Cell, Vol.

79, 93-105,

October

7, 1994, Copyright

0 1994 by Cell Press

Assembly of Recombinant TFIID Reveals Differential Coactivator Requirements for Distinct Transcriptional Activators
Jin-Long Chen, Laura Donatella Attardi, C. Peter Verrijzer, Kyoko Yokomori, and Robert Tjian Howard Hughes Medical Institute Department of Molecular and Cell Biology University of California, Berkeley Berkeley, California 94720-3202 1991) led to the hypothesis that TBP must associate with one or more coactivators to mediate transcriptional activation. The principle features of this coactivator hypothesis were that coactivators would be required to mediate activation but would not be necessary for basal transcription and that coactivators would be directly targeted by activation domains via specific protein interfaces (Pugh and Tjian, 1990). Moreover, it was envisioned that different classes of activators might interact with and therefore require distinct coactivators. Thus, the relationship between TAFs and coactivators has became a focal point of our research. Critical evidence in support of the coactivator model was the demonstration that the TAFs in the TFIID complex (TAF,,250, TAF,,150, TAF,,l 10, TAF,,80, TAF,,GO, TAF,,40, TAF1,30a, and TAFa30P) were responsible for coactivator function. This was accomplished byantibodyaffinity purification of Drosophila and human TFIID to apparent homogeneity, followed by separation of the TAFs from TBP with mild denaturants. Reconstitution of purified TBP with immunoaffinity-purified subunits established that the TAFs could provide coactivator function to diverse promoterspecific transcription factors such as Spl , neurogenic element-binding transcription factor 1 (NTF-I), and CCAATbinding transcription factor (CTF) (Dynlacht et al., 1991; Tanese et al., 1991). Subsequent studies confirmed that several other activators, includingzta, GAL4-El A, GAL4VP1 6, and GAL4-AH, which had been previously shown to contact basal factors such as TBP, TFIIB, or both directly, also required TAFs for activation (Chiang et al., 1993; Choy and Green, 1993; Zhou et al., 1992). To decipher the molecular basis of coactivator functions, a major task was the purification, molecular cloning, and expression of the TAF subunits from Drosophila and human TFIID. To date, cDNA clones and expressed proteins have been obtained for each of the eight TAFs found in the Drosophila TFIID complex (Dynlacht et al., 1993; Goodrich et al., 1993; Hoey et al., 1993; Kokubo et al., 1993a, 1993b, 1993c, 1994; Verrijzer et al., 1994; Weinzierl et al., 1993a, 1993b; Yokomori et al., 1993a). In addition, several human TAFs (TAF11250, TAF,,130, TAF,,lOO, and TAF,,70) have also been partly characterized (Hisatake et al., 1993; Ruppert et al., 1993; Weinzierl et al., 1993b; N. Tanese and R. T., unpublished data). As expected, these subunits of TFIID appear to be evolutionarily conserved both in structure and in function. The availability of overexpressed recombinant TAFs has enabled detailed biochemical studies. In addition to various TAF-TAF and TAF-TBP interactions critical for the assembly of TFIID, we have identified specific interactions between individual TAFs and activation domains of various gene-specific transcription factors. The first of these studies established that TAF,,l 10 can interact selectively with the Gln-rich activation domains of Spl (Hoey et al., 1993). Furthermore, analysis of mutant Spl activation domains revealed a correlation between loss of transcriptional activation and failure to bind TAF,,l 10 (Gill et al.,

Summary We previously reported that transcriptional regulators can bind selected TAF subunits of the TFIID complex. However, the specificity and function of individual TAFs in mediating transcriptional activation remained unknown. Here we report the in vitro assembly and transcriptional properties of TBP-TAF complexes reconstituted from the nine recombinant subunits of Drosophila TFIID. A minimal complex containing TBP and TAFtI directs basal but not activator-responsive transcription. By contrast, reconstituted holoTFIID supports activation by an assortment of activators. The activator NTF-1, which binds TAFII~~O, stimulates transcription with a complex containing only TBP, TAF,,250, and TAF,,lLO, whereas Spl binds and additionally requires TAF,,llO for activation. Interestingly, TAF,,150 enhances Spl activation even though this subunit does not bind directly to Spl. These results establish that specific subcomplexes of TFIID can mediate activation by different classes of activators and suggest that TAFs perform multiple functions during activation. Introduction How do sequence-specific promoter- or enhancer-binding proteins communicate with the RNA polymerase II (pol II) transcriptional apparatus to stimulate mRNA synthesis? Several studies have implicated TFIID as a central player in this process (for review, see Gill and Tjian, 1992; Hernandez, 1993). Although the TATA box-binding protein (TBP) subunit of TFIID originally had been considered a likely target for activators, several observations have prompted a reevaluation of this simple and appealing model. It now seems probable that TBP rarely if ever exists as a free molecule with all of its potential interfaces exposed on the outside surface of the so-called saddle structure (for references, see Klug, 1993). Instead, in eukaryotic cells, TBP is predominantly found associated with sets of tightly bound subunits, or TBP-associated factors (TAFs), to form SLl, TFIID, or TFIIIB complexes representing essential components of the RNA pol I, pol II, and pol Ill transcription apparati, respectively (for review, see Goodrich and Tjian, 1994; Hernandez, 1993). The observation that TBP can direct basal levels of transcription in vitro yet requires a complement of TAF subunits to mediate activator-dependent transcription (Dynlacht et al., 1991; Pugh and Tjian, 1990; Tanese et al.,

Cell 94

IP- endoeenous dTFiiD 7gJ k

Figure plexes

1. In Vitro Assembly

of TBP-TAFCom-

qTAFII250
I__L -TAF,+O 11-1-TAFIIl10 -. -TAF$O 1 --I -TAF@ IgG -rl, TBP- - -. -. -TAF@, -TAFI,30P/u

TBP
1, 7+ TAFI@ HA-TAF@o

TAFI#O I,

(A) Endogenous Drosophila TFIID consists of nine subunits. The partially purified TFIID complex was immunoprecipitated with a monoclonal antibody directed against dTBP. The TAFs were selectively eluted from TBP with 1 M guanidine-HCI, separated by SDS-PAGE, and visualized by silver staining (lane 2). TBP remained bound to the antibodies (lane 1). (8) Schematic diagram of the TFIID complex. Indicated are dTBP and the eight major Drosophila TAFs as well as most of the specific TBP-TAF and TAF-TAF interactions we have identified thus far. This cartoon summarizes our current understanding of the architecture of the TFIID complex. However, it should be noted that, as discussed in the text, the complete picture is not yet at hand. (C) Strategy of in vitro assembly of the TFIID complex. All TFIID subunits were purified from extracts prepared from 5% cells infected with recombinant baculoviruses expressing either TBP or various TAFs. Drosophila or human TAF11250 fused to an N-terminal 8 amino acid HA-epitope peptide was immobilized on Protein A-Sepharose beads conjugated covalently with monoclonal antibodies directed against the HA-epitope. TAF,,250 was used as the foundation on which the complex was built by stepwise addition of TSP and the other TAFs. The order of assembly was as indicated in the cartoon. At each assembly step the partial TFIID complex was incubated with an excessof the next component. Unbound material was removed by extensive washes. Finally, recombinant holo-TFIID or partial complexes of interest were eluted under native conditions with the HA peptide and used in in vitro transcription reactions.

