©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Physical Interactions of the Putative Transcriptional Adaptor, ADA2, with Acidic Activation Domains and TATA-binding Protein (*)

(Received for publication, May 31, 1995)

Nickolai A. Barlev Reyes Candau Lian Wang Paula Darpino Neal Silverman (1) Shelley L. Berger (§)

From the Wistar Institute, Philadelphia, Pennsylvania 19104 and the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

RNA polymerase II transcription requires functional interactions between activator proteins bound to upstream DNA sites and general factors bound to the core promoter. Accessory transcription factors, such as adaptors and coactivators, have important, but still unclear, roles in the activation process. We tested physical interactions of the putative adaptor ADA2 with activation domains derived from acidic activator proteins and with certain general transcription factors. ADA2 associated with the herpesvirus VP16 and yeast GCN4 activation domains but not with the activation domain of yeast HAP4, which previously was shown to be independent of ADA2 function in vivo and in vitro. Furthermore, the amino terminus of ADA2 directly interacted with the VP16 activation domain, suggesting that ADA2 provides determinants for interaction between activation domains and the adaptor complex. Both TATA-binding protein (TBP) and TFIIB have previously been shown to interact directly with the VP16 activation domain in vitro (Stringer, K. F., Ingles, C. J., and Greenblatt, J.(1990) Nature 345, 783-786; Lin, Y. S., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R.(1991) Nature 353, 569-571). Interestingly, when binding was tested between VP16 and these general factors in yeast nuclear extracts, both factors interacted with VP16, but only the TBP/VP16 association was dependent on ADA2. In addition, ADA2 physically associated with TBP, but not with TFIIB. These results suggest that the role of ADA2 in transcriptional activation is to promote physical interaction between activation domains and TBP.


INTRODUCTION

Transcriptional activators are sequence-specific DNA-binding proteins, or protein complexes, which contain specific domains dedicated to mediating transcriptional activation. Activation domains are classified according to amino acid composition. Among them, acidic activation domains are highly conserved in function, since they activate transcription from yeast to mammals (Ptashne, 1988). A well-studied example is the herpes simplex virus VP16 activation domain (Gerster and Roeder, 1988; O'Hare et al., 1988; Preston et al., 1988; Triezenberg et al., 1988) whose function requires certain hydrophobic residues, in addition to overall negative charge (Cress and Triezenberg, 1991; Regier et al., 1993).

The mechanism by which the basal transcriptional machinery increases the rate of initiation upon activation involves protein-protein contacts between activation domains and specific basal factors (Ptashne, 1988; Ptashne and Gann, 1990; Roeder, 1991; Gill and Tjian, 1992). An important yet unresolved question is whether there is direct interaction between the activation domain and components of the basal transcriptional machinery (for review of basal factors see (Zawel and Reinberg, 1993)), or whether additional factors are essential for this signaling pathway. Biochemical approaches have shown that activation domains indeed are capable of direct contact with general factors. For example, the activation domain of VP16 has been shown to contact directly three general transcription factors, TBP (^1)(TATA-binding protein (Stringer et al., 1990)), TFIIB (Roberts et al., 1993) and TFIIH (Xiao et al., 1994). However, these interactions may not be sufficient for transcriptional activation, and several lines of evidence suggest that additional factors are required. For example, TBP is associated with factors (TAFs) in a complex required for transcriptional stimulation in vitro by several classes of activation domains (Rose and Botstein, 1983; Pugh and Tjian, 1990; Dynlacht et al., 1991; Reese et al., 1994). TAFs include several coactivators which make specific contacts with activation domains to assemble active transcription machinery (Chen et al., 1994).

Certain other proteins and multiprotein complexes distinct from general factors or TAFs have been shown to be involved in transcriptional activation, such as the PC4 (Ge and Roeder, 1994)/P11 (Kretzschmar et al., 1994) cofactor derived from the earlier described USA (Meisterernst et al., 1991). In yeast, products of the SWI/SNF genes are required for the enhancement of transcription by many transcriptional activators (for review see Winston and Carlson, 1992; Carlson and Laurent, 1994)), perhaps through functional interactions with chromatin (Cote et al., 1994; Imbalzano et al., 1994). Also in yeast are SRB proteins, which interact with the carboxyl-terminal tail of the largest subunit of RNA polymerase II and are required for activated transcription from many promoters (Thompson et al., 1993). These factors, along with several general factors, may constitute a holoenzyme RNA polymerase complex (Kim et al., 1994; Koleske and Young, 1994) which includes a transcriptional stimulatory activity (Kim et al., 1994).

Yeast cofactors required for activation in vitro appeared to be distinct from general transcription factors, and thus were termed mediators (Kelleher et al., 1990) or adaptors (Berger et al., 1990). A genetic selection in yeast (Berger et al., 1992) led to the identification of the putative adaptors ADA2 (Berger et al., 1992) and ADA3 (Piña et al., 1993) which had properties consistent with an adaptor role in transcriptional activation. Recently, employing the same genetic selection, two new genes were identified (Marcus et al., 1994), and one was identical to GCN5, which previously had been found to be required for activated transcription by the yeast activator GCN4 (Georgakopoulos and Thireos, 1992).

