Proc. Nati. Acad. Sci. USAVol. 88, pp. 7674-7678, September 1991Biochemistry

Evidence for a factor required for transcriptional stimulation bythe chimeric acidic activator GAL-VP16 in HeLa cell extracts

(acidic activating domain/transcriptional intermediary factor/transcriptional interference/squelching)

JOHN H. WHITE*t, CHRISTEL BRou*, JUN Wu*, NICOLAS BURTON*, JEAN-MARC EGLY*,AND PIERRE CHAMBON***Laboratoire de Gdndtique Moldculaire des Eucaryotes du Centre National de la Recherche Scientifique, Unit6 de Genie G6n6tique et de Biologie Mol6culairede 'Institut National de la Sant6 et de la Recherche M6dicale, Institut de Chimie Biologique, Facult6 de M6decine 11, Rue Humann, 67085 StrasbourgC6dex, France

Contributed by Pierre Chambon, May 28, 1991

ABSTRACT We provide biochemical evidence for the ex-istence of a transcriptional intermediary factor (TIF) in HeLawhole-cell extracts (WCE) that is distinct from the basictranscription factors and that is required for transcriptionalstimulation by the chimeric acidic activator GAL-VP16. Wehave fractionated HeLa WCE by heparin-agarose chromatog-raphy. Of transcriptionally active fractions eluting in a stepbetween 0.24 and 0.6M KCI, the initial fractions are refractoryto GAL-VP16 stimulation, whereas subsequent fractions arestrongly stimulated by the activator. Aliquots of GAL-VP16-responsive fractions efficiently complement refractory frac-tions for transcriptional stimulation. Aliquots of responsivefractions are also far more efficient than those of refractoryfractions in overcoming transcriptional inhibition that isbrought about by high concentrations -of GAL-VP16. Exper-iments performed with heat-treated WCE support the idea thatHeLa cells contain a TIF that is essential for GAL-VP16stimulation, but that is not required for basal transcription.Addition of recombinant yeast or human transcription factorTFID (rTFIDY and rTFIDH, respectively) to aWCE heatedat 48°C for 15 mE restores basal transcription, but in neithercase is the reconstituted system activated by GAL-VP16.However, a 45°C heat-treated WCE reconstituted with eitherrTFHDH or rTFUDY is stimulated by GAL-VP16, suggestingthat a HeLa TIF can be selectively inactivated by heating at48WC, but not at 45°C. Interestingly, a TFUD fraction partiallypurified from HeLa cell extracts, but not rTFIIDH, efficientlyrelieves transcriptional Inhibition by GAL-VP16, suggestingthat there may be an association between TIF(s) and TFJDand, moreover, that TIF(s) may be the direct target of theacidic domain ofGAL-VP16. In summary, our results supportthe existence ofa TIF that is not essential for basal transcriptionbut that is required to mediate the stimulatory activity of theacidic activator GAL-VP16.

Formation of a basal transcriptional initiation complex oneukaryotic class B (II) promoters requires several generaltranscription factors in addition to RNA polymerase B (II).Specific binding of transcription factor TFIID (also known asBTF1; see refs. 1 and 2) to the TATA box, perhaps in thepresence ofSTF (or TFIIA; see ref. 1), is required prior to theassembly of RNA polymerase and other general factors onthe promoter (3-5). Initiation complex formation is modu-lated in vivo and in vitro by transcriptional activators, whichrecognize the upstream element and enhancer sequences ofa given promoter. Numerous biochemical and moleculargenetic analyses have indicated that these factors can gen-erally be dissected into domains required for specific DNA

binding and transcriptional activation (see refs. 6-8 forreviews and refs. 9-11).One of the first identified classes of transcriptional acti-

vating domains was characterized by its high concentration ofacidic amino acids, which may be arranged to form amphi-pathic a-helices (12). The herpes simplex virus-encodedVP16 protein, which contains a highly acidic 78-amino-acidC-terminal region, has been one of the most intensivelystudied activators of this class (13-15). The VP16 acidicdomain functions in vivo when coupled to the DNA bindingdomains of either the yeast activator GAL4 (GAL-VP16; ref.16) or the human estrogen receptor [ER(C)-VP16; ref. 9].GAL-VP16 is also a transcriptional activator in vitro inextracts of yeast (17, 18) and HeLa (19) cells.The components of the basal initiation complex that are

