TIBS 25 – JUNE 2000

2770968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved. PII: S0968-0004(00)01596-6

TRANSCRIPTIONAL REGULATION INeukaryotes is achieved through multi-protein complexes assembled at the enhancer and promoter regions of tar-get genes. In the case of protein-codinggenes, RNA polymerase II (Pol II) and itsassociated general transcription factors(GTFs) (TFIIA, B, D, E, F and H) are suffi-cient for recognition and low levels ofaccurate transcription from commoncore promoter elements in vitro (basal transcription)1.

For the most part, the components ofthis basal transcription machinery arerequired globally. By contrast, a secondclass of transcription factors, the largegroup of transcriptional activators thattypically assemble at distal enhancersites, show a great deal of variability intheir cell-type and gene specificities andin the spectrum of factors bound to aparticular enhancer. A major outstand-ing issue concerns the mechanisms bywhich the activation potential of diverseenhancer-bound factors is translatedinto increased activity of the basal tran-scription machinery on target genes (activated transcription).

Despite the complexity of this basalmachinery (.40 polypeptides), its re-sponse to activators on specific targetgenes is still dependent on additionalfactors called coactivators, which haveemerged as a third class of transcription

factors that mediate, and might integrate,the effects of transcriptional activatorson the Pol II basal machinery2. As dis-cussed in this article, coactivators areexplicitly defined as factors that are re-quired for the function of DNA-bindingactivators, but not for basal transcrip-tion per se, and do not show site-specificbinding by themselves.

Earlier studies had emphasized therole of TBP (TATA-box-binding protein)-associated factors (TAFs) within TFIID(Refs 3,4; reviewed in Ref. 5) and compo-nents within the partially purified upstream stimulatory activity (USA)fraction (Ref. 6; reviewed in Ref. 7) as essential coactivators. More-recentdevelopments in the field have focusedattention on metazoan orthologs of theyeast Mediator, the coactivator compo-nent of the Pol II holoenzyme that hasproved to be central to transcriptionalregulation in yeast, and have led simul-taneously to a convergence of yeast andmetazoan coactivator studies.

The Yeast Mediator/RNA polymerase IIholoenzyme

The identification of a novel entity,called the Pol II holoenzyme, as the ulti-mate target of transcriptional activatorswas initially the outcome of biochemicaland genetic studies in yeast. Such studieswere spurred, in part, by the observationthat metazoan activators also function inyeast, thus indicating a high degree offunctional conservation of the transcrip-tion apparatus over eukaryotic evolution.

Biochemical analysis first pointed tothe existence of a coactivator activity,

termed Mediator, that was necessary tosupport activated transcription in vitroin conjunction with general initiationfactors8, but apparently distinct fromthe TAF (Ref. 5) and USA (Ref. 7) coacti-vators identified in metazoans (seebelow). Concurrent genetic analysisalso implicated two broad groups of pro-teins in transcriptional regulation invivo. The SRB (suppressor of RNApolymerase B) proteins first emergedfrom genetic screens for suppressors ofpartial truncations in the C-terminal domain (CTD) of the largest subunit ofPol II (Ref. 9). Other gene products, including GAL11, RGR1 and SIN4, emergedfrom genetic screens for regulatory factors that function in other path-ways10 (see below). Subsequent studiesaimed at purifying the Mediator activityand the SRB proteins resulted in theidentification of a Pol II holoenzyme thatcontained the 12-subunit core Pol II,SRBs, other genetically identifiedpolypeptides (including GAL11, RGR1,SIN4) and novel Mediator polypeptides(MEDs) in a single complex11–13. In somecases, the holoenzyme was also foundto contain a subset of GTFs12.

Together with the finding that SRBproteins are also direct targets for acti-vators14, these developments offered aunified hypothesis for transcriptionalactivation in yeast that invokes the inte-gration of activation pathways throughthe same molecular entity. Indeed, intranscription systems reconstitutedwith purified GTFs, the purified holo-enzyme (in contrast to the core Pol II)can support activated transcription12,13.The global significance of the SRB- andMED-containing holoenzyme was furtherillustrated by a genome-wide analysis ofgene expression in yeast, which revealed that the phenotype of a mutationin one of the subunits (SRB4) was virtuallyindistinguishable from that of a Pol IIsubunit mutation that resulted in a complete shut-off of transcription fromessentially the entire genome15.

The association of the SRB, MED andother proteins with Pol II is reversibleand of relevance to the case in meta-zoans (see below). A reversible associa-tion was first suggested by the release ofa free Mediator complex (SRB2–SRB4–SRB5–SRB6–SRB7–MED1–MED2–MED4–MED6–MED7–MED8–ROX3–CSE2–NUT1–NUT2–HRS1/PGD1–GAL11–RGR1–SIN4)after treatment of the Pol II holoenzymewith an anti-CTD antibody13 (Fig. 1a).Moreover, it now appears that signifi-cant amounts of free Mediator coexistwith the holoenzyme-bound form16.

REVIEWS

Transcriptional regulationthrough Mediator-like

coactivators in yeast andmetazoan cells

Sohail Malik and Robert G. Roeder

A novel multiprotein complex has recently been identified as a coactivator fortranscriptional control of protein-encoding genes by RNA polymerase II inhigher eukaryotic cells. This complex is evolutionarily related to the Mediatorcomplex from yeast and, on the basis of its structural and functional charac-teristics, promises to be a key target of diverse regulatory circuits.