Several lines of evidence suggest that ADA2 comprises part of a complex containing additional components. First, mutant strains in either ada3 (Piña et al., 1993) or gcn5 (Marcus et al., 1994) have properties similar to ada2 mutants. Also, yeast two-hybrid analysis and coimmunoprecipitation assays showed interactions between ADA2 and GCN5 (Marcus et al., 1994), ADA3 (Horiuchi et al., 1995), and between ADA2 and the VP16 activation domain (Silverman et al., 1994). One model for the role of such a complex is that it constitutes a physical link to allow productive interaction between the activation domain and the basal transcription apparatus. ADA2 may play a central role in this complex, since it selectively interacts in functional assays with certain acidic activation domains, such as those of VP16 and yeast GCN4, but not with others, such as yeast HAP4 (Berger et al., 1992; Piña et al., 1993). In this report we show specificity in physical associations of ADA2 with activation domains and with TBP. Furthermore, we detect direct interaction between ADA2 and the VP16 activation domain, suggesting that ADA2 may provide determinants within the adaptor complex for interaction with activation domains. These data support a model that the adaptor complex is required for physical interaction of activator proteins with the basal transcriptional machinery.


MATERIALS AND METHODS

Yeast Strains

beta-Galactosidase assays (Rose et al., 1988) were carried out in the wild type yeast strain PSY316 (MATalphaade2-101 Deltahis3-200 leu2-3, 112 lys2 ura3-53). GST fusion protein binding studies were done with nuclear extracts prepared from diploid wild type or ada2 strains, from crosses between PSY316 and BWG1-7A (MATaura3-52 leu2-3, 2-112 his4-519 ade1-100) or ADA2 disruption derivatives.

GST-Activation Domain Fusion Proteins

Fusion genes were made by cloning of polymerase chain reaction-generated fragments containing each activation domain bearing a BamHI restriction site at one end and BglII at the other end, into pGEX-3X (Pharmacia Biotechnol Inc.) digested with BamHI, followed by dideoxynucleotide sequencing (Sanger et al., 1977) to confirm each structure. Following transformation into Escherichia coli XL1-blue, protein expression was induced with 1 mM isopropyl-1-thio-beta-D-galactopyranoside for 3 h, the cells were harvested, resuspended in phosphate-buffered saline, and sonicated on ice. Coupling of GST fusion proteins to glutathione-Sepharose 4B beads (Pharmacia) was performed as described (Smith and Johnson, 1988). GST-ADA2 was prepared as described (Frangioni and Neel, 1993).

Binding of Yeast Protein to GST Fusions

80 µl (approximately 2 mg of protein) of yeast extract were incubated with 20-50 µl of beads in binding buffer (50 mM NaCl, 20 mM HEPES, pH 7.5, 10 mM MgCl, 5 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 µM pepstatin A, 0.6 µM leupeptin, 2 µg/ml chymostatin, 2 mM benzamidine), for 30 min at 4 °C with rotation. The amount of beads bearing specific fusion proteins was normalized to equal the amount of beads bearing GST. The total amount of beads was equalized using washed glutathione-Sepharose beads. After binding the beads were washed with 2.5 ml of binding buffer (approximately 50 volume of beads). Proteins were eluted as described in the legend for each experiment. In general proteins were eluted with 100 µl of either 0.5 or 1 M NaCl in binding buffer. Eluted proteins and material remaining on beads were analyzed by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose (Schleicher & Schuell).

In the case of purified GAL4-VP16 fusion proteins, binding was performed in a binding buffer with 100 mM NaCl supplemented with purified bovine serum albumin (50 ng/µl) as a competitor. Typically, 600 ng of purified protein was used in each binding reaction. The purification of GAL4-VP16 fusion proteins was described (Berger et al., 1990).

Protein blots were blocked in 5% skim milk, 1 phosphate-buffered saline, and 0.1% Tween20, for 90 min and were then washed in 1 phosphate-buffered saline, 0.1% Tween20 for 30 min, with several changes of buffer. The following antibodies were used in immunodetection: alpha-ADA2 rabbit polyclonal antisera (Silverman et al., 1994), alpha-TBP rabbit polyclonal antisera (Reddy and Hahn, 1991), alpha-TFIIB rabbit polyclonal anitsera (Pinto et al., 1994), and alpha-GAL4 monoclonal antisera (Santa Cruz) was used to detect GAL4-VP16 fusion proteins. All secondary antibodies used were conjugated with horseradish peroxidase (Sigma). Immunodetection was performed using enhanced chemiluminescence (ECL kit; Amersham Corp.).

