Repression of Transcription and Interference with DNA Binding of TATA-Binding Protein by C-Terminal Alternatively Spliced p53

Experimental Cell Research 279, 248 –259 (2002) doi:10.1006/excr.2002.5596

Repression of Transcription and Interference with DNA Binding of TATA-Binding Protein by C-Terminal Alternatively Spliced p53 Hua Huang, Shinsuke Kaku, 1 Chad D. Knights, 2 Byung S. Park, 3 Jane Clifford, 4 and Molly Kulesz-Martin 2,5 Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263

The protein encoded by C-terminal alternatively spliced p53 mRNA (p53as) has been shown previously to occur naturally in mouse cells and to bind sequence-specifically to DNA more efficiently than p53 (p53r, regular form). In the current study, p53as and p53r proteins ectopically expressed in p53-deficient cells each transactivated reporter plasmids containing p53 binding sites. However, p53as consistently was more efficient in transcriptional repression of promoters lacking p53 binding sites and in concentrationdependent repression of the p21 WAF1/Cip-l/Sdi promoter sequence. The p53as protein, like p53r, associated with TATA-binding protein (TBP), indicating that this interaction does not require the last 26 amino acids of p53. Consistent with its stronger repression effects, p53as interfered with TBP binding to a TATA-containing DNA sequence more efficiently than p53r protein. Taken together, these in vitro and in vivo results demonstrate a novel role in transcriptional repression for a naturally occurring C-terminal variant form of mouse p53 protein associated with differences in DNA binding properties and interference with transcription factor binding. © 2002 Elsevier Science (USA) Key Words: p53; transactivation; transcriptional repression; Waf-1; TATA-binding protein.

INTRODUCTION

The tumor suppressor gene p53 is mutated or inactivated frequently in human cancer [1]. The p53 protein functions in large part by sequence-specific DNA binding 1

Current address: Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., 403-1 Yoshino-cho, Omiya, Saitama, 330-8530, Japan. 2 Current address: Department of Dermatology, OP06, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098. 3 Current address: Biostatistics/Informatics Shared Resources, Cancer Institute, CR145, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098. 4 Current address: Department of Biochemistry, MCP Hahnemann School of Medicine Philadelphia, PA 19102. 5 To whom reprint requests should be addressed. Fax: (503) 4022817. E-mail: [email protected]. 0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

and transcriptional regulation of downstream genes including GADD45 [2], mdm 2 [3, 4], muscle creatine kinase (MCK) [5, 6], cyclin G [7], and p21 WAF1/Cip-1/Sdi [8, 9]. There are four functional domains within the p53 protein: (1) an acidic N-terminal portion responsible for transactivation, (2) a central hydrophobic domain determining p53 conformation and sequence-specific DNA binding, (3) an oligomerization domain [10], and (4) a C-terminal domain implicated in regulation of DNA binding [11, 12], recognition of damaged DNA [13], reannealing of single-stranded DNA and RNA [14, 15], and transcriptional repression [16 –18]. p53 protein exists in active and latent forms, depending on posttranslational modifications that regulate DNA binding and transcription [11, 12, 19]. Activating modifications include phosphorylation by CKI [20], raf kinase [21], MAP kinase [22], ATM [23], and DNA-PK [24] at the N terminus or by CKII [25], PKC [26], cdk [27], and cdc2 at the C terminus [28] and acetylation (by p300 and pCAF) or deactylation at the p53 C terminus [29, 30]. Posttranslational modifications of p53 protein also can repress or interfere with gene transcription. Mutations of the CKII phosphorylation site, for example, result in loss of transcriptional repression activity for the c-fos and SV40 promoter, targets for p53-dependent repression in vivo [31, 32]. Activation of p53 for sequence-specific DNA binding also can be accomplished through protein interactions at the C terminus. The C-terminal antibody PAb421 acts as an activator of p53 DNA binding activity and is thought to relieve a negative regulatory domain, since truncation of the last 30 amino acids (p53 ⌬30) also constitutively activates p53 for sequence-specific DNA binding [11, 33]. Transcriptional repression by p53r protein has been reported for a variety of promoters lacking p53 consensus sequences. Induction of p53 correlates with repression of the cell proliferation genes c-fos [34], c-myc [35], c-ets-1 [36], cyclin A [37, 38], and PCNA [39]. Transcriptional repression, but not activation, by p53 can be antagonized by the adenovirus E1B 19K protein and the cellular protein bcl-2 [16, 40, 41]. Transcriptional repression has been demonstrated in cell-free systems [35], suggesting that it is not simply a consequence of