S. Malik and R.G. Roeder are in theLaboratory of Biochemistry and MolecularBiology, The Rockefeller University, New York,NY 10021, USA.Email: maliks@rockvax.rockefeller.edu

REVIEWS TIBS 25 – JUNE 2000

278

Apart from the core Pol II subunits andGTFs, the free Mediator also lacks SRB8,SRB9, SRB10 and SRB11 (Ref. 16). Thesehave only been found in some prepara-tions of Pol II holoenzyme and, consis-tent with the physiological relevance ofthis variation, their presence correlates

with metabolic-state-dependent fluctua-tions in the intracellular levels of theseproteins15.

Some of the polypeptides (e.g. GAL11,RGR1, SIN4 and HRS1/PGD1) found in theMediator had been identified geneticallyas either positive or negative modulators

of transcription10. Because of this, theMediator/holoenzyme complex has oftenbeen interpreted as a multicomponent‘control panel’ that can integrate a vari-ety of positive and negative regulatorysignals.

Metazoan Mediator complexesGiven the impetus from the findings

in yeast, several Pol II holoenzymeswere subsequently described in meta-zoans (reviewed in Ref. 17). Theseholoenzyme preparations contained, insome cases, homologs of SRB7, SRB10or SRB11, in addition to a wide range ofGTFs, putative coactivators [includingCREB-binding protein (CBP)] and fac-tors implicated in nuclear processesother than transcription (e.g. DNA re-pair). However, the exact relevance ofthese preparations to transcriptional ac-tivation has remained unclear. Thus, al-though it became apparent that Pol IIcan associate with many factors in iso-lated extracts and chromatographicfractions – not unexpected in view of theprotein–protein interactions that are in-trinsic to the formation of the preiniti-ation complex – a coactivator moietyanalagous to the relatively discreteMediator component of the yeastholoenzyme was not revealed throughthis line of inquiry.

However, more-recent studies of thisproblem using different approacheshave uncovered a set of mammalianMediator-like coactivator complexes.The best characterized of these, an SRB-and MED-containing cofactor complexdesignated SMCC, was isolated on thebasis of resident homologs to Mediatorand holoenzyme components (from celllines stably expressing epitope-taggedhuman SRB7, SRB10 or SRB11), and anability to mediate activation by GAL4derivatives18. SMCC is a 1.5 MDa com-plex of ~25 polypeptides that includehuman orthologs of yeast SRB10, SRB11,SRB7, MED6, MED7, NUT2 and RGR1(Figs 1b,2). SMCC also contains a homolog of yeast SOH1, a positive regu-lator of transcription that interacts genetically with HPR1, a putative Pol II-interacting protein, as well as with TFIIBand subunits of Pol II (reviewed inRef. 18). However, consistent with the re-versible association of yeast Mediatorand Pol II, the most stringently purifiedSMCC does not contain Pol II (see below).Importantly, SMCC contains additionalpolypeptides with no discernible similarityto yeast proteins.

Remarkably, some of these polypep-tides (including TRAP240, TRAP230,

Ti BS

TRAP170/RGR1

p36

SRB11/cyc C

TRAP220

TRAP230

TRAP100

TRAP240

TRAP97

TRAP93

TRAP95

TRAP150βMED7

p78

p24

p22

p12

SRB10/CDK8

TRAP80

NUT2

TRFP

SOH1

SRB7

p37

MED6

RGR1

GAL11

SRB5

MED1

SIN4MED7

MED9

CSE2

SRB10/CDK8

HRS1/PGD1NUT2

MED8SRB7

ROX3

MED6

SRB4

SRB2

SRB6

MED2

SRB8

SRB9

SRB11/cyc C

MED4

(a)

(b)

Yeast Mediator

Human Mediator (TRAP/SMCC)

Figure 1Modular structure of the Mediator complex. (a) Yeast Mediator has various modules, includ-ing a core, that have been identified through genetic and biochemical studies. The coreMediator subunits are further organized into two submodules. The RGR1 submodule(RGR1–MED1–MED2–MED4–MED7–MED8–MED9–CSE2–NUT2–SRB7) is shown in light red;the other submodule (MED6–SRB2–SRB4–SRB5–SRB6–ROX3) is green. The dissociableGAL11–SIN4–HRS1/PGD1 module, dedicated to specialized activators, is shown in light purple. RGR1, which interacts with the GAL11–SIN4–HRS1/PGD1 submodule, is shown asone of the core subunits, as is MED2, whose interaction with the Mediator is dependentupon HRS1/PGD1 (Ref. 35). SRB8, SRB9, SRB10 and SRB11 constitute another module(yellow) but they are variably associated with the holoenzyme and potentially with the freeMediator complex, reflecting the variation in their metabolic-state-dependent intracellular levels. (b) The metazoan Mediator (TRAP/SMCC) core subunits, defined as those invariablyfound in PC2 (Fig. 2) are colored either light red or dark red and include TRAP170/RGR1,TRAP150b, TRAP95, TRAP80, p78, p37, MED7, p24, p22, SRB7, SOH1, NUT2 and p12.MED6 (green) might represent a vestige of the yeast MED6–SRB4 submodule. Relatively labile subunits that, like TRAP220, might constitute additional specialized submodules areshown in blue. SRB10/CDK8 and SRB11/cyc C, which, as in yeast, are variably associatedwith the metazoan complex and might reflect physiological variations, are in yellow. Subunitsin yellow, green or light red are conserved, in whole or in part, in yeast.

