THE JOURNAL OF Vol. 268, No. 18. of June 25, pp. 13062 ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1903 by The American Society for Biochemistry and Molecular Biology, Inc. Vol.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1903 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 268, No. 18. Issue of June 25, pp. 13062-13067. 1993

Printed in U. S.A.

p53 Binds to the TATA-binding Protein-TATA Complex*

(Received for publication, October 22, 1992, and in revised form, February 1, 1993)

David W. Martin$& Ruben M. Munoz$B, Mark A. Sublerl, and Sumitra Debl From the Department of Microbiology, University of Texas Health Science Center, San Antonio, Texas 78284

Earlier reports show that p53, both wild type and mutants, may affect transcription. Wild-type p53 activates promoters with p53-binding sites while inhib- iting promoters without binding sites. Mutant p53, on the other hand, has been shown to activate transcription from specific promoters. These observations suggest that both wild-type and mutant p53 may interact with a general transcription factor@). In this report, we have shown that the cloned TATA-binding protein (TBP) from human and yeast interacts with human p63. TBP coimmunoprecipitates with wild-type or mutant human p53 when incubated with the pS3-specific monoclonal antibody and Protein A-agarose. Wild-type murine p53 has also been found to interact with human TBP. Protein blot assays have demonstrated that the interaction between p53 and human TBP is direct, By gel retention analysis, we have shown that the complex of TBP and p53 (both wild type and mutant) can bind to the TATA box. The similar qualitative binding ca- pability of wild-type and mutant p53 with human TBP and the similarity of the two complexes in binding to the TATA box suggest that the functional discrimination between wild-type and mutant p53 may not lie in their ability to bind TBP. The nature of the p53.TBP or p63*TBP*TATA complex may determine the suc- cess of transcription.

The nuclear phosphoprotein p53 was first identified in association with simian virus 40 (SV40) large T antigen (1, 2). Expression of wild-type p53 has been demonstrated to negatively control cellular proliferation. Several lines of evidence indicate that the wild-type protein is a tumor suppressor. Wild-type p53 inhibits proliferation of transformed cells, suppresses oncogene-mediated cell transformation, and elim- inates the tumorigenic potential of tumor-derived cell lines (3-13). On the other hand, tumor-derived mutant p53 cDNA clones were found to immortalize primary cells and cooperate with the ras oncogene in transformation of primary cells (9, 14, 15). p53 gene mutations are the most frequently reported genetic defects in human cancer (4, 16-20).

*This work was supported by grants from the Elsa U. Pardee Foundation, by Grant 91-37204-6820 from the United States Depart- ment of Agriculture, and by a research grant from the March of Dimes (to S. D.). This work was done by Sumitra Deb during the tenure of an established investigatorship of the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ All three authors contributed equally to the project. 8 Supported in part by National Institutes of Health Training

Grant AI07271-08 in microbial pathogenesis. ll To whom correspondence should be addressed Dept. of Micro-

biology, University of Texas Health Science Center a t San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284. Tel.: 512-567-3984; Fax: 512-567-6612.

p53 has been found to be associated with several viral- transforming proteins. SV40 T antigen (1, Z), adenovirus 5 E1B (21), and E6 of human Papillomavirus (22) bind to wild- type p53 presumably to inactivate p53’s function some way leading to transformation or tumorigenesis (23). Wild-type p53 inhibits SV40 DNA replication in uiuo and in uitro, presumably by binding to T antigen (24-27). E6 proteins of oncogenic human Papillomavirus can degrade p53 in vitro (28). Cellular proteins have also been found associated with p53. Among these are the heat shock protein hsc7O and two protein kinases, ~ 3 4 ‘ ~ ’ and casein kinase I1 (29-34). More recently, and of great significance, is the discovery that the product of the mdm-2 oncogene forms a tight complex with the p53 protein (35,36). mdm-2 has been found to be amplified frequently in sarcomas, and it has been proposed that the amplification of mdm-2 in sarcomas leads to escape from p53- regulated cell growth (36).