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p53-mediated growth suppression. The wide spectrum of promoters repressed by p53 suggests that interaction with the general transcription machinery is responsible for repression. The p53 protein binds the general transcription factor TFIID subunit TBP [35, 42, 43] and components of TFIIH [44]. p53 protein may repress many genes containing TATA boxes by sequestering these proteins [45]. The p53 protein interacts with other transcription factors, including CCAAT binding factor and ICE DNA elements (five inverted CCAAT elements), to suppress the transcription of hsp70 and topoisomerase II␣, respectively [46, 47]. The current study compares the transcriptional induction and repression activities of two naturally occurring forms of the p53 protein, p53r and p53as. As reported previously, the p53as variant is generated by mRNA alternative splicing of the wild-type p53 gene in normal and transformed mouse tissues and cell lines [48, 49]. The C terminus of p53as protein differs from p53r by substitution of 17 amino acids after the codon 365 Ser (encoded within the splice junction) and truncation of 9 amino acids due to a stop codon after p53as codon 381 Cys [48, 50]. Biochemical and functional similarities between p53as and p53r include the formation of tetramers in solution, suppression of cell growth, activation of genes containing p53-responsive elements (e.g., waf-1 and bax), and inhibition of bcl-2 expression [19, 51]. p53as differs from p53r protein in constitutive activity for sequence-specific DNA binding; p53r depends on activation at the C terminus, e.g., by binding to PAb421 [11, 19, 51]. While p53as is transcriptionally active in cells and in in vitro transcription assays, the current results indicate that p53as is more effective than p53r in transcriptional repression and interference with TBP binding to its TATA motif. These effects of p53as further define the region required for repression of transcription as upstream of the alternative splice junction 365 Ser and indicate that transcriptional repression by p53 protein forms is mediated at least in part through interaction with TBP and potentially other cellular proteins. MATERIALS AND METHODS Plasmids and DNA probe. The full-length mouse p53r cDNA and p53as cDNA were each subcloned into a pBR322-derived expression vector under the control of the cytomegalovirus immediate-early promoter (CMV promoter) or the retroviral long terminal repeat promoter (LTR promoter). The reporter plasmids pWWPLuc [8], PG 13CAT, and MG 15CAT [52] were provided by Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD). pWWPLuc contains an endogenous waf-1 promoter upstream of a luciferase gene; PG 13CAT contains 13 repeats of wild-type p53 consensus binding sites, and MG 15CAT contains 15 repeats of mutated p53 binding sites upstream from a chloramphenicol acetyltransferase (CAT) gene. The pWT30tk-luc plasmid containing the cyclin G promoter was a gift of D. Beach (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) [7]. Plasmid p50-2 containing the MCK promoter was provided by A.