TIBS 25 – JUNE 2000

279

TRAP220, TRAP150, TRAP100, TRAP97,TRAP93, TRAP80 and TRAP170, whichdisplays regions of sequence similarityto yeast RGR1) proved to be identical tocomponents previously found in the thyroid-hormone-receptor-associated protein (TRAP) coactivator complex18–20.The TRAP complex had been purifiedseveral years earlier on the basis of itsintracellular association and copurifica-tion with ligand-bound thyroid-hormonereceptor (TR) and its ability to functionas a cognate coactivator20 with USA-derived cofactors20,21.

Subsequent analysis revealed a nearidentity between the subunit compos-itions of SMCC and the TRAP complex,including, in addition to the above-mentioned proteins, a human homologof TBP-related-factor-proximal protein(TRFP) and additional proteins provi-sionally designated p37, p36, p24, p22and p12 (Ref. 19). Consistent with theseobservations, the SMCC and TRAP com-plexes were found to be functionallyequivalent in their ability to support ac-tivation by a number of activators thatinclude TR, p53 and herpesvirus virionprotein 16 (VP16) (Refs 18,19). Hence,TRAP and SMCC can be regarded asequivalent entities. Accordingly, werefer to the human Mediator-like com-plex as TRAP/SMCC. This convergenceof distinct coactivator studies not onlytraces the original description of astructurally defined human Mediator-like complex back to the identificationof the TRAP complex20 but also re-inforces the significance of TRAP/SMCCfor activator function in vivo as well asin vitro. In still another case of conver-gence, we have now found that the pre-viously described USA-derived cofactorPC2 (Refs 6,22) is also a Mediator-likecomplex23 (Figs 1b,2).

Several other Mediator-like com-plexes have been reported recently, in-cluding DRIP (Ref. 24), ARC (Ref. 25),NAT (Ref. 26), murine Mediator27, CRSP(Ref. 28) and a human SUR2-containingcomplex whose molecular compositionhas not been fully defined but that ap-pears to contain SRB10, SRB11 andMED7 (Ref. 29). Some of these com-plexes, like the TRAP complex, were iso-lated on the basis of their ability to interact with specific activators [DRIPthrough ligand-bound vitamin-D recep-tor, ARC through SREBP (sterol-response-element-binding protein) and VP16, andthe SUR2 complex through E1A] andwere subsequently shown to mediateactivator function in various in vitroassays. Others, like SMCC, were isolated

REVIEWS

Ti BS

TRAP/SMCC

PC2 DRIP ARC CRSP muMED NAT

240

230

220

250 250

240 240

205 200 200

230

170 170 150 150 150 110 150

150 α

150 β 150 β 130 130 130 140

105

100 100 100 100 100 95

97 97 (97) 96a

95 95 92 96b 90

80 80 77 77 77 78 70

93 93 (92) 85

78 78 70 70 70

56 56

42 55 45

36/28

37 34

36 3636/28 34/33 37

37

34 34 34 34 33 36 36

32

3133

32 33 33 32 33

32 28b 30

26 26 28a 23

24

22

24

22

22

21

19 19

15

18

21 17

18

15

1212

*RGR1

SUR2

TIG1

TFIIS-related

*SRB10/CDK8

*SRB11/CycC

*MED7

*MED6

*SRB7

TRFP

*SOH1

*NUT2/MED10

+

+

14

+

160b

160a

+

+

+

+

+

Figure 2Comparative chart showing the relationships in polypeptide composition of variousMediator-like complexes: TRAP/SMCC (Refs 18,19); PC2 (Ref. 23); DRIP (Ref. 24); ARC(Ref. 25); CRSP (Ref. 28); murine Mediator (muMED)27; and NAT (Ref. 26). Each column de-picts the components of a single complex, whereas each row indicates the presence (blue box)or clearly established absence (yellow box) of a given subunit within the different complexes.Boxed numbers indicate the molecular weights (in kDa) of subunits whose identities havebeen firmly established from reported amino acid sequences or immunoblot analyses; un-boxed numbers indicate the molecular weights (in kDa) of polypeptides whose sequenceshave not yet been reported. Horizontal lines linking the boxes indicate that the subunits areidentical. Other designations for selected subunits are given to the left. The 6 signs in PC2and CRSP indicate subunits that are variably associated. Asterisks mark subunits with ho-mology to yeast Mediator subunits. For p36 in TRAP/SMCC and PC2, and for p34 inmuMED, corresponding degradation products are also indicated. For TRAP/SMCC, 150aand 150b refer to two polypetides that are both present in the band previously referred toas TRAP150 (Refs 19,20).

REVIEWS TIBS 25 – JUNE 2000

280

on the basis of specific intrinsic subunits related to yeast Mediator com-ponents (SRB10/CDK8 for NAT, SRB7 andMED7 for murine Mediator). Only CRSPand PC2 were first observed as coactiva-tor activities [CRSP for Sp1 (Ref.28), andPC2 both for a variety of natural activa-tors, including Sp1 (Refs 6,30), and for GAL4-based activators22] and lateridentified as Mediator-like complexes.

Preparations of NAT (and SMCC underselective assay conditions18) have beenreported to regulate activated transcrip-tion negatively26. Although this has beenascribed to an SRB10/CDK8 kinase func-tion (consistent with results in yeast31),the affinity methods used for selectingNAT and SMCC (through SRB10/CDK8)also select a distinct SRB10/CDK8–SRB11/cyclin C complex that might havecontributed to the negative regulation.Nonetheless, these results are notinconsistent with NAT having an (as-yetunreported) coactivator function as wellas an intrinsic inhibitory activity, espe-cially in light of the coexistence of posi-tive and negative functions in the samecomplex in yeast10. A coactivator or co-repressor function has not been de-scribed for the murine Mediator27.

