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Volume 270, Number 8, Issue of February 24, 1995 pp. 3631-3637
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a HeLa Cell Factor Which Negatively Regulates Transcriptional Activation in Vitro by Transcriptional Enhancer Factor-1 (TEF-1) (*)

(Received for publication, October 19, 1994; and in revised form, December 15, 1994)

Sunita Chaudhary (§),  ,  Laszlo Tora ,  Irwin Davidson (¶)

From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, B.P. 163-67404 Illkirch Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A novel negatively acting factor has been identified and partially purified from HeLa and BJA-B cell extracts by chromatographic fractionation. Addition of this factor to HeLa cell extracts or to a reconstituted HeLa cell transcription system repressed transcriptional activation by a chimeric activator, GAL-TEF-1, containing the activation function of transcriptional enhancer factor-1 (TEF-1). In contrast, this factor did not repress transactivation by the chimeric GAL-VP16 activator. Repression of transactivation by GAL-TEF-1 could be alleviated by the addition of immunopurified HeLa cell TFIID, but not by increased quantities of GAL-TEF-1. These observations suggest that this negatively acting factor represses transactivation by interfering with the function of, or competing for, the TATA-binding protein-associated coactivators which mediate the activity TEF-1.

INTRODUCTION

The mechanism by which transcriptional activator proteins enhance transcription initiation by RNA polymerase II is at present poorly understood. Using in vitro transcription systems derived from HeLa cells, accurate and regulated initiation by RNA polymerase II requires two classes of factors. The first class comprises the factors TFIIB, TFIIE, TFIIF, TFIIH, and the TATA-binding protein (TBP) (^1)(reviewed in (1, 2, 3, 4) ). These proteins or a subset of these proteins (5, 6) are necessary and sufficient to direct basal transcription from a variety of TATA-containing promoters. In addition to these, others factors such as TFIIA and TFIII may be required for transcription in the presence of certain repressor proteins or for transcription from TATA-less promoters (for reviews, see (1, 2, 3, 4) ). Interactions between transactivators and several basal transcription factors have been reported (reviewed in (7) and references therein), and at least some of these interactions may be necessary for the transactivation process. The second class of factors, coactivators, are not required for basal transcription but are required for stimulation by transcriptional activators in vitro. Some of these coactivators, such as USA or ACF(8, 9, 10) , are separable from the basal transcription factors, but others are tightly associated with TBP in the TFIID complex (reviewed in (11, 12, 13, 14) ). The requirement for interaction between transactivators and basal transcription factors and for the presence of coactivators indicates that activation may be a multistep process(15, 16) .

The HeLa cell TFIID factor is a multiprotein complex comprising TBP, which mediates specific binding to the TATA element, and possibly up to 13 TBP-associated factors (TAFs)(17, 18, 19, 20, 21) . TBP has been shown to support basal transcription, but unlike the native TFIID complex, it does not mediate stimulation by transactivators ( (17) and references therein). This observation implies that one or several of the TAFs in the TFIID complex are required for the function of transcriptional activators. Several lines of evidence indicate that the activity of activators with different classes of activation functions (i.e. rich in prolines, glutamines, acidic, etc.) may be mediated by distinct TAFs. It has been shown that the acidic activation function of the herpes simplex virus protein VP16 interacts with Drosophila (d)TAF40, whereas the glutamine-rich activation function of SP1 interacts with dTAF110(22, 23, 24) . In the case of SP1 a partially reconstituted TFIID complex containing TBP, dTAF250, and dTAF110 was shown to mediate stimulation in vitro by this activator(25) . It has also been reported that transactivation by a chimera (GAL-TEF-1) comprising the DNA binding domain of the yeast activator GAL4 and the activation function of transcriptional enhancer factor-1 (TEF-1) can be mediated by two chromatographically separable TFIID complexes(17) . One of these, eluting in the phosphocellulose (PC) 1.0 M KCl (PC1.0) fraction also supported activation by GAL-VP16 and GAL-ER(EF) (i.e. the activation function 2 (AF-2) of the human estrogen receptor (ER)), whereas the other eluting in the PC0.3 fraction functioned selectively with GAL-TEF-1. Moreover, we have recently cloned and expressed human (h)TAF30, which is present in the PC1.0-derived TFIID, and shown that hTFIID can be separated into two populations either containing or lacking hTAF30(26) . The TFIID population containing hTAF30 is required for the activity of the ER AF-2 in vitro, and a subdomain of the ER AF-2 interacts directly with hTAF30. These results support the idea that the activity of different transactivators is mediated by direct and selective interaction with distinct TAFs.

The function of transcriptional activators in vivo also appears to require the action of coactivators, alternatively designated transcriptional intermediary factors(27, 28, 29) . Overexpression of some transcriptional activators results in a squelching or transcriptional interference effect leading to diminished transactivation(30) . These results suggest that high concentrations of the activation functions of these transactivators titrates, or in some other way interferes with, the activity of a limiting factor(s) required for the stimulation of transcription. Moreover, selective interference between heterologous activators suggests the existence of several transcriptional intermediary factors specific for different classes of activation functions(28, 29) .