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Levine (The Rockefeller University, New York, NY) [6]. The p0.5CAT, kindly provided by G. Lozano (M. D. Anderson Cancer Center, Houston, TX), contains the murine p53 promoter sequence from nucleotide ⫺320 to ⫹202. The pCAT-control (Promega) plasmid is a CAT reporter driven by the SV40 promoter and enhancer. The MLPCAT contains an insert of ⫺174 to ⫹33 of the adenovirus majorlate promoter in pUC18CAT, upstream from the CAT gene. DHFRCAT contains nucleotides ⫺210 to ⫺23 of the hamster dihydrofolate reductase (DHFR) promoter region upstream from the CAT gene [53]. Plasmids pBSp53 and pBSp53as contain full-length mouse p53 or p53as cDNA, respectively, controlled by the T3 phage promoter for generating p53 and p53as proteins by in vitro transcription and translation [51]. The luciferase control RNA (Promega) was used for in vitro translated control protein. The DNA fragment for electrophoretic mobility shift assays (EMSA) of TBP binding contains a TBP consensus TATA sequence derived from ⫺41 to ⫺18 of the adenovirus major late promoter (5⬘-GAAGGGGGGCTATAAAAGGGGGTG-3⬘, a gift of T. Beerman, Roswell Park Cancer Institute, Buffalo, NY). Double-stranded DNA oligonucleotide (100 ng) was end-labeled using 30 ␮Ci of [␥- 32P]ATP (3000Ci/mmol, NEN) and bacteriophage T4 polynucleotide kinase (New England Biolabs) at 37°C for 1 h and separated from unincorporated [␥- 32P]ATP using a Quickspin column (Boehringer-Mannheim). An unlabeled DNA sequence modified within the TATAA region, 5⬘-TAGAGAA-3⬘, was used as a mutated probe for nonspecific competition [54]. Proteins. Full-length mouse p53as and p53r cDNA were subcloned into baculovirus vector pfastBAC HTb (Invitrogen) to construct recombinant p53 proteins. Baculovirus vectors were transfected into Sf-9 insect cells to generate high-titer baculovirus stocks. Purified recombinant p53r and p53as proteins were obtained from SF9-infected cells purified by means of PAb421 antibody column for p53r and polyclonal antibody ApAs for p53as (Oncogene Research Products) [49]. Purified TBP used in EMSA was purchased from Promega, Inc. The GST–TBP fusion protein was obtained from Santa Cruz Biotechnology, Inc. Cell culture and transfection. The p53-null mouse fibroblast (10)1 cell line (gift of A. Levine) was maintained in Dulbecco/Vogt modified Eagle’s minimal essential medium (DMEM) containing 10% calf serum. 10.1 cells were cotransfected with 3 ␮g of reporter plasmid [with the exception of pCAT-control (2 ␮g) and MG 15CAT (10 ␮g)] and the indicated amounts of p53 expression vectors (p53as and p53r) or vector control each using the calcium phosphate precipitation method [55]. The total transfected DNA was adjusted to 10 ␮g per plate with pCMV vector (except MG 15CAT was adjusted to 11 ␮g). p53 and p21 proteins were measured in equal aliquots of protein from transfected cell lysates separated by means of SDS-PAGE and immunoblotted with monoclonal antibody m21-22 (mouse p21 WAF1/Cip-1/Sdi protein) or as indicated. CAT and luciferase assays. Transfected cells were harvested 40 to 48 h posttransfection and lysed in 200 ␮l of reporter lysis buffer (Promega). CAT activity was measured by means of the diffusion method [56], while luciferase activity was detected by means of an LB9501 luminometer (Promega). Results are representative of at least three independent experiments that confirmed the trend of increased or decreased transcriptional activation or repression where each graph in Figs. 1 and 3 represents a single experiment displaying this consistent trend. CAT and luciferase activities were standardized to the total cell protein assayed using a MicroBCA protein kit (Pierce). Experimental values obtained were divided by the control vector value (reporter ⫹ vector control) to obtain “fold of vector control.” This value was converted to log fold and plotted on a standard arithmetic scale, so activation data were above the x axis and repression data were below the x axis. The y axis was converted using the antilog value so ⫹1 is relabeled as ⫹10 (fold of activation) and ⫺1 is labeled as ⫺10 (fold of repression). This representation of data allows for visual equality of activation and repression events that display the same magnitude.

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Protein binding assay. Recombinant p53as or p53r proteins extracted from baculovirus-infected Sf-9 cells in lysis buffer [50 mM Tris–C1 (pH 8.0), 150 mM NaCl, 1 mM DDT, 1% NP-40, 1 mM PMSF, 5 mM leupeptin, and 10 mg/ml pepstatin A] were clarified by centrifugation at 10,000 g, and used directly in the binding assays. Glutathione Sepharose beads (Pharmacia) (1:1, v/v) alone or plus 2 ␮g glutathione-S-transferase (GST) or 2 ␮g GST–TBP fusion protein in lysis buffer (supplemented with BSA at 100 mg/ml) were incubated at 4°C for 30 min followed by incubation with noninfected Sf-9 cell lysate used to inhibit nonspecific binding. Sf-9 cell extracts infected with p53as or p53r or noninfected Sf-9 lysate control were added to the washed beads and incubated on ice for 1.5 h. The beads were washed four times with lysis buffer and once with NETN buffer [20 mM Tris–Cl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% NP-40] and eluted in SDS sample buffer. The proteins were separated by 7.5% SDS–PAGE and transferred to a nitrocellulose membrane. Detection of bound p53as or p53r was done by blotting with monoclonal antibody PAb242 (which recognizes both p53as and p53r) followed by detection using chemiluminescence. EMSA. A 10-ng aliquot of TBP was incubated with 10,000 cpm of end-labeled DNA fragment at 30°C for 40 min in a reaction buffer consisting of 20 mM Hepes–KOH (pH 7.9), 25 mM KCl, 10% glycerol, 0.025% NP-40, 100 mg/ml bovine serum albumin, 5 mg/ml poly[d(G– C)], 0.5 mM DTT, 2 mM spermidine, 0.1 mM EDTA, and 2 mM of MgCl 2 in a final volume of 20 ␮l, including, where indicated, recombinant p53as or p53r proteins and 200 ng of anti-p53 or anti-p53as antibodies. The mixtures were separated by electrophoresis in a 4% native polyacrylamide gel followed by gel fixation in 10% acetic acid, gel dehydration, and visualization by autoradiography. Statistical analysis. The two-way classification model with interaction [57] was used to test the statistical significance of the differences between p53as and p53r for transactivation or repression of the indicated reporter plasmids. The response variable used was log 2(intensity) with two main factors, p53 expression plasmids (p53as and p53r normalized to CMV control) and p53 plasmid concentrations (0.01, 0.1, and 1.0 ␮g). The t test [57] was employed to test the statistical significance of the differences in transactivation values between p53as or p53r and the CMV control. The orthogonal contrast technique [57] was used to demonstrate a quadratic relationship between the log 2(intensity) of the pWWPLuc reporter over the p53as and p53r plasmid concentrations 0.01, 0.1, 0.5, 1.0, and 2.5 ␮g performed in triplicate. A quantitative trend relationship between log 2(intensity) of p53as and p53r over these concentrations was shown using the polynomial response function for response curves [57]. Statistical data analysis was performed using SAS (Statistical Analysis System) Version 8.1 and Statistica Version 6.0 software.