1994). The acidic activation domain of GAL4-VP16 was shown to interact with TAFI140, and antibodies directed againstTAFIAOinhibitedVP16-mediated activation but not basal transcription in vitro (Goodrich et al., 1993). Moreover, certain TAFs such as TAF,,l 10 and TAF,,40 interact directly with other components of the basal apparatus, including TFIIA and TFIIB (Yokomori et al., 1993b; Goodrich et al., 1993). Together, these results provided the initial evidence that specific activators make contact with distinct TAFs in the TFIID complex and that these proteinprotein interactions may transmit the activation signal to the basal machinery. Although these studies are consistent with the coactivator hypothesis, direct evidence for the role of individual TAFs in mediating transcriptional activation remained elusive. In particular, the experiments reported thus far could not demonstrate decisively that the interaction between TAFs and activators is a requisite step leading to transcriptional activation. Conclusive evidence for specific TAF requirements by different types of activators required reconstitution of distinct partial TBP-TAF complexes using purified subunits. Here we report the in vitro assembly of both complete TFIID and a variety of partial TBP-TAF complexes from

recombinant Drosophila TBP and TAFs. The role of individual TAFs in mediating activator-responsive transcription was tested by assaying the activity of various enhancer proteins bearing different classes of activation domains, such as Spl and NTF-1, in combination with distinct TBP-TAF complexes lacking specific subunits. Finally, we have tested the ability of an assembled holoTFIID containing TBPand the eight major DrosophilaTAFs to support activation by several unrelated transcriptional activators, including Spl, NTF-1, CTF, VP16, and cJun (activating protein 1 [AP-11). Our results provide evidence for the role of individual TAFs as coactivators capable of interacting with selected activation motifs to mediate transcription. Results Assembly of TBP-TAF Complexes in Vitro Endogenous Drosophila TFIID is a multiprotein complex comprised of TBP and at least eight TAFs (Figure 1A). Having recently obtained cDNAs for all the major Drosophila TAFs, we have begun to identify many of the proteinprotein interactions involved in the assembly of TFIID (Dynlacht et al., 1993; Verrijzer et al., 1994; Weinzierl et

Activator-Specific 95

TBP-TAF

Complexes

M (kD1 zoo-

1178(t

12345678 III

IO

Figure

2. The Dimeric

TBP-TAF,250

Complex

Supports

Basal but Not Activated

Transcription

(A) Silver-stained polyacrylamide gel of TBP-TAF,,250 complexes. A complex consisting of dTBP and either Drosophila TAFtI (dTAF,,250, lane 2) or human TAFf1250 (hTAF,,250, lane 3) was assembled using the strategy described in Figure 1C. Lane 1 shows a similar amount of dTBP alone. (B) A TFIIDdependent Drosophila fractionated transcription system was supplemented with either no protein (lanes 1 and 2) dTBP alone (5 ng, lanes 3 and 4) or a similar molar amount of either dTBP-dTAF,,250 (lanes 5 and 6) or dTSP-hTAF,,250 (lanes 7 and 6). As a control, endogenous, partially purified Drosophila TFIID (Ct.3 fraction) was added to reactions in lanes 9 and 10. Purified recombinant Spl (about 30 ng) was added to samples in the even-numbered 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, immunopurified to near homogeneity (lanes 5 and 6) was tested in either the absence (lanes 1, 3, and 5) or presence (lanes 2, 4. and 6) of 30 ng of purified Spl in a TFIID-dependent human fractionated transcription system. Transcription products were detected by primer extension.

al., 1993a; Weinzierl et al., 1993b; Yokomori et al., 1993a; J.-L. C., K. Y., and R. T., unpublished data). Our current view of the TFIID subunit architecture is summarized in Figure 1 B. Some TAFs, such as TAFI1250, TAF,,150, and TAF,,30a, are in direct contact with TBP, whereas other TAFs are brought into the complex via TAF-TAF interactions. In particular, the largest subunit of TFIID (TAF,,250) appearstofunctionasan importantscaffold formanyother TAFs. For example, TAFI1150, TAF,,l 10, TAF@, TAFI130a, and TAF,,308 all efficiently associate with TAF,,250. Furthermore, TAFII1 10 helps recruit TAF1180, while TAFI180 allows TAFtI to associate stably with the TFIID complex. In addition to these relatively robust interactions, there are also significantly weaker interactions between TFIID subunits. For example, TAFI180 binds inefficiently to TAF,,150, while TAF,,60 associates weakly with TBP. These results reveal that the TFIID complex is held together by a plethora of TAF-TAF interactions and a limited number of TAF-TBP interactions. We have used the results of these subunit interaction studies to design a strategy for assembling TBP-TAF complexes in vitro. Our goals were to reconstitute functional TBP-TAF complexes containing various combinations of TAFs and ultimately to assemble a recombinant holoTFIID. All TFIID subunits were purified from extracts prepared from Sf9 cells infected with recombinant baculoviruses expressing the TAFs or TBP. Through empirically driven trial and error, we arrived at a stepwise assembly

protocol in which successive subunits of TFIID were incorporated onto an immobilized core subunit (Figure 1C). We chose TAFI1250 as the foundation for building TFIID, because it appears to provide a scaffold contacted by many of the other TAFs. We immobilized hemagglutinin (HA)tagged full-length TAF,,250 on protein A-Sepharose beads covalently conjugated with anti-HA antibodies. This strategy was chosen so that we could conveniently elute transcriptionally active complexes with HA peptide under native conditions. Next, TBP was incubated with the immobilized TAF11250, and free TBP was removed by extensive washes, resulting in a dimeric TBP-TAFtI complex. This assembly process was repeated for each successive TAF in order to build up the TFIID complex. At certain stages during the reconstitution process, the order in which the TAFs were added proved to be important. For example, TAFII~ 10 was not able to associate stably with TBP-TAF complexes in the absence of TAF,,250 (data not shown). Likewise, TAFI140 only efficiently entered the complex after TAF,,GO was present. Furthermore, TAF,,80 was only able to join the complex after TAF,,llO and TAFI1150 had been assembled and had to be assembled before TAFI130a (data not shown). After various combinations of TAFs and TBP had been assembled, they were eluted with HA peptides and subjected to SDS-polyacrylamide gel electrophoresis (PAGE), followed by silver staining (see Figures 2A, 3A, 6A, and 7A) and Western blotting (data not shown). Taking into