Smaller polypeptides (,30 kDa) werenot reported for the DRIP, ARC and CRSPcomplexes. Apart from this, a compari-son of the polypeptide compositions ofthe various complexes (Fig. 2) revealsthat, in essence, they represent eitherthe same or a very similar cellular entityor derivatives thereof. This is in appar-ent contrast to the situation with TFIIDand SAGA, which are discrete multipro-tein complexes that share a subset ofTAFs but otherwise contain distinct sub-units (reviewed in Ref. 32). Yet, despitetheir overall similarity (Fig. 2) and onthe basis of their reported sizes andcompositional complexity, the variouscomplexes tend to fall into two maingroups. The first includes the largerTRAP/SMCC, ARC, DRIP and NAT com-plexes, and the second includes PC2,CRSP and the murine Mediator. Themost notable difference in the firstgroup is the lack of the SRB10–SRB11 ki-nase–cyclin pair in ARC and DRIP, whichis not surprising in view of the reportedvariable association of the correspond-ing yeast factors15. Hence, as in yeast,any heterogeneity in subunit composi-tion might best be attributed to an intrinsic modular organization of thecomplex (see below) and variations thatreflect different physiological states of the cell or different purification procedures, or both.

Structural and functional modularity of theMediator

The modular organization of the yeastMediator first became apparent fromcombined genetic and biochemical stud-ies of components that had originallybeen identified as dedicated transcrip-tion factors. For example, GAL11 andSIN4 were originally identified in screensexamining galactose utilization andmating-type switching, respectively10.Like the SRB8, SRB9, SRB10 and SRB11proteins, which form one subcomplex(cited in Ref. 33), GAL11, SIN4 and an-other genetically defined polypeptide,HRS1/PGD1, were subsequently found toconstitute another dissociable subcom-plex within the Mediator, to which theyare anchored through RGR1 (Ref. 34).The inactivation and actual physical sep-aration of the GAL11–SIN4–HRS1/PGD1subcomplex by mutation (deletion ofSIN4 or of the RGR1 C-terminus) appearsto have no effect on basal transcriptionand only selective effects on activatedtranscription, which is consistent with aspecialized role for the constituentpolypeptides in regulating the targetgenes for which they were selected34–36.This further suggested that the Mediatorcore is composed of the remainingpolypeptides (reviewed in Ref. 33) (Fig. 1a).

The modular organization of the yeastMediator is also evident from purely bio-chemical analyses, which revealed twosubmodules37: RGR1–GAL11–HRS1/PGD1–SIN4–MED1–MED2–MED4–MED7–CSE2–SRB7 and MED6–SRB2–SRB4–SRB5–SRB6–ROX3 (Fig. 1a). A subcomplex (SRB2–SRB4–SRB5–SRB6) has recently been re-constituted from recombinant proteins38.The functional relevance of these mod-ules might lie in either their ability toserve as targets for different classes ofactivator38 or their global function inbasal transcription35,36 (see below).

The heterogeneity of the various re-ported complexes (Fig. 2) suggests thatthe metazoan Mediator complex is alsoorganized in a modular fashion. PC2(like CRSP and the murine Mediator) ismissing not only the SRB10–SRB11 kinase–cyclin pair but also additionalpolypeptides that include at leastTRAP240 and TRAP230. Given this, wepostulate that PC2 consists of integralMediator subunits and might represent arelatively stable coactivator core of thelarger Mediator-like complex (Fig. 1b).Furthermore, TRAP220, TRAP100, MED6and p36 are variably associated with thePC2 complex23. Potentially related to thisobservation, MED6 is preferentially lostfrom holoenzyme preparations from

yeast med6ts mutants39. It is thus possi-ble that the accretion of several regula-tory modules (the SRB10–SRB11 kinase–cyclin pair, TRAP220, etc.) aroundthe PC2 core could generate a pluripo-tent entity (TRAP/SMCC) that can re-spond to a variety of cellular signals. Inlight of this hypothesis, it is also intrigu-ing that a mitogen-activated nuclear ki-nase, RING3, was found associated withthe murine Mediator27. Similarly, theTIG1 subunit of the ARC complex is encoded by one of many genes that are induced by the effector 12-o-tetrade-canoylphorbol-13-acetate25. Furthermore,consistent with a likely role of SRB7 as acore subunit, disruption of the ubiqui-tously expressed mouse SRB7 gene wasfound to be embryonic lethal40.

None of the metazoan Mediator-likecomplexes described to date containreadily apparent homologs of yeastSRB2, SRB4, SRB5 or SRB6. As mentioned,these constitute a distinct submodule to-gether with MED6 (Refs 37,38) and arecritical for activator interactions38, al-beit not sufficient for activator func-tion35. Given that the subunit composi-tions of metazoan complexes indicate acloser resemblance to the yeast RGR1submodule, it is possible that a bona fideMED6–SRB2–SRB4–SRB5–SRB6 moduleremains to be discovered.

The low-resolution three-dimensionalstructures of the yeast and murineMediators deduced from electron-micro-scopic image analysis display a remark-able degree of similarity41, especiallygiven that only a small subset of thepolypeptides in the murine Mediator areactually related in amino acid sequenceto the yeast proteins. The retention of aconserved three-dimensional shapethrough evolution could further reflectconstraints imposed by the highly con-served shape of Pol II, with which theMediator interacts.

