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termed , 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) 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 products, including GAL11, RGR1 and SIN4, emerged from genetic screens for regulatory A novel multiprotein complex has recently been identified as a for factors that function in other path- transcriptional control of -encoding 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 (Ͼ40 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 TRAP150␤, 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 that interacts labile subunits that, like TRAP220, might constitute additional specialized submodules are shown in blue. SRB10/CDK8 and SRB11/cyc C, which, as in yeast, are variably associated genetically with HPR1, a putative Pol II- with the metazoan complex and might reflect physiological variations, are in yellow. Subunits interacting protein, as well as with TFIIB in yellow, green or light red are conserved, in whole or in part, in yeast. and subunits of Pol II (reviewed in Ref. 18). However, consistent with the re- versible association of yeast Mediator Apart from the core Pol II subunits and with metabolic-state-dependent fluctua- and Pol II, the most stringently purified GTFs, the free Mediator also lacks SRB8, tions in the intracellular levels of these SMCC does not contain Pol II (see below). SRB9, SRB10 and SRB11 (Ref. 16). These proteins15. Importantly, SMCC contains additional have only been found in some prepara- Some of the polypeptides (e.g. GAL11, polypeptides with no discernible similarity tions of Pol II holoenzyme and, consis- RGR1, SIN4 and HRS1/PGD1) found in the to yeast proteins. tent with the physiological relevance of Mediator had been identified genetically Remarkably, some of these polypep- this variation, their presence correlates as either positive or negative modulators tides (including TRAP240, TRAP230, 278 TIBS 25 – JUNE 2000 REVIEWS

TRAP220, TRAP150, TRAP100, TRAP97, TRAP/ PC2 DRIP ARC CRSP muMED NAT TRAP93, TRAP80 and TRAP170, which SMCC displays regions of sequence similarity to yeast RGR1) proved to be identical to 240 250 250 components previously found in the thyroid-hormone-receptor-associated 230 240 240 160a 230 protein (TRAP) coactivator complex18–20. + 220 205 200 200 160b The TRAP complex had been purified several years earlier on the basis of its *RGR1 170 170 150 150 150 110 150 intracellular association and copurifica- 150 α tion with ligand-bound thyroid-hormone 140 receptor (TR) and its ability to function SUR2 150 β 150 β 130 130 130 as a cognate coactivator20 with USA- 20,21 TIG1 105 derived cofactors . + + Subsequent analysis revealed a near 100 100 100 100 100 95 identity between the subunit compos- 97 + (97) 96a itions of SMCC and the TRAP complex, 97 including, in addition to the above- 95 95 92 96b 90 mentioned proteins, a human homolog + + of TBP-related-factor-proximal protein 93 93 (92) 85 (TRFP) and additional proteins provi- 80 77 77 77 78 70 sionally designated p37, p36, p24, p22 80 and p12 (Ref. 19). Consistent with these TFIIS-related 78 78 70 70 70 observations, the SMCC and TRAP com- plexes were found to be functionally *SRB10/CDK8 56 56 equivalent in their ability to support ac- 42 55 45 tivation by a number of activators that include TR, p53 and herpesvirus virion 37 37 34 protein 16 (VP16) (Refs 18,19). Hence, + 37 TRAP and SMCC can be regarded as 36/28 36/28 36 36 34/33 equivalent entities. Accordingly, we *MED7 34 34 34 34 33 36 36 refer to the human Mediator-like com- plex as TRAP/SMCC. This convergence *SRB11/CycC 33 31 of distinct coactivator studies not only + *MED6 traces the original description of a 32 32 33 33 32 33 structurally defined human Mediator- 32 28b 30 like complex back to the identification of the TRAP complex20 but also re- TRFP 26 26 28a 23 inforces the significance of TRAP/SMCC 24 24 22 for activator function in vivo as well as 22 21 in vitro. In still another case of conver- 22 gence, we have now found that the pre- *SRB7 21 17 viously described USA-derived cofactor 19 19 PC2 (Refs 6,22) is also a Mediator-like *SOH1 18 18 complex23 (Figs 1b,2). Several other Mediator-like com- *NUT2/MED10 15 15 14 plexes have been reported recently, in- 12 12 cluding DRIP (Ref. 24), ARC (Ref. 25), NAT (Ref. 26), murine Mediator27, CRSP Ti BS (Ref. 28) and a human