RESULTS

Sequence-Specific Transcriptional Effects of p53 Alternative Spliced Forms The p53as protein can transcriptionally activate reporter plasmids containing p53 responsive elements from the waf-1 and cyclin G promoters, as we initially reported [19]. Similarly, transient transfections of p53 null (10)1 fibroblasts with p53r or p53as expression plasmids resulted in activation of the mouse MCK promoter, the PG13luc reporter containing 13 repeats of a wild-type p53 consensus binding sequence, and the p53 promoter segment p0.5CAT (Fig. 1). Transactivation intensity values of p53as and p53r compared with CMV control were statistically significant (P ⬍ 0.05)

for all promoter elements represented in Fig. 1, with the exception of 1.0 ␮g p53r for the p0.5CAT and pWWPLuc reporters. Activation reached a peak as p53r or p53as plasmid concentrations increased, then began to diminish at 0.1 or 1 ␮g input plasmid DNA (Fig. 1). These results indicated that p53as protein consistently activated transcription of each of the reporter plasmids with p53 binding sites and that p53as displayed up to twofold weaker activation than p53r at the same input plasmid concentrations. The expression of p53as and p53r proteins was verified by immunoblotting using polyclonal antibody CM-5 that recognizes both p53 forms (Fig. 2A). p53as expression, over the range of expression vector used (0.01, 0.1, and 1.0 ␮g), was approximately threefold less than that of p53 protein (2.9-, 2.7-, and 2.8-fold, respectively), which was reproducible among experiments. This correlated with less activation of the reporters by p53as than p53r in transient transfection assays (for MCK and waf-1 shown in Fig. 1, and for cyclin G as shown previously [19]). The p53r and p53as expression induced endogenous p21 protein expression, as expected from p53as protein activation of transcription of the endogenous waf-1 gene (Fig. 2B). This supports previous findings where inducible p53as in stably transfected p53 null fibroblasts increased p21 WAF1/Cip-1/Sdi protein and mRNA [19]. It has been demonstrated that p53as is constitutively active for sequence-specific DNA binding, while p53r requires an activating event such as PAb421 binding or phosphorylation. Yet both p53r and p53as proteins generated from expression plasmids were transcriptionally active for the p53 reporter target and endogenous p21. However, the ability of p53as at higher levels to increase endogenous p21 levels, yet repress expression driven from a reporter construct containing the p21 promoter, may be explained by the stability of endogenous p21 protein. As the levels of p53as build, the expression of endogenous p21 continues to rise. Once levels of p53as reach a level that can induce repression, p21 protein has already accumulated and can become stabilized by interacting with other cellular proteins, making it difficult to see the repressive effects of p53as on an endogenous protein [58]. Biphasic Effect of p53as and p53r on the Waf-1 Promoter While p53as tended to be weaker than p53r for transcriptional activation, this trend was reversed for transcriptional repression. The WWP plasmid contains the 2.4-kb promoter region derived from the endogenous p21 WAF1/Cip-1/Sdi gene that has been defined as a major downstream target of p53. While activation of the waf-1 reporter (Fig. 1D) occurred at the lower p53r and p53as plasmid concentrations tested including 0.1 ␮g,

