tfii

8/2/2019 TFII

1/13

Cell, Vol. 79, 93-105, October 7, 1994, Copyright 0 1994 by Cell Press

Assembly of Recombinant TFIIDReveals Differential Coactivator Requirementsfor Distinct Transcriptional ActivatorsJin-Long Chen, Laura Donatel la Attardi ,C. Peter Verr ijzer, Kyoko Yokom ori , and Robert Tj ianHoward Hughes Medical Insti tuteDepartment of Molecular and Cel l BiologyUniversi ty of Cal i fornia, BerkeleyBerkeley, California 94720-3202

SummaryWe previous ly reported that transcriptional regulatorscan bind selected TAF subunits of the TFIID complex.Howeve r, the specif ic i ty and function of individualTAFs in mediating transcr iptional activation remainedunknown. Here we report the in vi tro assemb ly andtranscr iptional properties of TBP-TAF complexes re-consti tuted from the nine recombinant subunits ofDrosophi la TFIID . A minimal complex containing TBPand TAF tI directs basal but not activator-respon-sive transcr iption. By contrast, reconsti tuted holo-TFII D supports activation by an assortment of activa-tors. The activator NTF-1, which binds TAFII~~O,stimulates transcr iption with a complex containingonly TBP, TAF,,250, and TAF,,lLO, whereas Spl bindsand additionally requires T AF,,llO fo r activa tion. Inter-estingly, T AF,,150 enhances Spl activation eventhough this subunit does not bind directly to Spl.These results establ ish that speci f ic subcomplexes ofTFII D can mediate activation by di fferent classes ofactivators and suggest that TAFs perform mult iplefunctions dur ing activation.IntroductionHow do sequence-specif ic promoter- or enhancer-bindingproteins com municate with the RNA polymerase II (pol II)transcr iptional apparatus to stimulate mRN A synthesis?Several studies have impl icated TFII D as a central playerin this process ( for review, see Gi ll and Tj ian, 1992; Her-nandez, 1993). Al though the TATA box-binding protein(TBP) subunit of TFII D or iginal ly had been considered al ikely target for activators, several observations haveprompted a reevaluation of this simple and appeal ingmodel. It now seem s probable that TBP rarely i f ever existsas a free molecule with al l of its potential interfaces ex-posed on the outside surface of the so-cal led saddle struc-ture (for references, see Klug, 1993). Instead, in eukaryoticcel ls, TBP is predominantly found associated with sets oft ightly bound subunits, or TBP-associated factors (TAFs),to form SLl, T FIID , or TFIIIB complexes representing es-sential compone nts of the RNA pol I, pol II , and pol I lltranscr iption apparati , respectively ( for review, see Good-r ich and Tj ian, 1994; Hernandez, 1993).

The observation that TBP can direct basal levels of tran-scr iption in vi tro yet requires a complement of TAF sub-units to mediate activator-dependent transcr iption (Dyn-lacht et al ., 1991; Pugh and Tjian, 1990; Tanese et al .,

1991) led to the hypothesis that TBP m ust associate withone or more coactivators to mediate transcr iptional activa-t ion. The pr inciple features of this coactivator hypothesiswere that coactivators would be required to mediate acti -vation but would not be necessary for basal transcr iptionand that coactivators would be directly targeted by activa-t ion domains via speci f ic protein interfaces (Pugh andTj ian, 1990). Moreover, i t was envisioned that di fferentclasses of activators might interact with and therefore re-quire dist inct coactivators. Thus, the relationship betweenTAFs and coactivators has became a focal point of ourresearch.

Cri t ical evidence in support of the coactivator model w asthe demonstration that the TAFs in the TFII D complex(TAF, ,250, TAF, ,150, TAF, , l 10, TAF, ,80, TAF, ,GO, TAF, ,40,TAF1,30a, and TAFa30P) were responsible for coactivatorfunction. This was accompl ished byantibodyaff ini ty pur i fi -cation of Drosophi la and human TFII D to apparent homo-geneity, fol lowed by separation of the TAFs from TBP withmild denaturants. Reconsti tution of pur i fied TBP with im-munoaff ini ty-pur i f ied subunits establ ished that the TAFscould provide coactivator function to diverse promoter-speci f ic transcr iption factors such as Spl , neurogenic ele-ment-binding transcr iption factor 1 (NTF-I) , and CCAA T-binding transcr iption factor (CTF) (Dynlacht et al ., 1991;Tanese e t al ., 1991). Subsequent studies confirmed thatseveral other activators, includingzta, GAL4-El A, GAL4-VP1 6, and GAL4-AH, which had been previously shownto contact basal factors such as TBP, TFIIB , or both di-rectly, also required TAFs for activation (Chiang et al .,1993; Choy and Green, 1993; Zh ou et al ., 1992).

To decipher the molecular basis of coactivator func-t ions, a major task was the pur if ication, molecular cloning,and expression of the TAF subunits from Drosophila andhuman TFII D. To date, cDNA clones and expressed pro-teins have been obtained for each of the eight TAFs foundin the Drosophi la TFII D complex (Dynlacht et al ., 1993;Goodrich et al ., 1993; Hoey et al ., 1993; Kokubo et al .,1993a, 1993b, 1993c, 1994 ; Verr i jzer et al ., 1994; Wein-zier l et al ., 1993a, 1993b; Yokomori et al ., 1993a). In addi-t ion, several human TAFs (TAF11250 , TAF,,130 , TAF,,lOO,and TAF,,70) have also been partly character ized (Hisa-take e t al ., 1993; Ruppert et al ., 1993; Weinzierl et al .,1993b; N. Tanese and R. T., unpubl ished data). As ex-pected, these subunits of TFII D appear to be evolutionar i lyconserved both in structure and in function.

The avai labi li ty of overexpressed recombinant TAFs hasenabled detailed biochem ical studie s. In addition to vari-ous TAF-TAF and TAF-TBP interactions cr i t ical for theassembly of TFII D, we have identif ied specif ic interactionsbetween individual TAFs and activation domains of var i-ous gene-specif ic transcr iption factors. The f i rst of thesestudies establ ished that TAF,,l 10 can interact selectivelywith the Gln-rich activation domains of Spl (Hoe y et al .,1993). Furthermore, analysis of mutant Spl activation do-mains revealed a correlation between loss of transcr ip-tional activation and failure to bind TAF ,,l 10 (Gill et al.,

8/2/2019 TFII

2/13

Cell94

A BIP- endoeenousdTFiiD7gJ

k #qTAFII250I__L -TAF,+O

11-1 TAFIIl10-. -TAF$O1 --I -TAF@

IgG -rl,TBP- - -. -TAF@,

-. -TAFI,30P/u

TBP1,

HA-TAF@o

TAFI#O

7+I,

TAFI@

1994). The acidic activation domain of GAL4-VP16 wasshown to interact with TAFI140, and antibodies directedagainstTAFIAOinhibi tedVP16-mediated activation but notbasal transcription in vitro (Goodrich et al., 1993). More-over, certain TAFs such as TAF,,l 10 and TAF,,40 interactdirectly with other components of the basal apparatus,including TFIIA and TFIIB (Yokomori et al ., 1993b; G ood-r ich et al ., 1993). Together, these resu lts provided the ini-t ial evidence that speci f ic activators make contact withdistinct TAFs in the TFIID complex and that these protein-protein interactions may transmit the activation signal tothe basal machinery.Although these studies are consistent with the coactiva-tor hypothesis, direct evidence for the role of individualTAFs in mediating transcr iptional activation remained elu-sive. In part icular, the exper iments reported thus far couldnot demonstrate decisively tha t the interaction betweenTAFs and activators is a requisite step leading to transcr ip-t ional activation. Conclusive evidence for speci f ic TAF re-quirements by di fferent types of activators required recon-sti tution of dist inct part ial TBP-TAF complexes usingpur i f ied subunits.Here we report the in vi tro assembly of both completeTFIID and a var iety of part ial TBP-TAF complexes from