SUR2-containing complex whose molecular composition Figure 2 has not been fully defined but that ap- Comparative chart showing the relationships in polypeptide composition of various Mediator-like complexes: TRAP/SMCC (Refs 18,19); PC2 (Ref. 23); DRIP (Ref. 24); ARC pears to contain SRB10, SRB11 and 27 MED7 (Ref. 29). Some of these com- (Ref. 25); CRSP (Ref. 28); murine Mediator (muMED) ; and NAT (Ref. 26). Each column de- picts the components of a single complex, whereas each row indicates the presence (blue box) plexes, like the TRAP complex, were iso- or clearly established absence (yellow box) of a given subunit within the different complexes. lated on the basis of their ability to Boxed numbers indicate the molecular weights (in kDa) of subunits whose identities have interact with specific activators [DRIP been firmly established from reported amino acid sequences or immunoblot analyses; un- through ligand-bound vitamin-D recep- boxed numbers indicate the molecular weights (in kDa) of polypeptides whose sequences tor, ARC through SREBP (sterol-response- have not yet been reported. Horizontal lines linking the boxes indicate that the subunits are Ϯ element-binding protein) and VP16, and identical. Other designations for selected subunits are given to the left. The signs in PC2 and CRSP indicate subunits that are variably associated. Asterisks mark subunits with ho- the SUR2 complex through E1A] and mology to yeast Mediator subunits. For p36 in TRAP/SMCC and PC2, and for p34 in were subsequently shown to mediate muMED, corresponding degradation products are also indicated. For TRAP/SMCC, 150␣ activator function in various in vitro and 150␤ refer to two polypetides that are both present in the band previously referred to assays. Others, like SMCC, were isolated as TRAP150 (Refs 19,20). 279 REVIEWS TIBS 25 – JUNE 2000 on the basis of specific intrinsic Structural and functional modularity of the yeast med6ts mutants39. It is thus possi- subunits related to yeast Mediator com- Mediator ble that the accretion of several regula- ponents (SRB10/CDK8 for NAT, SRB7 and The modular organization of the yeast tory modules (the SRB10–SRB11 MED7 for murine Mediator). Only CRSP Mediator first became apparent from kinase–cyclin pair, TRAP220, etc.) around and PC2 were first observed as coactiva- combined genetic and biochemical stud- the PC2 core could generate a pluripo- tor activities [CRSP for Sp1 (Ref. 28), and ies of components that had originally tent entity (TRAP/SMCC) that can re- PC2 both for a variety of natural activa- been identified as dedicated transcrip- spond to a variety of cellular signals. In tors, including Sp1 (Refs 6,30), and tion factors. For example, GAL11 and light of this hypothesis, it is also intrigu- for GAL4-based activators22] and later SIN4 were originally identified in screens ing that a mitogen-activated nuclear ki- identified as Mediator-like complexes. examining galactose utilization and nase, RING3, was found associated with Preparations of NAT (and SMCC under mating-type switching, respectively10. the murine Mediator27. Similarly, the selective assay conditions18) have been Like the SRB8, SRB9, SRB10 and SRB11 TIG1 subunit of the ARC complex is reported to regulate activated transcrip- proteins, which form one subcomplex encoded by one of many genes that are tion negatively26. Although this has been (cited in Ref. 33), GAL11, SIN4 and an- induced by the effector 12-o-tetrade- ascribed to an SRB10/CDK8 kinase func- other genetically defined polypeptide, canoylphorbol-13-acetate25. Furthermore, tion (consistent with results in yeast31), HRS1/PGD1, were subsequently found to consistent with a likely role of SRB7 as a the affinity methods used for selecting constitute another dissociable subcom- core subunit, disruption of the ubiqui- NAT and SMCC (through SRB10/CDK8) plex within the Mediator, to which they tously expressed mouse SRB7 gene was also select a distinct SRB10/CDK8– are anchored through RGR1 (Ref. 34). found to be embryonic lethal40. SRB11/cyclin C complex that might have The inactivation and actual physical sep- None of the metazoan Mediator-like contributed to the negative regulation. aration of the GAL11–SIN4–HRS1/PGD1 complexes described to date contain Nonetheless, these results are not subcomplex by mutation (deletion of readily apparent homologs of yeast inconsistent with