Cell 96

B
Spl -TAF,1250 -TAFl11SO I I

C
A A 4 Spl - A
+

- A

117-

,-TAF11110

Figure

3. The TBP-TAFII~~O-TAF~,IBO-TAF~~~

10 Complex

Mediates

Activation

by Spl

(A) Silver-stained gel of distinct partial 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 vitro assembly was as described in Figure 1C. (8) Approximately equimolar amounts (5 ng of TBP) of these complexes were tested in the Drosophila fractionated system. Transcription reactions with 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) were performed 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 ability to support activation by Spl in the human transcription system. Reactions were performed in either the absence (lanes 1 and 4) or the presence of 10 ng of Spl (lanes 2 and 5) or 30 ng of Spl (lanes 3 and 6).

account the inefficient staining of TAFI1250 (Weinzierl et al., 1993a), we estimated that the purified complexes contain approximately stoichiometric amounts of the recombinant TFIID subunits. The integrity of eluted complexes was verified by reimmunoprecipitation using antibodies directed against different subunits (data not shown). These experiments established that once formed, both partial and complete TBP-TAF complexes remain stably associated. Using this strategy, we were able to reconstitute a recombinant holo-TFIID complex successfully. The TBP-TAF11250 Complex Supports Basal Transcription but Not Activation We first tested whether the minimal TBP-TAFr1250 complex would be sufficient to mediate activation in vitro. To address this question, the transcriptional properties of TBP alone were compared with those of a minimal complex comprised of TBP and either Drosophila or human TAFrr250 (Figure 2A). Purified TBP-TAF950 complexes were tested for their ability to support transcriptional activation by the activator Spl in reconstituted reactions derived from either Drosophila or HeLa fractionated transcription components (Figures 28 and 2C, respectively). The purified TBP-TAF,,PBO complexes supported basal transcription to a level comparable to that observed with TBP alone, whereas no transcription was detected in the absence of exogenousiy added TBP or TBP-TAF complexes. We did not detect any significant inhibition or stimulation of basal transcription, even though stoichiometric

amounts of full-length TAFe250 were present in these complexes. However, the presence of TAF11250 also did not restore responsiveness to the activator, Spl . By contrast, endogenous holo-TFIID directed high levels of Spl activation. These results indicate that the minimal complex containing TBP and TAFI1250 is capable of allowing basal levels of transcription, but fails to support activation by Spl . A Four-Subunit Complex Supports Spl Activation It was not surprising that a minimal complex containing TBP-TAF11250 failed to mediate Spl activation, since our previous studies showed that Spl binds directly to TAF,,llO (Hoey et al., 1993). Therefore, the TAFtI 10 subunit of TFIID might serve as an essential coactivator for Spl-dependent transcription. However, when we previously tested a ternary complex containing TBP, TAFr,l 10, and a truncated version of TAFI1250, only weak activation by Spl was observed in vitro (Weinzieri et al., 1993a). We had hypothesized that this weak response may be due to the use of a truncated form of TAFr,250. A more interesting alternative was that otherTAFs, in addition toTAFI1250 and TAF,,l 10, play some role in Spl activation. We therefore assessed the ability of the triple complex containing fulilength dTAF,,250 or hTAFrr250 to mediate Spl activation (Figure 3). Our data indicate that incorporating full-length TAF,,250 into the triple complex is not sufficient to support robust activation by Spl (Figure 38, lanes l-3), consistent with the notion that an additional component of TFIID might be necessary.

Activator-Specific 97

TBP-TAF

Complexes

An attractive candidate for one such missing subunit(s) is TAFl1150, since it was recently shown to bind DNA and recognize specific core promoter sequences (Verrijzer et al., 1994). We therefore reasoned that TAF,,l50 might play an important role in mediating transcription by certain activators. To test this hypothesis, we compared the transcriptional properties of a quadruple complex (TBP-TAF,,250TAFI1150-TAF11110) with those of the triple complex lacking TAF,,l 10 (TBP-TAF,,250-TAFI1150) (Figure 3). When the immunopurified quadruple complex was assayed in the fractionated Drosophila transcription reaction, we observed a dramatic activation by Spl (a-to lo-fold) (Figure 38, lanes 7-9). Importantly, activation was critically dependent on the presence of TAF,,l 10, since omission of this subunit abolished Spl responsiveness (Figure 38, lanes 4-6). Using the human fractionated transcription system, we confirmed a similar behavior of these complexes in supporting Spl activation (Figure 3C). These experiments establish that a subset of TAFs can mediate activation by Spl in vitro and that TAFII~ 10 serves as an essential coactivator. Spl Binds TAFlll 10 but Not TAF,,250 or TAFll150 Our functional studies of the quadruple complex suggest that, in addition to TBP, three TAFs are required for activation by Spl. Since TAF,,llO is directly contacted by Spl and is essential for activation, its behavior fits the definition of a coactivator. However, the role of the other two TAFs in the activation process is less clear. To address this question, we determined whether Spl might also directly contact TAFI1150, TAF,,250, or both. Biotinylated oligonucleotides containing Spl-binding sites were coupled to a streptavidin-agarose resin. Purified recombinant Spl was loaded onto the column and the unbound material removed. A control DNA affinity resin that lacked Spl protein was also prepared and run in parallel. Partially purified TAF,,250, TAF,,150, and TAF,,l 10 from baculovirus expression extracts were incubated with each resin, and after extensive washing, the bound proteins were eluted with a high salt buffer (1 M KCI). The input and eluted material were subsequently analyzed by SDS-PAGE, followed by immunoblotting (Figure 4A). As expected, TAF,,llO bound avidly to the Spl affinity resin. Interestingly, neither TAF,,250 nor TAF,,150 was retained on the Spl-containing resin. It is worth noting that the same source of factors was used here as in the assembly of transcriptionally active complexes, indicating that these TAFs are correctly folded and functional. None of the proteins tested bound to the control resin lacking Spl. As an additional test of Spl binding specificity, we loaded crude Sf9 extracts containing TAF,,l 10 onto the Spl affinity column and the control column. After extensive washing, all bound material was eluted with high salt and directly analyzed by silver staining of SDS gels. Only three major species were detected: full-length Spl, a truncated Spl polypeptide, and a prominent protein of 110 kDa that comigrates with TAFlll 10 (Figure 48) and cross-reacts with anti-TAF,,llO (data not shown). As expected, no TAF,,llO was retained on the control matrix. Thus, although TAF,,l 10 constitutes less than 0.1% of the total