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FIG. 1. Transcriptional effects of p53as and p53r on reporter plasmids p50-2 (MCK promoter), PG13Luc, pWWPLuc (waf-1), and p0.5CAT (p53). (A) p50-2, (B) PG13Luc (PG), (C) p0.5CAT, and (D) pWWPLuc plasmid were transfected with pCMVp53as, pCMVp53r, or pCMV vector, except as otherwise indicated, into (10)1 p53-null fibroblasts. Indicated CAT or luciferase activities were standardized to total cellular protein and pCMV vector control (one fold). Magnitude of activation or repression is represented in the bar graphs as log(test vector value/control vector value), then converted to the antilog value for direct reading on the y axis as 1-fold activity. Each graph shows the results of a single experiment out of three experiments showing identical trends. Differences between p53as and p53r were statistically significant in (B)–(D) (P ⬍ 0.01) but not (A); statistical analyses were based on data from at least three experiments per reporter plasmid comparing p53r, p53as, and control plasmid concentrations 0.01, 0.1, and 1.0 ␮g (see Materials and Methods). Error bars shown in (D) are the standard deviation about the mean of triplicate fold intensity values.

increasing plasmid concentrations resulted in a decrease in activation and increase in transcriptional repression, with p53as exerting stronger repressive effects than p53r. The effects of p53as and p53r on this promoter were defined by a statistical trend demonstrating dose-dependent decrease of pWWP reporter intensity over the extended concentration span 0.01– 2.5 ␮g (P ⬍ 0.0001). This trend was replicated in three additional experiments using three plasmid concentrations (0.01, 0.1, and 1.0 ␮g) that also demonstrated statistically significant differences between p53as and p53r. Lower concentrations of p53as or p53r activated the WWP reporter gene while higher concentrations of plasmids (at about 0.5 ␮g of p53as and 1 ␮g of p53r) repressed transcription (Fig. 1D). It is also notable that the stronger, concentration-dependent repressive effects of p53as were exerted despite the weaker p53as protein expression evident in Fig. 2A,

implying that these results underestimate the differences in transcriptional repression by p53as compared with p53r protein. Stronger Repressive Effect of p53as on Promoters without p53 Binding Sites The repressive effects of p53 observed in the transient transfection experiments for promoters with p53 binding sites lead us to test several reporters lacking p53 binding sites. We showed previously that p53as did not activate the MG 15CAT plasmid, which is similar in structure to PG 13CAT but with mutated p53 binding sites [19]. Figure 3 shows that p53as significantly repressed not only MG 15CAT (Fig. 3A) but also three other promoter sequences lacking p53 binding sites: pCAT control, adenovirus major late promoter MLP, and the DHFR promoter (Figs. 3B, 3C, and 3D, respec-

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tively) (with up to 40-fold repression at 0.1 to 1 ␮g p53as plasmid), while p53r showed variable and weaker effects (up to 10-fold repression). Interactions Between p53as and TBP

FIG. 2. Immunoblotting of p53 and p21 proteins. (A) p53as (AS) and p53r (R) protein expression after transfection of indicated amounts of pCMV vector (vec), pCMV53as, and pCMV53r plasmids. An equal amount of protein from transfected cell lysates was applied to each lane and separated by means of 7.5% SDS–PAGE. Polyclonal antibody CM-5 was used to detect both p53as and p53r proteins. (B) p21 WAF1/Cip-1/Sdi protein expression was detected using monoclonal antibody m21-22.

TBP has been reported to be a p53 N- and C-terminal interacting protein and a key factor in basal and regulated transcription [59 – 62]. Therefore, we tested the p53as or p53r proteins in Sf-9 insect cells for physical association with TBP and functional interference with TBP-specific DNA binding. The p53as and p53r proteins were precipitated by a GST–TBP fusion protein (Fig. 4, lanes 5 and 6), while little or no p53as or p53r proteins bound to glutathione beads alone (lanes 1 and 2) or to GST protein bound to beads (lanes 3 and 4). There was no detectable p53 protein bound to GST– TBP in noninfected Sf-9 cell lysates (lane 7). Since p53as and p53 proteins each can interact with TBP, they were then tested for their ability to interfere with the binding of TBP to its consensus TATA sequence. EMSAs demonstrated a single band representing TBP bound to the TATA consensus sequence probe in the

FIG. 3. Repression of transcription from plasmids lacking intact p53 binding sites by p53as and p53r. (A) MG 15CAT plasmid (MG, containing 15 mutated p53 binding sites), (B) pCATcontrol plasmid, (C) MLPCAT (MLP), or (D) DHFRCAT (DCAT) were cotransfected with the indicated amounts of pCMV53as, pCMV53r, or pCMV vector control into (10)1 cells. CAT activity was standardized to the protein amount and is shown relative to pCMV vector (one fold). Each graph shows the results of a single experiment out of three experiments showing identical trends. The stronger repressive effects of p53as compared with p53r were statistically significant for all the reporter plasmids shown (P ⬍ 0.01), based on data from at least three experiments per plasmid, analyzed as described under Materials and Methods.