Construction of GAL4 DNA-binding Domain Fusions and beta-Galactosidase Assays

VP16 activation domain fragments were prepared by polymerase chain reaction as described for the GST fusions above and cloned into the BglII site of pPC97 (Chevray and Nathans, 1992). The structure of each derivative was confirmed by dideoxynucleotide sequencing. Yeast strains were transformed by the lithium acetate method (Ito et al., 1983). Transcriptional activity was determined by assay of beta-galactosidase produced from a reporter plasmid containing the GAL1-10 promoter region driving expression of the bacterial LacZ gene (Guarente et al., 1982). Unit activity was normalized to total protein in whole cell extracts of each sample (Rose and Botstein, 1983). The amount of each fusion protein in the wild type strain was similar, as judged by gel retardation assay using a GAL4-binding site as probe (not shown).

Transcription Inhibition Assay

The templates, nuclear extracts, and other methods were described (Berger et al., 1990). Equivalent amounts of each activator (by Bradford assay (Bradford, 1976)) were rechecked by gel shift and Western blot analysis to be certain that functionally equal amounts were used in the activation and inhibition experiments.


RESULTS

ADA2 Interacts with VP16 and GCN4 Activation Domains, but Not with That of HAP4

We have previously shown that certain acidic activation domains, such as herpesvirus VP16 and yeast GCN4, require ADA2 for activation both in vivo and in vitro, while other activation domains, such as yeast HAP4, are relatively independent of ADA2 (Berger et al., 1992; Piña et al., 1993). We wished to determine whether the functional dependence correlates with physical association between ADA2 and activation domains. Proteins containing GST (Smith and Johnson, 1988) fused to activation domains derived from VP16, GCN4, and HAP4 were coupled to glutathione-Sepharose beads. The region of each activation domain included in the GST fusions has previously been shown to be sufficient for activation both in vivo and in vitro (Berger et al., 1992; Piña et al., 1993).

Yeast nuclear extract (Lue and Kornberg, 1987) was incubated in separate reactions with equal amounts of beads bearing one of the three activation domains fused to GST, or with beads containing GST alone (Fig. 1A). Proteins remaining bound to beads after extensive washes were eluted with 0.5 M salt buffer, and then both the eluted material and material remaining on beads were analyzed by immunoblotting for the presence of ADA2 (Fig. 1B). The profiles of ADA2 interaction were similar for both 0.5 M-eluted material (Fig. 1B, left panel) and material remaining bound to beads after the salt wash (Fig. 1B, right panel). ADA2 did not bind to the control beads containing GST alone but bound to and eluted from beads containing either GST-VP16 or GST-GCN4. However, ADA2 was not detected in eluate from beads bearing GST-HAP4. Thus, ADA2 physically interacted with acidic activation domains previously found to be functionally dependent on ADA2 activity, i.e. VP16 and GCN4, and did not bind to an acidic activation domain found to be independent of ADA2 activity, i.e. HAP4.


Figure 1: Binding of ADA2 to acidic activation domains from herpes virus VP16, yeast GCN4, and yeast HAP4. A, Coomassie Blue staining of GST-activation domain fusion proteins. A sample of each bead preparation is shown that was equal to the amount used for the binding assay described in panel B. Lane 1, GST alone; lane 2, GST-VP16, VP16 amino acid residues 413-490 (Triezenberg et al., 1988); lane 3, GST-GCN4, GCN4 amino acid residues 9-172 (Hope and Struhl, 1986); lane 4, GST-HAP4, HAP4 amino acid residues 330-554 (Forsburg and Guarente, 1989). Protein size standards are shown on the left. B, immunoblot analysis of yeast protein associating with GST activation domain fusions using alpha-ADA2 antisera. Yeast extract was incubated with GST fusions to activation domains (panel A). Proteins that bound to, and eluted from, each column were immunoprobed with alpha-ADA2 antisera. The left panel shows protein that eluted from the column in wash buffer containing 0.5 M NaCl. The right panel shows protein remaining on the column after the high salt wash. Lane 1, GST alone; lane 2, GST-VP16; lane 3, GST-GCN4; lane 4, GST-HAP4; lane 5, (left panel) bacterially expressed ADA2. Protein size standards are depicted on the left.



ADA2 Associates with Two Distinct Regions of the VP16 Activation Domain

As the next step we wished to determine which subregion of VP16 interacted with ADA2. Previous results from deletion mapping and mutagenesis of the VP16 activation domain identified two separate subdomains sufficient for activation in animal cells: one between residues 440 and 450 (Cress and Triezenberg, 1991) and the second between residues 470 and 480 (Regier et al., 1993; Walker et al., 1993; Kunzler et al., 1994). In addition, we have detected a third subregion, between 450 and 470, which has transcriptional activity in yeast, (^2)Based on these results, GST was fused to subregions of VP16 comprising amino acid residues 413-450, 413-470, or 470-490 (Fig. 2A). Affinity matrixes were prepared bearing each of these fusions and equal amounts of each preparation (Fig. 2B) were incubated with yeast nuclear extract, washed, and bound proteins were eluted and immunoprobed for the presence of ADA2.