targets of GALVP16 and other transcriptional activatorshave been the subject of intensive scrutiny recently (see ref.20 and references therein). Analysis of these target factorshas been given impetus by the observation in vivo that highconcentrations of a transcriptional activator will squelch (21)or interfere (22) with transcription stimulated by anotheractivator with an unrelated DNA binding site. This raises thepossibility that the competing activator is sequestering fac-tors required for transcriptional activation. A systematic invivo study of this kind in HeLa cells has suggested thatdifferent classes of activators may be coupled to componentsof the basal transcription machinery through specific tran-scriptional intermediary factors (TIFs) (10). Squelching byGAL-VP16 has been reproduced in vitro in yeast extracts(17, 18). Kornberg and coworkers (17) have exploited thisphenomenon to isolate a yeast fraction that relieves tran-scriptional inhibition by GALVP16. This effect cannot bereproduced by addition ofRNA polymerase B (II) or generaltranscription factors, thus indicating the existence of a TIFthat represents an additional component of the yeast tran-scription apparatus.Evidence for such factor(s) in extracts of higher eukaryotic

cells has so far been indirect; it has largely been limited to theobservation that recombinant TFIID can be used to recon-stitute basal, but not stimulated, transcription in heterolo-gous in vitro transcription systems (23, 24). In this report, weprovide biochemical evidence for a TIF in HeLa whole cellextracts (WCE) and demonstrate that this factor is notessential for basal transcription and is thus distinct frompreviously identified general transcription factors.

Abbreviations: TIF, transcriptional intermediary factor; WCE,whole-cell extract(s); rTFIIDH, recombinant human TFIID; rTFI-IDY, recombinant yeast TFIID; Ad2MLP, adenovirus 2 major latepromoter; BT1M, Blue Trisacryl 1 M KCI fraction.TPresent address: Department of Physiology, McGill University,McIntyre Medical Sciences Building, 3655 Drummond Street, Mon-treal, P.Q., H3G 1Y6, Canada.tTo whom reprint requests should be addressed.

7674

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Proc. Natl. Acad. Sci. USA 88 (1991) 7675

MATERIALS AND METHODSRecombinant Plasmids and Expression in Escherichia coil.

Plasmid 17M5/pAL7 contains five 17-mer binding sites (seeref. 9) recognized by the DNA binding domain of GAL4inserted in the Bgl II site (-65) upstream of the TATA region(-34 to +33) of the adenovirus 2 major late promoter(Ad2MLP). GAL-VP16 (9) was expressed in E. coli frompET-3C by using the bacteriophage T7 expression system(25); was purified from supernatants of sonicated cells bychromatography over heparin-agarose, phenyl-Sepharose,and DNA-cellulose columns; and was 80o pure asjudged byCoomassie brilliant blue-stained SDS/polyacrylamide gels.Recombinant human TFIID (rTFIIDH) and recombinantyeast TFIID (rTFIIDY; formerly known as BTF1Y; see ref.1) were expressed in E. coli from pET-3A (25); their purifi-cation to homogeneity will be described elsewhere.

Fractionation of HeLa WCE by Heparin-Agarose Chroma-tography. Two hundred milligrams ofHeLaWCE was loadedon a 40-ml heparin-agarose column equilibrated in 25 mMTrisHCl, pH 7.9/5 mM MgCl2/0.5 mM dithiothreitol/0.1mM EDTA/10% (vol/vol) glycerol. Bound protein waseluted with steps of 0.24, 0.6, and 1 M KCl (see ref. 26).Aliquots (8 /l) ofthe 0.6M KCl fractions (-4 ml) were testedfor transcriptional activity as described below (see alsolegends to figures).