Figure 1. In Vitro Assembly of TBP-TAFCom-plexes(A) Endog enous Drosophi la TFIID consists ofn ine subunits. The partia lly purif ied TFIID com-plex was immunoprecip itated with a mono-clonal antibody directed against dTBP. TheTAFs were selectively e luted from TBP with1 M guanid ine-HCI, separated by SDS-PA GE,and visualized by silver stainin g (lane 2). TBPremained bound to the antibodies ( lane 1).(8) Schematic d iagram of the TFIID complex.Indicated are dTBP and the e ight major Dro-sophila TAFs as well as most of the specificTBP-TAF and TAF-TAF interactions we haveident ified thus far. This cartoon summarizesour current understan ding of the architectureof the TFIID complex. However, it should benoted th at, as discussed in the text, the com-plete p icture is not yet at hand.(C) Strategy of in vitro assem bly of the TFIIDcomplex. All TFIID subunits were purif ied fromextracts prepared from 5% cells infected withrecombin ant baculoviruses expressing eitherTBP or various TAFs. Drosophila or humanTAF11250 fused to an N-terminal 8 amino acidHA-epitope peptide was immobilize d on Pro-tein A-Sephar ose beads conjugate d cova-lently with monoclonal antibodies d irectedagainst the HA-epitope. TAF,,250 was used asthe foundation on which the complex was builtby stepwise addition of TSP and the otherTAFs. The order of assembly was as indicat edin the cartoon. At each assembly step the par-tia l TFIID complex was incubated with an ex-cessof the next component. Unbound materia lwas removed by extensive washes. Finally , re-combinant holo-TFIID or partia l complexes ofinterest were eluted under native conditionswith the HA peptide and used in in v itro tran-scription reactions.

recombinant Drosophi la TBP and TAF s. The role of indi-vidual TAF s in mediating activator-responsive transcr ip-t ion was tested by assaying the activi ty of var ious en-hancer proteins bear ing di fferent classe s of activationdomains, such as Spl and NTF-1, in combination withdistinct TBP-TAF complexes lacking specif ic subunits. Fi-nal ly, we have tested the abi li ty of an assembled holo-TFIID containing TBPand the eight major Drosophi laTAFsto support activation by several unrelated transcr iptionalactivators, including Spl, NTF-1, CTF , VP16, and cJun(activating protein 1 [AP-11). Our results provide evidencefor the role of individual TAFs a s coactivators capable ofinteracting with selected activation moti fs to mediate tran-scr iption.ResultsAssem bly of TBP-TAF Complexes in Vi troEndogenous Drosophi la TFIID is a mult iprotein complexcomprised of TBP and at least eight TAFs (Figure 1A).Having recently obtained cDN As for al l the major Drosoph-i la TAFs, we have begun to identi fy many o f the protein-protein interactions involved in the assemb ly of TFIID(Dynlacht et al ., 1993; Verr ijzer et al ., 1994; Weinzier l et

8/2/2019 TFII

3/13

Activator-Specific TBP-TAF Complexes95

M (kD1zoo-117-

8(t

I ? 3 12345678 Y IOIII

Figure 2. The Dimeric TBP-TAF,250 Complex Supports Basal but Not Activated Transcription(A) Silver-sta ined polyacrylamide gel of TBP-TAF,,250 complexes. A complex consisting of dTBP and either Drosophila TAFtI (dTAF,,250, lane2) or human TAFf1250 (hTAF,,250, lane 3) was assembled using the strategy described in Figure 1C. Lane 1 shows a s imilar amount of dTBPalone.(B) A TFIIDdependent Drosophila fractionated transcription system was supplemented with e ither no prote in ( lanes 1 and 2) dTBP alone (5 ng,lanes 3 and 4) or a s imilar molar amount of e ither dTBP-dTAF ,,250 (lanes 5 and 6) or dTSP-hTAF,,250 (lanes 7 and 6). As a contro l, endogenous,partia lly purif ied Drosophila TFIID (Ct.3 fraction) was added to reactions in lanes 9 and 10. Purif ied recombinant Spl (about 30 ng) was added tosamples in the even-numbe red lanes. Transcription was assayed by primer extension.(C) The transcriptional activity of dTBP (lanes 1 and 2) dTBP-hTAFI1250 (lanes 3 and 4) and endogenous human HA-TFIID, immunopurif ied tonear homogeneity ( lanes 5 and 6) was tested in e ither the absence (lanes 1, 3, and 5) or presence (lanes 2, 4. and 6) of 30 ng of purif ied Splin a TFIID-de pendent huma n fractionated transcription system. Transcription products were detected by primer extension.

al., 1993a; W einzierl et al ., 1993b; Yokomori et al ., 1993a;J.-L. C ., K. Y., and R . T., unpublished data). Our currentview of the TFII D subunit archi tecture is summarized inFigure 1 B. Some TAF s, such as TAFI1250, TAF,,150 , andTAF,,30a , are in direct contact with TBP, whereas otherTAFs are brought into the complex via TAF-TAF interac-t ions. In part icular, the largest su bunit of TFIID (TAF,,250)appearstofunctionasan importantscaffold formanyotherTAFs. For example, TAFI1150, TAF, , l 10, TAF@, TAFI130a,and TAF,,308 al l eff ic iently associate with TAF,,250 . Fur-thermore, TAFII1 10 helps recrui t TAF1180, whi le T AFI180al lows TAFtI to associate stably with the TFII D complex.In addit ion to these relatively robust interactions, thereare also signi f icantly weaker interactions between TFII Dsubunits. For example, TAFI180 binds ineff ic iently toTAF,,150 , whi le TAF,,60 associates weakly w ith TBP.These results reveal that the TFII D complex is held to-gether by a plethora of TAF-TAF interactions and a l imitednumber of TAF-TBP interactions.

We have used the results of these subunit interactionstudies to design a strategy for assembl ing TBP-TAF com-plexes in vi tro. Our goals were to reconsti tute functionalTBP-TAF complexes containing var ious combinations ofTAFs and ul t imately to assemble a recombinant holo-TFIID . Al l TFIID subunits were pur i f ied from extracts pre-pared from Sf9 cel ls infected with recombinant baculovi-ruses expressing the TAFs or TBP. Through empir icallydr iven tr ial and error, we arr ived at a stepwise assembly

protocol in which su ccessive subunits of TFII D were incor-porated onto an immobi l ized core subunit (Figure 1C). Wechose TAFI1250 as the foundation for bui lding TFII D, be-cause i t appears to provide a scaffold contacted by manyof the other TAF s. We immobi l ized hemagglutinin (HA)-tagged ful l- length TAF,,250 on protein A-Sepharose beadscovalently conjugated with anti -HA antibodies. This strat-egy was chosen so that we could conveniently elute tran-scr iptional ly active complexes with HA peptide under na-t ive condit ions. Next, TBP was incubated with theimmobi l ized TAF11250, and free TBP was removed by ex-tensive washe s, result ing in a dimeric TBP-TA FtI com-plex. This assemb ly process was repeated for each suc-cessive TAF in order to bui ld up the TFIID complex. Atcertain stages dur ing the reconsti tution process, the orderin which the TAFs we re added proved to be important.For example, TAFII~ 10 was no t able to associate stablywith TBP-TAF complexes in the absence of TAF,,250 (datanot shown). Likewise, TAFI140 only eff ic iently entered thecomplex after TAF,,GO was present. Furthermore, TAF,,80was only able to join the complex after TAF,,l lO andTAFI1150 had been a ssembled and had to be assembledbefore TAFI130a (data not shown).