TRANSCRIPTIONAL REPRESSION BY p53as SPLICE VARIANT

FIG. 4. Association between p53as and p53r proteins and TBP. The p53as (AS) and p53r (R) recombinant baculovirus-infected Sf-9 cell lysates and noninfected Sf-9 cell lysate (Ctrl) were incubated with either glutathione beads alone (lanes 1 and 2), beads with GST protein bound (lanes 3 and 4) or with GST–TBP fusion protein bound (lanes 5–7), followed by detection of retained p53 proteins by immunoblotting with PAb242. An aliquot of lysate was immunoblotted directly to indicate equivalent amounts of p53as and p53r proteins used in the binding reactions (lanes 8 and 9).

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To determine whether the p53 proteins could affect TBP after it had already bound to DNA, TBP was incubated with the DNA probe for 20 min to allow for complex formation (Fig. 6B, lane 1), and then p53as (lane 3) or p53r (lane 7) was added to the reaction mixture and incubated for an additional 20 min. Under these conditions p53as and p53r had a negligible effect on preformed TBP–DNA complexes, compared with interference with TBP binding when p53 and TBP proteins were added together (lanes 2 and 6 compared with lane 1). Increasing the amount of TBP protein could overcome the interfering effect of p53as or p53r (lanes 4, 5, and 8, respectively, compared with lane 2), suggesting that the ratio of TBP to p53 proteins determined the extent of TBP binding. DISCUSSION

absence of p53 protein (Fig. 5A, lane 1). The binding was specific, based on competition with unlabeled probe DNA (lane 2) but not with mutated probe sequence (lane 3) and an observed supershift with antiTBP antibody but not with IgG control (lanes 4 and 5, respectively). There was no shift of DNA probe when TBP protein was excluded from the reaction (lane 6), and neither p53as nor p53r bound to the TATA sequence (data not shown). When p53as protein was included in the reactions, there was a concentrationdependent interference with the specific binding of TBP to DNA (Fig. 5B, lanes 2– 4 compared with lane 1 and Fig. 5C). The p53r protein had comparatively minimal effects in the same concentration range (Fig. 5B, lanes 1 and 5–10 and Fig. 5C). The p53r protein interfered with TBP binding to the TATA sequence at higher concentration, requiring 270 ng of p53r protein to achieve the same level of inhibition as 40 ng of p53as protein (Fig. 5C). Inclusion of specific antibodies in the EMSA reactions indicated that p53r and p53as were not present in the complex with TBP and its DNA consensus sequence. Figure 6A shows that the TBP–DNA complexes were shifted to the same degree by monoclonal antibody against TBP whether the complexes formed in the absence (lane 2 compared with lane 1) or presence (lanes 6 and 9) of p53as or p53r, respectively. Further, TBP–DNA complexes could not be supershifted by p53r antibody PAb421 (lane 10 compared with lane 8) or p53as antibody ApAs (lane 7 compared with lane 5). Also, the addition of PAb421 results in increased p53r DNA binding activity, changing p53r to a protein conformation that more closely resembles p53as [51]. The reduction in TBP binding to its TATA sequence in Fig. 6, lane 10, may be attributable to this change in p53r conformation. These results indicate that interactions between p53as and TBP proteins interfered with TBP binding to its consensus TATA sequence.

The ability of p53 protein to bind specifically to sequences within the promoters of growth-inhibitory, antiangiogenesis, and apoptotic genes underlies its role as a transcription factor. The properties of the naturally occurring murine alternative splice variant p53as predicted that it would be a transcriptional activator of p53 downstream genes because p53as exhibits strong DNA binding to p53 consensus sequences by EMSA [19] and because p53as protein binds to the same target sequences in DNA as p53 protein [63]. Other properties of p53as, its tetramerization [51], its ability to induce p21 and bcl-2 protein expression in cells, and its induction in cells by DNA damage [19], suggest that it would behave as a transcription factor in transient transfection experiments. We previously showed that p53as activates transcription of endogenous p21WAF1/Cip-1/Sdi and waf-1- and cyclin G-driven reporter plasmids in cells expressing a stable p53as transfectant [19]. The current studies show that p53as protein positively regulates transcription from additional promoters containing p53 consensus binding sites, including the MCK promoter, and sequences that contain DNA elements similar to the p53 consensus binding site, including the p53 gene promoter itself, which has previously been shown responsive to p53r [64]. The transactivation is sequencespecific and dependent on wild-type p53 binding sites. Overall, p53as protein proved to be a naturally activated p53 protein form capable of constitutive sequence-specific DNA binding and transcriptional activity. However, while stronger DNA binding activities might have been expected to relate to stronger transcriptional activities in cells, p53as consistently showed weaker transcriptional activity than p53r at equivalent plasmid DNA concentrations. Almog et al. recently confirmed transactivation by p53as of p53 binding sequences from the cyclin G and waf-1 promoters and, in addition, demonstrated activation of apo-1, mdm-2, and GADD45 promoters [65]. As in our study,