Figure 2: Binding of yeast ADA2 to GST-VP16 and to deletion derivatives of VP16. A, schematic of GST fusions to the VP16 activation domain and its deletion derivatives. The full-length activation domain of VP16 comprises amino acids 413-490 (Triezenberg et al., 1988), and deleted versions are shown relative to the full-length activation domain. B, Coomassie Blue staining of GST-VP16 fusion proteins. A sample from each bead preparation is shown that was equal to the amount used for the binding assay described in panel C. Lane 1, GST alone; lane 2, GST-VP16, amino acid residues 413-490; lane 3, GST-VP16, amino acid residues 470-490; lane 4, GST-VP16, amino acid residues 413-470; lane 5, GST-VP16, amino acid residues 413-450. An arrow indicates the expected size of the 413-450 deletion derivative. Protein size standards are shown on the left. C, immunoblot analysis of yeast proteins associating with GST-VP16 fusions using alpha-ADA2 antisera. Yeast nuclear extract was incubated with the GST-VP16 fusions shown in panel B. Proteins which bound to, and eluted from, each column in wash buffer containing 1 M NaCl were immunoprobed with alpha-ADA2 antisera. A similar profile of interaction between ADA2 and subregions of VP16 was seen when the remaining protein bound to beads was boiled and immunoprobed for ADA2 (data not shown). Lane 1, GST alone; lane 2, GST-VP16; lane 3, GST-VP16; lane 4, GST-VP16; lane 5, GST-VP16. Protein size standards are depicted on the left.



Consistent with the previous result (Fig. 1B), ADA2 was detected in eluate from beads containing the full-length VP16 activation domain (Fig. 2C). ADA2 was also found in eluate from beads bearing the VP16 distal region, between residues 470 and 490. The region of VP16 between 413 and 470 also bound ADA2, albeit more weakly than the 470-490 distal region. ADA2 was nearly undetectable in the eluate from beads bearing the proximal VP16 region, between 413 and 450. Thus two regions of VP16 may be important for interaction with ADA2, the region between 470 and 490 and the region between 450 and 470.

Since two distinct regions of the VP16 activation domain associate with ADA2 in the binding assay, we determined the level of transcriptional activity of these identical regions of VP16 in yeast. Gene fusions were generated between subregions of VP16 and the GAL4 DNA-binding domain (amino acids 1-147). The transcriptional activity of the GAL4-VP16 fusions was tested using a bacterial lacZ reporter gene, whose expression was driven by a GAL4-inducible promoter (Guarente et al., 1982). As expected, GAL4-VP16 containing the full-length activation domain was a potent activator (Table 1). The GAL4-VP16 fusion was a weak activator. GAL4-VP16 was intermediate, and the GAL4-VP16 fusion constituted a strong activator. Thus the strength of each activation region in the in vivo assay as GAL4 DNA-binding domain fusions paralleled their ability to bind to ADA2 as GST fusions (Table 1), suggesting that strength of activation in vivo is related to the ability of activator to associate with ADA2 in vitro.



The Carboxyl Terminus, but Not the Amino terminus, of VP16 Can Inhibit Activated Transcription from a Heterologous Promoter

We previously have shown that GAL4-VP16 was capable of strongly inhibiting transcription at a heterologous test promoter in yeast nuclear extracts (Berger et al., 1990). The strong transcriptional inhibition, resulting in transcription well below the basal level (e.g. compare lanes 5 and 6 in Fig. 3B), apparently was caused by sequestration of both adaptors and general factors from the test promoter. This conclusion was based on the following observation: adding an oligonucleotide containing a GAL4 DNA-binding site into the inhibition assay caused GAL4-VP16 to inhibit only activated transcription at the test promoter, without affecting basal transcription. It was surmised that under the latter conditions, i.e. inhibition of activated transcription, an adaptor shared by the test promoter was sequestered by GAL4-VP16. Since, in our current study, GST fusions to carboxyl-terminal regions of VP16 were capable of pulling ADA2 out of a yeast extract, we wished to test whether GAL4 fusions to the carboxyl terminus of VP16 were capable of specific inhibition of activated transcription, in a manner similar to the full-length VP16 activation domain in the previous study.


Figure 3: Transcriptional activation and inhibition by GAL4-VP16 or deletion derivatives. A, DNA templates used in the in vitro transcription reactions. The templates are identical except for the activator-binding sites upstream of the core promoter, as described (Berger et al., 1990). B, primer extension assay of RNA synthesized in vitro in yeast nuclear extract. The DNA templates used in each reaction were: lanes 1-4, gal.3; lanes 5-14, dA/dT. The GAL4-VP16 activation domain fusions used were: lanes 4 and 5, none; lanes 1 and 12-14, C = GAL4-VP16; lanes 2 and 9-11, N = GAL4-VP16; lanes 3 and 6-8, FL = GAL4-VP16. An oligonucleotide containing a GAL4 DNA-binding site was included: lanes 7, 10, and 13, 300 ng; lanes 8, 11, and 14, 600 ng. The amount of GAL4-VP16 protein used in the activation control (lanes 1-3) was 5 pmol, and the amount used in the inhibition experiment (lanes 6-14) was 10 pmol. The level of transcription observed at the gal.3 template without addition of exogenous activator (lane 4) was identical to the level of transcription observed at a basal template, without upstream binding sites (data not shown), as shown previously (Berger et al., 1990).