The HeLa cell protein TEF-1 is an example of a transactivator whose activity is mediated by a limiting factor(s) both in vivo and in vitro. TEF-1 was first identified by its ability to bind to two enhansons within the enhancer of simian virus 40(31, 32, 33) . Overexpression of TEF-1 in HeLa cells does not stimulate transcription from cognate reporter plasmids to a level above that generated by the endogenous HeLa cell TEF-1, but rather results in a repression of the activity of endogenous HeLa cell TEF-1(34, 35) . Similarly, transactivation by a GAL-TEF-1 chimera is optimal at low concentrations, whereas diminished activity is observed at higher concentrations. The dominant negative phenotype of TEF-1 does not require site-specific binding of TEF-1 to the promoter of the reporter gene, but does require the regions of the protein necessary for transactivation in the context of GAL-TEF-1 chimeras(34, 35) . Together these results suggest that transactivation in vivo by TEF-1 requires the action of a TIF(s) which is highly limiting in HeLa cells. Such a factor also appears to be present in limiting amounts in keratinocytes, HepG2 liver cells, COS cells, and SiHa cervical carcinoma cells(36) . In contrast, the activation function of TEF-1 is inactive in lymphoid cells suggesting that a TIF required for its activity is absent or repressed in these cells(34, 36) .

Analogous observations concerning transactivation and transcriptional interference using TEF-1 or GAL-TEF-1 chimeras have been made in vitro. Addition of purified recombinant TEF-1 to HeLa cell extracts repressed activation by the TEF-1 endogenously present in these extracts(34) . Similarly, low concentrations of a GAL-TEF-1 chimera activated transcription in HeLa cell extracts, but this activation was diminished with higher concentrations of the chimera. GAL-TEF-1 stimulated transcription efficiently in HeLa cell extracts, whereas much lower activity was observed in extracts from BJA-B lymphoid cells(37) . The activity of GAL-TEF-1 in BJA-B cell extracts could be dramatically increased by the addition of immunopurified HeLa cell TFIID, implying that TBP-associated factors required for GAL-TEF-1 activity were lacking or present in reduced amounts in these extracts. However, immunopurification and chromatography experiments indicated that the low activity of GAL-TEF-1 was not due to the lack of the appropriate TBP-associated factors, but rather to the presence of a cell-specific activity, NEF-1, in BJA-B extracts which repressed transactivation by GAL-TEF-1(37) .

During the chromatographic fractionation of HeLa cell extracts, we identified a factor which repressed transactivation by GAL-TEF-1. This novel activity, whose chromatographic properties and cell specificity are distinct from those of NEF-1 described above, was also detected in extracts from BJA-B cells. In this study we have partially purified and characterized this HeLa cell factor (NEF-2) which negatively regulates transactivation in vitro by GAL-TEF-1. Addition of this factor to HeLa cell extracts or a reconstituted HeLa cell transcription system repressed transactivation by GAL-TEF-1, but not by GAL-VP16. The negative effect of NEF-2 could be alleviated by the addition of immunopurified TFIID, but not by addition of increased amounts of GAL-TEF-1. These results suggest that this factor exerts its inhibitory effect on transcriptional activation by selectively interfering with the function of, or competing for, a TBP-associated factor(s) required for the activity of TEF-1.

MATERIALS AND METHODS

Preparation of Cell Extracts and Chromatography

Nuclear extracts from HeLa cells or whole cell extracts from BJA-B cells, grown in suspension, were prepared as described previously(17, 37, 38, 39) . All chromatography steps (performed at 4 °C) were essentially as described previously(17) . Approximately 250 ml of HeLa cell nuclear extract (500 mg of protein were applied onto a 300-ml heparin ultrogel column equilibrated in buffer B (50 mM Tris-HCl, pH 7.9, 20% (v/v) glycerol, 0.5 mM dithiothreitol, 0.2 mM EDTA) containing 0.1 M KCl. The column was washed extensively with buffer B containing 0.1 M KCl and then with buffer B containing 0.24 M KCl. The bound protein was then eluted with buffer B containing 0.6 M KCl. The 0.6 M KCl fraction was then diluted until the conductivity was less than 0.4 M KCl and adjusted to 2.26 M ammonium sulfate. The solution was stirred for 1 h and then centrifuged at 9000 rpm for 20 min in a Beckman JA-10 rotor. The protein pellet was then resuspended in buffer C (50 mM Tris-HCl, pH 7.3 (at 25 °C), 0.2 mM EDTA, 0.5 mM dithiothreitol, and 20% (v/v) glycerol), diluted until the conductivity was equivalent to that of 0.1 M KCl, and loaded on a 150-ml phosphocellulose P11 column. The phosphocellulose column was then sequentially eluted with buffer C containing 0.3, 0.5, and 1.0 M KCl. 100 ml of BJA-B WCE (approximately 100 mg of protein) were chromatographed essentially as described above using correspondingly smaller volumes of resin. Peak protein fractions were collected and dialyzed against buffer B containing 0.05 M KCl prior to in vitro transcription.