Potential mechanisms for Mediator functionas a coactivator

The available evidence seems to becompatible with TRAP/SMCC and the re-lated metazoan complexes acting asadaptors. The original, and so far themost compelling, evidence for the phys-iological relevance of interactions of ac-tivators with TRAP/SMCC came from thefinding that TRAPs could be isolatedfrom extracts of ligand-treated (but notuntreated) cells in association with TR(Ref. 20). Direct physical interactions ofTRAP220 with TR (Ref. 42) and ofTRAP80 with the activators p53 andVP16 (Ref. 19), along with correlations

TIBS 25 – JUNE 2000

281

between these interactions and corre-sponding activator functions, also im-plies that contact with the activator isimportant at some point in the activationpathway. Other examples of activator in-teractions (DRIP with the vitamin-D receptor, ARC with SREBP and VP16,and SUR2 with EIA) have already beenmentioned above. The fact thatTRAP/SMCC can be isolated as a com-plex with liganded TR further suggests anovel pathway in which pre-existingcoactivator–activator complexes arebrought into contact with the promoter19,20.

Furthermore, although direct physi-cal interactions between TRAP/SMCCand Pol II have yet to be demonstrated,there is strong circumstantial evidencefor such an interaction. First, substoi-chiometric levels of Pol II can be foundassociated with TRAP/SMCC under rela-tively low-stringency purification condi-tions18 and with the TR–TRAP complexeven under higher-stringency condi-tions (C-X. Yuan and R.G. Roeder, unpub-lished); the latter finding suggests an activator (TR)-induced stabilization of TRAP–Pol II interactions. Second, aPol II-containing holoenzyme complexcan be immunoprecipitated with anti-bodies against SRB7 (Ref. 43). Finally,NAT has been reported to bind to Pol II(Ref. 26). Taken together, these obser-vations can be interpreted in terms of asimple recruitment model44 in which ac-tivators expedite entry of Pol II (andGTFs) into the preinitiation complexthrough the agency of TRAP/SMCC.

Even though the original insights intothe existence of a Pol II-interactingSRB–MED complex came from studies ofthe Pol II CTD (Refs 11,13), the complexmight not interact with Pol II exclusivelythrough the CTD. In fact, the negativemodulatory functions of both NAT(Ref. 26) and SMCC, as well as the coacti-vator function of SMCC (Ref. 18), werefound to be independent of the CTD,which is in apparent contrast to the activation function of the yeastMediator12,13. In the case of NAT, directphysical interactions with Pol II canoccur independently of the CTD, al-though prior phosphorylation of the CTDprevents this interaction. This was inter-preted as resulting from phosphoryl-ation-induced conformational changes inthe body of Pol II, which could be respon-sible for the bulk of the interaction27.Indeed, the low-resolution three-dimen-sional structure of the yeast Mediatorcomplexed with Pol II has revealed an extensive interface, with the region of

contact on Pol II extending beyond theCTD to the DNA-binding channel41. Theobservation that the yeast homolog ofthe TRAP/SMCC subunit SOH1 shows ge-netic interactions with the RPB1 and RPB2subunits of Pol II (Ref. 45) indicates thatSOH1 might contribute to this interaction18.

Consistent with the mechanisms con-sidered above for metazoan complexes,a major mechanistic step in transcrip-tional activation in vivo is believed to berecruitment of Mediator-containing holo-enzyme components by activators tothe promoter38,44. However, physical re-cruitment of the yeast holoenzymemight not be sufficient to achieve acti-vation. Thus, although a DSRB5 holoen-zyme is unable to carry out basal andactivated transcription, it neverthelessretains the ability to interact with VP16(Ref. 36). Additionally, the functional defects cannot be overcome by artificialrecruitment of DSRB5 holoenzyme (Ref. 36). Furthermore, the wild-typeholoenzyme displays a high basal tran-scription activity relative to the corePol II (Ref. 13), which suggests that me-diation of an activator response mightalso be intrinsically linked to processes(such as promoter melting, isomeriza-tion, initiation and promoter clearance)that govern basal transcription (see dis-cussion in Ref. 35; Ref. 36).

Possibly related to one of these pro-cesses, the yeast Mediator stronglystimulates the CTD kinase activity ofTFIIH (Refs 13,16). Although equivalentactivities remain to be tested rigorouslyfor TRAP/SMCC or other metazoan com-plexes, the analogy with the yeastMediator implies a more active role for these complexes in activation func-tions, most probably in addition to their(passive) recruitment functions. Indeed,a recruitment-type function might be fulfilled by the specialized (activator-dedicated) substructures, with a more-kinetic, post-recruitment role (such asisomerization) being carried out by acore complex (e.g. PC2).

Gene-specific activation mechanisms andcoactivator redundancy

The structural modularity ofTRAP/SMCC has implications for tissue-and gene-specific activation mecha-nisms, and also lends itself to modelsboth for differential activation by cer-tain activators and for synergistic activa-tion of a given gene by multiple activators.Indeed, for the latter situation, thedemonstration that TRAP80 (p53, VP16)and TRAP220 (TR, VDR) are targets for diverse activators suggests how

multiple enhancer-bound activatorscould channel their combined activationpotential to the preinitiation complex44

(Fig. 3). This is reminiscent of the situ-ation in yeast, in which certain submod-ules are thought to constitute special-ized targets for a subset of activators.

The potential existence of a substruc-ture within TRAP/SMCC that is dedicatedto a subset of activators (including TR) isborne out by recent results from anothermouse knockout study46. Although dis-ruption of the mouse TRAP220 gene, likethat of SRB7, is embryonic lethal, viable fi-broblasts can be isolated from mouse em-bryos prior to death. These cells supportnormal activation by a number of acti-vators (including VP16 and p53) but notby one (TR) that predominantly targetsTRAP220 (Ref. 42). In another variation ofthe mechanism of synergy, the glucocorti-coid receptor, which, like other steroidhormone receptors, possesses two dis-tinct activation domains (AF1 and AF2)that function synergistically in some cel-lular contexts, was shown to interact withTRAP220/DRIP205 through the AF2 do-main but with RGR1/TRAP170/DRIP150through the AF1 domain47.