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FIG. 5. Interference with TBP sequence-specific DNA binding by p53as and p53r. (A) TBP and TATA-containing oligonucleotide ( 32P-AdML) were incubated without (lane 1) or with unlabeled competitor wild-type sequence (wt) oligonucleotide (lane 2) or mutated (mu) oligonucleotide (lane 3) and were supershifted by antibody to TBP (lane 4) but not IgG control (lane 5) as visualized by autoradiography. (B) Interfering effect of indicated amounts of p53as and p53r on TBP-specific binding to 32P-AdML oligonucleotide. Increasing amounts of purified recombinant p53as or p53r proteins were included in the reactions as indicated. (C) Quantitation by densitometry of signal intensities of data shown in (B) relative to control lanes lacking p53.

p53as transactivation was weaker than that of p53r, with the exception of the mdm-2 promoter used in their study only. The use of a range of plasmid concentrations in the current study showed that p53as protein can repress transcription of the waf-1 reporter and that its repression effect was more efficient than that of p53r. Further, promoters lacking p53 binding sites revealed a striking differential repression by p53as compared with p53r. The stronger repression by p53as than p53r at higher concentrations of input plasmid was evident for the waf-1 promoter in the same experiments in which p53r showed stronger transcriptional activation than p53as at lower concentrations, suggesting that it involved an intrinsic difference in the proteins. The repression was not simply due to limiting factors in cells because p53r continued to activate transcription via the waf-1 promoter at the same concentration at which p53as began to show repression. The stronger repressive effects of p53as probably explain

the apparently weaker activation effects in the transient transfection assays demonstrated in our results and by Almog et al. [65], as a consequence of more potent repression of the CMV promoter in the expression plasmids. This would result in the lower steadystate levels of p53as protein observed (approximately 50% of p53 protein) from the same concentration of expression plasmid. The report by Almog et al. [65] that endogenous p53as protein may be more stable than p53 in cells supports the conclusion that less p53as protein is being expressed from the transfected plasmids. While p53as was active for transcription in cells, it is still possible that post-translational modifications at the N terminus are required for activity, or that C-terminal activating steps within the last 26 amino acids are required for full activation of p53 protein transcriptional activity. Recent studies of p300 interaction with p53 protein have suggested that acetylation of histones rather than of p53 is critical for

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FIG. 6. Identification of components in TBP–DNA complexes by specific antibodies, and interference of p53as protein with TBP–DNA complex formation but not preformed complexes. EMSA was performed as in Fig. 5 except that (A) 200 ng of the indicated antibodies was added to the reactions, and (B) p53as or p53r protein was added 20 min later than TBP (bold AS and R) in the reactions shown in lanes 3 and 7. Increasing amounts of TBP included in the reactions shown in lanes 4, 5, and 8 overcame interference by AS and R proteins. The intensities of the binding complexes were measured by densitometry and standardized to control lacking p53 (lane 1). Preimmune serum (Pre-I) is a control for ApAs polyclonal antibody to p53as. PAb421 (421) is specific to p53r, and IgG is a control for PAb421.