Analytical and preparative (HeLa extracts only) scale HPLC was performed on either TSK-phenyl 5PW or heparin 5PW columns (3.3 ml, 0.75 cm (internal diameter) times 7.5 cm; 50 ml, 2.15 times 15 cm, respectively) using the Beckman Gold system as described previously(17) . 20 mg of the PC0.3 fractions were loaded on the 3.3-ml columns. The phenyl 5PW columns were eluted with linear 5- or 7-column volume gradients (0.9-0.0 M ammonium sulfate), and 28-32 fractions were collected. The heparin 5PW columns were eluted with a linear 5-column volume 0.2-0.8 M KCl gradient, and 28 fractions were collected.

For the further purification of HeLa cell NEF-2, 250 mg of the PC0.3 fraction were loaded on a 50-ml phenyl 5PW column, and the bound protein was eluted with a 7-column volume gradient. The NEF-2-containing fractions were then pooled, dialyzed, and loaded on a 3.3-ml heparin 5PW column. The bound protein was eluted with a 7-column volume 0.15-0.6 M KCl gradient. Due to limiting amounts of material the BJA-B cell NEF-2 could not be purified beyond the third chromatographic step.

HeLa cell TFIID was partially purified from the PC0.3 and 1.0 fractions by chromatography on phenyl 5PW columns. The TBP-containing fractions were identified by immunoblot analysis. In the PC0.3 fraction the TBP containing fractions were separated from those containing NEF-2, and both of these factors could be recovered from the same chromatography preparation.

HeLa cell NERF-1 was purified as described previously(40) . Briefly, the H0.6-derived PC1.0 fraction was chromatographed on a phenyl 5PW column using a linear 0.9-0 M ammonium sulfate gradient. The NERF-1-containing fractions were identified by in vitro transcription assays and pooled.

TFIIA (STF) was purified from the heparin 0.1 M KCl flow-through fraction as described previously(17) .

Overexpression and Purification of Recombinant Proteins

GAL(1-147)-VP16 and GAL-(1-147)-TEF-1(166-426) and recombinant human TBP were overexpressed in Escherichia coli using the pET T7 expression system (41) and purified by ion exchange and/or site-specific DNA affinity chromatography as described previously(17, 34, 40, 42) . The concentration of the recombinant proteins was estimated by staining SDS-polyacrylamide gels with Coomassie Brilliant Blue.

In Vitro Transcription

In vitro transcriptions were performed essentially as described(43, 44) . 25-µl reactions contained 25 ng of 17M5/pAL7 and 25 ng of pG1, as an internal control, along with the partially purified fractions or HeLa WCE (60 µg), as indicated in the figure legends. Where indicated the reactions also contained 50-60 ng of purified activator proteins. After 15-min preincubation at 25 °C with all of the components, transcription was initiated by the addition of NTP (500 µM final concentration), and incubation was continued for 45 min at 25 °C. RNA transcripts were detected by quantitative S1 nuclease mapping as described previously(43, 45) .

Immunopurification of HeLa Cell TFIID

Monoclonal antibody 3G3 was purified from ascites fluid by caprylic acid and ammonium sulfate precipitation as described previously(17, 44) . Immunoprecipitations (IP) were performed as described previously(17, 37) . Briefly, the phenyl 5PW fractions from the PC0.3 or PC1.0 fractions containing TFIID were dialyzed against IP buffer (50 mM Tris-HCl, pH 7.9, 5 mM MgCl(2), 10% (v/v) glycerol, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, and 0.1 M KCl) and were first preincubated at 4 °C for 1 h with protein G-Sepharose in the absence of antibody. The protein G-Sepharose was then removed by centrifugation at 2000 rpm for 5 min. Independently the antibody was also preincubated at 4 °C for 1 h with protein G-Sepharose, and the resulting complexes were centrifuged as above and extensively washed four times with IP buffer. The coupled antibody-protein G-Sepharose and precleared phenyl 5PW fractions were then mixed and incubated with rotation for 2 h at 4 °C. The complexes were then collected by centrifugation and washed five times with IP buffer. The immunoprecipitated TFIID complexes were eluted by the addition of a 1000-fold excess of the peptide PA81 (corresponding to the first 17 amino acids of human TBP; see Refs. 17 and 44) for 3 h at 4 °C with rotation. The samples were then centrifuged at 2000 rpm for 5 min and the supernatant collected and frozen at -80 °C. Routinely the TFIID from 1-2 ml of the pooled phenyl 5PW fractions was precipitated using 100-200 µl of protein G-Sepharose and 10-50 µg of purified antibody. The TFIID was then eluted in a 100-200-µl volume.