After var ious combinations of TAFs and TBP had beenassembled, they were eluted with HA peptides and sub-jected to SDS-polyacrylamide gel electrophoresis (PAGE),fol lowed by si lver staining (see Figures 2A, 3A, 6A, and7A) and Western blott ing (data not shown). Taking into

8/2/2019 TFII

4/13

Ce l l96

A B CSpl - A - A - 4 Spl - A - A

+ITAF,1250-TAFl11SO I

117- ,-TAF11110

Figure 3. The TBP-TAFII~~O-TAF~,IBO-TAF~~~ 10 Complex Mediates Activation by Spl(A) Silver-sta ined gel of d istinct partia l TFIID complexes consisting of dTBP-hTAF,,250-dTAF,r150 (lane 1). dTBP-hTAFI1250-dTAFIIl IO (lane 2)or dTBP-hTAFI1250-dTAFII150-dTAFIII 10 ( lane 3). The in v itro assembly w as as described in Figure 1C.(8) Approxi mately equim olar amounts (5 ng of TBP) of these complexes were tested in the Drosoph ila fractionated system. Transcription reactionswith dTBP-hTAFI,250-dTAFI,l 10 ( lanes l-3), dTBP-hTAF11250-dTAFI11 50 (lanes 4-6) or dTBP-hTAF,,SCO-dTAF,,l 50-dTAFI11 10 (lanes 7-9) wereperformed in either the absence (lanes 1, 4, and 7) or presence of 10 ng (lanes 2, 5, and 6) or 30 ng (lanes 3, 6, and 9) of purified Spl(C) The dTBP-hTAFI,250-dTAFI1150 (lanes l-3) or dTSP-hTAF,,250-dTAFI,150-dTAFI11 10 (lanes 4-6) complexes were tested for their abil ity tosupport activation by Sp l in the huma n transcription system. Reactions were performed in either the absence (lanes 1 and 4) or the presence of10 ng of Spl ( lanes 2 and 5) or 30 ng of Spl ( lanes 3 and 6).

account the ineff ic ient staining of TAFI1250 (Weinzier l etal ., 1993a), we estimated that the pur i fied complexes con-tain approximately stoichiometr ic amounts of the recom-binant TFIID subunits. The integr ity of eluted com plexeswas ver i f ied by reimmunoprecipitation using antibodiesdirected against di fferent subunits (data not shown).These exper iments establ ished that once formed, bothpartial and complete TBP-TAF complexes remain stablyassociated. Using this strategy, we were able to reconsti -tute a recombinant holo-TFIID complex successfu l ly.The TBP-TAF11250 Complex Supports BasalTranscr iption but Not ActivationWe fi rst tested whether the minimal TBP-TAFr1250 com-plex would be suff icient to mediate activation in vi tro. Toaddress this question, the transcr iptional properties ofTBP alone were compared with those of a minimal com-plex comprised of TBP and either Drosophi la or humanTAFrr250 (Figure 2A). Pur i fied TBP-TAF950 complexeswere tested for their ability to suppo rt transc riptional acti-vation by the activator Spl in reconsti tuted reactions de-rived from either Drosophila or HeLa fractionated tran-scr iption compone nts (Figures 28 and 2C, respectively) .The pur i fied TBP-TAF,,PBO complexes supported basaltranscr iption to a level comparable to that observed withTBP alone, whereas no transcr iption was detected in theabsence of exogenousiy added TBP or TBP-TAF com-plexes. We did not detect any signi f icant inhibi t ion or st im-ulation o f basal transcr iption, even though stoichiometr ic

amounts of ful l- length TAFe250 were present in these com-plexes. Howeve r, the presence of TAF11250 also did notrestore responsiveness to the activator, Spl . By contrast,endogenous holo-TFIID directed high levels of Spl activa-t ion. These results indicate that the minimal complex con-taining TBP and TA FI1250 is capable of al lowing basal lev-els of transcr iption, but fai ls to support activation by Spl .A Four-Subunit Complex Supports Spl ActivationIt was not surpr ising that a minimal complex containingTBP-TAF11250 fai led to mediate Spl activation, since ourprevious stud ies showed that Spl binds directly toTAF,,l lO (Hoey et al ., 1993). Therefore, the TAF tI 10 sub-unit of TFII D might serve as an essential coactivator forSpl-dependent transcr iption. Howeve r, when we previ-ously tested a ternary complex containing TBP, TAFr,l 10,and a truncated version of TAFI1250, only weak activationby Spl was observed in vi tro (Weinzier i et al ., 1993a). W ehad hypothesized that this weak response may be due tothe use of a truncated form of TAFr,250. A more interestingalternative was that otherTAFs, in addit ion toTAFI1250 andTAF,,l 10, play some role in Spl activation. We thereforeassessed the abi li ty of the tr iple complex containing ful i-length dT AF,,250 or hTAFrr250 to mediate Spl activation(Figure 3 ). Our data indicate that incorporating full-lengthTAF,,250 into the tr iple complex is not suff ic ient to supportrobust activation by Spl (Figure 38, lanes l -3) , consistentwith the notion that an addit ional component of TFIIDmight be necessary.

8/2/2019 TFII

5/13


An attractive candidate for one such missing subunit(s)is TAFl1150, since i t was recently shown to bind DNA andrecognize specif ic core promoter sequences (Verri jzer etal ., 1994). We therefore reasoned that TAF,,l50 might playan importan t role in mediating transcription by certain a cti-vators. To test this hypothesis, we compared the transcr ip-t ional properties of a quadruple complex (TBP-TAF,,250-TAFI1150-TAF11110) with those of the triple complex lackingTAF,,l 10 (TBP-TAF,,250-TAFI1150) (Figure 3). When theimmunopuri f ied quadruple complex was assayed in thefractionated Drosophi la transcr iption reaction, we ob-served a dram atic activatio n by Spl (a-to lo-fold) (Figure38, lanes 7-9). Im portantly, activation was cr i t ical ly de-pendent on the presence of TAF,,l 10, since omission ofthis subunit abolished Spl responsiveness (Figure 38,lanes 4-6). Using the human fractionated transcr iptionsyste m, we confi rmed a simi lar behavior of these com-plexes in supporting Spl activation (Figure 3C). Theseexper iments establ ish that a subset of TAFs can mediateactivation by Spl in vi tro and that TA FII~ 10 serves as anessential coactivator.Spl Binds TAFl l l 10 but Not TAF,,250 or TAFl l150Our functional studies of the quadruple complex suggestthat, in addit ion to TBP, three TAF s are required for activa-t ion by Spl. Since T AF,,l lO is directly contacted by Spland is essential for activation, i ts behavior f i ts the defini tionof a coactivator. Howeve r, the role of the other two TAFsin the activation process is less clear. To address thisquestion, we determined whether Spl might also directlycontact T AFI1150, TAF,,250 , or both. Biotinylated ol igonu-cleotides containing Spl-binding sites were coupled to astreptavidin-agarose resin. Pur i fied recombinant Spl wasloaded onto the column and the unbound mater ial re-moved. A control DNA aff ini ty resin that lacked Spl proteinwa s also prepared and run in parallel. Partially purifiedTAF,,250 , TAF,,150, and TAF,,l 10 from baculovirus expres-sion extracts were incubated with each resin, and afterextensive washing, the bound proteins were eluted witha high sal t buffer (1 M KCI). The input and eluted mater ialwere subsequently analyzed by SDS-PAGE, fol lowed byimmunoblott ing (Figure 4A). As expected, TAF,,l lO boundavidly to the Spl aff ini ty resin. Interestingly, nei therTAF,,250 nor TAF,,150 was retained on the Spl-containingresin. It is worth noting that the same source of factors wasused here as in the assembly of transcr iptional ly activecomplexes, indicating that these TAF s are correctly foldedand functional. None of the proteins tested bound to thecontrol re sin lacking Spl.

