL11, RGR1 and SIN4, emerged from genetic screens for regulatory A novel multiprotein complex has recently been identified as a coactivator for factors that function in other path- transcriptional control of protein-encoding genes by RNA polymerase II in ways10 (see below). Subsequent studies higher eukaryotic cells. This complex is evolutionarily related to the Mediator aimed at purifying the Mediator activity complex from yeast and, on the basis of its structural and functional charac- and the SRB proteins resulted in the teristics, promises to be a key target of diverse regulatory circuits. identification of a Pol II holoenzyme that contained the 12-subunit core Pol II, SRBs, other genetically identified TRANSCRIPTIONAL REGULATION IN factors that mediate, and might integrate, polypeptides (including GAL11, RGR1, eukaryotes is achieved through multi- the effects of transcriptional activators SIN4) and novel Mediator polypeptides protein complexes assembled at the on the Pol II basal machinery2. As dis- (MEDs) in a single complex11–13. In some enhancer and promoter regions of tar- cussed in this article, coactivators are cases, the holoenzyme was also found get genes. In the case of protein-coding explicitly defined as factors that are re- to contain a subset of GTFs12. genes, RNA polymerase II (Pol II) and its quired for the function of DNA-binding Together with the finding that SRB associated general transcription factors activators, but not for basal transcrip- proteins are also direct targets for acti- (GTFs) (TFIIA, B, D, E, F and H) are suffi- tion per se, and do not show site-specific vators14, these developments offered a cient for recognition and low levels of binding by themselves. unified hypothesis for transcriptional accurate transcription from common Earlier studies had emphasized the activation in yeast that invokes the inte- core promoter elements in vitro (basal role of TBP (TATA-box-binding protein)- gration of activation pathways through transcription)1. associated factors (TAFs) within TFIID the same molecular entity. Indeed, in For the most part, the components of (Refs 3,4; reviewed in Ref. 5) and compo- transcription systems reconstituted this basal transcription machinery are nents within the partially purified with purified GTFs, the purified holo- required globally. By contrast, a second upstream stimulatory activity (USA) enzyme (in contrast to the core Pol II) class of transcription factors, the large fraction (Ref. 6; reviewed in Ref. 7) as can support activated transcription12,13. group of transcriptional activators that essential coactivators. More-recent The global significance of the SRB- and typically assemble at distal enhancer developments in the field have focused MED-containing holoenzyme was further sites, show a great deal of variability in attention on metazoan orthologs of the illustrated by a genome-wide analysis of their cell-type and gene specificities and yeast Mediator, the coactivator compo- gene expression in yeast, which in the spectrum of factors bound to a nent of the Pol II holoenzyme that has revealed that the phenotype of a mutation particular enhancer. A major outstand- proved to be central to transcriptional in one of the subunits (SRB4) was virtually ing issue concerns the mechanisms by regulation in yeast, and have led simul- indistinguishable from that of a Pol II which the activation potential of diverse taneously to a convergence of yeast and subunit mutation that resulted in a enhancer-bound factors is translated metazoan coactivator studies. complete shut-off of transcription from into increased activity of the basal tran- essentially the entire genome15. scription machinery on target genes The Yeast Mediator/RNA polymerase II The association of the SRB, MED and (activated transcription). holoenzyme other proteins with Pol II is reversible Despite the complexity of this basal The identification of a novel entity, and of relevance to the case in meta- machinery (.40 polypeptides), its re- called the Pol II holoenzyme, as the ulti- zoans (see below). A reversible associa- sponse to activators on specific target mate target of transcriptional activators tion was first suggested by the release of genes is still dependent on additional was initially the outcome of biochemical a free Mediator complex (SRB2–SRB4– factors called coactivators, which have and genetic studies in