A
TAF,,lIll TAF,,lSll TAF,,ZSO

b+

- sp1 -

&p + - sp1 -

,eq+ - Sp1 h,IL,,, -L,l

I23

I,

x9

Immunohlot

\ M(kD) + J+
zoo117XO-

TAFnllO

+TAF[,l

IO

**

Spl

50-

+ - + Spl I 2 3 .l
Silver Stain
Figure 4. Spl TAF,, 150 Directly Binds to TAF,,llO but Not lo TAF,,250 or

(A) Partially purified TAF,,llO (lanes l-3), TAF,,l50 (lanes 4-6), or TAFtI (lanes 7-9) were loaded onto an agarose GC box DNA affinity column either with (lanes 2, 5, and 6) or without (lanes 3, 6, and 9) DNA-bound Spl. After extensive washes, bound proteins ware eluted with a high salt buffer. As well as the eluted protein fractions, 10% of the input material (lanes 1, 4, and 7) was separated by SDS-PAGE and visualized by Western blot analysis. (B) The interaction between Spl and TAF,,llO is highly specific. A crude extract prepared from Sf9 cells infected with TAF,,llOexpressing recombinant baculoviruses (lanes 2 and 3) was applied to an agarose DNA affinity column with (lanes 2) or without Spl (lane 3). Also an Sf9 control extract was loaded onto the Spl-coated column (lane 4). After extensive washing, the bound proteins were eluted with high salt and resolved by SDS-PAGE and silver staining. Lane 1 shows 5%of theTAF,,l lo-containing inputextract. TAF,,l 10, Spl, andan Spl proteolytic degradation product (asterisk) are indicated. The identity of these protein bands was verified by Western immunoblot analysis (data not shown).

protein in the input extract, it is selectively purified (over lOOO-fold) by binding to Spl. These experiments confirmed the highly specific natureof the interaction between Spl and TAFlll 10. Intriguingly, the other two TAFs that are required to potentiate Spl activation (TAFa250 and TAF,,lSO) do not contact Spl directly on their own. These results suggest that activation may require multiple TAFs, some that make direct contact with activators, while others have distinct functions in the activation process.

C
TFIID + +TAFDl50 + , MOW
-200 -117 - 97 TBP-* TAFn40 -- 66

NTF-l+

#r

Autoradiogram

Silver stain

Immunoblot

B
M W) 20011780-

TAFII~SOAC

TAFDISOAN ~

TAFnllO -

d Lip - +

TAFD80 -

TAFn60 -

TAFn40 ~

6
4-

B- +

6- +

6-8 +

2- +
g-

NTF-1 Activation Domain

4-

12 Figure 5. The Ile-Rich

3 Minimal

456 Activation

7 Domain

89 of NTF-1

10 11 12 Interacts

13 14 15

16 17 18

Selectively

with TAFI115Cl and TAF&O

(A) NTF-1 binds to the Drosophila TFIID (dTFIID) complex. The dTFllD complex was immunopurified from a partially purified dTFllD fraction (Q.3) using antibodies directed against TBP and then incubated with radiolabeled NTF-1. The silver-stained gel shows the TAF pattern obtained upon immunopurification of the TFIID complex. NTF-1 binding was detected by autoradiography. NTF-1 was not immunoprecipitated by TBP antibodies in 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. Radiolabeled dTAF,,l5OAC, dTAF,,l50AN, dTAF,,llO, dTAFrr80, dTAF,,GO, and dTAF,,40 were incubated with beads alone or beads covalently coupled to a peptide corresponding to the 56 amino acid minimal activation domain of NTF-1. The input amounts correspond to 20% of the total protein used in the binding experiment. (C) NTF-1 binds to dTAF,,150. Baculovirus-expressed dTAF,,150 was immunopurified using anti-dTAF,,lBO antibodies. Recombinant vaccinia virusproduced NTF-1 was incubated with the immunopurified dTAFrrl50, and bound NTF-1 was detected by Western blot analysis. NTF-1 was not coimmunoprecipitated by a control baculovirus extract without dTAF,,150

The Isoleucine-Rich Activator NTF-1 Contacts TAF,,150 and TAF,,GO In this and preceding studies, we have provided a compelling body of evidence that TAF,,llO can serve as a bona fide coactivator for Spl . What about the TAF requirements for other activators? One attractive possibility is that different types of activators might require distinct coactivators. Spl and the Drosophila transcription factor NTF-1 were originally used to show that the TAFs in the TFIID complex can function ascoactivators(Dynlacht et al., 1991). Recent in vitro and in vivo studies mapped the activation domain of NTF-1 to a 56 amino acid region that contains a preponderance of hydrophobic residues, in particular, isoleucines (Attardi and Tjian, 1993). Therefore, it seemed appropriate to compare the TAF requirements for activation by these two apparently distinct types of transcriptional activators. As an initial step in identifying which TAFs might serve as coactivators for NTF-1, we determined whether NTF-1 makes direct contact with the TFIID complex. First, TFIID was immunopurified with a resin coated with anti-TBP antibodies, and the resulting affinity matrix was used to bind in

vitro translated, radiolabeled NTF-1. We found that NTF-1 bound efficiently to the TFllD affinity resin, indicating that one or more of the TFIID subunits can interact directly with NTF-1 (Figure 5A). Next, we tested the ability of the minimal activation domain of NTF-1 to bind individual TAFs. For these experiments, an affinity resin containing only the 56 amino acid NTF-1 activation domain was generated. Binding assays showed that the N-terminal region of TAF11150 contains an interaction surface for this activation domain (Figure 58). Of all the other TAFs tested for binding to NTF-1, TAFr60 was the only other subunit that interacted specifically with the affinity resin. As expected, none of the TAFs bound to the control resin lacking the activator peptide. We also carried out protein binding experiments with recombinant TAF11150 and full-length NTF-1. First, TAF,,lSO was immunopurified from Sf9 extracts, and the affinity resin containing TAF,,l50 was mixed with recombinant NTF-1. The isolated complexes were subsequently analyzed by SDS-PAGE and the presence of NTF-1 detected by immunoblot analysis. Our results indicate that NTF-1 can indeed bind selectively toTAF,,150