Purified samples of three GAL4 DNA-binding domain fusions to VP16 were compared in their ability to inhibit activated transcription at the test promoter in vitro. The fusions contained either the full-length activation domain (413-490; FL), the amino-terminal region (413-456; N), or the carboxyl-terminal region (452-490; C). As an initial control to show that all three domains were active in vitro, low noninhibiting concentrations of the GAL4-VP16 fusions were compared in their ability to activate transcription at a promoter containing three GAL4-binding sites (template gal.3 in Fig. 3A). Equal amounts of protein activated transcription to a similar extent (Fig. 3B: compare activated transcription in lanes 1-3 to basal transcription in lane 4).

The second assay tested the ability of each GAL4-VP16 fusion to inhibit activated transcription at the test promoter (template dA/dT in Fig. 3A). Activation at the dA/dT template (Fig. 3B, lane 5) is caused by an endogenous activator in the extract (Lue et al., 1989) and results in approximately a 5-fold increase in transcription over the basal level (lane 4 and see Berger et al.(1990) for details). In this assay, as was seen previously (Berger et al., 1990), GAL4-VP16 strongly inhibited activated transcription at the dA/dT template (compare lane 6 to lane 5). Also as seen previously, GAL4-VP16 specifically inhibited activated transcription when the oligomer containing the GAL4 DNA-binding site was included in the reaction (i.e. transcription in lanes 7 and 8 equal basal transcription in lane 4). Interestingly, the profile of inhibition by GAL4-VP16(C) was comparable to that of FL (lanes 12-14 are similar to lanes 6-8), while GAL4-VP16(N) was markedly different (lanes 9-11). In fact, GAL4-VP16(N) was incapable of inhibiting activated transcription at all (lanes 10 and 11 are the same as lane 5). Thus, in this inhibition assay, the carboxyl terminus of VP16 sequestered the adaptor activity. This correlates with its ability to associate with ADA2 in the GST pull-down assay (Fig. 2). In contrast, the amino terminus of VP16 failed to sequester the adaptor activity in the inhibition assay and did not associate with ADA2 in the GST assay. Thus, these data point to the carboxyl terminus of the VP16 activation domain as crucial for physical associations with cofactors required for activation in yeast.

ADA2 and the VP16 Activation Domain Directly Interact

Since GST-VP16 was able to associate with endogenous ADA2 in yeast extract, we tested whether ADA2 could interact directly with the VP16 activation domain. GST-ADA2 was constructed and an affinity matrix was prepared and incubated with GAL4-VP16 fusion proteins. We compared the ability of GAL4-VP16, GAL4-VP16, or GAL4-VP16 to bind to GST-ADA2. GST was used as a negative control, and both GST-TBP and GST-TFIIB were used as positive controls, since they previously have been shown to bind directly to VP16 (Stringer et al., 1990; Roberts et al., 1993). Similar amounts of each GST fusion (Fig. 4A) were used to test binding of the purified GAL4-VP16 fusions. Following extensive washes, material remaining bound to beads was eluted and immunoblotted using alpha-GAL4 antisera (Fig. 4B).


Figure 4: Direct binding between the VP16 activation domain and ADA2. A, Coomassie Blue staining of GST fusion proteins. A sample from each bead preparation is shown that was equal to the amount used for the binding assay described in panel B. Lane 1, GST alone. Lane 2, GST-TBP (Eisenmann et al., 1989; Hahn et al., 1989). Lane 3, GST-TFIIB (Pinto et al., 1992). Lane 4, GST-ADA2 (Berger et al., 1992). Protein size standards are shown on the left. Apparent difference in mobility of GST protein here and in Fig. 2B relative to size standards (See Blue, Pharmacia Biotech Inc.) was due to the use of different concentrations of electrophoresis separating gel. B, immunoblot analysis of purified GAL4-VP16 proteins binding to GST fusions to ADA2, TBP, and TFIIB. Purified GAL4-VP16 protein was incubated with the GST fusions shown in panel A. Protein which eluted from each column was immunoprobed with alpha-GAL4 monoclonal antisera. Lane input represents 80% of total material used in GST fusion binding assay. GST fusions were the same as shown in A. GAL4-VP16 fusion proteins contain the region of the VP16 activation domain stated for each. C, schematic map of GST fusions to ADA2 and its deletion derivatives. The full-length ADA2 comprises amino acids 1-434. Deleted versions of ADA2, 1-176 (N), 176-333 (M), and 333-434 (C), are shown relative to full-length. D, binding of purified GAL4-VP16 to GST-ADA2. Immunoblot analysis of purified GAL4-VP16 binding to GST fusions to ADA2 (full-length and deletion derivatives as in panel C). Material remaining on beads was immunoprobed with alpha-GAL4 antisera. Lane input represents 80% of total material used in the binding. Protein size standards are depicted on the left.



None of the GAL4-VP16 fusion proteins interacted with GST alone (Fig. 4B). All three of the GAL4-VP16 fusion proteins interacted with both GST-TBP and GST-TFIIB (Fig. 4B). These data are consistent with previous observations that TBP (Stringer et al., 1990) (^3)and TFIIB (Goodrich et al., 1993; Walker et al., 1993) bind to the VP16 activation domain and to both amino- and carboxyl-terminal subregions.