NAT having an (as-yet SIN4 or of the RGR1 C-terminus) appears SRB2, SRB4, SRB5 or SRB6. As mentioned, unreported) coactivator function as well to have no effect on basal transcription these constitute a distinct submodule to- as an intrinsic inhibitory activity, espe- and only selective effects on activated gether with MED6 (Refs 37,38) and are cially in light of the coexistence of posi- transcription, which is consistent with a critical for activator interactions38, al- tive and negative functions in the same specialized role for the constituent beit not sufficient for activator func- complex in yeast10. A coactivator or co- polypeptides in regulating the target tion35. Given that the subunit composi- repressor function has not been de- genes for which they were selected34–36. tions of metazoan complexes indicate a scribed for the murine Mediator27. This further suggested that the Mediator closer resemblance to the yeast RGR1 Smaller polypeptides (Ͻ30 kDa) were core is composed of the remaining submodule, it is possible that a bona fide not reported for the DRIP, ARC and CRSP polypeptides (reviewed in Ref. 33) (Fig. 1a). MED6–SRB2–SRB4–SRB5–SRB6 module complexes. Apart from this, a compari- The modular organization of the yeast remains to be discovered. son of the polypeptide compositions of Mediator is also evident from purely bio- The low-resolution three-dimensional the various complexes (Fig. 2) reveals chemical analyses, which revealed two structures of the yeast and murine that, in essence, they represent either submodules37: RGR1–GAL11–HRS1/PGD1– Mediators deduced from electron-micro- the same or a very similar cellular entity SIN4–MED1–MED2–MED4–MED7–CSE2– scopic image analysis display a remark- or derivatives thereof. This is in appar- SRB7 and MED6–SRB2–SRB4–SRB5–SRB6– able degree of similarity41, especially ent contrast to the situation with TFIID ROX3 (Fig. 1a). A subcomplex (SRB2– given that only a small subset of the and SAGA, which are discrete multipro- SRB4–SRB5–SRB6) has recently been re- polypeptides in the murine Mediator are tein complexes that share a subset of constituted from recombinant proteins38. actually related in amino acid sequence TAFs but otherwise contain distinct sub- The functional relevance of these mod- to the yeast proteins. The retention of a units (reviewed in Ref. 32). Yet, despite ules might lie in either their ability to conserved three-dimensional shape their overall similarity (Fig. 2) and on serve as targets for different classes of through evolution could further reflect the basis of their reported sizes and activator38 or their global function in constraints imposed by the highly con- compositional complexity, the various basal transcription35,36 (see below). served shape of Pol II, with which the complexes tend to fall into two main The heterogeneity of the various re- Mediator interacts. groups. The first includes the larger ported complexes (Fig. 2) suggests that TRAP/SMCC, ARC, DRIP and NAT com- the metazoan Mediator complex is also Potential mechanisms for Mediator function plexes, and the second includes PC2, organized in a modular fashion. PC2 as a coactivator CRSP and the murine Mediator. The (like CRSP and the murine Mediator) is The available evidence seems to be most notable difference in the first missing not only the SRB10–SRB11 compatible with TRAP/SMCC and the re- group is the lack of the SRB10–SRB11 ki- kinase–cyclin pair but also additional lated metazoan complexes acting as nase–cyclin pair in ARC and DRIP, which polypeptides that include at least adaptors. The original, and so far the is not surprising in view of the reported TRAP240 and TRAP230. Given this, we most compelling, evidence for the phys- variable association of the correspond- postulate that PC2 consists of integral iological relevance of interactions of ac- ing yeast factors15. Hence, as in yeast, Mediator subunits and might represent a tivators with TRAP/SMCC came from the any heterogeneity in subunit composi- relatively stable coactivator core of the finding that TRAPs could be isolated tion might best be attributed to an larger Mediator-like complex (Fig. 1b). from extracts of ligand-treated (but not intrinsic modular organization of the Furthermore, TRAP220, TRAP100, MED6 untreated) cells in association with TR complex (see below) and variations that and p36 are variably