Activator-Specific 99

TBP-TAF

Complexes

kD zoo117XO-

ct

Ga14....e..- T41;,,250

NTF-1

-A

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

so-

Figure

6. Two Different

Trimeric

Complexes

Mediate

Activation

by GAL4-NTF-1

but Not by Spl

(A) Silver-stained gel of the TBP-TAFIr250-TAF1160 complex. This complex was assembled by addition of TAFrr60 to the TBP-TAF,,250 complex as 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 DNA-binding domain fused to the 56 amino acid NTF-1 minimal activation domain (GAL4-NTF-1). Approximately equimolar amounts (5 ng TBP) of TBP-TAFrr250 (lanes l-3) TBP-TAF,,250-TAF,,150 (lanes 4-6) TBP-TAF,,250-TAF,r150-TAkrl lO (lanes 7-Q), or TBP-TAFr,250-TAF,,60 (lanes 10-12) were supplemented 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 either 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 established that a well-defined minimal activation region of the Drosophila activator NTF-1 can bind and directly contact TAFll150 as well as TAF,,GO. Our findings also suggest that the activator interface of TAF,,lSO is located within the N-terminal third of the molecule, while the C-terminal region contains surfaces that interact with TBP and TAFI1250 (Verrijzer et al., 1994). These results point to the possibility that NTF-1 activation may be in part mediated by interactions with TAF,,l50 and TAF,,GO. Two Distinct Ternary Complexes Support Activation by NTF-1 but Not Spl To investigate the functional relevance of the NTF-l-TAF interactions, we tested whether a minimal TFIID complex containing either TAFM150 or TAF,,GO could mediate activation by NTF-1 (Figure 6). For these experiments, the 56 amino acid activation domain of NTF-1 was fused to the GAL4 DNA-binding region (GAL4-NTF-1). As expected, the minimal TBP-TAF11250 complex was not sufficient to mediate activation by GAL4-NTF-1 (Figure 6B). However, the ternary complex containing TAFI1150 supported strong activation by GAL4-NTF-1 (-/-fold). Conversely, this trimerit complex was not responsive to Spl (see Figures 38 and 3C), thus correlating transcriptional activity and specific activator-TAF interactions. Recall, however, that a quadruple complex containing both TAF,,150 and

TAFrrliO responded efficiently to activation by Spl. Importantly, this quadruple complex also efficiently supported activation by GAL4-NTF-1. Taken together, these results support the notions that different types of activators require distinct coactivators and that some partial complexes can mediate activation by multiple activators. Our protein interaction studies established that the core activation domain of NTF-1 not only binds TAF,,150 but also interacts with TAF,,GO. To assess the coactivator potential of TAFrr60, a different trimeric complex, containing TBP, TAFI1250, and TAFrr60, was assembled (Figure 6A) and tested for its ability to support activation (Figure 6B). Interestingly, the trimeric TBP-TAFa250-TAF,r60 complex, containing TAF,,GO instead of TAF,,150, also responded to GAL4-NTF-1 activation (5-fold, lanes 1O-l 2) but was unable to support transcriptional activation by Spl (lanes 13-15). This result further confirms the differential coactivator requirements for GAM-NTF-1 and Spl. Together, these experiments establish that binding of NTF-1 to either TAF11150 or TAF,,GO is a functionally relevant interaction that can direct activation. Recombinant Holo-TFIID Mediates Transcriptional Activation by Several Different Activators Encouraged by our success in assembling active partial TFIID complexes, we next attempted to reconstitute holoTFIID containing TBP and all eight TAF subunits that we

Cdl 100

4 4 kD M s
200116 :; L, q b$ y r 1 ,;;,$a j * J.

=TAFn250 -TAF,$SO -TAF=llO

rTAF$O -TAFn60 -TBP -TAF,+O

43-

rTFIID
Gal4Wlfic GaMCTF Gal4Jun Gal4

NTF-1 - A j

- 4

-A

-4-A

-A

12 Figure

34 Holo-TFIID

56 Supports

10 by Several

12 Distinct

3 Activators

4 5

7 8

10 11 12

7. Recombinant

Activation

(A) Silver-stained gel of the in vitro assembled holo=TFIID complex containing dTBP and the eight major Drosophila TAFs. Reconstitution was performed as illustrated in Figure 1C. All subunits and a TAF,,60 proteolytic breakdown (asterisk) are indicated. TAF1130a and TAF,,306 were not separated in this gel (6%). The identity of each subunit was verified by Western blot analysis (data not shown). (8) Recombinant TFIID supports activation by either Spl or GAL4-NTF-1 to a level comparable to that obtained with endogenous TFIID. A TFllDdependent human transcription system was supplemented with approximatelyequimolar amountsof either recombinant Drosophila holo-TFIID (rTFIID, lanes 1, 2, and 5-7) or immunopurified endogenous human HA-tagged TFIID (endo. TFIID, lanes 3, 4, and 6-10). Transcription reactions were performed either in the absence of an activator (lanes 1, 3, 5, and 6) or in the presence of 30 ng of Spl (lanes 2 and 4) or 10 ng (lanes 6 and 9) or 30 ng of GAL4-NTF-1 (lanes 7 and 10). (C) rTFllD mediates activation by various distinct activators. Several chimeric activators were constructed by fusing distinct well-characterized activation domains to the GAL4 DNA-binding domain. Transcription was tested in the absence (lanes 1,4, 7, and 10) or presence of GAL4-VPl6c (VPt6c is the C-terminal half of the acidic activation domain of VP16) (10 ng and 30 ng, lanes 2 and 3, respectively), GAL4-CTF (10 ng and 30 ng, lanes 5 and 6), GAL4-Jun (10 ng and 30 ng. lanes 6 and 9). or the GAL4 DNA-binding domain by itself (IO ng and 30 ng, lanes 1 I and 12). Transcription products were detected by primer extension.

have isolated. Using the strategy described in Figure lC, we eventually succeeded in building a recombinant TFIID complex containing approximately stoichiometric amounts of the nine TFIID subunits (Figure 7A). Next, we compared the ability of recombinant and endogenous TFIID to support activation by both Spl and NTF-1. As shown in Figure 76, recombinant holo-TFIID mediates strong activation by Spl (12-fold) and NTF-1 (8-fold). Activation by recombinant TFIID was indistinguishable from that directed by a comparable amount of endogenous TFIID. Thus, at least under these conditions, our reconstituted TFIID appears to be fully functional for the Gln-rich activator Spl and the Ile-rich activator NTF-1. We extended these studies to test whether recombinant TFIID also was functionally competent to support activation by several other types of activators. Activation domains representing different classes such as the acidic domain of VP18 or the proline-rich domain of CTF, as well as the unclassified Jun A2 domain, were fused to

the GAM-DNA-binding region and tested for activation in reactions containing recombinant TFIID. Figure 7C shows that reconstituted TFIID efficiently supports activation by all these different activators. As a control, in the absence of an activation domain, the GAL4 DNA-binding domain alone failed to enhance transcription. Moreover, these activators were not responsive in transcription reactions supplemented with TBP instead of recombinant TFIID (data not shown). Taken together, these results indicate that purified recombinant TFIID is able to recapitulate the transcriptional properties of native TFIID, at least for the set of activators we have tested. Discussion Our early studies suggested that one or more of the TAFs that make up the TFIID complex is responsible for mediating transcriptional activation by sequence-specific promoter- and enhancer-binding factors (Dynlacht et al.,

Activator-Specific 101

TBP-TAF

Complexes

A minimal TBPlTAF complex fails to support activation.