As an addit ional test of Spl binding specif ic i ty, weloaded crude Sf9 extracts containing TAF,,l 10 onto theSpl aff ini ty column and the control column. After exten-sive washing, al l bound mater ial was eluted with high sal tand directly analyzed by si lver staining of SDS gels. Onlythree major species were detected: ful l- length Spl, a trun-cated Spl polypeptide, and a prominent protein of 110 kDathat comigrates with TAFl ll 10 (Figure 48) and cross-reactswith anti -TAF,,l lO (data not shown). As expected, noTAF,,l lO was retained on the control m atr ix. Thus, al-though TAF,,l 10 consti tutes less than 0.1% of the total

ATAF,,lIll TAF,,lSll TAF,,ZSO

sb+ - sp1 s&p + - sp1 s,e q+ - Sp1h, L,,,- - - -L,l

I23 1 5 I, 7 x9Immunohlot

B \M(kD)+ + - TAFnllO

zoo-117- +TAF[,l IO

XO-

50-

I

** - Spl*

+ - + SplI 2 3 .l

Silver StainFigure 4. Spl Directly Binds to TAF,,l lO but Not lo TAF,,250 orTAF,, 150(A) Partia lly purif ied TAF,,l lO (lanes l-3), T AF,,l50 ( lanes 4-6), orTAFtI ( lanes 7-9) were loaded onto an agarose GC box DNA aff ini tycolumn either with ( lanes 2, 5, and 6) or without ( lanes 3, 6, and 9)DNA-bound Spl. After extensive washes, bound prote ins ware e lutedwith a h igh salt buffer. As well as the e luted prote in fractions, 10% ofthe input m ateria l ( lanes 1, 4, and 7) was separated by SDS-PA GEand visualized by Western blot analysis.(B) The interaction between Spl and TAF,,l lO is h ighly specific . Acrude extract prepared from Sf9 cells infected with TAF,,llO -expressing recombin ant baculoviruses (lanes 2 and 3) was appl ied toan agarose DN A aff ini ty column with ( lanes 2) or without Spl ( lane3). Also an Sf9 contro l extract was loaded onto the Spl-coated column(lane 4). After extensive washing, the bound prote ins were e luted withhigh salt and resolved by SDS-PA GE and silver sta in ing. Lane 1 shows5%of theTAF,,l lo-contain ing inputextract. TAF,,l 10, Spl, andan Splproteolytic degrada tion product (asterisk) are indicated . The identity ofthese protein bands was verified by Western imm unob lot analysis(data not shown).

protein in the input e xtrac t, it is selectiv ely purified (overlOOO-fold) by binding to Spl. These exper iments con-fi rmed the highly speci f ic natureof the interaction betweenSpl and TAFl l l 10. Intr iguingly, the other two TAFs thatare required to potentiate Spl a ctivation (TAFa250 andTAF,,lSO) do not contact Spl directly on their own . Theseresults suggest that activation may require m ult iple TAF s,some that make direct contact with activators, whi le othershave distinct functions in the activation process.

8/2/2019 TFII

6/13

CTFIID + - +-

TBP-*TAFn40 -- I

Autoradiogram Silver sta in Immunoblot

B TAFII~SOAC TAFDISOAN TAFnllO TAFD80 TAFn60 TAFn40~ - - - ~

TAFDl50 + - MO W, -200NTF-l+ #r -117

- 97

- 66

dLip - + B6 + 6 -M W) $ 6-8 Q2 - 3+ + g- + NTF-1Activation200-

Doma in117- 4-80- 4-

12 3 456 7 89 10 11 12 13 14 15 16 17 18Figure 5. The Ile-Rich Minim al Activation Domain of NTF-1 Interacts Selectively with TAFI115Cl and TAF&O(A) NTF-1 binds to the Drosophila TFIID (dTFIID) complex. The dTFllD complex was immunopurif ied from a partia lly purif ied dTFllD fraction (Q.3)using antibodies d irected against TBP and then incubated with radio labeled NTF-1. The silver-sta ined gel shows the TAF pattern obtained uponimmunopurif ication of the TFIID complex. NTF-1 binding was detected by autoradiography. NTF-1 was not immunoprecip itated by TBP antibodiesin the absence of dTFIID.(B) The minimal activation domain of NTF-1 interacts with the N-terminus of dTAFrrl50 and with dTAF#J but not with other TAFs. Radio labele ddTAF,,l5OAC, dTAF,,l50AN, dTAF,,l lO, dTAFrr80, dTAF,,GO, and dTAF,,40 were incubated with beads alone or beads covalently coupled to a peptidecorresponding to the 56 amino acid mini mal activation domain of NTF-1. The input amounts correspond to 20% of the tota l prote in used in thebinding experiment.(C) NTF-1 binds to dTAF,,150. Baculovirus-expressed dTAF,,150 was immunopurif ied using anti-dTAF,,lBO antibodies. Recombinant vaccinia v irus-produced NTF-1 was incubated with the immunopurif ied dTAFrrl50, and bound NTF-1 was detected by Western b lot analysis. NTF-1 was notcoimm unoprec ipitated by a control baculovirus extract withou t dTAF ,,150

The Isoleucine-Rich Activator NTF-1 Contac tsTAF,,150 and TAF,,GOIn this and preceding studies, we have provided a compel-l ing body of evidence that TAF,,l lO can serve as a bonafide coactivator for Spl . What about the TAF requirementsfor other activators? One attractive possibi l ity is that di ffer-ent types of activators might require distinct co activators.Spl and the Drosophi la transcr iption factor NTF-1 wereor iginal ly used to show that the TAF s in the TFIID complexcan function asco activators(Dynlacht et al ., 1991). Recentin vi tro and in vivo studies mapped the activation domainof NTF-1 to a 56 amino acid region that contains a prepon-derance of hydropho bic residue s, in particular, isoleucines(Attardi and Tjian, 1993). Therefore, it seem ed appropriateto compare the TAF requirements for activation by thesetwo apparently dist inct types of transcr iptional activators.

As an ini tial step in identi fying which TAFs m ight serveas coactivators for NTF-1, we determined whether NTF-1makes direct contact w ith the TFIID complex. First, TFIIDwas immunopuri f ied with a resin coated with anti -TBP anti -bodies, and the resulting aff ini ty m atr ix was used to bind in

vi tro translated, radiolabeled NTF-1. We found that NTF-1bound e ff ic iently to the TFl lD aff ini ty resin, indicating thatone or more of the TFIID subunits can interact directlywith NTF-1 (Figure 5A). Next, we tested the abi l i ty of theminimal activation domain of NTF-1 to bind individualTAFs . For these exper iments, an aff ini ty resin containingonly the 56 amino acid NTF-1 activation domain was gen-erated. Binding assays showed tha t the N-terminal regionof TAF11150 contains an interaction surface for this activa-t ion domain (Figure 58). Of al l the other T AFs tested forbinding to NTF-1, TAFr60 was the only other subunit thatinteracted specif ical ly with the aff ini ty resin. As expected,none of the TAF s bound to the control resin lacking theactivato r peptide. We also carried out protein bindingexper iments with recombinant TAF11150 and ful l- lengthNTF-1. First, TAF,,lSO was immunopuri f ied from Sf9 ex-tracts, and the aff ini ty resin containing TAF,,l50 was mixedwith recombinant NTF-1. The isolated com plexes weresubsequently analyzed by SDS-PAGE and the presenceof NTF-1 detected by immunoblot analysis. Our resultsindicate that NTF-1 can indeed bind selectively toTAF,,150

8/2/2019 TFII

7/13


A B

c t Ga14-kD ....e.. T41;,,250 NTF-1 -Azoo-117-

XO- ew.# lNC vi*- -TM,,60

so-- -TBP

Figure 6. Two Different Trimeric Complexes Mediate Activation by GAL4-NTF-1 but Not by Spl(A) Silver-sta ined gel of the TBP-TAFIr250-TAF1160 complex. This complex was assembled by addition of TAFrr60 to the TBP-TAF,,250 complexas described in Figure IC.(B) Distinct partial TFIID complexes were tested for their ability to support activation by a chimeric activator consisting of the GAL4 D NA-b indingdomain fused to the 56 amino acid NTF-1 minim al activation domain (GAL4-NTF-1). Approximately equimolar amounts (5 ng TBP) of TBP-TAFrr250(lanes l-3) TBP-T AF,,250-T AF,,150 (lanes 4-6) TBP-TA F,,250-TAF,r15 0-TAkrl lO (lanes 7-Q), or TBP-T AFr,250-TA F,,60 (lanes 10-12) weresupplemented to a TFIID-dependent human transcription system. Either no activator ( lanes 1, 4, 7, and 10) or approximately 10 ng (lanes 2, 5,8, and 11) or 30 ng of GAL4-NTF-1 (lanes 3, 6, 9, and 12) was added. The TBP-TAFr,250-TAFrr60 complex was also tested in e ither the absence(lane 13) or presence of 10 ng (lane 14) or 30 ng (lane 15) of Spl. Transcription products were detected by primer extension.