associated with the (Ref. 20). Direct physical interactions of reflect different physiological states PC2 complex23. Potentially related to this TRAP220 with TR (Ref. 42) and of of the cell or different purification observation, MED6 is preferentially lost TRAP80 with the activators p53 and procedures, or both. from holoenzyme preparations from VP16 (Ref. 19), along with correlations 280 TIBS 25 – JUNE 2000 REVIEWS between these interactions and corre- contact on Pol II extending beyond the multiple enhancer-bound activators sponding activator functions, also im- CTD to the DNA-binding channel41. The could channel their combined activation plies that contact with the activator is observation that the yeast homolog of potential to the preinitiation complex44 important at some point in the activation the TRAP/SMCC subunit SOH1 shows ge- (Fig. 3). This is reminiscent of the situ- pathway. Other examples of activator in- netic interactions with the RPB1 and RPB2 ation in yeast, in which certain submod- teractions (DRIP with the vitamin-D subunits of Pol II (Ref. 45) indicates that ules are thought to constitute special- receptor, ARC with SREBP and VP16, SOH1 might contribute to this interaction18. ized targets for a subset of activators. and SUR2 with EIA) have already been Consistent with the mechanisms con- The potential existence of a substruc- mentioned above. The fact that sidered above for metazoan complexes, ture within TRAP/SMCC that is dedicated TRAP/SMCC can be isolated as a com- a major mechanistic step in transcrip- to a subset of activators (including TR) is plex with liganded TR further suggests a tional activation in vivo is believed to be borne out by recent results from another novel pathway in which pre-existing recruitment of Mediator-containing holo- mouse knockout study46. Although dis- coactivator–activator complexes are enzyme components by activators to ruption of the mouse TRAP220 gene, like brought into contact with the the promoter38,44. However, physical re- that of SRB7, is embryonic lethal, viable fi- promoter19,20. cruitment of the yeast holoenzyme broblasts can be isolated from mouse em- Furthermore, although direct physi- might not be sufficient to achieve acti- bryos prior to death. These cells support cal interactions between TRAP/SMCC vation. Thus, although a ⌬SRB5 holoen- normal activation by a number of acti- and Pol II have yet to be demonstrated, zyme is unable to carry out basal and vators (including VP16 and p53) but not there is strong circumstantial evidence activated transcription, it nevertheless by one (TR) that predominantly targets for such an interaction. First, substoi- retains the ability to interact with VP16 TRAP220 (Ref. 42). In another variation of chiometric levels of Pol II can be found (Ref. 36). Additionally, the functional the mechanism of synergy, the glucocorti- associated with TRAP/SMCC under rela- defects cannot be overcome by artificial coid receptor, which, like other steroid tively low-stringency purification condi- recruitment of ⌬SRB5 holoenzyme hormone receptors, possesses two dis- tions18 and with the TR–TRAP complex (Ref. 36). Furthermore, the wild-type tinct activation domains (AF1 and AF2) even under higher-stringency condi- holoenzyme displays a high basal tran- that function synergistically in some cel- tions (C-X. Yuan and R.G. Roeder, unpub- scription activity relative to the core lular contexts, was shown to interact with lished); the latter finding suggests an Pol II (Ref. 13), which suggests that me- TRAP220/DRIP205 through the AF2 do- activator (TR)-induced stabilization diation of an activator response might main but with RGR1/TRAP170/DRIP150 of TRAP–Pol II interactions. Second, a also be intrinsically linked to processes through the AF1 domain47. Pol II-containing holoenzyme complex (such as promoter melting, isomeriza- On the related issue of coactivator re- can be immunoprecipitated with anti- tion, initiation and promoter clearance) dundancy and alternative activation bodies against SRB7 (Ref. 43). Finally, that govern basal transcription (see dis- pathways, Mediator-like complexes NAT has been reported to bind to Pol II cussion in Ref. 35; Ref. 36). function mostly in concert with, rather (Ref. 26). Taken together, these obser- Possibly related to one of these pro- than instead of, other kinds of coactiva- vations can be