Figure 8. A Model for Transcriptional Activation by Different Promoter-Specific Transcription Factors

Two distinct trimeric complexes can support activation by Gal4-NTF-1, but not by Spl.

A tetrameric complex, containing TAFIIllO can mediate Spl-responsive activation.

Holo-TFIID supports activation by an assortment of activators.

1991; Tanese et al., 1991). Furthermore, we showed that certain activators directly interact with specific TAFs (Hoey et al., 1993; Goodrich et al., 1993). However, direct evidence for the role of individual TAFs during activation of transcription was lacking. Here, we report the assembly and purification of various functional TBP-TAF complexes reconstituted from the nine subunits of TFIID. Different activators required distinct subunits for transcriptional activation, while reconstituted holo-TFIID was able to support transcription by a variety of different activators. These results establish that TBP plus the TAFs is sufficient to replace endogenous TFIID to support activated transcription in vitro. The Role of Individual TAFs in the TFIID Complex An in vitro assembly strategy allowed us to determine the function of partial complexes and analyze the requirements for specific TAFs. These studies revealed how some activators communicate with TFIID to mediate transcription (Figure 8). Neither TEP alone nor a minimal complex containing TBP and TAF1,250 is able to mediate activation by either Spl or GAL4-NTF-1 (Figure 8A). By

contrast, if either TAF,,150 or TAFI180 is incorporated into the complex, GAL4-NTF-1 efficiently activates transcription (Figure 88). As might be predicted, both of theseTAFs bind directly to the NTF-1 activation domain, defining a novel set of activator-TAF interactions. Moreover, these results demonstrate that GAL4-NTF-1 can activate transcription via two distinct partial TFIID complexes, indicating the potential for dual or multiple pathways by which a single activator can mediate transcription. Importantly, these trimeric complexes that were active for GAL4NTF-1 failed to support activation by Spl (Figure 8s). Instead, Spl-dependent activation required the presence of TAF,,llO (Figure 8C). A complex containing TBPTAF,,250-TAF,,lSO-TAF,l l O appears to be the minimal oligomer responsive to activation by Spl. Interestingly, this tetrameric complex was also able to mediate NTF-1 activation, since it contains TAF,,lBO. Finally, when all the major TAFs (TAFI1250, TAF,,150, TAF,,llO, TAFe80, TAF,,GO, TAF,,40, TAF,130a, and TAF,,308) and TBP were incorporated into a holo-TFIID, the recombinant complex was able to support activation by an assortment of different classes of activators. Thus, although we cannot exclude

Cdl 102

a role for potential minor TAFs in the native TFIID complex, our recombinant TFIID seems to be completely functional. Our subunit assembly studies confirmed that TBPTAF,,250 can serve as a core TFIID complex. Although this minimal complex is incapable of mediating activation by the upstream activators we tested, the specific activity of this complex in basal transcription is indistinguishable from TBP alone. This result is noteworthy in light of previous studies that reported that the presence of TAFI1250 actually abolished basal transcription by preventing the binding of TBP to the TATA box (Kokubo et al., 1993a). However, in these previous studies, increasing amounts of free, denatured, and renatured TAFI1250 were added to the transcription reactions, perhaps resulting in nonspecific inhibitory interactions. By contrast, in our case, the TBP-TAF11250 complex is preassembled and purified by virtue of an affinity resin directed against TAF,,250. Therefore, it is highly unlikely that there is an excess of free TBP in the transcription reaction to account for the observed basal transcription. Coactivator Requirements for Spl Activation Our studies revealed that TAF,,llO behaves in a manner expected of a bona fide coactivator for Spl . It is required for activated but not basal transcription, and it is directly contacted by Spl . Since we have demonstrated the requirement for TAFlll 10 in reconstituted transcription reactions, it seems reasonable to conclude that TAF,,llO serves as an adaptor to link Spl to the basal transcriptional apparatus. A simple mechanism by which Spl might work is to recruit TFIID to the DNA template by binding specifically to TAFlll 10, thus increasing the number of preinitiation complexes. Alternatively, Spl might inducesomeallosteric change of the TFIID structure by contacting TAFlll 10, thereby enhancing the assembly of a functional initiation complex and increasing the rate of initiation. Whether the function of TAFlll 10 is merely to serve as a binding target for Spl or whether this subunit participates in additional steps during activation remains to be determined. We were surprised to find that TAFI1150 was also important to reconstitute Spl activation. Unlike TAF,,llO, TAF,,150 fails to contact Spl. Moreover, TAF,,150 is not necessary for the incorporation of TAFtI 10 into theTFllD complex. Interestingly, it appears that other TAFs (i.e., TAFI160) cannot substitute for TAFI1150, since several partial complexes containing TAFII1 10 but lacking TAFI1150 failed to support Spl-dependent transcription (J.-L. C. and Ft. T., unpublished data). What, then, is the function of TAFlll 50 during Spl activation? Since TAFlll 50 can recognize and bind core promoter sequences (Verrijzer et al., 1994) it is possible that this subunit helps stabilize the binding of TFIID to certain promoters. Alternatively, it may be that the presence of an activator contacting TFIID might induce TAF,ll50 to bind DNA. Consistent with this interpretation, a recent study using endogenous TFIID showed that Spl can help TFIID but not TBP bind to the initiator (Kaufmann and Smale, 1994). It is also possible that TAFI1150 functions during some subsequent step in the assembly of an initiation complex. Our preliminary findings