Partial Purification of TFIID (BTF1) Activity from HeLaCells. HeLa WCE were fractionated by heparin-agarosechromatography as described above. The 0.6M KCi step wasdiluted 6-fold in 25 mM Tris HCl, pH 7.9/0.5 mM dithiothrei-tol/0.1 mM EDTA/10%o (vol/vol) glycerol (TDEG10) andloaded on a 2.5 x 15 cm2 sulfopropyl 5-PW HPLC column(Toyosoda) equilibrated in TDEG10 containing 100mM KCl.Bound protein was eluted with 350 mM (SP350 fraction) and1 M KCi. TFIID activity was assayed by using a heat-treatedWCE (ref. 27; see below) and was found in the SP350.Ninety-five milliliters of SP350 fraction (80 mg) was equili-brated in TDEG10 containing 50 mM KCl and loaded on a13-ml DEAE-cellulose column. Bound protein was elutedwith a 90-ml gradient from 50 to 500 mM KCl and collectedin 15 6-ml fractions. A single fraction containing TFIIDactivity was equilibrated in TDEG10 containing 50 mM KCland loaded on a 3-ml Blue Trisacryl HPLC column. Boundprotein was eluted with steps of 0.6 M, 1 M, and 2 M KCi.TFIID activity was found in the 1 M KCl step. This fractionwas dialyzed against 25 mM Tris-HC1, pH 7.9/0.5 mMdithiothreitol/0.1 mM EDTA/20% glycerol/50 MM KCI andstored in 100-Al aliquots at -1980C.In Vitro Transcription. Reactions (20-25 ,ul), incubated at

250C, were performed essentially as described (28). Aliquotsof 200-500 1.d of WCE were heated prior to each experimentin 1.5-ml Eppendorf tubes for exactly 15 min at 450C or 480Cas indicated in the figures. Ten microliters of heat-treatedWCE, 8-104ul of untreated WCE, or 8 /l of heparin fractionssupplemented with 1 /l of STF (DEO.35; ref. 26) was used ineach reaction (see also legends to figures). Purified RNA (28)was analyzed by quantitative SI nuclease analysis (9).

RESULTSFractionation of HeLa WCE into GAL-VP16-Responsive

and -Refractory Fractions. To monitor GAL-VP16-depen-dent transcription we used the plasmid 17M5/pAL7, whichcontains five 17-mer GAL4 DNA recognition sites recog-nized by the DNA binding domain of GAL4 inserted up-stream of the TATA region (-34 to +33) of Ad2MLP (Fig.IA). GAL-VP16 stimulates transcription from this promoter7- to 10-fold in HeLa WCE (Figs. 2-4). This stimulationrequires the YP16 acidic domain and is strictly dependent onthe presence of 17-mers in the test promoter (data not

A (-1)BgIll 5xl7mer BgIll TATAAAA (+1)

17M5/pAL7 f 1m IT4 -(-65) (-34) (+33) (-9) (+60)

SSl Probe

B Fraction 40 (I)0 3 6_-- _

..A4 0@ *0.(Ad2MLPd*2 _*

- -j.- - - tr - -t- - + - 4- - + - + - + GAL-L___j ____ I,_ ,j _ j VP1632 33 34 35 36 37 38 40 42

Heparin Fraction

z0C0

0<a, E= CO

mL

Fai - + -33 (8 l t

Fraction 33 (8gl)

z0F

CD(3

HEPARIN FRACTION

FIG. 1. Fractionation of HeLa WCE by heparin-agarose chro-matography. (A) Schematic representation of the test promoter ofplasmid 17M5/pAL7 containing five 17-mer binding sites recognizedby the DNA binding domain of GAL4 inserted (in the Bgl II site at-65) upstream of the TATA region (-34 to +33) ofAd2MLP. pAL7is related to pAL10 (29), but it lacks the 17 RNA polymerasepromoter located upstream of the Ad2MLP. Also shown is the probeused for quantitative S1 nuclease analysis, which is located at +60of the promoterless globin gene and protects 110 nucleotides of atranscript originating from the Ad2MLP start site. (B Left) Quanti-tative S1 nuclease analysis of transcriptionally active fractionseluting in a step between 0.24 and 0.6 M KCl from a heparin-Ultrogelcolumn tested in the absence and presence of GAL-VP16 using the17M5/pAL7 test promoter (Ad2MLP + 1). Reaction mixtures (20/l)contained 8 ,ul of the heparin fraction supplemented with 1 Al ofSTF(DEO.35; ref. 26). (B Right) Complementation of heparin fraction 33(8 Al) with 3 or 6 Al of heparin fraction 40 in the presence of 1 Al ofSTF. GALVP16 (1.6 pmol) was added as indicated. Note that thesmall stimulation of fraction 33 seen in the absence of fraction 40 isnot reproducible (for example, see lanes corresponding to fraction 33in B Left). (C) Graphic representation of the transcription reactionspresented in B Left, including levels of basal transcription (e),transcriptional stimulation by GAL-VP16 (o), and TFIID activity (v)of each fraction as measured using a heat-treated WCE (see Mate-rials and Methods and Fig. 3 for details of the assay).