GAL4-VP16 bound to the beads bearing GST-ADA2 (Fig. 4B, top panel). GAL4-VP16 also interacted with GST-ADA2 (Fig. 4B, lower panel). In contrast, GAL4VP16 interacted poorly with GST-ADA2 (Fig. 4B, center panel). These data indicate that the VP16 activation domain can interact directly with ADA2. The results also show that the carboxyl terminus of VP16 interacted with ADA2, in agreement with the GST-VP16 binding data (Fig. 2C) and GAL4-VP16 inhibition assay (Fig. 3B).

Thus, ADA2 interacts directly with the VP16 activation domain. To determine the region of ADA2 which interacts with VP16, GST fusions were prepared containing subregions of ADA2 (Fig. 3C). Bead preparations were incubated with purified recombinant GAL4-VP16 protein. GAL4-VP16 specifically interacted with the amino terminus of ADA2, but not with either the middle region or the carboxyl terminus of ADA2 (Fig. 3D). This result further demonstrates specificity of the interaction between ADA2 and the activation domain of VP16.

ADA2 Associates with TBP and Is Required for VP16/TBP Interaction in Yeast Extract

Previous data, as well as those shown here, demonstrate that the VP16 activation domain interacts directly with TBP and TFIIB (Stringer et al., 1990; Lin et al., 1991). We tested whether endogenous TBP or TFIIB in yeast extract associated with the VP16 activation domain and, if so, whether the interaction required ADA2. Thus, we compared the ability of TBP and TFIIB to bind GST-VP16 in the presence or absence of ADA2. Nuclear extracts from either a wild type strain or an isogenic derivative strain lacking ADA2 were prepared. As a control, the relative amounts of TBP or TFIIB were determined in the two extracts and were found to be similar, or somewhat higher in the ada2 extract (Fig. 5A). These extracts were then incubated with GST, GSTVP16, or GST-VP16, and eluates from each bead matrix were immunoblotted to detect TBP or TFIIB. Both TBP and TFIIB were capable of binding to the full-length activation domain or to the carboxyl-terminal region (Fig. 5B). Strikingly, much less TBP was found in the eluate from GST-VP16 beads after incubation with ada2 extract compared to the wild type extract (Fig. 5B). TBP also was undetectable in the eluate from the GST-VP16 beads. Thus, in these conditions, TBP association with VP16 was dependent upon ADA2. In contrast, TFIIB binding to full-length VP16 or the 470-490 subregion was only slightly reduced in incubations with the ada2 extract compared to the wild type extract (Fig. 5B). Thus, the consequence of ADA2 loss was different for TBP compared to TFIIB, suggesting that ADA2 is required for TBP but not for TFIIB binding to VP16.


Figure 5: Binding to GST-VP16 of yeast TBP and TFIIB derived from wild type and ada2 extract. A, immunoblot analysis of TBP and TFIIB in wild type and ada2 extract. TBP or TFIIB was probed in wild type or ada2 yeast nuclear extract using alpha-TBP antisera (upper) or alpha-TFIIB (lower) antisera. B, immunoblot analysis of TBP or TFIIB associating with the VP16 activation domain in wild type or ada2 extract. Yeast nuclear extract was incubated with the GST fusions listed (proteins are shown in Fig. 2B), and material was eluted in binding buffer containing 1 M NaCl. The GST fusions used were: lanes 1 and 4, GST alone; lanes 2 and 5, GST-VP16, VP16 residues 413-490; lanes 3 and 6, GST-VP16, VP16 residues 470-490. The extracts used were: lanes 1-3, extract from wild type cells; lanes 4-6, extract from ada2 cells. alpha-TBP (upper) or alpha-TFIIB (lower) polyclonal antisera were used for detection.



This dependence of VP16/TBP interaction on ADA2 could indicate that ADA2 itself is required for activation domain/basal factor association, or the dependence could be an indirect consequence of the absence of ADA2 in the mutant extract. In order to distinguish between these alternatives, we tested the ability of TBP or TFIIB to interact with ADA2 in yeast extract. Beads containing comparable amounts of GST alone, GST-TBP, or GST-TFIIB (Fig. 4A) were incubated with yeast extract, and eluates were immunoblotted to detect ADA2. ADA2 did not bind to GST alone (Fig. 6A, right panel). ADA2 interacted with GST-TBP, but not GST-TFIIB (Fig. 6A, left panel). Another experiment, which included GST-VP16 and GST-HAP4 activation domain fusions as positive and negative controls, respectively, also showed association of ADA2 with GST-TBP, but not with GST-TFIIB (Fig. 6A, right panel). Thus, since ADA2 associated with TBP (Fig. 6A), and since ADA2 was required for VP16 interaction with TBP (Fig. 5B), we conclude that ADA2 itself may be physically required for VP16 interaction with TBP in a yeast nuclear extract.