RESULTS

Extracts from HeLa and BJA-B Lymphoid Cells Contain a Factor Which Negatively Regulates Transactivation by GAL-TEF-1

We have previously reported a protocol for the chromatographic fractionation of HeLa cell extracts which allowed the isolation of two separable TFIID complexes ((17) , Fig. 1, and see ``Materials and Methods''). HeLa cell nuclear extracts were first fractionated on a Heparin ultrogel column. The heparin 0.6 M KCl (H0.6) fraction was subsequently chromatographed on a phosphocellulose column. The H0.6-derived phosphocellulose 0.5 M KCl (PC0.5) fraction (see Fig. 1and ``Materials and Methods''), when supplemented with partially purified TFIIA, supports basal transcription from templates containing the adenovirus major late promoter (AdMLP) TATA element or the beta-globin promoter ( (17) and see AdMLP+1 and Glob+1 in lane 1Fig. 2, A and C). In this system transcription from the AdMLP reporter, (17M5/pAL7) which contains five binding sites for the yeast activator GAL4 inserted upstream of the AdMLP TATA element, was stimulated approximately 3-fold by the addition of a purified chimeric activator, GAL-TEF-1, comprising the DNA binding domain of GAL4 (residues 1-147) and the activation function of TEF-1 (residues 166-426) (lanes 1 and 2Fig. 2, A and C). Increased levels of GAL-TEF-1-activated transcription were observed when these fractions were combined with partially purified or immunopurified TFIID (see ``Materials and Methods''), but not purified recombinant TBP ( (17) and see below). During the purification of TFIID from the phosphocellulose 0.3 M KCl (PC0.3) fraction, we noted the existence of an activity which was separable from TFIID and which appeared to negatively affect transactivation by GAL-TEF-1. When the PC0.3 fraction was further separated by HPLC chromatography on heparin or phenyl 5PW columns ( Fig. 1and ``Materials and Methods'') an activity was eluted which reduced transactivation by GAL-TEF-1 (compare transactivation in lanes 1-6 with that observed in the presence of fractions 6-12 in lanes 7-14 in Fig. 2A and in lanes 1 and 2 and 9 and 10 with fractions 4-8 in lanes 3-8 in Fig. 2B). Similarly, a factor with analogous properties was observed eluting in the equivalent chromatography fractions derived from extracts of BJA-B lymphoid cells (see fractions 6-12 in lanes 7-14 in Fig. 2C and fractions 4-8 in lanes 3-8 in Fig. 2D).

Figure 1: Chromatographic protocol used to fractionate cell extracts. HeLa and BJA-B cell extracts were first chromatographed over heparin Ultrogel and phosphocellulose P11. FT indicates the flow-through fraction in 0.1 M KCl. The concentrations of KCl used to elute the columns are indicated. The ammonium sulfate used to concentrate the H0.6 fraction is abbreviated to AS. The PC0.3 fraction was subsequently separated by HPLC on Phenyl or heparin 5PW columns. The columns were eluted with linear gradients (indicated by the arrows) between the salt concentrations shown at each end of the arrows. Although NEF-2 activity could not be measured in the phosphocellulose fraction, the subsequent phenyl 5PW fractions contained less than 10% of the starting protein. This fraction was enriched a further 10-fold by the HPLC-heparin step.


Figure 2: A and B, the HeLa PC0.3 fraction contains a factor which inhibits the activity of GAL-TEF-1. The H0.6-derived PC0.3 fraction was separated by HPLC on heparin (A) or phenyl (B) 5PW columns as indicated under ``Materials and Methods.'' In A 28 fractions were collected, and each alternate fraction was tested. Transcription reactions contained 6 µl of the PC0.5 fraction, 1 µl of the partially purified TFIIA fraction, and 10 µl of the PC0.3 fractions indicated above each lane along with 25 ng of each of the DNA templates. The presence or absence of 60 ng of purified GAL-TEF-1 is also indicated above each lane. The positions of the correctly initiated transcripts from the reporters containing the AdMLP TATA element or beta-globin promoters are indicated to the left of the figure. The NEF-2 activity eluted between 0.22 and 0.28 M KCl. In B 30 fractions were collected. Transcription reactions were constituted as described in A. The NEF-2 activity eluted between 0.8 and 0.7 M ammonium sulfate. C and D, the BJA-B PC0.3 fraction contains a factor which inhibits the activity of GAL-TEF-1. HPLC was performed as described under ``Materials and Methods'' and in A and B. The transcription reactions were constituted as described in A and B.


To further characterize this factor, designated negative factor-2 (NEF-2), the HeLa cell phenyl 5PW fractions containing this activity were combined and further chromatographed on a HPLC heparin 5PW column (see Fig. 1and ``Materials and Methods''). The fractions containing the peak of NEF-2 activity were then pooled. To test the effect of the NEF-2-containing fractions on transactivation by GAL-TEF-1, they were added to a HeLa WCE in the presence or absence of GAL-TEF-1. Addition of increasing concentrations of the NEF-2 fraction resulted in a reduction in the levels of activation by GAL-TEF-1 (compare activation in lanes 1 and 2 with lanes 3-8 in Fig. 3A).