In addition to interaction with viral and cellular proteins, several other interesting biochemical properties of p53 have also been identified. Wild-type (but not mutant) p53 binds to the SV40 early promoter (37), to the human ribosomal gene cluster (38), and to other genomic fragments (39,40). p53 also binds to the murine muscle creatine kinase gene regulatory region (41), which was found to be p53-responsive (41, 42). The consensus sequence for its binding has been determined recently (39).

A growing body of experimental evidence indicates involvement of p53 in transcription. Initially, a p53-GAL4 fusion protein was shown to activate transcription from promoters containing GAL4-binding sites (43, 44). The transactivation domain was found to be contained wit.hin the N-terminal42 amino acids (45). More recently, the wild-type protein (but not the mutant) has been demonstrated to be a sequence- specific transactivator for a promoter having synthetic up- stream p53-binding sites in vivo and in uitro (40, 84, 85). Weintraub et al. (42) first demonstrated that the murine muscle creatine kinase enhancer could be activated by wild- type p53. Recently, Zambetti et al. (41) detailed binding of wild-type p53 to the murine muscle creatine kinase enhancer region and showed a relationship between p53-mediated activation and p53 binding. The in uivo transactivation of the murine muscle creatine kinase enhancer by wild-type p53 was inhibited by mdm-2 protein (351, presumably by forming a complex with p53. This suggests that mdm-2 may interfere with the normal function of the tumor suppressor.

Another group of experimental results suggests that over- expression of wild-type human p53 leads to the inhibition of gene expression in uiuo for a number of cellular and viral promoters (46-51). Interestingly, wild-type p53 inhibited the human proliferating cell nuclear antigen and the multiple drug resistance gene (MDR1) promoter activities, while a few mutants activated the promoters in uiuo (47, 51). Wild-type p53 also inhibited retinoblastoma gene promoter function (52). A mutational analysis of the retinoblastoma promoter showed a part of the basal promoter to be susceptible to p53

13062

p53 and the TATA-binding Protein 13063

regulation. This suggests that p53 exerts its negative effect on transcription through inhibition of basal promoter activity (51). Using synthetic minimal promoter-chloramphenicol ace- tyltransferase constructs and in uiuo transfection assays, we demonstrated that a minimal promoter containing a lone TATA box could be successfully inhibited by wild-type p53 and activated by a mutant (p53-281G) (52). It is possible that p53 may be acting through some component of the general transcriptional machinery.

The association of general transcription factors with promoter sequences is an ordered process led by TFIID, which associates with the TATA motif and provides a recognition site for association of the other factors, and RNA polymerase I1 (53,54). The TATA-binding protein (TBP),' a single polypeptide of 38 kDa (55-57), forms a core part of the TFIID multisubunit protein complex (58-61). Studies have indicated that TBP can interact with DNA, different sets of TBP- associated factors, and several RNA polymerase I1 transcription factors (62, 63). There are also several reports that TBP can be contacted directly by several transcription factors including VP16 (64), E l a (65, 66), and ZTa (67). Factors interacting with TBP and repressing basal transcription have also been described (68-70).

In the present communication we show that the cloned TBP from human and yeast interacts with p53. TBP coimmunoprecipitates with wild-type or mutant p53 when incubated with a p53-specific monoclonal antibody and Protein A-agarose. Protein blot assays showed that the interaction between p53 and TBP was direct. Murine p53 was also found to interact with TBP by coimmunoprecipitation. By gel retention analysis with a radiolabeled TATA box-containing probe, we demonstrate that the complex of TBP and p53 (both wild- type and the mutant) could bind to the TATA box. The interaction between p53 and the TBP.TATA complex may mediate the influence of p53 on transcription.

