The Tumor Suppressor Protein p53 Requires a Cofactor to Activate Transcriptionally the Human BAX Promoter*

Edward C. Thornborrow and James J. ManfrediDagger

From The Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029

Received for publication, December 22, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An important regulator of the proapoptotic BAX is the tumor suppressor protein p53. Unlike the p21 gene, in which p53-dependent transcriptional activation is mediated by a response element containing two consensus p53 half-sites, it previously was reported that activation of the BAX element by p53 requires additional sequences. Here, it is demonstrated that the minimal BAX response element capable of mediating p53-dependent transcriptional activation consists of two p53 half-sites plus an adjacent 6 base pairs (5'-GGGCGT-3'). This GC-rich region constitutes a "GC box" capable both of binding members of the Sp family of transcription factors, including Sp1 in vitro, and of conferring Sp1-dependent transcriptional activation on a minimal promoter in cells. Mutations within this GC box abrogated the ability of p53 to activate transcription without affecting the affinity of p53 for its binding site, demonstrating that these 6 bases are required for p53-dependent activation. In addition, a positive correlation was observed between the ability of p53 to activate transcription in cells and the ability of Sp1 to bind this response element in vitro. Mutations that inhibited Sp1 binding also blocked the ability of p53 to activate transcription through this element. Together, these results suggest a model in which p53 requires the cooperation of Sp1 or a Sp1-like factor to mediate transcriptional activation of the human BAX promoter.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The BCL-2 family of proteins are key mediators of the apoptotic response. One member of this family is the proapoptotic BAX. Preceding apoptosis, cytosolic BAX translocates to the mitochondria and homodimerizes. Homodimeric BAX then is thought to cause the release of cytochrome c (1-3) which subsequently functions as a coactivator of Apaf-1 in the cleavage of pro-caspase-9, initiating programmed cell death (4). BAX exists in equilibrium with two of its homologs, BCL-2 and BCL-XL. Unlike BAX, these two homologs exert antiapoptotic effects by heterodimerizing with BAX in the mitochondria, blocking its ability to release cytochrome c (5, 6). Thus, an important determinant of the apoptotic response of a cell is the balance between the levels of BAX and BCL-2/BCL-XL. In this regard, regulation of the level of expression of BAX protein is key.

An important regulator of BAX gene expression is the tumor suppressor protein p53 (7, 8). The p53 protein has been implicated in several growth-related pathways, including apoptosis and cell cycle arrest (9, 10). The ability of p53 to function as a sequence-specific DNA-binding protein appears to be central to its role as a tumor suppressor (11, 12). At its amino terminus, the protein contains a potent transcriptional activation domain (13) that is linked to a central core domain that mediates sequence-specific DNA binding (14-16). Both of these domains have been shown to be important for p53-mediated growth suppression (17).

A DNA consensus sequence through which p53 binds and activates transcription has been identified. This sequence consists of two palindromic decamers of 5'-RRRCWWGYYY-3' (where R is a purine; Y is a pyrimidine; and W is an adenine or thymine) separated by 0-13 base pairs, forming four repeats of the pentamer 5'-RRRCW-3' alternating between the top and bottom strands of the DNA duplex (18, 19). Through sequences similar to this consensus, p53 has been shown to activate the transcription of many genes, including BAX, p21, mdm2, gadd45, IGF-BP3, and cyclin G (8, 20-26). When compared with alternate p53 targets, studies demonstrate that the BAX gene is differentially regulated by wild-type p53 in a cell type-specific manner (7, 27, 28). In the mouse, p53-dependent regulation of BAX expression following ionizing radiation is seen in the prostate, thymus, spleen, small intestine, and lung, as well as sympathetic, Purkinje, and olfactory cortical neurons. In the kidney, heart, liver, and brain, however, no p53-dependent regulation of BAX is observed (7, 27). Furthermore, the myeloid leukemia ML-1, Burkitt's lymphoma WMN and AG876, and lymphoblastoid NL2 and FWL cell lines induce BAX following ionizing radiation, whereas the fibroblast AG1522 and WI38, colorectal carcinoma RKO, and osteosarcoma U2-OS cell lines fail to do so (28). In addition, several tumor-derived p53 mutants have been identified that are capable of activating transcription through the promoter of the p21 gene but not through the BAX promoter (29-32). This correlates with an inability of these mutants to trigger apoptosis (29, 31, 32), suggesting that a failure in the ability of p53 to transactivate the BAX gene may play an important role in tumor formation and progression. Supporting this, Yin et al. (33) demonstrated that BAX is an obligatory downstream effector for the p53-mediated apoptosis that attenuates choroid plexus tumor growth in the TgT121 mouse model. Thus, a complete understanding of the transcriptional regulation of the BAX promoter by p53 may yield important information relevant to our understanding of tumorigenesis.