yeast. Such studies SRB5–SRB6–SRB7–MED1–MED2–MED4– emerged as a third class of transcription were spurred, in part, by the observation MED6–MED7–MED8–ROX3–CSE2–NUT1– that metazoan activators also function in NUT2–HRS1/PGD1–GAL11–RGR1–SIN4) yeast, thus indicating a high degree of after treatment of the Pol II holoenzyme S. Malik and R.G. Roeder are in the 13 Laboratory of Biochemistry and Molecular functional conservation of the transcrip- with an anti-CTD antibody (Fig. 1a). Biology, The Rockefeller University, New York, tion apparatus over eukaryotic evolution. Moreover, it now appears that signifi- NY 10021, USA. Biochemical analysis first pointed to cant amounts of free Mediator coexist Email: [email protected] the existence of a coactivator activity, with the holoenzyme-bound form16. 0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved. PII: S0968-0004(00)01596-6 277 REVIEWS TIBS 25 – JUNE 2000 of transcription10. Because of this, the (a) MED9 Mediator/holoenzyme complex has often ROX3 MED1 been interpreted as a multicomponent CSE2 SRB4 ‘control panel’ that can integrate a vari- MED2 ety of positive and negative regulatory MED8 HRS1/PGD1 SRB8 SRB7 NUT2 signals. SRB5 Metazoan Mediator complexes SRB2 MED7 SIN4 SRB9 MED4 Given the impetus from the findings RGR1 in yeast, several Pol II holoenzymes were subsequently described in meta- SRB11/ MED6 cyc C zoans (reviewed in Ref. 17). These holoenzyme preparations contained, in SRB6 GAL11 some cases, homologs of SRB7, SRB10 SRB10/ CDK8 or SRB11, in addition to a wide range of GTFs, putative coactivators [including Yeast Mediator CREB-binding protein (CBP)] and fac- tors implicated in nuclear processes (b) other than transcription (e.g. DNA re- pair). However, the exact relevance of TRAP230 TRAP240 these preparations to transcriptional ac- p22 TRAP95 tivation has remained unclear. Thus, al- p37 p24 though it became apparent that Pol II TRFP can associate with many factors in iso- TRAP100 SRB7 p12 TRAP80 lated extracts and chromatographic fractions – not unexpected in view of the TRAP97 protein–protein interactions that are in- NUT2 TRAP150β SRB11/ MED7 trinsic to the formation of the preiniti- cyc C TRAP93 ation complex – a coactivator moiety TRAP170/RGR1 analagous to the relatively discrete p78 p36 Mediator component of the yeast SRB10/ MED6 SOH1 holoenzyme was not revealed through CDK8 TRAP220 this line of inquiry. However, more-recent studies of this problem using different approaches Human Mediator (TRAP/SMCC) Ti BS have uncovered a set of mammalian Mediator-like coactivator complexes. The best characterized of these, an SRB- Figure 1 Modular structure of the Mediator complex. (a) Yeast Mediator has various modules, includ- and MED-containing cofactor complex ing a core, that have been identified through genetic and biochemical studies. The core designated SMCC, was isolated on the Mediator subunits are further organized into two submodules. The RGR1 submodule basis of resident homologs to Mediator (RGR1–MED1–MED2–MED4–MED7–MED8–MED9–CSE2–NUT2–SRB7) is shown in light red; and holoenzyme components (from cell the other submodule (MED6–SRB2–SRB4–SRB5–SRB6–ROX3) is green. The dissociable lines stably expressing epitope-tagged GAL11–SIN4–HRS1/PGD1 module, dedicated to specialized activators, is shown in light human SRB7, SRB10 or SRB11), and an purple. RGR1, which interacts with the GAL11–SIN4–HRS1/PGD1 submodule, is shown as ability to mediate activation by GAL4 one of the core subunits, as is MED2, whose interaction with the Mediator is dependent 18 upon HRS1/PGD1 (Ref. 35). SRB8, SRB9, SRB10 and SRB11 constitute another module derivatives . SMCC is a 1.5 MDa com- (yellow) but they are variably associated with the holoenzyme and potentially with the free plex of ~25 polypeptides that include Mediator complex, reflecting the variation in their metabolic-state-dependent intracellular human orthologs of yeast SRB10, SRB11, levels. (b) The metazoan Mediator (TRAP/SMCC) core subunits, defined as those invariably SRB7, MED6, MED7, NUT2 and RGR1 found in PC2 (Fig. 2) are colored either light red or dark red and include TRAP170/RGR1, (Figs 1b,2). SMCC also contains a TRAP150b, TRAP95, TRAP80, p78, p37, MED7, p24, p22, SRB7, SOH1, NUT2 and p12. homolog of yeast SOH1, a positive regu- MED6 (green) might represent a vestige of the yeast MED6–SRB4 submodule. Relatively lator of transcription