interpreted in terms of a cesses, the yeast Mediator strongly tor. In in vitro systems, there is a per- simple recruitment model44 in which ac- stimulates the CTD kinase activity of sistent requirement for certain USA- tivators expedite entry of Pol II (and TFIIH (Refs 13,16). Although equivalent derived cofactors for TRAP/SMCC GTFs) into the preinitiation complex activities remain to be tested rigorously coactivator function18–21. With the iden- through the agency of TRAP/SMCC. for TRAP/SMCC or other metazoan com- tification of PC2 as a Mediator-like com- Even though the original insights into plexes, the analogy with the yeast plex in its own right23, two broad classes the existence of a Pol II-interacting Mediator implies a more active role of USA cofactors can now be distin- SRB–MED complex came from studies of for these complexes in activation func- guished: one class represented by PC2 the Pol II CTD (Refs 11,13), the complex tions, most probably in addition to their and by a more-complete TRAP/SMCC might not interact with Pol II exclusively (passive) recruitment functions. Indeed, complex that appears to lack mainly through the CTD. In fact, the negative a recruitment-type function might be SRB10 and SRB11 (Ref. 23), and a second modulatory functions of both NAT fulfilled by the specialized (activator- class that includes PC1/polyADP-ribose- (Ref. 26) and SMCC, as well as the coacti- dedicated) substructures, with a more- polymerase, PC3/topoisomerase I, PC4 vator function of SMCC (Ref. 18), were kinetic, post-recruitment role (such as and PC52 (Refs 2,7). The second class found to be independent of the CTD, isomerization) being carried out by a includes relatively abundant nuclear which is in apparent contrast to the core complex (e.g. PC2). proteins that are involved in processes activation function of the yeast other than transcription, and function Mediator12,13. In the case of NAT, direct Gene-specific activation mechanisms and only at relatively high factor : template physical interactions with Pol II can coactivator redundancy ratios. These latter cofactors are thus occur independently of the CTD, al- The structural modularity of thought to provide an architectural func- though prior phosphorylation of the CTD TRAP/SMCC has implications for tissue- tion in stabilizing the preinitiation com- prevents this interaction. This was inter- and gene-specific activation mecha- plex2. The observed synergism between preted as resulting from phosphoryl- nisms, and also lends itself to models PC2 and PC3 plus PC4 on the one hand23 ation-induced conformational changes in both for differential activation by cer- and between TRAP/SMCC and PC4 on the body of Pol II, which could be respon- tain activators and for synergistic activa- the other18,19,21 thus nicely reflects their sible for the bulk of the interaction27. tion of a given gene by multiple activators. apparently different modes of action. Indeed, the low-resolution three-dimen- Indeed, for the latter situation, the Control by a tissue-specific coactivator sional structure of the yeast Mediator demonstration that TRAP80 (p53, VP16) such as OCA-B (which regulates B-cell- complexed with Pol II has revealed an and TRAP220 (TR, VDR) are targets specific expression of immunoglobin- extensive interface, with the region of for diverse activators suggests how gene promoters through interactions 281 REVIEWS TIBS 25 – JUNE 2000

taining TRAP/SMCC, Pol II and GTFs, once the chromatin has been opened up by the action of HATs. Given that the HATs and TRAP/SMCC both function TR with a wide range of activators, this model could be more generally applica- UAS ble. In this regard, an ARC preparation p53 for which efficient function was demon- strable only with chromatin templates has been purified through its interaction PCs with the activator SREBP, which also binds significant amounts of CBP TRAP220 (Ref. 25). Chromatin templates could TRAP80 thus impose more-stringent require- ments for both types of coactivator. TRAP/SMCC TAFs, however, appear to provide an Mediator exception to the generalization that Mediator-like coactivators do not bypass other coactivator requirements. Con- IIH sistent with genetic studies showing that TAFs IIE at least some of the TFIID-specific TAFs IID are not generally required for activator IIF Pol II function in yeast (reviewed in Ref. 50), IIA