EXPERIMENTAL PROCEDURES

Expression and Purification of Wild-type and Mutant Human p53s and Wild-type Murine p53-Recombinant baculoviruses expressing wild-type or mutant (248W, 281G) human p53 proteins were generated using linearized baculoviral DNA (Pharmingen) and the transfer vector pVL1392 (Invitrogen) containing cloned wild-type or mutant human p53 cDNAs (71). The wild-type or mutant human p53 protein was isolated from a cellular extract 48 h after infection of SF9 cells with the virus. The protein was purified by immunoaffinity chromatography using a column of Affi-Gel 10 coupled with purified monoclonal antibody against p53 (PAb421) as described before (72). As judged by SDS-polyacrylamide gel electrophoretic analysis using Coo- massie Blue stain, the preparation is 80-90% pure (see Fig. l). Wild- type murine p53 was also purified using a similar method with a recombinant baculovirus (a generous gift from Dr. Louis Miller, University of Georgia) (73) expressing wild-type murine p53. Differ- ent columns were used for each respective protein to prevent cross- contamination. Confirmation of p53 was done by Western blot analysis using PAb421 to detect the protein.

Expression of Wild-type Humanp53, Human TBP, and Yeast TBP in Vitro-Wild-type human p53, human TBP, and yeast TBP were expressed by in vitro transcription and translation using the TNT system (Promega). All constructs were under the control of the T7 promoter. The wild-type human p53 cDNA clone was a generous gift from Dr. Arnold J. Levine (71). The human TBP and yeast TBP clones were the kind gifts of Drs. Horikoshi and Roeder, Rockefeller University. Aliquots from programmed extracts were analyzed on 10% SDS-polyacrylamide gels. Gels were treated with Enhance (Du Pont-New England Nuclear) and the 35S-labeled proteins were visualized by autoradiography.

Immunoprecipitation-Immunoprecipitation using wild-type or mutant human p53 or wild-type and murine p53 were performed by adding 100 ng of immunoaffinity-purified protein to 2 pl of human TBP-programmed extracts under conditions as described by Lee et

The abbreviation used is: TBP, TATA-binding protein.

al. (66). Incubations were done in a 20-p1 reaction volume for 30 min at 30 "C. PAb421 hybridoma supernatant was then added to the reaction and incubated at room temperature for 1 h. The immune complexes were precipitated by gently rocking for 2 h at room temperature with Protein A-agarose. Pellets were washed as described (66) and resuspended in 2 X Laemmli (74) sample buffer, boiled 10 min, and run on 10% SDS-polyacrylamide gels. Gels were treated with Enhance (Du Pont-New England Nuclear) and visualized by autoradiography. Labeled human TBP in 2 X Laemmli (74) buffer was boiled 10 min and run in parallel with the reactions to mark the position of migration of the precipitated bands. Coimmunoprecipita- tion with yeast TBP was done as described with human TBP.

Direct Contact Binding-50-100 ng of bacterially expressed, purified human TBP (Promega) or 250 ng of immunoaffinity-purified p53 were run on 10% SDS-polyacrylamide gels and blotted to nitrocellulose (Bio-Rad, 0.2 pm) The blot was denatured and then renatured essentially as described by Lee et al. (66). The blot was incubated with 100 pl of programmed lysate containing 35S-labeled wild- type p53 (for human TBP blots) or 50 pl of programmed lysate containing 35S-labeled human TBP (for p53 blots) for 12 h at room temperature. The blot was washed, dried, and exposed for autoradiography as described (66).

Gel Retardation Assays-Gel shift assays were performed as described by Hoopes et al. (75). A TATA-containing probe (5'-

GAGGCATGCTATAAAAGTCGACGAGCTTCCA-3') was derived from the TATA-only construct (76), containing the TATA box from the adenovirus major late promoter. The fragment for DNA binding was generated by Hind111 digestion and labeled with [32P]dCTP. Human TBP was a bacterially expressed product acquired from Promega. Gel shift experiments to test the effect of wild-type or mutant human p53 on TBP.TATA complex formation (see Fig. 6) made use of 20 ng of human TBP. Two levels of p53 were used, 4 and 20 ng. The control lane used 20 ng of p53 incubated along with the TATA-containing probe. No complex with the TATA-containing probe was seen for either the wild-type or mutant p53-281G. The antibody shift experiment contained 10 ng of human TBP and 20 ng of wild-type human p53 in the presence of 7.5 pg pf PAb421 (specific for p53) or PAblOl (specific for T antigen).