(Figure 5C). These results establ ished that a wel l -definedminimal activation region of the Drosophi la activatorNTF-1 can bind and directly contact TAFl l150 as wel l asTAF,,G O. Our f indings also suggest that the activator inter-face of TAF,,lSO is located within the N-terminal third ofthe molecule, whi le the C-terminal region contains sur-faces that interact with TBP and TAFI1250 (Verri jzer et al .,1994). These results point to the possibi l ity that NTF-1activation may be in part mediated by interactions withTAF,,l50 and TAF,,GO .Two Distinct Ternary Complexes SupportActivation by NTF-1 but Not SplTo investigate the functional relevance of the NTF-l-TAFinteractions, we tested whether a minimal T FIID complexcontaining ei ther TAFM 150 or TAF,,GO could mediate activa-t ion by NTF-1 (Figure 6). For these expe r iments, the 56amino acid activation domain of NTF-1 was fused to theGAL4 DNA-binding region (GAL4-NTF-1). As expected,the minimal T BP-TAF11250 complex was not suff ic ient tomediate activation by GAL4-NTF-1 (Figure 6B ). Howeve r,the ternary complex containing TAFI1150 supported strongactivation by GAL4-NTF-1 (- /-fold) . Conversely, this tr i-meri t co mplex was not responsive to Spl (see Figures38 and 3C), thus correlating transcr iptional activi ty andspecif ic activator-TAF interactions. Recal l , however, thata quadruple complex containing both TAF,,150 and

TAFrr l iO responded eff ic iently to activation by Spl. Im -portantly, this quadruple complex also eff ic iently sup-ported activation by GAL4-NTF-1. Taken together, theseresults support the notions that di fferent types of activatorsrequire distinct coa ctivators and that some partial com-plexes can mediate activation by mult iple activators.

Our protein interaction studies establ ished that the coreactivation domain of NTF-1 not only binds TAF,,150 butalso interacts with TAF,,G O. To assess the coactivator po-tential of TAFrr60, a di fferent tr imer ic complex, containingTBP, TAFI1250, and TAFrr60, was assembled (Figure 6A)and tested for i ts abi l ity to support activation (Figure 6B).Interestingly, the tr imeric TBP-TAFa250-TAF,r60 com-plex, containing TAF,,GO instead o f TAF,,150 , also re-sponded to GAL4-NTF-1 activation (5-fold, lanes 1O-l 2)but was unable to support transcr iptional activation by Spl( lanes 13-15). This result fur ther confi rms the di fferentialcoactivator requirements for GAM-NT F-1 and Spl. To-gether, these exper iments establ ish that binding of NTF-1to ei ther TAF11150 or TAF,,GO is a functional ly relevant inter-action that can direct activation.Recombinant Holo-TFIID Mediates Transcr iptionalActivation by Several Different ActivatorsEncouraged by our succes s in assembl ing active partialTFIID complexes, we next attempted to reconsti tute holo-TFIID containing TBP and all eight TAF subunits that we

8/2/2019 TFII

8/13

8/2/2019 TFII

9/13

Activator-Specific TBP-TAF Complexes10 1

A minimal TBPlTAF complexfails to support activation.

Figure 8. A Model for Transcriptional Activa-tion by Different Promote r-Specific Transcrip-tion Factors

Two distinct trimeric complexescan support activationby Gal4-NTF-1, but not by Spl.

A tetrameric complex, containingTAFIIllO can mediateSpl-responsive activation.

Holo-TFIID supports activationby an assortment of activators.

1991; Tanese et al ., 1991). Furthermore, we showed thatcertain activators directly interact with speci f ic TAFs (Hoeyet al ., 1993; Goodrich et al ., 1993). Howeve r, direct evi-dence for the role of individual TAFs dur ing activation oftranscr iption was lacking. Here, we report the assemblyand pur i f ication of var ious functional TBP-TAF complexesreconsti tuted from the nine subunits of TFIID . Differentactivators required distinct subunits for transcr iptional ac-t ivation, wh i le reconsti tuted holo-TFIID was able to supporttranscr iption by a var iety of di fferent activators. These re-sul ts establ ish that TBP plus the TAFs is suff ic ient to re-place endogenous TFIID to support activated transcr iptionin vitro.The Role of Individual TAFs in the TFIID Comp lexAn in vi tro assembly strategy al lowed us to determine thefunction of part ial c omplexes and analyze the require-ments for speci f ic TAF s. These stud ies revealed howsome activators com municate with TFIID to mediate tran-scr iption (Figure 8). Neither TEP alone nor a minimal com-plex containing TBP and TAF1,250 is able to mediate acti -vation by ei ther Spl or GAL4-NTF-1 (Figure 8A). By

contrast, i f ei ther TA F,,150 or TAFI180 is incorporated intothe complex, GAL4-NTF-1 eff ic iently activates transcr ip-t ion (Figure 88). As might be predicted, both of theseTA Fsbind directly to the NTF-1 activation domain, defining anovel set of activator-TAF interactions. Moreover, theseresults demonstrate that GAL4-NTF-1 can activate tran-scr iption via two distinct p art ial TF IID complexes, indicat-ing the potential for dual or mult iple pathways by whicha single activator can mediate transcr iption. Importantly,these tr imeric complexes that were active for GAL4-NTF-1 failed to support activation by Spl (Figure 8s). In-stead, Spl-dependent activation required the presence ofTAF,,l lO (Figure 8C ). A complex containing TBP-TAF,,250-TAF,,lSO-TAF,l l O appears to be the minimalol igomer responsive to activation by Spl. Interestingly,this tetrameric complex was also able to mediate NTF-1activation, since i t contains TAF,,lBO. Final ly, when al l themajor TAFs (TAFI1250, TAF,,150 , TAF,,l lO, TAFe80,TAF,,G O, TAF,,40 , TAF,130a, and TAF,,308) and TBP wereincorporated into a holo-TFIID, the recombinant complexwas able to support activation by an assortment of di fferentclasses of activators. Thus, al though we cannot exclude

8/2/2019 TFII

10/13

Cd l10 2

a role for potential minor TA Fs in the native TFII D com plex,our recombinant TFIID seems to be completely functional.

Our subunit assembly studies confi rmed that TBP-TAF,,250 can serve a s a core TFIID co mplex. Al thoughthis minimal com plex is incapable of mediating activationby the upstream activators we tested, the specif ic activi tyof this comp lex in basal tran scription is indistinguishablefrom TBP alone. This result is noteworthy in l ight of previ-ous studies that reported that the presence of TAFI1250actua lly abolished basal transcription by preventing thebinding of TBP to the TATA box (Kokubo et al ., 1993a).Howeve r, in these previous studies, increasing amountsof free, denatured, and renatured TAFI1250 were addedto the transcr iption reactions, perhaps result ing in nonspe-ci f ic inhibitory interactions. By contrast, in our case, theTBP-TAF11250 complex is preassembled and pur i fied byvir tue of an aff ini ty resin directed against TAF,,250 . There-fore, i t is highly unl ikely that there is an excess of freeTBP in the transcr iption reaction to account for the ob-served basal transcr iption.Coactivator Requirements for Spl ActivationOur studies revealed that TAF,,l lO behaves in a mannerexpected of a bona f ide coactivator for Spl . I t is requiredfor activated but not basal transcr iption, and i t is directlycontacted by Spl . Since we have demonstrated the re-quirement for TAFl l l 10 in reconsti tuted transcr iption reac-t ions, i t seems reasonable to conclude that TAF,,l lOserves as an adaptor to l ink Spl to the basal transcriptionalapparatus. A simple mechan ism by which Spl might workis to recrui t TFIID to the DNA template by binding specif i -cal ly to TAFl l l 10, thus increasing the number of preinit ia-t ion complexes. Al ternatively, Spl might inducesomeal lo-ster ic change of the TFII D structure by contactingTAFl l l 10, thereby enhancing the assemb ly of a functionalinitiation com plex and increasing the rate of initiation.Whether the function of TAFl l l 10 is merely to serve as abinding target for Spl or wheth er this subun it p articipatesin additional step s during activatio n remains to be deter-mined.