Figure 3: A, NEF-2 inhibits transactivation by GAL-TEF-1 in a HeLa WCE. Transcription reactions contained 12.5 ng of each of the DNA templates, 10 µl (60 µg) of HeLa WCE, and the quantities of the NEF-2 fraction indicated above each lane. The presence or absence of 60 ng of GAL-TEF-1 is indicated above each lane. Transactivation was reduced from 8-fold in lanes 1 and 2 to 2.5-fold in lanes 7 and 8. B, NEF-2 inhibits transactivation by GAL-TEF-1 in a reconstituted HeLa cell transcription system. Transcription reactions contained 25 ng of each DNA template along with 6 µl of the PC0.5 fraction, 1 µl of the TFIIA fraction, and the quantities of the NEF-2 fractions indicated above each lane. In addition, the reactions in lanes 1-8 contained 5 µl of the phenyl 5PW fraction (derived from the PC1.0 fraction) containing TFIID (as described under ``Materials and Methods''); the reactions in lanes 9-16 contained 5 µl of the TFIID-containing phenyl 5PW fractions from the PC0.3 fraction. Transactivation was reduced from greater than 10-fold in lanes 7 and 8 or 1-2- to 3-fold in lanes 5 and 6 (when normalized to transcription from the globin promoter), or from 7-fold in lanes 9 and 10 to less than 2-fold in lanes 15 and 16. C, NEF-2 does not inhibit transactivation by GAL-VP16. Transcription reactions contained 25 ng of each DNA template, 10 µl of a PC0.5 fraction, 1 µl of the TFIIA fraction, 5 µl of a TFIID-containing phenyl 5PW fraction derived from the PC1.0 fraction, and the quantities of the NEF-2 fraction indicated above each lane. The presence of 60 ng of either GAL-TEF-1 or GAL-VP16 is indicated above each lane. D, transcription reactions were constituted as described in lanes 1-8 of B and contained the quantities of the BJA-B NEF-2 fraction indicated above each lane. The NEF-2 was derived from the BJA-B PC0.3 fraction by chromatography on a phenyl 5PW column. The NEF-2-containing fractions were pooled and added in the quantities indicated above each lane. Transactivation was reduced from 5-fold in lanes 1 and 2 to less than 2-fold in lanes 5 and 6.


The addition of the phenyl 5PW fractions containing TFIID derived from the PC1.0 or PC0.3 fractions (see ``Materials and Methods'') to the PC0.5 + TFIIA fractions allows efficient transactivation by GAL-TEF-1 (lanes 7 and 8 and 9 and 10 in Fig. 3B). As observed above using the HeLa WCE, addition of increasing amounts of the NEF-2 fraction to this system inhibited transactivation by GAL-TEF-1, but at these concentrations, did not affect basal transcription from the AdMLP TATA element (compare lanes 7 and 8 with lanes 1-6 and lanes 9 and 10 with lanes 11-16 in Fig. 3B and see figure legends). Similar results were obtained with NEF-2 which had been further purified from the heparin 5PW fractions on a HPLC DEAE column (data not shown) and with independent NEF-2 preparations (see Fig. 3C, 4, and 5).

Analogous experiments were performed using NEF-2 partially purified from BJA-B WCEs by the same protocol (see ``Materials and Methods''). As observed above using the HeLa cell fractions, addition of increasing amounts of the BJA-B NEF-2 fractions resulted in inhibition of activation by GAL-TEF-1 (see lanes 3-6 in Fig. 3D). Together these results indicate that extracts from both HeLa and BJA-B cells contain an activity which negatively modulates transactivation in vitro by GAL-TEF-1.

To address the specificity of inhibition by NEF-2, its ability to inhibit transactivation by the chimeric acidic activator GAL-VP16 was examined. Both GAL-TEF-1 and GAL-VP16 efficiently stimulate transcription when the PC0.5 and TFIIA fractions are supplemented with TFIID derived from the PC1.0 fraction. Addition of increasing quantities of the NEF-2 fraction inhibited transactivation by GAL-TEF-1 (lanes 3-6 in Fig. 3C). In contrast, in the presence of the same amounts of NEF-2 efficient transactivation by GAL-VP16 was observed (lanes 7-10, Fig. 3C).

In some of the above experiments the addition of the NEF-2 fractions resulted in an inhibition of transcription from the AdMLP and beta-globin promoters. This effect was particularly evident using the cruder heparin or phenyl 5PW fractions (see appropriate lanes in Fig. 2, A-D). Nevertheless, using the more purified fractions little or no inhibition of transcription from the AdMLP was observed at NEF-2 concentrations which efficiently repressed transactivation by GAL-TEF-1 (see appropriate lanes in Fig. 3B), suggesting that the inhibition of basal transcription seen with the cruder fractions was not due to the NEF-2 factor. However, some repression of transcription from the beta-globin promoter was observed. In contrast to the 17M5/pAL7 template which contains only the TATA element from the AdMLP, plasmid pG1 contains not only the minimal beta-globin TATA element, but also its upstream regulatory elements. Thus, transcription from this promoter may be activated by cognate factors endogenously present in the chromatography fractions. This idea is further supported by the fact that transcription from this promoter is strongly stimulated by the addition of TFIID, which can mediate transactivation, but only weakly by TBP, which does not mediate transactivation, whereas the opposite is true for the AdMLP (see (17) and Fig. 5). Consequently, the inhibition of transcription from the beta-globin promoter may reflect the ability of NEF-2 to negatively regulate transactivation by a factor(s) binding to these upstream elements.