AGCTTCGAATCTAGATCTGCAGATCGATGATCAGAATTCTC-

RESULTS

Recombinant Baculovirus Expression and Immumaffinity Purification of Human and Murine p53"We have generated recombinant baculoviruses expressing wild-type p53 or mutant (248W, 281G) human p53 proteins using linearized baculoviral DNA and the transfer vector pVL1392 (Invitrogen) containing cloned wild-type or mutant human p53 cDNAs (71). The wild-type or mutant p53 protein was isolated from a cellular extract 48 h after infection of SF9 cells with the virus. The protein was purified by immunoaffinity chromatography using a column of Affi-Gel 10 coupled with purified monoclonal antibody against p53 (PAb421) as described before (72). As judged by SDS-polyacrylamide gel electrophoretic analysis using Coomassie Blue stain, the preparation is 80-9096 pure (Fig. 1). Wild-type murine p53 was also purified using a similar method with a recombinant baculovirus (a generous gift from Dr. Louis Miller, University of Georgia) (73) expressing wild-type murine p53.

A B C D

kD 200-

97-

68-

43- c c -

Wild-type 248 281 Murine

FIG. 1. SDS-polyacrylamide gel electrophoretic analysis of wild-type and mutant p53. Recombinant baculoviruses expressing wild-type or mutant p53 were used to generate protein that was purified by immunoaffinity chromatography. Samples were run on SDS-polyacrylamide gels and visualized by staining with Coomassie Blue. A, wild-type p53; B, p53-248W; C, p53-281G; and D, murine p53. Arrows indicate the position of p53.

13064 p53 and the TATA-binding Protein

Coimmunoprecipitation of p53 and TBP-Because of the involvement of p53 in transcriptional regulation, we tested the possibility that p53 may bind TBP by performing an in vitro immunoprecipitation assay. For this experiment, we used baculovirus-expressed, immunoaffinity-purified wild-type human p53 and in vitro synthesized human TBP. I n vitro synthesis of human TBP (35S-labeled) was carried out using a cDNA clone of human TBP (a kind gift from Drs. Horikoshi and Roeder) (57) under a T7 polymerase promoter using a commercial reticulocyte lysate system that was capable of synthesizing the RNA and protein in the same preparation (TNT lysate system, Promega). As shown in Fig. 2 A , a major polypeptide that migrated identically with the purified bacterially expressed human TBP (Promega) (position of migration of purified TBP shown by an arrow in the figure) could be visualized by polyacrylamide gel electrophoresis and autoradiography. An aliquot of the synthetic human TBP was mixed with an increasing amount of baculovirus-expressed, immunoaffinity-purified human wild-type p53 and immunoprecipitated by the monoclonal antibody PAb421 against p53 and Protein A-agarose as described under “Experimental Pro- cedures.’’ Results presented in Fig. 2B show that as the amount of added p53 was increased, an increasing amount of TBP was precipitated (lanes 2 4 ) . TBP was not precipitated by PAb421 in the absence of p53 (lane 1 ). These results indicate that p53 forms a complex with TBP resulting in the coimmunoprecipitation of TBP and p53 by PAb421.

Direct Interaction of TBP and p53”Protein blotting experiments were performed to provide experimental evidence to demonstrate a direct interaction between TBP and p53. Fig. 3 shows the results of such experiments. As a probe for human TBP, we expressed wild-type human p53 in vitro as discussed under “Experimental Procedures.” An aliquot of the 3sS- labeled protein was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The arrow shows the major product corresponding to p53 (Fig. 3A). In one experiment

A B

kD 68- p53(ng) - (00 zm a

hTBP + + + + 43- - - kD 68-

28-

43-

c

28- 1 2 3 4

FIG. 2. Coimmunoprecipitation of human TBP and p53. A , expression of human TBP. Human TBP was expressed by in oitro transcription and translation using the TNT system (Promega) as described under “Experimental Procedures.” An aliquot of the 35S- labeled material was analyzed on an SDS-polyacrylamide gel. The arrow indicates the position of a bacterially expressed human TBP run in parallel and stained with Coomassie Blue. B, coimmunoprecipitation of human TBP by p53. Increasing amounts of immunoaffinity- purified p53 were added to 2 p1 of human TBP-programmed extracts under conditions as described by Lee et al. (66). Incubations were done in a 20-pl reaction volume for 30 min at 30 “C. PAb421 hybridoma supernatant was then added to the reaction and incubated at room temperature for 1 h. The immune complexes were precipitated by gently rocking for 2 h a t room temperature with Protein A-agarose. Pellets were washed as described (66), resuspended in 2 X Laemmli (74) sample buffer, boiled 10 min, and run on 10% SDS-polyacrylamide gels. The arrow indicates the migration of labeled human TBP run in parallel.