We were surpr ised to f ind that TAFI1150 was also im-portant to reconsti tute Spl activation. Unl ike TAF,,l lO,TAF,,150 fai ls to contact Spl. Moreover, TAF,,150 is notnecessary for the incorporation of TAF tI 10 into theTFl lDcomplex. Interestingly, i t appears that other TAFs ( i .e.,TAFI160) cannot substi tute for TAFI1150 , since several par-t ial complexes containing TAFII1 10 but lacking TA FI1150fai led to support S pl-dependent transcr iption (J.-L. C. andFt. T., unpubl ished data). What, then, is the function ofTAFlll 50 during S pl a ctivation? Since TAFlll 50 can recog-nize and bind core promoter seque nces (Verrijzer et al.,1994) i t is possible that this subunit helps stabi lize thebinding of TFIID to certain promoters. Al ternatively, i t maybe that the presence of an activator contacting TFIID mightinduce TAF,l l50 to bind DNA. Cons istent with this interpre-tation, a recent stud y using endogenous TFIID showedthat Spl can help TFIID but not TBP bind to the init iator(Kaufmann and Smale, 1994). It is also possible thatTAFI1150 functions dur ing so me subsequent step in theassembly of an ini tiat ion complex. Our prel iminary f indings

indicate that TAF,,150 may interact with some o f the basalfactors directly (K. Y., C. P. V., and Ft. T., unpubl isheddata), consistent with the idea that TAFII150 may contrib-ute to events other than making direct contact with activa-tors. Interestingly, a recent study suggested a role forTAFs in the recrui tment of basal factors dur ing activation(Choy and Green, 1993).

The minimal com plex that is responsive to Spl containsnot only TBP, TAFl l l 10, and TAFI1150, bu t also TAFI1250.A wel l -establ ished function of TAF,,250 is to serve as acore subunit contacted by TBP and other TAFs , includingTAFl l l10 (Weinzierl et al ., 1993a). Is the function ofTAFI1250 dur ing Spl a ctivation pr imarily a structural one?Although i t is not directly contacted by Spl , i t is possiblethat TAFtI plays a more active role dur ing transcr ip-t ional activation. Indirect evidence supporting this idea isprovided by the observation that a temperature-sensit ivemutation of TAFtI leads to a cel l cycle arrest phenotypeand affects transcr iptional activation both in vi tro and invivo (Wang and Tj ian, 1994). Moreover, TAF,,250 appearsto remain a ssociated with a complete TFII D complex atthe nonpermissive temperature (E. H. Wang and Ft. T.,unpubl ished data). Thus, TAFI1250 may play some role intransmitt ing the activation signal once TAF,,l lO is con-tacted by Spl .Activator-Specif ic TBP-TAF ComplexesAn important conclusion from the exper iments descr ibedin this paper is that dist inct classes of activators targetdi fferent TA Fs, thereby increasing the specif ic i ty and di-versi ty through combinator ial interactions. Activation do-mains of di fferent transcr iption factors suc h as the Gln-richdomains of Spl, the Pro-rich CTF activation domain, theacidic activation domain of VP16 (reviewed by Mitchel land Tj ian, 1969), and the I le-r ich moti f of NTF-1 (Attardiand Tj ian, 1993) have been loosely classi f ied on the basisof their character ist ic amino acid composit ion. Here, wehave identif ied TAFII150 and TAFI160 as the direct targetsof NTF-1 . Importantly, the NTF-1 minimal activation do-main fai ls to interact selectively with TAF,,l 10, and, con-versely, Spl does no t bind TAF,,150. We also fai led todetect interaction of NTF-1 w ith TAFI140 and of VP16 withTAF,,150 (data not shown). Most importantly, we foundthat the TAFs involved in these specif ic TAF-activator in-teraction s are also required fo r transcriptional activa tion.Taken together, these data lend strong support to the ideaof a mechan ism in which an integral step in the processof transcr iptional activation involves di fferent c lasses ofactivators targeting distinct TA Fs in the TFII D complex.

Can we general ize these observations? Recent studiesindicate that TAFe l 10 binds several different Gln-rich acti-vation domains, including Spl regions A and B (Hoey et al .,1993), CAMP response element-binding protein (CREB)(Ferreri et al., 1994) and the Drosoph ila transcription fac-tor buttonhe ad (F. Sauer, unpublished data). Similarly, anumber o f di fferent acidic activation domains were foundto interact selectively with TAF1 140 (C. Thut and R. T., un-publ ished data). We also suspect that other activation do-mains, related to the Ile-r ich activator of NTF-1, wi l l targetTAFI1150. Thus, i t seems l ikely that mult iple activators of

8/2/2019 TFII

11/13


a given class wi l l target a part icular TAF. It is importantto note, how ever, that the preponderance of a part icularset of amino acids within activation domains does not nec-essar i ly predidt a speci f ic interaction with a given TAF .For example, a number of Gin-rich transcr iption factorsfai led to bind TAFI~l10 (Hoey e t al ., 1993). Th erefore, thebasis for the specif ic i ty of interactions between activatorsand TAFs remains unclear. Identi fying the targets of otheractivators may help determine which features of theseproteins are critical for recognition and transcription (seeTj ian and Maniatis, 1994). In addit ion, such studies m ayresult in a more functional classi f ication of activation do-mains.

Our studies with NTF-1 revealed that some activatorsmay interact with mult iple TAF s ( i .e., TAFI1150 andTAF,,GO ), and that di fferent co mplexes containing distinctcoactivators are able to support transcr iptional activation.An important lesson from these results is that a single,relatively smal l (56 amino acids) activation domain canwork via two distinct pathway s. This observation alsoraises the possibi l ity that such mult iple contacts may playsome role dur ing synergistic activation (reviewed byPtashne, 1988). At present, we have not addressed thisintriguing ques tion. It is also plausible that a single TAFcan provide interfaces for interaction with di fferent typesof activators. For example, i t has recently been determinedthat a region of TAF,,l lO, dist inct from the Spl-bindingdomain, interacts with ElA (J. V. Geisberg, J.-L. C., andR. P. Ricciardi , unpubl ished data). As discussed above,there need not be a simple one-on-one relationship be-tween activators and TAFs. W e have shown that a singleactivator can contact mult iple T AFs and, conversely, thata given TAF can interact with more than one activator.This provides the necessary combinator ial complexi ty toal low communication between di fferent activators andT F I ID .

Final ly, an important con clusion from ourstudieson TAFrequirements for activation by Spl and NTF-1 is thatTAF,,lSO might have two distinct roles in the activationprocess. In the case of NTF-1, i t is directly contacted bythe activation domain and might serve asimi larfunction asTAF,,l 10 in Spl activation. Howeve r, al though not directlycontacted by Spl, TAF,,lSO is also cr i t ical for activation bythis transcr iption factor. Thus, a single TAF may performdifferent tas ks dur ing activation by di fferent enhancer pro-teins.TFIID: A Molecular Central Processing UnitEukaryotic promoter and enhancer regions are often com-plex and designed to bind a large array of different se-quence-specif ic transcr iption factors. The expression ofa given gene therefore depends on the interplay betweenvar ious DNA bound enhancer factors and the basal tran-scr iption machinery. The discovery of TAFs and the stud-ies reported here provide the most direct evidence for thenotion tha t mult iple subunits of TFIID , each with distinctstructural character ist ics, can provide unique and specif icsurfaces for interactions with di fferent upstream activa-tors. This arrangement provides the opportuni ty for mult i -ple activators and repressors to interact simultaneously

with componen ts of the transcr iptional machinery. Wetherefore propose that TFII D may function as a centralprocessing unit to integrate the information from a mult i -tude of enhancer and promoter-bound factors to controlthe level of transcription initiation. No w that it is possibleto reconsti tute TFIID in vi tro, future research may unravelthe molecular mechan isms of transcr iptional activation inmore detai l and provide a cr i t ical test to this general hy-pothesis.