shown). HeLa WCE were fractionated by heparin-agarosechromatography to analyze factor(s) that are necessary forGAL-VP16 action. The factors required for accurate initia-tion from Ad2MLP elute between 0.24 and 0.6 M KCl, withthe exception of STF (TFIIA), which is in the flow-through(26). Fractions from a 0.24-0.6 M KC1 step supplementedwith STF (DEO.35; ref. 26) were tested for TFIID (BTF1)activity, basal transcription, and GAL-VP16 stimulation(Fig. 1 B and C). Strikingly, the first two transcriptionallyactive fractions (fractions 32 and 33) are not stimulated byGAL-VP16. In contrast, subsequent fractions (fractions 34-42) are stimulated by the activator to levels as high as 30-fold(Fig. 1 B and C). These results suggest that fractions 32 and33 may lack factor(s), found in fractions 34-42, that are notrequired for basal transcription, but that are necessary forGAL-VP16 stimulation.To test for such factor(s) in GAL-VP16-responsive frac-

tions, aliquots of fraction 40 were used to complementfraction 33, which is refractory to stimulation by GAL-VP16.

Biochemistry: White et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

Addition of fraction 40 had little effect on basal transcriptionbut resulted in a >10fold increase in transcription in thepresence of GAL-VP16 (Fig. 1B Right). This result is im-portant for two reasons. First, it indicates that GAL-VP16stimulation of the low basal transcription of fraction 40 is notsimply due to counteraction of the effects of inhibitors (forexample, histones; see refs. 30 and 31), which could possiblybe present- in the fraction. If this were the case, it would beexpected that addition of fraction 40 to fraction 33 wouldinhibit basal transcription. Instead, addition of fraction 40causes an -2-fold increase in basal transcription (Fig. 18Right). Second, this result supports the proposal that fraction40 contains a factor(s) necessary for GAL-VP16 action, sincethe stimulation observed with combined fractions 33 and 40is at least 4-fold greater than that expected from an additiveeffect.GAL-VP16-Responsive Fractions Relieve Transcriptional

Inhibition by High Concentrations of the Activator. Additionof 1.6 pmol of GAL-VP16 to a WCE generally produces a 7-to 10-fold stimulation in transcription (Fig. 2A, lanes 1 and 2;Figs. 3 and 4; and data not shown). Increasing quantitiesabove 1.6 pmol generally stimulate less and less such that

A WCE+

r--_ lFraction 32 1 Fraction 40(1l)2 4 6 82 4 6 8 2 4 6 8 2 4 6 8

1 2 3 4 5 6 7 8 9 1001 11

0 1.640 o 40

B

z

0

4=

CO,a-j

0

1

Iat 1W1AAd2MLP+1

11 12 13 14 15 16 17 18 19

Jl GAL-VP16

(pmol)

FIG. 2. Relief of transcriptional inhibition caused by excessGAL-VP16 by transcriptionally active heparin-Ultrogel fractions.(A) Quantitative S1 nuclease analysis of transcription from thepromoter of 17M5/pAL7 (AdMLP +1; see Fig. 1A) by a HeLa WCEin the absence (lane 1) or in the presence of 1.6 pmol (lane 2) or 40pmol (lane 3) of GAL-VP16. HeLa WCE were also complementedwith increasing volumes of heparin fraction 32 (lanes 4-11) orfraction 40 (lanes 12-19; see Fig. 1 for characterization of heparinfractions) as indicated, in the absence (lanes 4-7 and 12-15) or

presence of 40 pmol of GAL-VP16 (lanes 8-11 and 16-19). (B)Graphic representation of the transcription reactions presented in A.(Left) Plot of relative transcription obtained in lanes 1-3 ofA in thepresence of the GAL-VP16 concentrations indicated. (Right) Re-sults of complementation experiments of lanes 4-19 of A withincreasing quantities of heparin fraction 32 (Fr 32) in the absence (o)or presence (o) of 40 pmol of GA1-VP16 or with increasing quan-tities of heparin fraction 40 (Fr 40) in the absence (v) or presence (A)of 40 pmol of GALVP16.