A B C

kD 208-

105-

.i ?.

kD 105 68- “ C

68-

43- - 43- . kD 43- .+ 28- 28-

28- 18- 18-

FIG. 3. A , expression of p53 in vitro. Wild-type p53 was expressed by in oitro transcription and translation using the TNT system (Promega) as described under “Experimental Procedures.” An aliquot of ”S-labeled material was subjected to SDS-polyacrylamide gel analysis and visualized by autoradiography. The arrow indicates the position of the major product, which corresponds to p53. B, direct binding of p53 to human TBP. 50-100 ng of bacterially expressed, purified human TBP (Promega) was run on a 10% SDS-polyacrylamide gel and blotted to nitrocellulose. The blot was denatured and then renatured essentially as described by Lee et al. (66). The blot was incubated with 100 pl of programmed lysate containing 3sS- labeled wild-type p53 for 12 h at room temperature. The blot was washed, dried, and exposed for autoradiography as described (66). The arrow indicates the position of labeled human TBP run in parallel. C, direct binding of human TBP to p53. 250 ng of immunoaffinity-purified p53 was immobilized on nitrocellulose and probed with 50 p1 of programmed lysate containing 3sS-labeled human TBP in a way similar to what has been described above.

(shown in Fig. 3B), we ran bacterially expressed human TBP (Promega) along with molecular weight markers in a 10% SDS-polyacrylamide gel. The proteins were then electroblot- ted onto nitrocellulose paper, subjected to a denaturation and renaturation protocol (66), and incubated with in vitro translated, 35S-labeled wild-type human p53 overnight. The blot was subsequently washed and exposed to x-ray film. The figure representing the autoradiogram indicated the presence of a band at an identical position to 3sS-labeled TBP run in parallel (not shown). This result suggests direct interaction between human TBP and p53.

The converse experiment was also performed (Fig. 3C). In this experiment, baculovirus-expressed, immunoaffinity-purified human p53 was run in an SDS-polyacrylamide gel, blotted to nitrocellulose filter paper, and probed with in vitro translated, 3sS-labeled human TBP. The results in Fig. 3C show that a band becomes visible at a position in the gel where purified human p53 migrated (data not shown). Taken together, these results show a direct interaction between p53 and TBP.

Interaction of Human TBP with Transforming Mutants of Human p53 and Murine Wild-type p53-Immunoprecipita- tion experiments were carried out using recombinant baculovirus-expressed, immunoaffinity-purified transforming mutants of human p53 (248W and 281G) and in vitro synthesized human TBP (35S-labeled) in a manner similar to the experiment described in the previous section. Fig. 4A shows the results of this immunoprecipitation experiment. The presence of a band in lanes 2-4, representing reaction mixtures with different p53 proteins, at a position where TBP alone migrated (as depicted by an arrow), indicates successful association of wild-type human p53 (lane 2 ) and transforming mutants of p53 (p53-248W, lane 3; and p53-281G, lane 4 ) with TBP. It is important to note that the transforming mutants activate the proliferating cell nuclear antigen and MDRl promoters (47, 51) significantly. In fact, for its trans- activating role, p53-281G requires only a TATA box (51). This suggests the possibility that mutant p53s contact TBP and exert a positive influence on transcription. Results in Fig. 4B show coimmunoprecipitation of synthetic TBP and purified murine p53 by PAb421 and Protein A-agarose. Thus, murine p53 can also interact with human TBP, as expected by the similar roles played by human and murine p53s (23). The successful coimmunoprecipitation of human TBP with

