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

Evidence for a factor required for transcriptional stimulation by the 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 Mol6culaire de 'Institut National de la Sant6 et de la Recherche M6dicale, Institut de Chimie Biologique, Facult6 de M6decine 11, Rue Humann, 67085 Strasbourg C6dex, France

Contributed by Pierre Chambon, May 28, 1991

binding and transcriptional activation (see refs. 6-8 for reviews and refs. 9-11). One of the first identified classes of transcriptional activating domains was characterized by its high concentration of acidic amino acids, which may be arranged to form amphipathic a-helices (12). The herpes simplex virus-encoded VP16 protein, which contains a highly acidic 78-amino-acid C-terminal region, has been one of the most intensively studied activators of this class (13-15). The VP16 acidic domain functions in vivo when coupled to the DNA binding domains 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 in extracts of yeast (17, 18) and HeLa (19) cells. The components of the basal initiation complex that are targets of GALVP16 and other transcriptional activators have been the subject of intensive scrutiny recently (see ref. 20 and references therein). Analysis of these target factors has been given impetus by the observation in vivo that high concentrations of a transcriptional activator will squelch (21) or interfere (22) with transcription stimulated by another activator with an unrelated DNA binding site. This raises the possibility that the competing activator is sequestering factors required for transcriptional activation. A systematic in vivo study of this kind in HeLa cells has suggested that different classes of activators may be coupled to components of the basal transcription machinery through specific transcriptional intermediary factors (TIFs) (10). Squelching by GAL-VP16 has been reproduced in vitro in yeast extracts (17, 18). Kornberg and coworkers (17) have exploited this phenomenon to isolate a yeast fraction that relieves transcriptional inhibition by GALVP16. This effect cannot be reproduced by addition of RNA polymerase B (II) or general transcription factors, thus indicating the existence of a TIF that represents an additional component of the yeast transcription apparatus. Evidence for such factor(s) in extracts of higher eukaryotic cells has so far been indirect; it has largely been limited to the observation that recombinant TFIID can be used to reconstitute basal, but not stimulated, transcription in heterologous in vitro transcription systems (23, 24). In this report, we provide biochemical evidence for a TIF in HeLa whole cell extracts (WCE) and demonstrate that this factor is not essential for basal transcription and is thus distinct from previously identified general transcription factors.

We provide biochemical evidence for the exABSTRACT istence of a transcriptional intermediary factor (TIF) in HeLa whole-cell extracts (WCE) that is distinct from the basic transcription factors and that is required for transcriptional stimulation by the chimeric acidic activator GAL-VP16. We have fractionated HeLa WCE by heparin-agarose chromatography. Of transcriptionally active fractions eluting in a step between 0.24 and 0.6 M KCI, the initial fractions are refractory to GAL-VP16 stimulation, whereas subsequent fractions are strongly stimulated by the activator. Aliquots of GAL-VP16responsive fractions efficiently complement refractory fractions for transcriptional stimulation. Aliquots of responsive fractions are also far more efficient than those of refractory fractions in overcoming transcriptional inhibition that is brought about by high concentrations -of GAL-VP16. Experiments performed with heat-treated WCE support the idea that HeLa cells contain a TIF that is essential for GAL-VP16 stimulation, but that is not required for basal transcription. Addition of recombinant yeast or human transcription factor TFID (rTFIDY and rTFIDH, respectively) to a WCE heated at 48°C for 15 mE restores basal transcription, but in neither case is the reconstituted system activated by GAL-VP16. However, a 45°C heat-treated WCE reconstituted with either rTFHDH or rTFUDY is stimulated by GAL-VP16, suggesting that a HeLa TIF can be selectively inactivated by heating at 48WC, but not at 45°C. Interestingly, a TFUD fraction partially purified from HeLa cell extracts, but not rTFIIDH, efficiently relieves transcriptional Inhibition by GAL-VP16, suggesting that there may be an association between TIF(s) and TFJD and, moreover, that TIF(s) may be the direct target of the acidic domain of GAL-VP16. In summary, our results support the existence of a TIF that is not essential for basal transcription but that is required to mediate the stimulatory activity of the acidic activator GAL-VP16.

Formation of a basal transcriptional initiation complex on eukaryotic class B (II) promoters requires several general transcription factors in addition to RNA polymerase B (II). Specific binding of transcription factor TFIID (also known as BTF1; see refs. 1 and 2) to the TATA box, perhaps in the presence of STF (or TFIIA; see ref. 1), is required prior to the assembly of RNA polymerase and other general factors on the promoter (3-5). Initiation complex formation is modulated in vivo and in vitro by transcriptional activators, which recognize the upstream element and enhancer sequences of a given promoter. Numerous biochemical and molecular genetic analyses have indicated that these factors can generally be dissected into domains required for specific DNA

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

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Biochemistry: White et al.