Figure 5: A, inhibition by NEF-2 can be alleviated by immunopurified HeLa cell TFIID. In lanes 1-6 the transcription reactions were reconstituted as described for lanes 1-8 in Fig. 3B. + above each lane indicates the presence of either 10 µl of a NEF-2 fraction or 60 ng of GAL-TEF-1. In lanes 5 and 6 the reactions comprised 10 µl of immunopurified HeLa cell TFIID in addition to the TFIID-containing phenyl 5PW fractions. The addition of the NEF-2 fraction reduced transactivation from 8-fold in lanes 1 and 2 to 2.5-fold in lanes 3 and 4. Addition of the E-TFIID resulted in the recovery of a 6-fold stimulation in lanes 5 and 6. In lanes 7-10 the TFIID-containing phenyl 5PW fractions were omitted and replaced by 50 ng of recombinant human TBP. B, the transcription reactions were reconstituted as described in lanes 1-8 in Fig. 3B. + above each lane indicates the presence of 10 µl of a NEF-2 fraction or 60 ng of GAL-TEF-1. Lane 6 contains 200 ng of GAL-TEF-1. C, NEF-2 does not inhibit transactivation when added after the formation of preinitiation complexes. Transcription reactions were reconstituted as described in lanes 1-8 in Fig. 3B, except that 75 ng of each DNA template were used to compensate for the fact that only one cycle of transcription was allowed. Note that as described previously(43) , with higher concentrations of DNA template the addition of the activator results in diminished leves of transcription from the beta-globin promoter due to competition for a limiting transcription factor. In lanes 1-4 the DNA template was added to the mixture of the transcription factors in the presence or absence of GAL-TEF-1 and NEF-2 as indicated above each lane. The reactions were incubated for 45 min at 25 °C, and transcription was then initiated by the addition of the NTP solution. After a further 5 min the reactions were terminated, and the RNA was extracted. In lanes 5 and 6 the DNA templates were added to the transcription factors in either the presence or absence of GAL-TEF-1 and incubated for 30 min at 25 °C. The NEF-2 fraction was then added and incubation was continued for a further 15 min prior to the addition of the NTP solution. After a further 5 min the reactions were terminated and the RNA extracted.


NEF-2 Exhibits Properties Distinct from Those of Previously Identified Transcriptional Inhibitors

The existence of factors which negatively affect transcription has been reported previously. Proteins such as NC1, NC2, Dr1, Dr2 (topoisomerase I, Refs. 10 and 46-48), histone H1(49) , and NERF-1 (topoisomerase II, (40) ) have all been shown to inhibit transcription in vitro. In contrast to NEF-2, these proteins are reported to preferentially inhibit basal transcription and inhibit activated transcription only at higher concentrations. To compare the effect of one of these inhibitors to that of NEF-2, increasing quantities of NEF-2 or NERF-1 (human topoisomerase II) were added to the PC0.5 + TFIIA fractions supplemented with partially purified TFIID. Increasing quantities of NEF-2 reduced the levels of GAL-TEF-1-activated transcription, but did not significantly reduce basal transcription from the AdMLP template (compare AdMLP + 1 in lanes 1 and 7 and the transactivation in lanes 1 and 2 and 7 and 8 in Fig. 4). In contrast, addition of topoisomerase II preferentially reduced basal transcription resulting in an apparent increase in transactivation (see lanes 1-2 and 9-12Fig. 4). At the highest concentrations of topoisomerase II activated transcription was reduced, but basal transcription was barely detectable. Thus, these two inhibitory activities have distinct effects on basal and GALTEF-1-activated transcription.

Figure 4: Comparison of the properties of NEF-2 and another transcriptional repressor NERF-1 (topoisomerase II). Transcription reactions were reconstituted as described in lanes 1-8 of Fig. 3B along with increasing amounts of the NEF-2 or NERF-1 (see ``Materials and Methods'') fractions.


The Inhibitory Effect of NEF-2 Can Be Alleviated by Immunopurified TFIID

Two possible mechanisms may account for the inhibitory effect of NEF-2. First NEF-2 could interact with and modify or mask the TEF-1 activating domain. Alternatively, NEF-2 may exert its effect not on GAL-TEF-1 itself, but rather on one of the coactivators required for the activity of this protein. To distinguish between these possibilities, increased quantities of GAL-TEF-1, or immunopurified TFIID comprising coactivators mediating the activity of GAL-TEF-1, were added to transcription reactions where activation was inhibited by the addition of NEF-2 (see lanes 1 and 2 and 3 and 4Fig. 5, A and B). Adding three times the normal amount of GAL-TEF-1 to the transcription reactions did not relieve inhibition by NEF-2 (lanes 5 and 6, Fig. 5B). In contrast, addition of HeLa cell TFIID, immunopurified using an anti-TBP monoclonal antibody (see ``Materials and Methods'') was both necessary and sufficient to alleviate inhibition by NEF-2 (compare lanes 1 and 2 with lanes 3 and 4 and 5 and 6 in Fig. 5A). These results strongly suggest that the target for repression by NEF-2 is a TBP-associated factor(s) required for transactivation by GAL-TEF-1, rather than the activation function of TEF-1 itself. As we have reported previously (17) addition of purified recombinant TBP to the reconstituted transcription system increased basal transcription from the 17M5/pAL7 reporter, but no further increase in transcription was seen in the presence of GAL-TEF-1 (see lanes 1 and 2 and 7 and 8Fig. 5A). Under these conditions the addition of NEF-2 had no effect on transcription either in the presence or in the absence of GALTEF-1 (see lanes 7 and 8 and 9 and 10 in Fig. 5A).