with 40 pmol ofGALVP16 no stimulation is observed (Fig.2A, lane 3; Fig. 2B; and data not shown). This effect isreminiscent of the transcriptional inhibition (squelching) ob-served with high concentrations of GAL-VP16 in vivo inyeast and mammalian cells (10, 17) and in vitro in yeastextracts (17, 18).We tested the relative abilities of the GAL-VP16-

refractory fraction 32 and -responsive fraction 40 to over-come this inhibition (Fig. 2). Fractions 32 and 40 were chosenbecause they produce similar levels of basal transcription(Fig. 1 B and C). In the presence of 40 pmol of GAL-VP16,addition of increasing amounts of fraction 32 to a WCEproduced a <2-fold increase in transcription, which is sig-nificantly less than the observed 4-fold increase in basaltranscription (Fig. 2A, lanes 8-11 and 4-7, respectively; Fig.2B). In contrast, in the presence of 40 pmol of GAL-VP16,addition of increasing quantities offraction 40 led to an 8-foldincrease in transcription, which is similar to that obtainedwith optimal concentrations of GALVP16 (Fig. 2A, lanes16-19, and compare with lanes 1 and 2; Fig. 2B). Thisstimulation is 4-fold greater than the effect of fraction 40 onbasal transcription (Fig. 2A, lanes 12-15 and Fig. 2B). Takentogether these data suggest that transcriptional inhibition byGAL-VP16 sequesters a factor(s), present in fraction 40, thatis essential for GAL-VP16 stimulation and that is lacking infraction 32.A 48C Heat-Treated WCE Complemented with Either

rTFHIDH or rTFIDY Is Not Stimulated by GAL-VP16. Weused heat-treated WCE to further analyze the factor(s) re-quired for GAL-VP16 stimulation. WCE heated at 47TC for15 min are transcriptionally inactive, but transcription can berestored by adding a source ofTFIID (27). A WCE heated at480C for 15 min can still be complemented for basal tran-scription with rTFIIDH or rTFIIDY, but in both cases thereconstituted system is unresponsive to GALVP16 (Fig. 3,lanes 9-14). This observation supports the results obtained inFigs. 1 and 2 indicating that HeLa cells contain a factor, notrequired for basal transcription, but that is essential forGAL-VP16 stimulation.A 45°C Heat-Treated WCE Is Stimulated by GAL-VP16

When Complemented with Either rTFIIDH or rTFIIDY. Itwas then determined if basal transcription ofaWCE could beheat-inactivated without destroying the factor(s) required forGAL-VP16 activation. Heating aliquots of WCE at 45TCinstead of 48TC for 15 min still completely eliminated basal

rTFIID - H Y -- H YHeat ^ [I Fl

Treatment none 45CC 480CGAL-VP16 -+ - + +- _e- . +

Ad2MLP wpS W

1 2 3 4 5 6 7 8 9 10 1 12 13 14

FIG. 3. Complementation ofWCE heat-treated at 450C and 480Cby rTFIIDH (H) and rTFIIDY (Y). Quantitative S1 nuclease analysisof transcription reactions containing GAL-VP16 (1.6 pmol) as indi-cated. Lanes 1 and 2, transcription with an untreated WCE. Lanes3-8, transcription with a WCE heated at 450C for 15 min withoutcomplementation (lanes 3 and 4) or in the presence of 1 ,A1 ofrTFIIDH(lanes 5 and 6) or 1 jul of rTFIIDY (lanes 7 and 8). Lanes 9-14,transcription with a WCE heated at 480C for 15 min without com-plementation (lanes 9 and 10) or in the presence of 1 ;lI of rTFIIDH(lanes 11 and 12) or rTFIIDY (lanes 13 and 14). Samples (200-500CA)of HeLa WCE were heated for exactly 15 min at 450C or at 480C asindicated. Ten microliters of heat-treated extract was used for eachreaction.