Figure 6: Interaction between ADA2 and TBP or TFIIB. A, immunoblot analysis of ADA2 binding to GST-TBP or GST-TFIIB. Yeast nuclear extract was incubated with the GST fusion proteins listed (proteins are shown in Figs. 1A and 4A), and protein was eluted in binding buffer containing 1 M NaCl. Either GST alone, GST-VP16 = VP16 residues 413-490, GST-HAP4 = HAP4 residues 330-554, GST-TBP, or GST-TFIIB was used in each incubation. The GST fusion proteins were normalized relative to one another before use. The Western blot was immunoprobed with alpha-ADA2 antisera. B, immunoblot analysis of TBP associating with GST-ADA2. Bacterial extract containing TBP was incubated with the GST fusions listed (proteins are shown in Figs. 1A and 4A), and protein eluting in binding buffer containing 1 M NaCl, or material remaining on beads, was immunoprobed with alpha-TBP antisera.



Since endogenous ADA2 in yeast extract was capable of interacting with GST-TBP we wished to determine whether ADA2 and TBP could interact directly in the absence of other yeast proteins. Bacterially expressed TBP was incubated with GST alone, GST-VP16 as a positive control, or GST-ADA2, and eluates were immunoprobed for TBP. The results show that TBP was retained by GST-VP16 affinity matrix, but not by GST or GST-ADA2 (Fig. 6B). Thus, there was no interaction between TBP and ADA2 in these conditions, suggesting a requirement for additional yeast factors. Consistent with these data, reticulocyte-translated ADA2 and TBP did not coimmunoprecipitate using either alpha-ADA2 or alpha-TBP antisera (data not shown).^2


DISCUSSION

Previous work demonstrated that the putative adaptors ADA2, ADA3, and GCN5 are required for transcriptional activation. First, mutations in these genes lowered the activity of certain acidic activation domains, both in vivo and in vitro (Berger et al., 1992; Piña et al., 1993; Marcus et al., 1994), and, second, mutations in ADA2 reduced activated transcription without altering basal transcription in vitro (Berger et al., 1992). These functional effects could be a consequence of activation domain interactions with adaptor proteins, or adaptors could have an indirect effect on transcription. To distinguish between a direct or indirect role for ADA2, we have examined interactions between ADA2, activation domains, and basal transcription factors.

The data presented here support the model that ADA2 provides a physical function in transcriptional activation and may provide a physical link between certain activation domains and basal transcription factors. ADA2 interacted with activation domains derived from the transcriptional activators VP16 and yeast GCN4. These activators have been shown to require ADA2 for their function both in vivo and in vitro. A different approach previously showed that the VP16 activation domain associated with endogenous ADA2 in yeast extract (Silverman et al., 1994). In the present study we extended these observations with the finding that ADA2 binds poorly to yeast HAP4, an activation domain that previously was shown to be functionally independent of ADA2 (Berger et al., 1992; Piña et al., 1993). Thus, the specificity shown in this physical interaction assay supports the specificity previously observed in the functional assays. The consistency between these assays lends support to the model that ADA2 has a physical role in the transcription process. Furthermore, the lack of physical interaction between ADA2 and the HAP4 activation domain result raises the possibility that different cofactors or adaptors may be involved in HAP4 interactions with the basal machinery. Additional biochemical and genetic data support the notion that HAP4 uses different cofactors than VP16 or GCN4 (Wang et al., 1995).

The GST pull-down assay indicated a direct interaction between ADA2 and the VP16 activation domain. Interaction between the carboxyl-terminal region of VP16 and ADA2 was shown in two ways: the 470-490 region of VP16 (as a GST fusion) interacted with endogenous ADA2 in yeast extract and, second, ADA2 (as a GST fusion) interacted with the same region of VP16 purified as a GAL4-VP16 fusion. These results indicated that ADA2 provides determinants for direct interaction with VP16. Consistent with these results, we show that the carboxyl region of VP16, but not the amino region, can sequester factors required for activation in a transcriptional inhibition assay in vitro.

Further evidence that ADA2 provides specific determinants for direct interaction with VP16 was the demonstration that the amino terminus of ADA2, but not the remainder of the protein, bound VP16. This observation is particularly important, since a putative human homologue of ADA2 (^4)exhibits strong conservation in the amino terminus. Since VP16 is a higher eukaryotic protein, it is reasonable that the region of yeast ADA2 that interacts with VP16 would be conserved in the ADA2 human homologue.