Expression and Purification of Recombina nt TAFs and TB PAll recombinant TFIID subunits were expressed in the baculovirusexpression system. Most expression constructs contain ing full- lengthcDNAs have been described previously(dTBP , Weinzierl et al., 1993a;HA-hTAF,,250, Rupperl et a l., 1993; dTAF,,l50, Verrijzer et a l., 1994;dTAFIIl lO, Hoey et a l., 1993; dTAF,,80, Dynlacht et a l., 1993; dTAF,,GO,Weinrierl et a l., 1993b; dTAF,,40, Goodrich et a l., 1993; dTAF,,30a,Yokomori et a l., 1993a). Full- length dTAF,,250 was obtained from threeoverlapping cDNA clones. The complete coding sequence was sub-cloned into a modified version of the pVL1392 baculovirus expressionvector (Pharm ingen). The dTAF ,,30P expression vector was generate dby subcloning the Ndel fragment contain ing the fu ll- length cDNA intopVL1392. All recombinant v iruses were p laque purif ied and amplif ied.All prote in preparation, purif ication, and in v itro complex assemblywas carried out in buffer HEMG-ND (25 mM HEP ES [pH 7.61.0.1 mMEDTA, 12.5 mM MgCI,, 10% glycerol, 0.1% NP -40, 1.5 mM DTT, 0.2mM AEB SF (CalBiochem), 1 mM sodium metabisulf ite , 0.7 pglml pep-statin, 2 wglml leupeptin) contain ing variable concentrations of KCI. AllTFIID subunits were purified by convention al column chromatogra phy,fo llowed in some cases by prote in or DNA affin ity chromatography.Importantly, some of the TAFs were only partia lly purif ied ( i.e.,dTAF,,GO, 10% pure). How ever, the selectivity of the TFIID assemblyresulted in a near homogeneous complex.Assembly of TBP-TAF Complexes In VltroThe procedure for the assembly of the TFIID complex is outli ned inFigure 1C. Anti-HA monoclonal antibody 12CA5 was covalently l inkedto protein A-Sep harose as described (Zhou et al., 1992). The entireprocedure was carried out at 4OC in HEMG-ND contain ing 0.1 M orhigher concentrations of KCI. First, HA-TAF,,250 was incubated withthe a-HA affin ity column for l-2 hr, and free HA-TAFI1250 was thenremoved by extensive washing . Next, a molar excess (at least IO-fold)of each subsequent subunit was added. After 3 hr at 4OC, unboundsubunits as well as impurit ies were removed by repeatedly washingwith a lOO-fold excess of buffer. The assembly process resulted insuch an additional purif ication that the resulting complexes were nearhomoge neous. This procedure was repeated for each successive TAF.The resulting TFIID complexeswere e luted with HEMG-ND buffer con-ta in ing 1 mg/ml H A peptide (YPYD VPDY A) for 1 hr. The eluted com-plexes were analyzed by SDS-PA GE, fo llowed by s ilver sta in ing andWestern b lott ing and used in transcription reactions. Reimmunopre-cipitati on reactions were performed as previously described (Weinzierlet a l., 1993a).In Vitro Protein-Protein Interaction AssaysThe Spl -TAF interaction assay was carried out using Spl D NA affin itycolumn essentially as described previously (Hoey et al., 1993), withthe exceptions that the KCI concentration was 100 mM, and baculovi-rus-expressed TAFs were used instead of in vitro translated proteins.The NTF-1-TAF interactions were studied sim ilarly as described (Yo-komori et a l., 1993a). The TFIID complex was immunopurif ied as de-scribed (Hoey et a l., 1993). To generate an NTF-I minim al activationdomain affin ity resin, a synthetic 63-mer CGGEHQP-NTF-1 (aminoacids 169-228) was covalently attached to divinylsulfone-de rivatizedagarose via a th ioether l inkage (-1 mg of peptidell ml hydratedbeads). About 15 ~1 of beads with or without peptide was incubatedwith radio labele d TAFs for several hours at 4C. After extensive wash-ing with 0.1 M HEMG-ND, the beads were resuspended in samplebuffer and analyzed by SDS-PA GE fo llowed by autoradiography.

8/2/2019 TFII

12/13

in Vitro TranscriptionIn vitro transcription assays were performed in either the Drosophilaor HeLaTFiiDd epende nt fractionated transcription system. The DNAtemplates used included (GC)3BCAT (Dynlacht et al., 1991). whichcontains three S pl sites upstream of the El B TATA box, and GsBCAT(Lillie and Green, 1969), which contains five GALC binding sites. Splwas overexpressed in HeLa cell using a vaccinia virus-expressingvector a nd purified to near homogeneity as described (Hoey et al.,1993). GAL4 (residues l-147), GAL4-VP16c (residues 457-490),GAL4-NTF-1 (residues 173-226), GAL4Jun A2 (cJun activation do-main A2, residues 139-253), and GAL4-CTFwere expressed in Esche-richia coli and purified as previously described (Goodrich et al., 1993;Attardi and Tjian, 1993; Baichw al and Tjian, 1990; Tanese et al., 1991).The Drosophila fractionated transcription system was established es-sentially as described (Wampler et al., 1990; Dynlacht et al., 1991).A HeLa TFIID-dependent transcription system was prepared essen-tially as described (Dignam et al., 1963). Nuc lear extracts were fraction-ated on Phospho cellulose Pll (Whatman). The flowthrough (PC.l),0.3 M eluate (PC.3), 0.5 M eluate (PC.5), and 1 M eluate (PC1 .O) werecollected. The TFIID activity (PC1 .O) was further fractionated by on aDE52 column (Chiang et al., 1993; Meisterernst et al., 1991) and theflowthrough (DE.1; US A) and 0.3 M eluate (DE.3) collected. The PC.1,PC.5, a nd DE.1 fractions were reap plied to PI 1 and DE52 to removeany potentia l TFIID contamina tion. The transcription assays were per-formed as previously described (Pugh and Tjian, 1990). Typical reac-tions (25 ~1) contained 100 ng of DNA tem plate, 2 pg of PC.1, 1.4 pgof PC.5, and 100 ng of DE.1 (USA) fractions, and several nanogramsof either endogeno us HA-TFIID or assembled TBP-TAF complexes.HA-tagged huma n TFIID was immuno purified as described (Zhou etal., 1992). The reaction products were detected by primer extens ion.Products were visualized by autoradiography and quantifie d by Phos-pholmag er analysis (Molecular Dynamics). Each transcription reactionwas repeated several times, and representative data are shown.

We are grateful to D. King for the NTF-1 peptide resin, R. Weinzierlfor monoc lonal antibodies against TAFs, S. Ruppert for hTAF,,250-expressing baculovirus, J. Goodrich and A. Vasserot for GAL4-VP16cand GAL4Jun. and Y. Mul for Sf9 and H5 cell stocks. We thank H .Beckman, T. Hoey, and E. Wang for helpfu l technical advice and C.-M.Chiang for advice on anti-FLAG M2 purification, and G. Culter forexcellent computer artwork. Finally, we thank W. Herr, T. Hoey, S.McKnigh t, B. Meyer, E. OShea, G. Peterson, D. Reinberg, D. Rio, K.Yama moto, and members of the Tjian laboratory, especially R.Dikstein, G. Gill, J. Goodrich, D. King, M. Maxon, and E. Wang forcritical comments on the manuscript. C. P. V. is supported by a long-term European Molecular Biology Organization fellowship, and K. Y.by a Leukem ia Society Fellowship. This work was supported in partby a grant from th e National Institutes of Health to R. T.Received July 1, 1994; revised August 4, 1994.ReferencesAttardi, L. D., and T jian. R. (1993). Drosophila tissue-specific transcrip-tion factor NTF-1 contains a novel isoleucine-rich activation motif.Genes Dev. 7, 1341-1353 .Baichwa l, V. R., and Tjian, R. (1990). Control of cJun activity by inter-action of a cell-specific inhibitor with regulatory doma in 6 : differencesbetween v- and cJun. Cell 63, 815-625.Chiang, C.-M., Ge, H., Wang, Z., Hoffmann, A., and Roeder, R. G.(1993). UniqueT ATA-bin ding protein-containingcomplexe sand cofac-tors involved in transcription by RNA polymerases II and ill. EMBOJ. 12, 2749-2762.Choy, B., and Green, M. R. (1993). Eukaryotic activators function dur-ing multipl e steps of preinitiation complex assembly. Nature 366,531-536.Dignam , J. D., Martin, P. L., Shastry, B. S., and Roeder, R. G. (1983).Eukaryotic gene transcription with purified components. Meth. Enzy-mol. 707, 582-596.Dynlacht, B. D., Hoey, T., and Tjian, R. (1991). Isolation of coactivators