TBP IIB TAFs are not essential for yeast Pol II holoenzyme/Mediator function in DNA- TATA INR templated reactions containing TBP in Ti BS place of TFIID (Refs 12,13). In metazoan systems that used DNA templates, TFIID- Figure 3 specific TAFs were found not to be essen- A model for the activation of transcription through synergistic activator interactions with TRAP/SMCC (and other Mediator-like complexes). The preinitiation complex consisting of tial for the function of certain activators in 51 RNA polymerase II (Pol II) and general transcription factors (yellow) is assembled at the nuclear extracts or for activation by the core elements [the TATA box (TATA) and initiator (INR)] of a model promoter. A hypothetical TR–TRAP complex in a system reconsti- distally located enhancer element (UAS) containing binding sites for the activators p53 and tuted from partially purified factors21. the thyroid-hormone receptor (TR) (both green) is also shown. Arrows indicate interactions However, CRSP-mediated function of Sp1 of p53 and TR with TRAP80 and TRAP220, respectively, within the TRAP/SMCC complex depends on TAFs in an in vitro transcrip- (blue), shown here bound to Pol II. Two other potential cofactors are also highlighted: TFIIA tion system reconstituted from homoge- and the TBP-associated components (TAFs) in TFIID (light purple), and architectural factors 28 such as PC4 (red). Concerted interactions, primarily between activators and TRAP/SMCC, neous GTF preparations , just as some but also involving other coactivators, general transcription factors and Pol II, are thought to activators (e.g. GAL–VP16) that do not lead to elevated transcription levels. require TAFs in nuclear extracts do require TAFs in systems reconstituted with (preselected) purified GTFs and with the DNA-binding activator Oct-1) nuclear receptors21 in which there is general cofactors2. might be superimposed upon regulation synergism between the two kinds of These seemingly discordant results by other (general) coactivators such as coactivators. In this model, chromatin on TAFs could be ascribed to differences PCs and Mediator complexes48. Thus, in templates are first made more accessible in the assay conditions and the nature retrospect, the previously demon- to the transcription machinery through of the activators, as well as to a possible strated dependence of OCA-B function the chromatin-remodeling activities of functional redundancy in cofactor re- on the USA-derived cofactors PC2 and activator-recruited coactivators like quirements. Nonetheless, it does appear PC4 can now be viewed in terms of an CBP/p300 and p160 family members. In that, although Mediator-like coactiva- interplay between several diverse types the second step, receptor-bound tors can override TAFs in some cases, of coactivators that include a tissue- TRAP/SMCC is predicted to exert more- the two kinds of coactivators might func- specific coactivator (OCA-B), a Mediator- direct effects on components of the tion synergistically in other cases. This, like complex (PC2) and an architectural preinitiation complex. once again, underscores the need to coactivator (PC4)48. Because interactions of both classes analyse coactivator requirements on a The coactivator CBP/p300 and other of coactivators with the receptor de- case-by-case basis, ideally under near- histone acetyl transferases (HATs), in- pend on the latter’s AF-2 domain, the physiological conditions that include cluding members of the p160/SRC-1 fam- second step is also predicted to entail the use of both chromatin templates ily that were originally reported as an exchange of coactivators on the re- and the normal complement of cellular nuclear-receptor-interacting proteins, are ceptor. Assuming that the interactions cofactors. also generally regarded as important of HATs and TRAP/SMCC with the AF-2 coactivators in vivo. They probably func- domain are reversible, this exchange Concluding remarks tion at the level of chromatin templates49. might, in turn, be driven by a shift in The discovery of TRAP/SMCC and We have previously proposed a two-step equilibrium towards a more energeti- related coactivator complexes has un- model for transcriptional activation by cally favored preinitiation complex con- covered another layer of control in the 282 TIBS 25 – JUNE 2000 REVIEWS

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