7676 Biochemistry: White et al.

Proc. Natl. Acad. Sci. USA 88 (1991) 7677

transcription (Fig. 3, lanes 3 and 4), which could be restored,as expected, by addition of rTFIIDH or rTFIIDY (Fig. 3,lanes 5 and 7). Interestingly, extracts thus complementedwith both rTFIIDH and rTFIIDY can be stimulated withGAL-VP16 (Fig. 3, compare lanes 5 and 6 and also lanes 7and 8).TFIID Activity Partially Purified from HeLa WCE but Not

rTFIIDH Efficiently Relieves Transcriptional Inhibition byGAL-VP16. We fractionated GAL-VP16-responsive heparinfractions by chromatography over a sulfopropyl columnfollowed by DEAE-cellulose and Blue Trisacryl columns(Materials and Methods) to further investigate the relation-ship between factors required for GALVP16 stimulation andTFIID. Fractions were analyzed for both TFIID activity andtheir ability to relieve transcriptional inhibition by GAL-VP16. The activity overcoming transcriptional inhibitioncopurified with TFIID (Fig. 4 and data not shown). The BlueTrisacryl 1 M KCI fraction (BT1M) efficiently relieved tran-scriptional inhibition by excess GALVP16. Addition of 5 ,lof BT1M stimulated basal transcription -2-fold (Fig. 4A,lanes 1-4), but stimulated 13-fold a WCE transcriptionallyinhibited by GAL-VP16 to a level greater than that obtainedwith an optimal concentration of the activator (Fig. 4, lanes5-9). In contrast, rTFIIDH similarly stimulated basal tran-scription and transcription inhibited by an excess of GAL-VP16 (2- to 3-fold; Fig. 4, lanes 10-16).The BT1M TFIID activity and rTFIIDH were compared

for their ability to complement a heat-treated WCE for basaltranscription (Fig. 4A, lanes 17-19). Clearly, 2 A.l ofrTFIIDHis more active in this assay than 5 Al of the partially purifiedTFIID activity (Fig. 4A, compare lanes 18 and 19). Takentogether the above results indicate that a factor other than

AGAL-VP16 0 3.6 70

(pmol) i- ir

TFIID Hela (BT1M)(uI) 0 1 3 5 0 0 1 3 5

Ad2MLP..* -_

111 2 3 4 5 6 7 8 9

WCE 450C0 3.6 70 If

= :2E.Coli (rTFIIDH)0120 012 0 2 5

- me - in

10111213141516 171819

B 40i-

z-0

TEIID BT1M rTFIIDH

GAL-VP1 6|uAd la(pmol) 3.6 70 0 3.6 70

FIG. 4. Comparison of the relief of transcriptional inhibition

caused by excess GAL-VP16 using either rTFIIDH or a TFIID

fraction (BT1M) partially purified from HeLa WCE. The BT1M

TFIID activity was partially purified by heparin-agarose, sulfopropyl

5-PW HPLC, DEAE-cellulose, and Blue Trisacryl HPLC chroma-

tography where it was found in the 1 M KCI fraction (see Materials

and Methods for details). (A) Lanes 1-16, quantitative Si nuclease

analysis of transcription reactions performed with HeLa WCE in the

absence (lanes 1-4 and 10-12) or in the presence of 3.6 pmol (lanes

5 and 13) or 70 pmol (lanes 6-9 and 14-16) of GAL-VP16. Reactions

were supplemented with increasing concentrations of TFIID BT1M

as indicated (lanes 2-4 and 7-9) or with rTFIIDH as indicated (lanes

11, 12, 15, and 16). Lanes 17-19, complementation of a 450C

heat-treated WCE (lane 17) with 2 ,u of rTFIIDH (lane 18) or with

5 of BT1M TFIID (lane 19). (B) Graphic representation of the

transcription obtained in lanes 1-16 of A. Note that each bar

corresponds to the lane immediately above in A.

TFIID is required to overcome transcriptional inhibition byGAL-VP16, consistent with the idea that factor(s) differentfrom those required for basal transcription are required forstimulation by GAL-VP16.