associated with the TATA-bin ding protein that media te transcriptionalactivation. Cell 66, 563-576.Dynlacht, B. D., Weinzierl, R. 0. J., Admo n, A., and Tjian, R. (1993).The dTAF,,80 subun it of Dmsophila TFIID contains beta-transducinrepeats. Nature 363, 176-179.Ferreri, K., Gill, G., and Montminy, M. (1994). The CAMP-reg ulatedtranscription factor CREB interacts with a compone nt of the TFIIDcomplex. Proc. Natl. Acad. Sci. USA 91, 1210-1213.Gill, G., and Tjian, R. (1992). Eukaryotic coactivators associated withthe TATA box binding protein. Curr. Opin. Genet. Dev. 2, 236-242.Gill, G., Pascal, E., Tseng, Z. H., andTjian , R. (1994). Aglutamine-richhydrophobic patch in transcription factor Spl contacts the dTAF,,l IOcomponen t of the Drosophila TFIID complex and mediates transcriptional activation. Proc. Na tl. Acad. Sci. USA 91, 192-196.Goodrich, J. A., and Tjian, R. (1994). TBP-TAF complexes: selectivityfactors for eukaryotic transcription. Curr. Opin. Cell Biol. 6, 403-409.Goodrich, J. A., Hoey, T., Thut, C. J., Admo n, A., and Tjian, R. (1993).Drosophila TAF,,40 interacts with both a VP1 6 activation doma in a ndthe basal transcription factor TFIIB. C ell 75, 519-530.Hernandez, N. (1993). TBP, a universal eukaryotic transcription fac-tor? Genes Dev. 7, 1291-1308.Hisatake, K., Hasegawa, S., Takada, R., Nakatani, Y., Horikoshi, M.,and Roeder, R. G. (1993). The ~2 50 subunit of native TATA box-bindin g factor TFIID is the cell-cycle regulatory protein CCGl. Nature362, 179-181.Hoey, T., Weinzierl, R. 0. J., Gill, G., Chen, J.-L., Dynlacht, 8. D.,and Tjian, R. (1993). Molecular cloning and functional analysis of Dro-sophila TAFllO reveal properties expected of coactivators. Cell 72,247-270.Kaufm ann, J., and Sma le, S. (1994). Direct recognition of initiator e le-ments by a componen t of the transcription factor IID complex. GenesDev. 8, 821-829.Klug, A. (1993). Opening the gateway. Nature 3 65, 486-487.Kokubo, T., Gong, D.-W., Yam ashita, S., Horikoshi. M., Roeder, R. G.,and Nakatani, Y. (1993a). Dmsophile 230-kD TFIID subunit, a func-tional homolog of the human ce il cycle gene product, negatively regu-lates DNA bindin g of the TATA b ox-binding subunit of TFIID. GenesDev. 7, 1033-1046.Kokubo, T., Gong, D-W., Yamas hita, S., Takada, R., Roeder, R. G.,Horikoshi, M., and Nakatani, Y. (1993b). Molecu lar cloning, expres-sion, and characterization of the Dmsophile 6Bkilod alton TFIID sub-unit . Mol. Cel l . B iol . 13, 7859-7863.Kokubo, T., Gong, D.-W., Roeder, R. G., Horikoshi, M., and Nakatani,Y. (1993~). The Dmsophi/a 1 O-kDa transcription factor TFIID subunitdirectly interacts with the N-terminal region of the 230-kDa subunit.Proc. Na tl. Acad. Sci. USA 90, 5696-5900.Kokubo, T., Gong, D.-W., Woottoon, J. C., Horikoshi, M., Roeder,R. G., and Nakatani, Y. (1994). Molecular cloning of Dmsophile TFIIDsubunits. Nature 367, 484-487.Lillie, J. W., and Green, M. R. (1989). Transcription activation by theadenovirus Ela protein. Nature 338, 39-44.Meisterernst, M., Roy, A. L., Lieu, H. M., and Roeder, R. G. (1991).Activation of class II gene transcription by regulatory factors is potenti-ated by a novel activity. Cell 66, 981-993.Mitchell, P. J., andTjian , R. (1989). Transcriptional regulation in mam-malia n cells by sequence-specific DNA bindin g proteins. Science 245,371-378.Ptashne, M. (1988). How eukaryotic transcriptional activators work.Nature 3 35, 683-689.Pugh, B . F., and Tjian, R. (1990). Mech anism of transcriptional activa-tion by Spl : evidence for coactivators. Cell 67, 1187-l 197.Ruppert, S., Wang, E. H., and Tjian, R. (1993). Clonin g and expressionof human TAF,,250: a TBP-associated factor implicated in cell-cycleregulation. Nature 362, 175-179.Tanese, N., Pugh, 8. F.,andTjian, R.(1991). Coactivatorsforaproline-rich activator purified from the multisubu nit human TFIID complex.Genes Dev. 5, 2212-2224 .Tjian, R.. and Maniatis,T. (1994).Transcriptionalactivation: acomplex

8/2/2019 TFII

13/13


puzzle with few easy pieces. Cell 7 7, 5-8.Verrijzer, C. P., Yokomori, K., Chen, J.-L., and Tjian, R. (1994). Dro-sophila TAFrr150: s imilarity to yeast gene TSM-1 and specific b indingto core promoter DNA. Science 264, 933-941,Wampler, S. L., Tyree, C. M., and Kadon aga, J. T. (1990). Fractionatio nof the general RNA polymerase II transcription factors from Drosoph ilaembryos. J. Bio l. Chem. 265, 21223-21231.Wang, E . H., and Tjian , R. (1994). Promoter-selective transcriptionaldefect in cell cycle mutant ts13 rescued by hTAF ,,250. Science 263.811-814.Weinzierl, A., Dynlacht, B. D., and Tjian, Ft. (1993a). Largest subunitof Drosophi le transcription factor IID directs assem bly of a complexcontain ing TBP and a coactivator. Nature 362, 51 l-517.Weinzierl, R. 0. J., Ruppert, S., Dynlacht, B. D., Tanese, N., and Tjian,R. (1993b). Clonin g and expression of Drosophila TAF,,80 and humanTAF,,70 reveal conserved interactions with other subunits of TFIID.EMBO J. 72, 5303-5309.Yokomori, K., Chen, J.-L., Admon, A., Zhou, S., and Tjian, R. (1993a).Molecular c loning and characterization of dTAF,,30o and dTAF,,308:two small subunits of Drosophila TFIID. Genes Dev. 7, 2587-2597.Yokomori. K., Admon, A., Goodrich, J. A., Chen, J.-L., and Tjian, R.(1993b). Drosoph ila TFIIA-L is processed into two subunits that areassociated with the TBP/TAF complex. Genes Dev. 7, 2235-2245.Zhou, Q., L ieberman, P. M., Boyer, T. G., and Berk, A. J. (1992).Holo-TFIID supports transcriptional stimu lation by diverse activatorsand from a TATA-less promoter. Genes Dev. 6, 1984-1974.

tfii

Documents