p53 and the TATA-binding Protein 13065

A 0

p u - wt 248281 p U - MU

hTBP + + + + hTBP + + kD 68

kD 43- -c

43- c

28-

18-

28-

18-

$ 2 3 4

FIG. 4. Coimmunoprecipitation of human TBP with mutant and murine p53. A, coimmunoprecipitation of human TBP and transforming mutants of p53. Immunoaffinity-purified wild-type p53, p53-248W, or p53-281G (100 ng each) was used for immunoprecipitation with the monoclonal antibody PAb421 and Protein A-agarose. The procedure was similar to that described in the legend to Fig. 2B. The arrow indicates the position of labeled human TBP run in parallel. B, coimmunoprecipitation of human TBP with murine p53. Immunoaffinity-purified murine p53 (100 ng) was used to perform immunoprecipitation of human TBP. The procedure was similar to that described in the legend to Fig. 2B. The arrow indicates the position of labeled human TBP run in parallel.

A B p53 - +

kD 68- YTSP + + 43- kD 43-

28- o c 28-

18-

c

FIG. 5. Coimmunoprecipitation of yeast TBP and p53. A, expression of yeast TBP. Yeast TBP was generated by in uitro transcription and translation using the TNT system (Promega) as described under “Experimental Procedures.” An aliquot of the 35S- labeled protein was analyzed on an SDS-polyacrylamide gel. The arrow indicates the major product. B , coimmunoprecipitation of yeast TBP by p53. Wild-type p53 (200 ng) was incubated with yeast TBP (2 pl of in uitro synthesized 35S-labeled protein) and precipitated by PAb421 and Protein A-agarose as described in the legend to Fig. 2B). The arrow indicates the position of labeled yeast TBP run in parallel.

wild-type murine p53 by PAb421 suggests that the association between TBP and p53 can occur across species.

Interaction of Yeast TBP with Wild-type H u m n p53- Since it has been demonstrated that human p53 expressed in yeast can act as a transcriptional modulator (78, 79), it was of interest to check whether yeast TBP can associate with human p53. Fig. 5A shows the electrophoretic mobility of the in vitro translated product of the yeast TBP gene (80) (a kind gift from Drs. Horikoshi and Roeder). The size of the polypeptide, as evident from its migration in the gel, matches the expected size of 28 kDa. Fig. 5B represents an immunoprecipitation experiment in which the synthetic yeast TBP gene product was incubated with purified wild-type p53 and then immunoprecipitated by PAb421 and Protein A-agarose. The immunoprecipitate was analyzed on an SDS-polyacrylamide gel followed by autoradiography. The figure shows the presence of a band that migrated to an identical position as in vitro synthesized yeast TBP run in parallel (not shown), indicating that yeast TBP can also bind to human wild-type p53. Positive association between yeast TBP and human p53 indicates that the p53-binding domain of TBP probably re- sides in the region of TBP that is conserved between human and yeast molecules, corresponding to residues 155-335 in human TBP (77). The interaction is also compatible with the

effect of human p53 on yeast transcription (78, 79). Influence of p53 on the Binding of TBP to the TATA Bor-

We used gel retardation analysis to determine whether the binding of p53 to TBP can affect the binding of TBP to the TATA box. After incubation at 30 “C as described previously (75) bacterially expressed human TBP formed specific retarded bands as shown in Fig. 6A with the TATA box- containing probe (5”AGCTTCGAATCTAGATCTGCAG-

- AGTCGACGAGCTTCCA-3’) (Fig. 6 , A and B, lunes 3; Fig. 6C, lane 2 ) . The complexes could be competed specifically by the TATA box-containing cold self-competitor but not by an identical DNA fragment lacking the TATA element (5’-

ATTCTCGAGGCATGCGTCGACGAGCTTCCA-3’) (data not shown). Next we co-incubated human TBP with increasing amounts of human wild-type p53 (Fig. 6A, lanes 4 and 5 ) or mutant p53-281G (Fig. 6B, lanes 4 and 5 ) and the TATA probe and performed gel retardation analysis. It is clear from the results presented in Fig. 6 that either wild-type p53 (Fig. 6A) or p53-281G (Fig. 6 B ) , although alone does not bind to the TATA probe (Fig. 6, A and B, lanes 2 ) , when added to the incubation mixture with the TATA probe and TBP, caused an upward shift of the retarded band. This suggests that p53 may form a complex with TATA-bound TBP. Complex formation takes place even with the transforming mutant p53- 281G, indicating that the ability to form a complex with TBP cannot be the sole criterion discriminating the functions of wild-type and mutant p53s.