indicate that TAF,,150 may interact with some of the basal factors directly (K. Y., C. P. V., and Ft. T., unpublished data), consistent with the idea that TAFII150 may contribute to events other than making direct contact with activators. Interestingly, a recent study suggested a role for TAFs in the recruitment of basal factors during activation (Choy and Green, 1993). The minimal complex that is responsive to Spl contains not only TBP, TAFlll 10, and TAFI1150, but also TAFI1250. A well-established function of TAF,,250 is to serve as a core subunit contacted by TBP and other TAFs, including TAFlll10 (Weinzierl et al., 1993a). Is the function of TAFI1250 during Spl activation primarily a structural one? Although it is not directly contacted by Spl , it is possible that TAFtI plays a more active role during transcriptional activation. Indirect evidence supporting this idea is provided by the observation that a temperature-sensitive mutation of TAFtI leads to a cell cycle arrest phenotype and affects transcriptional activation both in vitro and in vivo (Wang and Tjian, 1994). Moreover, TAF,,250 appears to remain associated with a complete TFIID complex at the nonpermissive temperature (E. H. Wang and Ft. T., unpublished data). Thus, TAFI1250 may play some role in transmitting the activation signal once TAF,,llO is contacted by Spl . Activator-Specific TBP-TAF Complexes An important conclusion from the experiments described in this paper is that distinct classes of activators target different TAFs, thereby increasing the specificity and diversity through combinatorial interactions. Activation domains of different transcription factors such as the Gln-rich domains of Spl, the Pro-rich CTF activation domain, the acidic activation domain of VP16 (reviewed by Mitchell and Tjian, 1969), and the Ile-rich motif of NTF-1 (Attardi and Tjian, 1993) have been loosely classified on the basis of their characteristic amino acid composition. Here, we have identified TAFII150 and TAFI160 as the direct targets of NTF-1. Importantly, the NTF-1 minimal activation domain fails to interact selectively with TAF,,l 10, and, conversely, Spl does not bind TAF,,150. We also failed to detect interaction of NTF-1 with TAFI140 and of VP16 with TAF,,150 (data not shown). Most importantly, we found that the TAFs involved in these specific TAF-activator interactions are also required for transcriptional activation. Taken together, these data lend strong support to the idea of a mechanism in which an integral step in the process of transcriptional activation involves different classes of activators targeting distinct TAFs in the TFIID complex. Can we generalize these observations? Recent studies indicate that TAFel 10 binds several different Gln-rich activation domains, including Spl regions A and B (Hoey et al., 1993), CAMP response element-binding protein (CREB) (Ferreri et al., 1994) and the Drosophila transcription factor buttonhead (F. Sauer, unpublished data). Similarly, a number of different acidic activation domains were found to interact selectively with TAF1140 (C. Thut and R. T., unpublished data). We also suspect that other activation domains, related to the Ile-rich activator of NTF-1, will target TAFI1150. Thus, it seems likely that multiple activators of

Activator-Specific 103

TBP-TAF

Complexes

a given class will target a particular TAF. It is important to note, however, that the preponderance of a particular set of amino acids within activation domains does not necessarily predidt a specific interaction with a given TAF. For example, a number of Gin-rich transcription factors failed to bind TAFI~l10 (Hoey et al., 1993). Therefore, the basis for the specificity of interactions between activators and TAFs remains unclear. Identifying the targets of other activators may help determine which features of these proteins are critical for recognition and transcription (see Tjian and Maniatis, 1994). In addition, such studies may result in a more functional classification of activation domains. Our studies with NTF-1 revealed that some activators may interact with multiple TAFs (i.e., TAFI1150 and TAF,,GO), and that different complexes containing distinct coactivators are able to support transcriptional activation. An important lesson from these results is that a single, relatively small (56 amino acids) activation domain can work via two distinct pathways. This observation also raises the possibility that such multiple contacts may play some role during synergistic activation (reviewed by Ptashne, 1988). At present, we have not addressed this intriguing question. It is also plausible that a single TAF can provide interfaces for interaction with different types of activators. For example, it has recently been determined that a region of TAF,,llO, distinct from the Spl-binding domain, interacts with ElA (J. V. Geisberg, J.-L. C., and R. P. Ricciardi, unpublished data). As discussed above, there need not be a simple one-on-one relationship between activators and TAFs. We have shown that a single activator can contact multiple TAFs and, conversely, that a given TAF can interact with more than one activator. This provides the necessary combinatorial complexity to allow communication between different activators and TFIID. Finally, an important conclusion from ourstudieson TAF requirements for activation by Spl and NTF-1 is that TAF,,lSO might have two distinct roles in the activation process. In the case of NTF-1, it is directly contacted by the activation domain and might serve asimilarfunction as TAF,,l 10 in Spl activation. However, although not directly contacted by Spl, TAF,,lSO is also critical for activation by this transcription factor. Thus, a single TAF may perform different tasks during activation by different enhancer proteins.
TFIID: A Molecular Central Processing Unit

with components of the transcriptional machinery. We therefore propose that TFIID may function as a central processing unit to integrate the information from a multitude of enhancer and promoter-bound factors to control the level of transcription initiation. Now that it is possible to reconstitute TFIID in vitro, future research may unravel the molecular mechanisms of transcriptional activation in more detail and provide a critical test to this general hypothesis.

Expression

and Purification

of Recombinant

TAFs and

TBP

TFIID subunits were expressed in the baculovirus expression system. Most expression constructs containing full-length cDNAs have been described previously(dTBP, Weinzierl et al., 1993a; HA-hTAF,,250, Rupperl et al., 1993; dTAF,,l50, Verrijzer et al., 1994; dTAFIIllO, Hoey et al., 1993; dTAF,,80, Dynlacht et al., 1993; dTAF,,GO, Weinrierl et al., 1993b; dTAF,,40, Goodrich et al., 1993; dTAF,,30a, Yokomori et al., 1993a). Full-length dTAF,,250 was obtained from three overlapping cDNA clones. The complete coding sequence was subcloned into a modified version of the pVL1392 baculovirus expression vector (Pharmingen). The dTAF,,30P expression vector was generated by subcloning the Ndel fragment containing the full-length cDNA into pVL1392. All recombinant viruses were plaque purified and amplified. All protein preparation, purification, and in vitro complex assembly was carried out in buffer HEMG-ND (25 mM HEPES [pH 7.61.0.1 mM EDTA, 12.5 mM MgCI,, 10% glycerol, 0.1% NP-40, 1.5 mM DTT, 0.2 mM AEBSF (CalBiochem), 1 mM sodium metabisulfite, 0.7 pglml pepstatin, 2 wglml leupeptin) containing variable concentrations of KCI. All TFIID subunits were purified by conventional column chromatography, followed in some cases by protein or DNA affinity chromatography. Importantly, some of the TAFs were only partially purified (i.e., dTAF,,GO, 10% pure). However, the selectivity of the TFIID assembly resulted in a near homogeneous complex.