On the related issue of coactivator re-dundancy and alternative activationpathways, Mediator-like complexesfunction mostly in concert with, ratherthan instead of, other kinds of coactiva-tor. In in vitro systems, there is a per-sistent requirement for certain USA-derived cofactors for TRAP/SMCC coactivator function18–21. With the iden-tification of PC2 as a Mediator-like com-plex in its own right23, two broad classesof USA cofactors can now be distin-guished: one class represented by PC2and by a more-complete TRAP/SMCCcomplex that appears to lack mainlySRB10 and SRB11 (Ref. 23), and a secondclass that includes PC1/polyADP-ribose-polymerase, PC3/topoisomerase I, PC4and PC52 (Refs 2,7). The second class includes relatively abundant nuclearproteins that are involved in processesother than transcription, and functiononly at relatively high factor : templateratios. These latter cofactors are thusthought to provide an architectural func-tion in stabilizing the preinitiation com-plex2. The observed synergism betweenPC2 and PC3 plus PC4 on the one hand23

and between TRAP/SMCC and PC4 onthe other18,19,21 thus nicely reflects theirapparently different modes of action.

Control by a tissue-specific coactivatorsuch as OCA-B (which regulates B-cell-specific expression of immunoglobin-gene promoters through interactions

REVIEWS

REVIEWS TIBS 25 – JUNE 2000

282

with the DNA-binding activator Oct-1)might be superimposed upon regulationby other (general) coactivators such asPCs and Mediator complexes48. Thus, inretrospect, the previously demon-strated dependence of OCA-B functionon the USA-derived cofactors PC2 andPC4 can now be viewed in terms of an interplay between several diverse typesof coactivators that include a tissue-specific coactivator (OCA-B), a Mediator-like complex (PC2) and an architecturalcoactivator (PC4)48.

The coactivator CBP/p300 and otherhistone acetyl transferases (HATs), in-cluding members of the p160/SRC-1 fam-ily that were originally reported as nuclear-receptor-interacting proteins, arealso generally regarded as importantcoactivators in vivo. They probably func-tion at the level of chromatin templates49.We have previously proposed a two-stepmodel for transcriptional activation by

nuclear receptors21 in which there is synergism between the two kinds ofcoactivators. In this model, chromatintemplates are first made more accessibleto the transcription machinery throughthe chromatin-remodeling activities ofactivator-recruited coactivators likeCBP/p300 and p160 family members. Inthe second step, receptor-boundTRAP/SMCC is predicted to exert more-direct effects on components of thepreinitiation complex.

Because interactions of both classesof coactivators with the receptor de-pend on the latter’s AF-2 domain, thesecond step is also predicted to entailan exchange of coactivators on the re-ceptor. Assuming that the interactionsof HATs and TRAP/SMCC with the AF-2domain are reversible, this exchangemight, in turn, be driven by a shift inequilibrium towards a more energeti-cally favored preinitiation complex con-

taining TRAP/SMCC, Pol II and GTFs,once the chromatin has been opened upby the action of HATs. Given that theHATs and TRAP/SMCC both functionwith a wide range of activators, thismodel could be more generally applica-ble. In this regard, an ARC preparationfor which efficient function was demon-strable only with chromatin templateshas been purified through its interactionwith the activator SREBP, which alsobinds significant amounts of CBP(Ref. 25). Chromatin templates couldthus impose more-stringent require-ments for both types of coactivator.

TAFs, however, appear to provide an exception to the generalization thatMediator-like coactivators do not bypassother coactivator requirements. Con-sistent with genetic studies showing thatat least some of the TFIID-specific TAFsare not generally required for activatorfunction in yeast (reviewed in Ref. 50),TAFs are not essential for yeast Pol IIholoenzyme/Mediator function in DNA-templated reactions containing TBP inplace of TFIID (Refs 12,13). In metazoansystems that used DNA templates, TFIID-specific TAFs were found not to be essen-tial for the function of certain activators innuclear extracts51 or for activation by theTR–TRAP complex in a system reconsti-tuted from partially purified factors21.However, CRSP-mediated function of Sp1depends on TAFs in an in vitro transcrip-tion system reconstituted from homoge-neous GTF preparations28, just as someactivators (e.g. GAL–VP16) that do not require TAFs in nuclear extracts do require TAFs in systems reconstitutedwith (preselected) purified GTFs andgeneral cofactors2.

These seemingly discordant resultson TAFs could be ascribed to differencesin the assay conditions and the natureof the activators, as well as to a possiblefunctional redundancy in cofactor re-quirements. Nonetheless, it does appearthat, although Mediator-like coactiva-tors can override TAFs in some cases,the two kinds of coactivators might func-tion synergistically in other cases. This, once again, underscores the need toanalyse coactivator requirements on acase-by-case basis, ideally under near-physiological conditions that includethe use of both chromatin templatesand the normal complement of cellularcofactors.