In order to demonstrate decisively that the complex evident in the gel retardation assay (in the presence of p53, TBP, and the TATA probe) actually contained p53, we added PAb421 to the incubation mixture and performed the retardation assay. As evident from Fig. 6C, PAb421 caused a supershift of the slower moving band in lane 4, indicating the presence of p53 in the complex. PAb421 alone did not affect the migration of the TATA probe or the TBP .TATA complex (Fig. 6D, lanes 1-3). Also, purified PAblOl, a monoclonal antibody against SV40 T antigen, could not generate this supershift (Fig. 6C, lane 4 ) . Therefore, p53 can bind to TATA- bound TBP. However, at this stage it is not yet clear whether the binding of p53 to TBP influences the affinity of TBP for the TATA box.

ATCGATGATCAGAATTCTCGAGGCATGCTATAAA-

AGCTTCGAATCTAGATCTGCAGATCGATGATCAGA-

DISCUSSION

In this communication we report that human wild-type p53 and two transforming mutants, p53-248W and p53-281G, can associate with human TBP. That transforming mutants can also interact with TBP suggests that the differential activity of wild-type and mutant p53 in vivo may not be explained simply by the ability (or lack thereof) to interact with TBP. That wild-type murine p53 can complex with human TBP suggests that the ability to mediate this interaction is conserved across species. Yeast TBP is also capable of associating with human wild-type p53. This is consistent with the fact that human wild-type p53 can function as a transactivator as well as a growth inhibitor in yeast (78, 79). The ability of yeast TBP to complex with human p53 implies that the p53- binding domain is probably located within the conserved region common to both yeast and human TBP (77).

With the association of p53 and TBP established, it was important to determine what effect p53 may have on the ability of TBP to bind the TATA box. We show that human TBP, even when complexed with wild-type or mutant human p53, can bind to a TATA-containing probe. p53 alone could not bind to the probe significantly. Thus, the functional difference between wild-type and mutant p53 does not lie in

13066 p53 and the TATA-binding Protein

A B hTBP (ng) - - 20 20 2O hTBP(ng) - - 20 20 20

p~ (ng) - 20 - 4 20 p53281G (ng) - 20 - 4 20

-TBP/p53 - -TBP

___y3 -Free Probe

. 4 -TBP/p53 u - -TBP

1 2 3 4 5 1 2 3 4 5

C

hTBP - + + + + p53 - - + + +

pABlOl - - - + - DAB421 - - - +

-TBp;p53. -TBP

D

hTBP - + + pAB421 + - +

- 1 2 3 4 5

1 2 3

FIG. 6. Effect of p53 on human TBP DNA binding. Gel shift assays were performed as described by Hoopes et al. (75). A TATA- containing probe (5'-AGCTTCGAATCTAGATCTGCAGATCGATGATCAGAATTCTCGAGGCATGCTATAAAAGTCGACGAGCTT- CCA-3') was derived from the TATA-only construct (78), containing the TATA box from the adenovirus major late promoter. The fragment for DNA binding was generated by Hind111 digestion and labeled with ["PIdCTP. Human T B P was bacterially expressed (Promega). TBP. TATA complex formation was achieved using 20 ng of human TBP. Two levels of p53 were used, 4 and 20 ng. The control lane used 20 ng of p53 incubated along with the TATA-containing probe. No complex was seen for either the wild-type or mutant p53-281. The antibody shift experiment contained 10 ng of human TBP and 20 ng of wild-type p53 in the presence of 7.5 pg pf PAb421. A, effect of wild-type p53 on human TBP binding to the TATA probe. The positions of the free probe, TBP complex, and TBP.p53 complex are shown. B, effect of mutant p53-281G on human TBP binding to the TATA probe. The positions of free probe, TBP complex, and TBP.p53 complex are shown. C, DNA-binding reactions with human TBP and p53 were tested with PAb421 (specific for p53) or PAblOl (specific for T antigen) to confirm the presence of p53 in the complex. The position of an additional complex, TBP.p53.PAb421, is shown. D, effect of PAb421 on complex migration. To confirm that PAb421 was not interacting with free probe or TBP, the TATA probe or TBP. TATA probe was incubated in the presence of PAb421. No additional complexes were evident.