All recombinant

Assembly

of TBP-TAF

Complexes

In Vltro

The procedure for the assembly of the TFIID complex is outlined in Figure 1C. Anti-HA monoclonal antibody 12CA5 was covalently linked to protein A-Sepharose as described (Zhou et al., 1992). The entire procedure was carried out at 4OC in HEMG-ND containing 0.1 M or higher concentrations of KCI. First, HA-TAF,,250 was incubated with the a-HA affinity column for l-2 hr, and free HA-TAFI1250 was then removed by extensive washing. Next, a molar excess (at least IO-fold) of each subsequent subunit was added. After 3 hr at 4OC, unbound subunits as well as impurities were removed by repeatedly washing with a lOO-fold excess of buffer. The assembly process resulted in such an additional purification that the resulting complexes were near homogeneous. This procedure was repeated for each successive TAF. The resulting TFIID complexeswere eluted with HEMG-ND buffer containing 1 mg/ml HA peptide (YPYDVPDYA) for 1 hr. The eluted complexes were analyzed by SDS-PAGE, followed by silver staining and Western blotting and used in transcription reactions. Reimmunoprecipitation reactions were performed as previously described (Weinzierl et al., 1993a).

Eukaryotic promoter and enhancer regions are often complex and designed to bind a large array of different sequence-specific transcription factors. The expression of a given gene therefore depends on the interplay between various DNA bound enhancer factors and the basal transcription machinery. The discovery of TAFs and the studies reported here provide the most direct evidence for the notion that multiple subunits of TFIID, each with distinct structural characteristics, can provide unique and specific surfaces for interactions with different upstream activators. This arrangement provides the opportunity for multiple activators and repressors to interact simultaneously

In Vitro

Protein-Protein

Interaction

Assays

The Spl -TAF interaction assay was carried out using Spl DNA affinity column essentially as described previously (Hoey et al., 1993), with the exceptions that the KCI concentration was 100 mM, and baculovirus-expressed TAFs were used instead of in vitro translated proteins. The NTF-1-TAF interactions were studied similarly as described (Yokomori et al., 1993a). The TFIID complex was immunopurified as described (Hoey et al., 1993). To generate an NTF-I minimal activation domain affinity resin, a synthetic 63-mer CGGEHQP-NTF-1 (amino acids 169-228) was covalently attached to divinylsulfone-derivatized agarose via a thioether linkage (-1 mg of peptidell ml hydrated beads). About 15 ~1 of beads with or without peptide was incubated with radiolabeled TAFs for several hours at 4C. After extensive washing with 0.1 M HEMG-ND, the beads were resuspended in sample buffer and analyzed by SDS-PAGE followed by autoradiography.

in Vitro Transcription In vitro transcription assays were performed in either the Drosophila or HeLaTFiiDdependent fractionated transcription system. The DNA templates used included (GC)3BCAT (Dynlacht et al., 1991). which contains three Spl sites upstream of the El B TATA box, and GsBCAT (Lillie and Green, 1969), which contains five GALCbinding sites. Spl was overexpressed in HeLa cell using a vaccinia virus-expressing vector and 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 domain A2, residues 139-253), and GAL4-CTFwere expressed in Escherichia coli and purified as previously described (Goodrich et al., 1993; Attardi and Tjian, 1993; Baichwal and Tjian, 1990; Tanese et al., 1991). The Drosophila fractionated transcription system was established essentially as described (Wampler et al., 1990; Dynlacht et al., 1991). A HeLa TFIID-dependent transcription system was prepared essentially as described (Dignam et al., 1963). Nuclear extracts were fractionated on Phosphocellulose Pll (Whatman). The flowthrough (PC.l), 0.3 M eluate (PC.3), 0.5 M eluate (PC.5), and 1 M eluate (PC1 .O) were collected. The TFIID activity (PC1 .O) was further fractionated by on a DE52 column (Chiang et al., 1993; Meisterernst et al., 1991) and the flowthrough (DE.1; USA) and 0.3 M eluate (DE.3) collected. The PC.1, PC.5, and DE.1 fractions were reapplied to PI 1 and DE52 to remove any potential TFIID contamination. The transcription assays were performed as previously described (Pugh and Tjian, 1990). Typical reactions (25 ~1) contained 100 ng of DNA template, 2 pg of PC.1, 1.4 pg of PC.5, and 100 ng of DE.1 (USA) fractions, and several nanograms of either endogenous HA-TFIID or assembled TBP-TAF complexes. HA-tagged human TFIID was immunopurified as described (Zhou et al., 1992). The reaction products were detected by primer extension. Products were visualized by autoradiography and quantified by Phospholmager analysis (Molecular Dynamics). Each transcription reaction was repeated several times, and representative data are shown.

We are grateful to D. King for the NTF-1 peptide resin, R. Weinzierl for monoclonal antibodies against TAFs, S. Ruppert for hTAF,,250expressing baculovirus, J. Goodrich and A. Vasserot for GAL4-VP16c and GAL4Jun. and Y. Mul for Sf9 and H5 cell stocks. We thank H. Beckman, T. Hoey, and E. Wang for helpful technical advice and C.-M. Chiang for advice on anti-FLAG M2 purification, and G. Culter for excellent computer artwork. Finally, we thank W. Herr, T. Hoey, S. McKnight, B. Meyer, E. OShea, G. Peterson, D. Reinberg, D. Rio, K. Yamamoto, and members of the Tjian laboratory, especially R. Dikstein, G. Gill, J. Goodrich, D. King, M. Maxon, and E. Wang for critical comments on the manuscript. C. P. V. is supported by a longterm European Molecular Biology Organization fellowship, and K. Y. by a Leukemia Society Fellowship. This work was supported in part by a grant from the National Institutes of Health to R. T. Received References Attardi, L. D., and Tjian. R. (1993). Drosophila tissue-specific transcription factor NTF-1 contains a novel isoleucine-rich activation motif. Genes Dev. 7, 1341-1353. Baichwal, V. R., and Tjian, R. (1990). Control of cJun activity by interaction of a cell-specific inhibitor with regulatory domain 6: differences between v- and cJun. Cell 63, 815-625. Chiang, C.-M., Ge, H., Wang, Z., Hoffmann, A., and Roeder, R. G. (1993). UniqueTATA-binding protein-containingcomplexesand cofactors involved in transcription by RNA polymerases II and ill. EMBO J. 12, 2749-2762. Choy, B., and Green, M. R. (1993). Eukaryotic activators function during multiple steps of preinitiation complex assembly. Nature 366,531536. Dignam, J. D., Martin, P. L., Shastry, B. S., and Roeder, R. G. (1983). Eukaryotic gene transcription with purified components. Meth. Enzymol. 707, 582-596. Dynlacht, B. D., Hoey, T., and Tjian, R. (1991). Isolation of coactivators July 1, 1994; revised August 4, 1994.

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