Concluding remarksThe discovery of TRAP/SMCC and

related coactivator complexes has un-covered another layer of control in the

Ti BSTATA

IIA

TR

IID

TAFs

IIB

INR

UAS

TBP

p53

PCs

Pol II

IIE

IIF

IIH

TRAP80TRAP220

TRAP/SMCCMediator

Figure 3A model for the activation of transcription through synergistic activator interactions withTRAP/SMCC (and other Mediator-like complexes). The preinitiation complex consisting ofRNA polymerase II (Pol II) and general transcription factors (yellow) is assembled at thecore elements [the TATA box (TATA) and initiator (INR)] of a model promoter. A hypotheticaldistally located enhancer element (UAS) containing binding sites for the activators p53 andthe thyroid-hormone receptor (TR) (both green) is also shown. Arrows indicate interactionsof p53 and TR with TRAP80 and TRAP220, respectively, within the TRAP/SMCC complex(blue), shown here bound to Pol II. Two other potential cofactors are also highlighted: TFIIAand the TBP-associated components (TAFs) in TFIID (light purple), and architectural factorssuch as PC4 (red). Concerted interactions, primarily between activators and TRAP/SMCC,but also involving other coactivators, general transcription factors and Pol II, are thought tolead to elevated transcription levels.

TIBS 25 – JUNE 2000

283

expression of metazoan genes and hasled to a pleasing convergence of dis-parate coactivator studies. These com-plexes, like the yeast Mediator to whichthey are evolutionarily related, display amodular organization and a consequentpotential for integrating diverse regulat-ory signals. However, because many ofthe subunits in the metazoan complexesare divergent, they could reflect addi-tional metazoan-specific control mecha-nisms. Therefore, the next challenges are(1) to determine how regulatory signalsimpinge on the Mediator complexes andare, in turn, relayed to the Pol II machin-ery in the context of natural genes and(2) to elucidate similarities and differ-ences (relating, for example, to organism-specific subunits) in the mechanisms ofthe yeast and metazoan complexes.

AcknowledgementsWe apologize to colleagues whose

work could not be cited directly owingto space limitations. We thank M. Ito andC-X. Yuan for permission to cite unpub-lished results. Our work was supported,in part, by NIH grants to R.G.R.

References1 Roeder, R.G. (1996) The role of general initiation

factors in transcription by RNA polymerase II. TrendsBiochem. Sci. 21, 327–335

2 Roeder, R.G. (1998) Role of general and gene-specificcofactors in the regulation of eukaryotic transcription.Cold Spring Harbor Symp. Quant. Biol. 63, 201–218

3 Pugh, F. and Tjian, R. (1990) Mechanism oftranscriptional activation by Sp1: evidence forcoactivators. Cell 61, 1187–1197

4 Hoffmann, A. et al. (1990) Highly conserved coredomain and unique N terminus with presumptiveregulatory motifs in a human TATA factor (TFIID). Nature346, 387–390

5 Verrijzer, C.P. and Tjian, R. (1996) TAFs mediatetranscriptional activation and promoter selectivity.Trends Biochem. Sci. 21, 338–342

6 Meisterernst, M. et al. (1991) Activation of class IIgene transcription by regulatory factors is potentiatedby a novel activity. Cell 66, 981–993

7 Kaiser, K. and Meisterernst, M. (1996) The humangeneral co-factors. Trends Biochem. Sci. 21, 342–345

8 Flanagan, P.M. et al. (1991) A mediator required foractivation of RNA polymerase II transcription in vitro.Nature 350, 436–438

9 Nonet, M.L. and Young, R.A. (1989) Intragenic andextragenic suppressors of mutations in theheptapepetide repeat domain of Saccharomyces

cerevisiae RNA polymerase II. Genetics 123, 715–72410 Carlson, M. (1997) Genetics and transcriptional

regulation in yeast: connections to the RNApolymerase II CTD. Annu. Rev. Cell Dev. Biol. 13, 1–23

11 Thompson, C.M. et al. (1993) A multisubunit complexassociated with the RNA polymerase II CTD and TATA-binding protein in yeast. Cell 73, 1361–1375

12 Koleske, A.J. and Young, R.A. (1994) An RNApolymerase II holoenzyme responsive to activators.Nature 368, 466–469

13 Kim, Y. et al. (1994) A multiprotein mediator oftranscriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77,599–608

14 Hengartner, J.C. et al. (1995) Association of anactivator with an RNA polymerase II holoenzyme.Genes Dev. 9, 897–910

15 Holstege, F.C.P. et al. (1998) Dissecting the regulatorycircuitry of a eukaryotic genome. Cell 95, 717–728

16 Myers, L.C. et al. (1998) The Med proteins of yeastand their function through the RNA polymerase IIcarboxy-terminal domain. Genes Dev. 12, 45–54

17 Parvin, J.D. and Young, R.A. (1998) Regulatory targetsin the RNA polymerase II holoenzyme. Curr. Opin.Genet. Dev. 8, 565–570

18 Gu, W. et al. (1999) A novel human SRB/MED-containing complex, SMCC, involved in transcriptionregulation. Mol. Cell 3, 97–108

19 Ito, M. et al. (1999) Identity between TRAP and SMCCcomplexes indicates novel pathways for the function ofnuclear receptors and diverse mammalian activators.Mol. Cell 3, 361–370

20 Fondell, J.D. et al. (1996) Ligand induction of atranscriptionally active thyroid hormone receptorcoactivator complex. Proc. Natl. Acad. Sci. U. S. A. 93,8329–8333

21 Fondell, J.D. et al. (1999) Thyroid hormone receptor-associated proteins and general positive cofactorsmediate thyroid hormone receptor function in theabsence of TATA box-binding protein-associatedfactors. Proc. Natl. Acad. Sci. U. S. A. 96, 1959–1964

22 Kretzschmar, M. et al. (1994) RNA polymerase IIcofactor PC2 facilitates activation of transcription byGAL4–AH in vitro. Mol. Cell. Biol. 14, 3927–3937