DISCUSSIONThe results presented here support the existence of a com-ponent of the transcription apparatus that we have termed atranscriptional intermediary factor, or TIF (10), which isessential for GAL-VP16 stimulation of transcription. Basaltranscription from WCE heated at 48TC for 15 min can berecovered by the addition of rTFIIDY or rTFIIDH (BTF1).However in neither case can the reconstituted heat-treatedsystem be stimulated by GAL-VP16, indicating that factor(s)required for GAL-VP16 action have been selectively inacti-vated by the heat treatment (Fig. 3). The observation thatheparin-agarose chromatography of HeLa WCE can resolvetranscriptional activity into GAL-VP16-responsive and -re-fractory fractions (Fig. 1) further supports the existence of afactor required for GAL-VP16 activation but not for basaltranscription. In addition, aliquots of responsive fractionsefficiently complement fractions refractory for GAL-VP16stimulation, under conditions where basal transcription is notsignificantly affected (Fig. 1B). These results also indicatethat transcriptional stimulation by GAL-VP16 is not due tothe counteraction of the effect of possible inhibitors, such ashistones (see refs. 30 and 31). GAL-VP16-responsive frac-tions are also much more efficient than refractory fractions atrelieving transcriptional interference/squelching by an ex-cess of the activator (Fig. 2), which suggests that the factorresponsible for stimulation is titratable by GAL-VP16. Takentogether, all of these results provide compelling evidence forthe existence of a TIF, which is distinct from the generaltranscription factors essential for basal transcription.

Partially purified TFIID activity restores stimulated tran-scription to WCE inhibited by excess GAL-VP16, indicatingthat TIF(s) copurify at least to some extent with TFIID. Theobservation that TFIID activity eluting from a heparin col-umn does not closely follow GAL-VP16 stimulation (Fig. 1)may indicate a partial dissociation of putative TIF-TFIIDcomplexes. It is also possible that there are several macro-molecular complexes of TFIID with slightly different chro-matographic properties-for example, containing other TIFsrecognizing different classes of transcriptional activatingdomains (see discussion of ref. 10). However, as notedabove, the addition of rTFIIDH does not relieve the inhibi-tion brought about by excess GAL-VP16, suggesting thatTFIID per se (as defined by rTFIID) may not be the directtarget of GAL-VP16. It has been shown that rTFIIDY bindsselectively to a VP16 affinity column (32). Any interactionbetween VP16 and TFIID may possibly be stabilized in ourreactions by the TIF(s).We have shown here that both rTFIIDH and rTFIIDY

restore basal transcription and, more importantly, transcrip-tional stimulation by GAL-VP16 to a 45°C heat-treatedWCE. These results are in apparent contrast to those ofPughand Tjian (23), who showed that heat-treated HeLa nuclearextracts complemented with rTFIIDH, but not rTFIIDY,were responsive to the transcriptional activator Spl. Thisdiscrepancy may simply be due to a difference in extractpreparation or, more intriguingly, to a difference in themechanism of action of Spl and GAL-VP16. The two acti-vators may possibly act through different TIF(s). The HeLaTIF(s) interacting with Spl may recognize the N-terminalregion of TFIID unique to the human protein, whereas thoserecognizing GAL-VP16 may interact with the conservedC-terminal region of TFIID. It has been shown recently thatrecombinant chimeric TFIID proteins in which the yeastN-terminal region has been replaced by that of the human

Biochemistry: White et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

protein are functional in yeast in vivo (33, 34). In addition,N-terminally truncated derivatives ofTFIIDY are functionalin yeast in vivo (albeit at reduced efficiency) and, moreover,are activated by the acidic activator GAL4 (33). It should alsobe noted in this regard that Spl is not a transcriptionalactivator in yeast (see ref. 24). Taken together, these obser-vations support the conclusion of Tasset et al. (10), whosuggested that different classes of transcriptional activatorsfunction by different mechanisms.

In summary, our results provide compelling evidence forthe existence ofa TIF required for transcriptional stimulationby the acidic activating domain of GAL-VP16 but that is notessential for basal transcription. Whether TIFs act by pro-viding a functional link between GAL-VP16 and the TATAbox binding protein, possibly by interacting directly withTFIID, remains to be demonstrated.

We are grateful to Drs. A. Berk (University of California, LosAngeles) and R. Tjian (University of California, Berkeley) for thegifts of rTFIIDH. We thank Dr. Bruno Cavallini for gift of recom-binant rTFIIDY and for useful discussion and also C. Werl6 for helpwith the figures. J.H.W. was supported by fellowships from theCentre National de la Recherche Scientifique (CNRS) and theFondation pour la Recherche Mddicale. J.W. and N.B. were sup-ported by fellowships from the Universite Louis Pasteur. This workwas supported by grants from the CNRS, Institut National de laSante et de la Recherche Midicale, Association pour la Recherchesur le Cancer, and the Fondation pour la Recherche Mddicale.

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