The above results suggest that NEF-2 interferes with a step in the activation process involving a TBP-associated factor(s) required for the activity of GAL-TEF-1. If this were the case, the addition of NEF-2 to the transcription reactions after the formation of preinitiation complexes (in the presence of GAL-TEF-1 and TFIID) should no longer inhibit transactivation by GAL-TEF-1. To test this possibility the transcription factors were incubated together with the DNA templates either in the presence or absence of GAL-TEF-1 for 30 min prior to the addition of the NEF-2 fraction. After the addition of the NEF-2 fraction, incubation was continued for a further 15 min before addition of the nucleoside triphosphates. Transcription reactions were terminated and the RNA extracted 5 min after the addition of the nucleoside triphosphates in order to limit the reinitiation of transcription. Addition of the NEF-2 fraction 30 min after the formation of preinitiation complexes no longer inhibited transactivation by GAL-TEF-1, whereas when the NEF-2 fraction was added along with the transcription factors at the beginning of the 30-min preincubation transactivation was inhibited (compare lanes 3 and 4 and 5 and 6 in Fig. 5C). These results indicate that NEF-2 acts during preinitiation complex formation to inhibit transactivation, but has no significant effect on the activity of preformed initiation complexes.

DISCUSSION

The results of the present study indicate that the PC0.3 fraction derived from HeLa cell extracts contains a factor which negatively modulates transactivation by GAL-TEF-1. A factor with analogous chromatographic and functional properties was also observed in the PC0.3 fraction derived from BJA-B cell extracts. The addition of this factor to HeLa WCE or a reconstituted HeLa cell transcription system inhibited transactivation by GAL-TEF-1, but had little effect on basal transcription from the AdMLP TATA element. The negative effect of NEF-2 was selective as concentrations of NEF-2 which inhibited transactivation by GAL-TEF-1 did not inhibit transactivation by GAL-VP16. Nevertheless, NEF-2 also appeared to inhibit activation of the beta-globin promoter by a factor(s) binding to its upstream regulatory elements. The ability of NEF-2 to inhibit transactivation by GAL-TEF-1, but not by GAL-VP16, may reflect the fact that the activation function of TEF-1 comprises regions rich in proline, serine, and threonine(35) , whereas in VP16 acidic and phenylalanine residues are involved in transactivation ( (50) and references therein). As described in the Introduction, it has been shown that distinct TAFs may be required by activators with different classes of activation function. Thus, these results together with the fact that immunopurified TFIID was necessary and sufficient to alleviate the negative effect of NEF-2 strongly suggest that NEF-2 interferes with the function of a TBP-associated factor required for the activity of GAL-TEF-1, but not GAL-VP16.

NEF-2 affects basal and activated transcription in a manner distinct from that of previously identified transcriptional repressors. NC1, NC2, Dr1, and Dr2 (topoisomerase I) are proteins which interact with TBP and alter its ability to interact with TFIIA and TFIIB, thus repressing preinitiation complex formation(10, 46, 47, 48) . Histone H1 and NERF-1 (topoisomerase II) inhibit transcription, at least in vitro, by binding to DNA virtually nonspecifically and precluding the binding of TBP/TFIID to the promoter(40, 49) . Each of these factors repress basal transcription, but repression can be partially overcome by transcriptional activators. Thus, due to the preferential repression of basal transcription these factors act to potentiate the effect of transcriptional activators in vitro. The effect of NEF-2 is clearly distinct from the above factors as a selective repression of GAL-TEF-1 activated transcription was observed.

One obvious possibility is that NEF-2 is the endogenous HeLa cell TEF-1 which would be a potent inhibitor of transactivation by GAL-TEF-1 (see Introduction). However, electrophoretic mobility shift assays using oligonucleotides comprising TEF-1 binding sites indicated that NEF-2 was separated from TEF-1 by chromatography on the HPLC heparin 5PW column. Also, endogenous HeLa cell TEF-1 undergoes extensive proteolytic degradation during chromatography on the HPLC phenyl 5PW column. (^2)Moreover, a factor with the chromatographic and functional properties of HeLa cell NEF-2 was detected in extracts from BJA-B lymphoid cells, which we have previously shown not to express TEF-1 mRNA.