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transcriptional activation in the chromatin context [66]. A comparison of the p53 splice variants may be useful for clarifying the role of p53 acetylation in transcriptional activation and repression. The current results suggest that, in addition to activation of its transcriptional properties through modifications at the C terminus, transcriptional repression by p53 proteins is at least in part due to posttranslational modifications or interactions with other proteins through the p53 C terminus. Several lines of evidence associate transcriptional repression with the C terminus of p53 within the last 75 residues [16 –18]. It has been suggested that truncation of the last 30 residues impairs the repressive effect of p53 [67]. However, the transcriptional repression was retained and even enhanced by the alterations within the last 26 residues due to alternative splicing in p53as, suggesting that the post-translational modifications within this region in p53r may be important not only in the regulation of sequence-specific DNA binding and transcriptional activation but in transcriptional repression. The p53as protein is just one example of an alternative p53 form. It has only been demonstrated to be expressed in rodents, although potential alternative splice sites exist in humans [68]. There are multiple modification sites on the p53r protein altered in p53as potentially involved in activation of sequence-specific DNA binding and transcription, RNA processing, export and ubiquitination, and stabilization. Mutation of the CKII phosphorylation site in murine p53r at Ser 389, absent in p53as, results in a loss of p53-mediated transcriptional repression of in vivo p53-dependent targets, including the c-fos and SV40 promoters [31, 32]. It is possible that phosphorylation of p53 protein at this or other C-terminal residues generates p53r forms that have repressive activities analogous to p53as protein. The repressive activity of p53as was a strong effect, being more than 40-fold basal transcriptional activity (Figs. 3A, 3D) compared with little more than 10-fold p53r repressive activity (Fig. 3D) or nearly 25-fold p53r transactivation (Fig. 1B). Repression by p53as protein was observed at the same input plasmid concentration as transcriptional activation by p53r in the case of the waf-1 promoter, implying a specific transcriptional effect due to the p53 C terminus, not simply generalized effects of limiting transcription factors. p53 protein’s ability to repress promoters with little sequence or structural homology suggests inhibition of transcription due to interference with basal transcription factors, including TBP and TAFs. The N- and C-terminal domains of p53 interact with the conserved carboxy terminus of TBP [18]. The interfering effect of p53as and p53r is most likely due to protein–protein interaction either blocking the DNA binding domain of TBP or causing a conformational change of TBP. It has been demonstrated that wild-type, but not mutant, p53

can interfere with TBP binding to its consensus TATA motif [35] and can repress transcription from many TATA-containing promoters [45]. While not as pronounced an effect, p53 also has the ability to inhibit transcriptional activity of TATA-less promoter sequences. The inability of overexpressed TBP to overcome repression of transcription from a TATA-containing promoter [62] suggests that the association between p53r and p53as and TBP proteins is not solely responsible for the transcriptional repression we observed for the waf-1 promoter. p53 also associates with other TFIID components: TAF II40 and TAF II60, the TBP-associated factors (TAFs) of Drosophila, and their human homologs TAF II31 and TAF II70 [69, 70]. TAFs play an essential role in regulated activation of transcription and in bridging between specific transcriptional activators and the basal transcriptional machinery. Besides TFIID, p53 protein interacts with three subunits (ERCC2, ERCC3, and p62) of the TFIIH complex, leading to modulation of the functions of both p53 and TFIIH. An example of this modulation is demonstrated in in vitro transcription assays where the addition of TFIIH can overcome p53-mediated transcriptional repression [44]. Others have reported that p53 could be detected in TBP–DNA complexes by anti-p53 antibodies [71], but we were unable to detect any p53 protein complexed with TBP–TATA. Discrepancies may be due to differences in oligonucleotide length and sequence or source of proteins. In cells, changes in the ratio of TBP/p53 may occur when p53as or p53r is induced relative to comparatively constant TBP concentrations, resulting in repressive effects dependent on the structural features of particular promoters. Repression or activation effects could vary dependent on whether TATA binding sites or p53 binding sites are present and occupied, affecting DNA conformation. In promoters with a TATA sequence, regardless of whether p53 binding sites are present, repression could occur through the ability of p53 to sequester and interfere with TBP binding. In promoters lacking both p53 and TATA binding sites, p53as or equivalently modified p53r forms could interact with TAFs TF II31 and TF II70, components of TFIIH, or other basal transcription factors to repress transcription. For transcriptional repression by p53 forms to have maximum physiological impact in control of the cellular response to DNA damage, it would potentially have specificity for particular sets of genes involved in growth arrest, DNA repair, or apoptosis. While p53 has been reported to repress transcription of a broad range of growth-related genes and oncogenes, induction of p53-activated genes is not uniform among all cell types or particular p53 inducing stimuli. Recently, we found that recombinant p53r proteins that are inactive for binding to multiple p53 binding sequences and the p53

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We are grateful to Laura Lee and Barbara Lisafeld for technical assistance and Jennifer Carlson and Michelle Bryant for help with the preparation of this manuscript. This work was supported by NCI CA31101, RPCI Core Grant CA16056, a research grant from Taisho Pharmaceutical Co., Ltd., Tokyo, Japan, OHSU Cancer Institute CA69533, and a Tartar Trust Fellowship of OHSU to C. D. Knights.

Chen, X., Farmer, G., Zhu, H., Prywes, R., and Prives, C. (1993). Cooperative DNA binding of p53 with TFIID (TBP): A possible mechanism for transcriptional activation Genes Dev. 7, 1837– 1849; Erratum: 7, 2652.

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