their ability to bind to TBP or in the ability of the TBP p53 complexes to bind to the TATA element. Perhaps a step subsequent to TBP binding to TATA is involved in the discrimination of functions of wild-type and mutants of p53.

Our observation that p53 can bind to TBP is a direct demonstration of p53's ability to interact with the transcription machinery. That both wild-type p53 and its mutants bind to TBP and that they have different transcriptional functions may not be mutually exclusive. Wild-type p53 activates promoters with p53-binding sites i n uiuo and in uitro (40-42,84, 85). On promoters without p53-binding sites, wild-type p53 has a strong inhibitory effect in uiuo (46-51), while mutants of p53, a t least for some promoters, have a strong activating role (47,51). Although not yet shown by i n uitro experimental results, it is possible that wild-type p53 directly inhibits transcription from some promoters that do not have p53- binding sites by interacting with TBP in a way detrimental to transcription initiation. Since wild-type and mutant p53 proteins display different conformations (81), one can speculate that the conformation of the wild-type p53. TBP complex is not favorable for interaction with other transcription factors and therefore inhibits transcription in general. It is possible that a conformation favorable for transcription could be achieved either by a mutation in the p53 protein or by direct DNA binding. Thus, a mutant p53 may interact with TBP and favor transcription in a general way because of its

altered conformation. For promoters with p53-binding sites, the situation may be different. The interaction of DNA-bound wild-type p53 with TBP may be facilitatory to transcription, being conducive to interaction with other general transcription factors. One can also imagine that because of the presence of p53-binding sites on these promoters and the ability of p53 to bind to TBP, p53 may be able to facilitate the nucleation of TBP on these promoters, enhancing transcriptional activity. In the case of mutant p53, defective in binding, such nucleation is not possible, and hence there is no preferential activation of these promoters. However, one may see a general activation of these promoters by the mutant. We observed that the mutant p53-281G activated a promoter with p53- binding sites i n uiuo, although the level of activation was lower than that produced by wild-type p53.2 Another possible explanation for the general inhibition of transcription by wild- type p53 is that it activates the expression of a gene encoding a transcriptional inhibitor. This promoter may have a p53- binding site for p53-mediated activation. We are still faced with one ambiguity: if wild-type p53 can inhibit many promoters why can it not inhibit transcription by promoters with p53-binding sites? One possible answer could be that because of the presence of p53-binding sites, TBP will nucleate at the initiation site of transcription, but at the time of initiation,

S. Deb, unpublished results.

p53 and the TAl

steric effects and/or relative affinities may force p53 to remain preferentially bound to its DNA-binding sites rather than to TBP, releasing the potential for inhibition of transcription by complexing with TBP at the site of transcription initiation. Future in vitro experiments should answer such questions.

Recent work has shown that TBP is required for transcription by RNA polymerase I1 on TATA-less promoters (82), by RNA polymerase I (62), and possibly by RNA polymerase I11 (59, 83). One may then speculate that p53 may be able to influence all three different types of transcriptional activities, regulating the synthesis of messenger, ribosomal, and transfer RNAs. Our finding that p53 can directly interact with TBP may, therefore, have interesting and far reaching implications.

Acknowledgments-We thank Dr. A. J. Levine for providing us with wild-type and mutant p53 constructs and Drs. M. Horikoshi and R. G . Roeder for TBP cDNA clones. We thank Swati Palit Deb, Doris R. Brown, and Kathy Partin for stimulating discussion and encouragement and Joyce Subler for assisting in literature searches and encouragement.

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