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

A

MATERIALS AND METHODS Recombinant Plasmids and Expression in Escherichia coil. Plasmid 17M5/pAL7 contains five 17-mer binding sites (see ref. 9) recognized by the DNA binding domain of GAL4 inserted 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 from pET-3C by using the bacteriophage T7 expression system (25); was purified from supernatants of sonicated cells by chromatography over heparin-agarose, phenyl-Sepharose, and DNA-cellulose columns; and was 80o pure as judged by Coomassie brilliant blue-stained SDS/polyacrylamide gels. Recombinant human TFIID (rTFIIDH) and recombinant yeast TFIID (rTFIIDY; formerly known as BTF1Y; see ref. 1) were expressed in E. coli from pET-3A (25); their purification to homogeneity will be described elsewhere. Fractionation of HeLa WCE by Heparin-Agarose Chromatography. Two hundred milligrams of HeLa WCE was loaded on a 40-ml heparin-agarose column equilibrated in 25 mM TrisHCl, pH 7.9/5 mM MgCl2/0.5 mM dithiothreitol/0.1 mM EDTA/10% (vol/vol) glycerol. Bound protein was eluted with steps of 0.24, 0.6, and 1 M KCl (see ref. 26). Aliquots (8 /l) of the 0.6 M KCl fractions (-4 ml) were tested for transcriptional activity as described below (see also legends to figures). Partial Purification of TFIID (BTF1) Activity from HeLa Cells. HeLa WCE were fractionated by heparin-agarose chromatography as described above. The 0.6 M KCi step was diluted 6-fold in 25 mM Tris HCl, pH 7.9/0.5 mM dithiothreitol/0.1 mM EDTA/10%o (vol/vol) glycerol (TDEG10) and loaded on a 2.5 x 15 cm2 sulfopropyl 5-PW HPLC column (Toyosoda) equilibrated in TDEG10 containing 100 mM KCl. Bound protein was eluted with 350 mM (SP350 fraction) and 1 M KCi. TFIID activity was assayed by using a heat-treated WCE (ref. 27; see below) and was found in the SP350. Ninety-five milliliters of SP350 fraction (80 mg) was equilibrated in TDEG10 containing 50 mM KCl and loaded on a 13-ml DEAE-cellulose column. Bound protein was eluted with a 90-ml gradient from 50 to 500 mM KCl and collected in 15 6-ml fractions. A single fraction containing TFIID activity was equilibrated in TDEG10 containing 50 mM KCl and loaded on a 3-ml Blue Trisacryl HPLC column. Bound protein 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 fraction was dialyzed against 25 mM Tris-HC1, pH 7.9/0.5 mM dithiothreitol/0.1 mM EDTA/20% glycerol/50 MM KCI and stored in 100-Al aliquots at -1980C. In Vitro Transcription. Reactions (20-25 ,ul), incubated at 250C, were performed essentially as described (28). Aliquots of 200-500 1.d of WCE were heated prior to each experiment in 1.5-ml Eppendorf tubes for exactly 15 min at 450C or 480C as indicated in the figures. Ten microliters of heat-treated WCE, 8-104ul of untreated WCE, or 8 /l of heparin fractions supplemented with 1 /l of STF (DEO.35; ref. 26) was used in each reaction (see also legends to figures). Purified RNA (28) was analyzed by quantitative SI nuclease analysis (9).

RESULTS Fractionation of HeLa WCE into GAL-VP16-Responsive and -Refractory Fractions. To monitor GAL-VP16-dependent transcription we used the plasmid 17M5/pAL7, which contains five 17-mer GAL4 DNA recognition sites recognized by the DNA binding domain of GAL4 inserted upstream of the TATA region (-34 to +33) of Ad2MLP (Fig. IA). GAL-VP16 stimulates transcription from this promoter 7- to 10-fold in HeLa WCE (Figs. 2-4). This stimulation requires the YP16 acidic domain and is strictly dependent on the presence of 17-mers in the test promoter (data not

7675

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a,010fold increase in transcription in the presence of GAL-VP16 (Fig. 1B Right). This result is important for two reasons. First, it indicates that GAL-VP16 stimulation of the low basal transcription of fraction 40 is not simply due to counteraction of the effects of inhibitors (for example, histones; see refs. 30 and 31), which could possibly be present- in the fraction. If this were the case, it would be expected that addition of fraction 40 to fraction 33 would inhibit basal transcription. Instead, addition of fraction 40 causes an -2-fold increase in basal transcription (Fig. 18 Right). Second, this result supports the proposal that fraction 40 contains a factor(s) necessary for GAL-VP16 action, since the stimulation observed with combined fractions 33 and 40 is at least 4-fold greater than that expected from an additive effect. GAL-VP16-Responsive Fractions Relieve Transcriptional Inhibition by High Concentrations of the Activator. Addition of 1.6 pmol of GAL-VP16 to a WCE generally produces a 7to 10-fold stimulation in transcription (Fig. 2A, lanes 1 and 2; Figs. 3 and 4; and data not shown). Increasing quantities above 1.6 pmol generally stimulate less and less such that A

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with 40 pmol of GALVP16 no stimulation is observed (Fig. 2A, lane 3; Fig. 2B; and data not shown). This effect is reminiscent of the transcriptional inhibition (squelching) observed with high concentrations of GAL-VP16 in vivo in yeast and mammalian cells (10, 17) and in vitro in yeast extracts (17, 18). We tested the relative abilities of the GAL-VP16refractory fraction 32 and -responsive fraction 40 to overcome this inhibition (Fig. 2). Fractions 32 and 40 were chosen because 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 WCE produced a