Electrophoretic mobility shift assays using oligonucleotides comprising GAL4 binding sites indicated that NEF-2 did not inhibit the binding of GAL-TEF-1, nor was there any evidence of proteolytic degradation of GAL-TEF-1 in the presence of the NEF-2 fraction.^2 In agreement with these observations, the negative effect of NEF-2 could be alleviated by the addition of immunopurified TFIID, but not by the addition of increased quantities of GAL-TEF-1. In addition, NEF-2 inhibited transactivation when added along with the transcription factors and GAL-TEF-1, but did not inhibit activation when added after the formation of preinitiation complexes. As noted above, these observations strongly suggest that NEF-2 affects transactivation by interfering with the action of one of the TBP-associated factors required by GAL-TEF-1. This interference may result from NEF-2 interacting with and masking or modifying such a TBP-associated factor. Alternatively, it cannot be excluded that NEF-2 may itself be a transcriptional activator which competes for the same TBP-associated factor mediating the activity of TEF-1. If this were the case, inhibition by NEF-2 would be analogous to previously reported in vitro transcriptional interference or squelching effects. Indeed, it has been shown that self-interference in vitro by elevated levels of GAL-VP16 or GAL-EIA could be alleviated by the addition of partially purified or immunopurified TFIID(42, 51) . These results, along with those in the present study, indicate that, at least in in vitro systems, TBP-associated factors can be the limiting coactivators which are titrated by high concentrations of their cognate transcriptional activators or may be targets for negative regulatory factors such as NEF-2. In addition, it is important to note that HeLa cell extracts contain numerous transcriptional activators; however, only one activity with the properties of NEF-2 was detected. Thus, the ability to efficiently repress transactivation by GAL-TEF-1 is a specific effect and not a general property of many different transactivators.

We have recently shown that although GAL-TEF-1 stimulates transcription 8-10-fold in HeLa cell extracts only a 2-3-fold stimulation was obtained in BJA-B cell extracts. In contrast, similar high levels of transactivation by GAL-VP16 were observed in extracts from each cell type(37) . Transactivation by GAL-TEF-1 could be obtained in BJA-B cell extracts upon addition of immunopurified HeLa cell TFIID implying that BJA-B cells lack, or contain lower quantities of, a TBP-associated factor(s) required for the activity of GAL-TEF-1. However, immunopurification experiments indicated that BJA-B cell TFIID supported efficient transactivation by GAL-TEF-1. In contrast, reconstitution of a BJA-B cell transcription system indicated the presence of an activity, NEF-1, in the PC0.3 fraction which inhibited transactivation by GAL-TEF-1(37) . NEF-1 is chromatographically separable from the NEF-2 activity detected in this study. NEF-1, copurified on phenyl 5PW columns with the TFIID in the PC0.3 fraction, but could be separated from TFIID when the TFIID was immunoprecipitated. Unlike NEF-2, NEF-1 appears to be cell-specific, as no analogous activity was detected in the equivalent HeLa fractions. Thus, the results presented here together with those of the above study (37) suggest that the low levels of GALTEF-1 activity in BJA-B cell extracts results from the combined negative effects of both NEF-1 and NEF-2 rather than from the absence of positively acting factors.

Although HeLa cells do not contain sufficient quantities of NEF-2 to totally inhibit the activity of TEF-1, it is, however, possible that the activity of TEF-1 in these cells may be partially repressed by the presence of NEF-2. Nevertheless, if there were developmental or cell cycle-induced changes in the levels of NEF-2, this would negatively modulate the activity of TEF-1. Thus, our results strongly suggest that competition for, or interference with the function of, TBP-associated coactivators may be a novel mechanism for regulating the activity of transcriptional activators in vivo. Consequently, in HeLa cells the activity of TEF-1 is regulated not only by limiting quantities of a positively acting factor(34, 35) , but also by the presence of the negatively acting NEF-2 factor. Several previous studies have underlined the potential physiological importance of such transcriptional interference effects in vivo (reviewed in (52) and references therein). The cloning and expression of NEF-2 will be required to further study its mechanism of action and to understand its potential role in the regulation of the activity of TEF-1 and other activators.

FOOTNOTES

*
This work was supported in part by grants from the CNRS, the INSERM, the Centre Hospitalier Universitaire Régionale, the Ministère de la Recherche et de la Technologie, the Fondation pour la Recherche Médicale, and the Association pour la Recherche Contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by fellowships from the Association pour la Recherche contre le Cancer and the CNRS. Present address: Biotechnology Research, Ciba Geigy Corp., P. O. Box 12257, Research Triangle Park, NC 27709-2257.

To whom correspondence should be addressed. Tel.: 33-88-65-34-40 (or 45); Fax: 33-88-65-32-01.

(^1)
The abbreviations used are: TBP, TATA-binding protein; TAF, TBP-associated factor; TEF-1, transcriptional enhancer factor-1; PC, phosphocellulose; ER, estrogen receptor; WCE, whole cell extract; HPLC, high performance liquid chromatography; IP, immunoprecipitation(s); AdMLP, adenovirus major late promoter.

(^2)
S. Chaudhary, L. Tora, and I. Davidson, unpublished data.

ACKNOWLEDGEMENTS

We thank P. Chambon for support and critical reading of the manuscript, V. Moncollin for providing the partially purified TFIIA, C. Brou for stimulating discussions, the cell culture group for the BJA-B and HeLa cells, the monoclonal antibody facility for the 3G3 antibody. We are also grateful to C. Werlé, B. Boulay, and J. M. Lafontaine for illustrations and photography.

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