23 Malik, S. et al. (2000) The USA-derived transcriptionalcoactivator PC2 is a submodule of TRAP/SMCC andacts synergistically with other PCs. Mol. Cell 5,753–760

24 Rachez, C. et al. (1999) Ligand-dependenttranscription activation by nuclear receptors requiresthe DRIP complex. Nature 398, 824–828

25 Näär, A.M. et al. (1999) Composite co-activator ARCmediates chromatin-directed transcriptional activation.Nature 398, 828–832

26 Sun, X. et al. (1998) NAT, a human complex containingSrb polypeptides that functions as a negative regulatorof activated transcription. Mol. Cell 2, 213–222

27 Jiang, Y-W. et al. (1998) Mammalian mediator oftranscriptional regulation and its possible role as anend-point of signal transduction pathways. Proc. Natl.Acad. Sci. U. S. A. 95, 8538–8543

28 Ryu, S. et al. (1999) The transcriptional cofactorcomplex CRSP is required for activity of the enhancer-binding protein Sp1. Nature 397, 446–450

29 Boyer, T.G. et al. (1999) Mammalian Srb/Mediatorcomplex is targeted by adenovirus E1A protein. Nature399, 276–279

30 Guermah, M. et al. (1998) Involvement of TFIID andUSA components in transcriptional activation of thehuman immunodeficiency virus promoter by NFkB and

Sp1. Mol. Cell. Biol. 18, 3234–324431 Hengartner, C.J. et al. (1998) Temporal regulation of

RNA polymerase II by SRB10 and Kin28 cyclin-dependent kinases. Mol. Cell 2, 43–53

32 Lee, T.I. and Young, R.A. (1998) Regulation of geneexpression by TBP-associated proteins. Genes Dev.12, 1398–1408

33 Myer, V.E. and Young, R.A. (1998) RNA polymerase IIholoenzymes and subcomplexes. J. Biol. Chem. 273,27757–27760

34 Li, Y. et al. (1995) Yeast global transcriptionalregulators Sin4 and Rgr1 are components of mediatorcomplex–RNA polymerase II holoenzyme. Proc. Natl.Acad. Sci. U. S. A. 92, 10864–10868

35 Myers, L.C. et al. (1999) Mediator protein mutationsthat selectively abolish activated transcription. Proc.Natl. Acad. Sci. U. S. A. 96, 67–72

36 Lee, Y.C. et al. (1999) An activator binding module ofyeast RNA polymerase II holoenzyme. Mol. Cell. Biol.19, 2967–2976

37 Lee, Y.C. and Kim, Y.J. (1998) Requirement for afunctional interaction between mediator componentsMed6 and Srb4 in RNA polymerase II transcription.Mol. Cell. Biol. 18, 5364–5370

38 Koh, S.S. et al. (1998) An activator target in the RNApolymerase II holoenzyme. Mol. Cell 1, 895–904

39 Lee, Y.C. et al. (1997) A transcriptional mediatorprotein that is required for activation of many RNApolymerase II promoters and is conserved from yeastto humans. Mol. Cell. Biol. 17, 4622–4632

40 Tudor, M. et al. (1999) Ubiquitous expression andembryonic requirement for RNA polymerase II coactivatorsubunit Srb7 in mice. Genes Dev. 13, 2365–2368

41 Asturias, F.J. et al. (1999) Conserved structures ofmediator and RNA polymerase II holoenzyme. Science283, 985–987

42 Yuan, C-X. et al. (1998) The TRAP220 component of athyroid hormone receptor-associated protein (TRAP)coactivator complex interacts directly with nuclearreceptors in a ligand-dependent fashion. Proc. Natl.Acad. Sci. U. S. A. 95, 7939–7944

43 Xiao, H. et al. (1999) The human homologue ofDrosophila TRF-proximal protein is associated with anRNA polymerase II-SRB complex. J. Biol. Chem. 274,3937–3940

44 Ptashne, M. and Gann, A. (1997) Transcriptionalactivation by recruitment. Nature 386, 569–577

45 Fan, H.Y. et al. (1996) Mutations in the RNApolymerase II transcription machinery suppress thehyperrecombination mutant hpr1D of Saccharomycescerevisiae. Genetics 142, 749–759

46 Ito, M. et al. (2000) Involvement of the TRAP220component of the TRAP/SMCC coactivator complex inembryonic development and thyroid hormone action.Mol. Cell 5, 683–693

47 Hittelman, A.B. et al. (1999) Differential regulation ofglucocorticoid receptor transcriptional activation via AF-1-associated proteins. EMBO J. 18, 5380–5388

48 Luo, Y. et al. (1998) Coactivation by OCA-B: definitionof critical regions and synergism with generalcofactors. Mol. Cell. Biol. 18, 3803–3810

49 Xu, L. et al. (1999) Coactivator and corepressorcomplexes in nuclear receptor function. Curr. Opin.Genet. Dev. 9, 140–147

50 Hahn, S. (1998) The role of TAFs in RNA polymerase IItranscription. Cell 95, 579–582

51 Oelgeschläger, T. et al. (1998) Transcription activationvia enhanced preinitiation complex assembly in ahuman cell-free system lacking TAFIIs. Mol. Cell 1,925–931

REVIEWS

Trends and BioMedNet Commentaries

The Trends Journals and BioMedNet are starting a new joint project. We have invited scientists tocontribute short up-to-the-minute articles on significant papers appearing in the primary literature.These commentaries will be published in the relevant Trends Journal and many will be selected to

appear daily in the News and Comment section of BioMedNet. In TiBS this exciting new section of thejournal will begin in the August issue, so watch this space!