Our data suggest that the role of the adaptor complex may be to promote association of VP16 with TBP. This conclusion rests on two observations. First, VP16 associated with both TBP and TFIIB derived from nuclear extracts. However, binding of TBP to VP16 was not detected in nuclear extracts lacking ADA2. Thus, VP16 association with TBP in this assay was strictly ADA2-dependent. In contrast, TFIIB association with VP16 was only moderately affected in extracts lacking ADA2, which may support the proposal that VP16/TFIIB interaction is direct (Lin et al., 1991; Roberts et al., 1993), although we cannot rule out the possibility that accessory factors are required for VP16/TFIIB interaction in vivo. Second, we detected physical association between GST-TBP and endogenous ADA2 in nuclear extract but not between GST-TFIIB and ADA2. Thus, since ADA2 in nuclear extract bound to the VP16 activation domain, and since ADA2 and VP16 interacted in a binding assay using purified proteins, we conclude that ADA2 may be required for VP16 interaction with TBP. We did not detect interaction between ADA2 and recombinant TBP under conditions where VP16 and TBP interact, leading to the conclusion that ADA2 may not interact directly with TBP. Since both ADA2 and TBP are part of multisubunit complexes, it is possible that one or many additional proteins are required for completion of the interaction pathway between VP16/ADA2 and TBP. These proteins could include ADA3 and GCN5, which form a complex with ADA2 (Marcus et al., 1994; Horiuchi et al., 1995), or TAFs, which occur in a complex with TBP (Reese et al., 1994), or other general factors that associate with TBP, such as TFIIA, which has been shown to be important for activation in the mammalian system (Ozer et al., 1994).

TBP interacted directly with VP16 (Stringer et al., 1990) and other activation domains in vitro, including that of the tumor suppressor p53 (Seto et al., 1992; Liu et al., 1993) and adenovirus E1A (Lee et al., 1991). One explanation for the requirement for ADA2 in yeast may be that adaptor proteins are required for efficient interaction of activation domains and general factors in vivo. Certain other factors have been identified which have important functions in transcriptional activation and, like ADA2, are not basal factors. TAFs, or coactivators, were recognized as essential components of the activation process in vitro but were dispensable for basal transcription. Many of the human and Drosophila melanogaster TAFs have been cloned, sequenced, and shown to interact with activators and basal factors (Dynlacht et al., 1993; Goodrich et al., 1993; Hoey et al., 1993; Ruppert et al., 1993; Weinzierl et al., 1993). TAF40 physically interacted with the carboxyl-terminal half of VP16, between residues 452 and 490 (Goodrich et al., 1993). This region includes both domains (450-470 and 470-490) which interacted with ADA2 in the data reported here. However, our experiments show that ADA2 association with VP16 was necessary for TBP, but not TFIIB, binding to VP16, whereas TAF40 appeared to interact with TFIIB. Also, there is no obvious similarity between TAF40 and ADA2. Thus, their pathways of action may be distinct.

Recent results indicate that, as in higher eukaryotes, TAFs are also associated with Saccharomyces cerevisiae TBP (Hisatake et al., 1993; Poon and Weil, 1993). Since ADA2 appears to be essential for VP16 interaction with TBP, it is possible that ADA2 is a TAF. Cloning and sequencing of TAFs have revealed only limited homology to adaptors, such as the bromodomain shared by yeast GCN5 and TAF 250 (Ruppert et al., 1993).

Recently a family of adaptor proteins has been identified in higher eukaryotes (Arany et al., 1995; Lundblad et al., 1995). These adaptors, CBP and p300, contain striking similarity to a putative zinc-binding motif in the amino terminus of ADA2 (Arany et al., 1994). A 133 amino acid region of CBP containing the ADA2-homologous zinc-binding domain has been shown to be sufficient for CBP interaction with TFIIB (Kwok et al., 1994). ADA2 did not interact with TFIIB in our assays, suggesting that the similar zinc fingers of CBP and ADA2 may have different roles in transcription.

Our data demonstrate that ADA2 can physically interact with activation domains and TBP. In these binding assays ADA2 has an essential role in stabilizing or recruiting TBP interaction with the activation domain of VP16. Further experiments should elucidate the nature of the interaction of ADA2 with VP16 and TBP and how this interaction contributes to transcriptional activation.


FOOTNOTES

*
This work was supported by Grant MCB9317243 from the National Science Foundation and a Junior Faculty Research Award from the American Cancer Society (to S. B.), Cancer Core Grant CA10815 from the National Institutes of Health and a grant from the Pew Charitable Trust to The Wistar Institute, and a grant from Ministerio de Educacion y Ciencia (Spain) (to R. C.). 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.

§
To whom correspondence should be addressed: The Wistar Institute, 3601 Spruce St., Rm. 358, Philadelphia, PA, 19104. Tel.: 215-898-3922; Fax: 215-898-0663; berger{at}wista.wistar.upenn.edu.

(^1)
The abbreviations used are: TBP, TATA-binding protein; TAFs, TATA binding factors; GST, glutathione S-transferase.

(^2)
R. Candau and S. L. Berger, unpublished data.

(^3)
S. Triezenberg, personal communication.

(^4)
R. Candau, P. Moore, L. Wang, N. A. Barlev, C. Ying, C. Rosen, and S. L. Berger, manuscript submitted for publication.


ACKNOWLEDGEMENTS

We gratefully acknowledge the generous contribution of purified GAL4-VP16 proteins by S. Triezenberg and colleagues. We thank S. Hahn and M. Hampsey for the generous gifts of TBP and TFIIB antisera. We thank G. Moore, B. Turcotte, F. Rauscher, III, and S. Triezenberg for comments on the manuscript.


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