Here is presented a detailed analysis of the p53 response element located in the promoter of the human BAX gene. The minimal BAX response element capable of mediating p53-dependent transcriptional activation is found to consist of two p53 half-sites plus an adjacent 6 base pairs (5'-GGGCGT-3') that demonstrate sequence-specific binding to the transcription factor Sp1. Mutational analysis of this "GC box" shows it to be required for p53-dependent activation, and a positive correlation between the ability of p53 to activate transcription in cells and the ability of Sp1 to bind this response element in vitro is observed. These results are consistent with a model in which p53 requires the cooperation of Sp1 or a Sp1-like factor to mediate transcriptional activation of the human BAX gene. This presents the intriguing possibility that regulation of this cofactor may represent a novel basis for the cell type-specific control of the proapoptotic BAX by wild-type p53.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells-- The osteosarcoma Saos-2 cell line was maintained in a humidified tissue culture incubator at 37 °C with 5% CO2. Cells were grown in Dulbecco's modified Eagle's medium, containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Drosophila SL2 cells were cultured at 25 °C in Schneider's Drosophila medium, containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Oligonucleotides-- For use in electrophoretic mobility shift assays and for subsequent cloning into luciferase reporter plasmids, complementary single-stranded oligonucleotides were annealed to produce double-stranded oligonucleotides with the following indicated sequences: BAX -113/-77, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGGGCTATATTGCTAGCGAATT; BAX -113/-83, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGGGCGCTAGCGAATT; BAX -113/-92, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGCTAGCGAATT; BAX -102/-83, AATTCGGTACCAGACAAGCCTGGGCGTGGGCGCTAGCGAATT; BAX-113/-83(sc -102/-93), AATTCGGTACCTCACAAGTTAGCTCACCTAAGGGGCGTGGGCGCTAGCGAATT; BAX(-113/-93)3, AATTCGGTACCTCACAAGTTAGAGACAAGCCTCACTGGTCACAAGTTAGAGACAAGCCTCACTGGTCACAAGTTAGAGACAAGCCTGCTAGCGAATT; BAX -92/-83, AATTCGGTACCGGGCGTGGGCGCTAGCGAATT; p21 5', AATTCGGTACCGAACATGTCCCAACATGTTGGCTAGCGAATT; BAX/p21 5' hybrid, AATTCGGTACCAGACAAGCCTCAACATGTTGGCTAGCGAATT; p21 5'/BAX hybrid, AATTCGGTACCGAACATGTCCGGGCGTGGGCGCTAGCGAATT; Sp1 consensus, ATTCGATCGGGGCGGGGCGAGC; BAXGG-92/-91AA, AATTCGGTACCTCACAAGTTAGAGACAAGCCTAAGCGTGGGCGCTAGCGAATT; BAXGG-85/-84AA, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGAACGCTAGCGAATT; BAXG-92A, AATTCGGTACCTCACAAGTTAGAGACAAGCCTAGGCGTGGGCGCTAGCGAATT; BAXG-91A, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGAGCGTGGGCGCTAGCGAATT; BAXG-90A, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGACGTGGGCGCTAGCGAATT; BAXC-89A, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGAGTGGGCGCTAGCGAATT; BAXG-88A, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCATGGGCGCTAGCGAATT; BAXT-87G, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGGGGGCGCTAGCGAATT; BAXG-86T, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTTGGCGCTAGCGAATT; BAXG-85T, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGTGCGCTAGCGAATT; BAXG-84T, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGGTCGCTAGCGAATT; BAXC-83A, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTGGGAGCTAGCGAATT; BAX sc -92/-83, AATTCGGTACCTCACAAGTTAGAGACAAGCCTTTTATGTTTAGCTAGCGAATT; BAX sc -86/-83, AATTCGGTACCTCACAAGTTAGAGACAAGCCTGGGCGTTTTAGCTAGCGAATT.

Plasmids-- The following synthetic double-stranded oligonucleotides were digested with KpnI and NheI and cloned into pGL3-E1bTATA (34), which also had been double-digested with KpnI and NheI to produce pTATA vectors with corresponding names: BAX -113/-83, BAX -113/-93, BAX -102/-83, BAX -113/-83(sc -102/-93), BAX(-113/-93)3, BAX -92/-83, BAX -133/-77, p21 5', BAXGG-92/-91AA, BAXGG-85/-84AA, BAXG-92A, BAXG-91A, BAXG-90A, BAXC-89A, BAXG-88A, BAXT-87G, BAXG-86T, BAXG-85T, BAXG-84T, BAXC-83A, BAX sc -92/-83, BAX sc -86/-83. pBAX -315/+51, pBAX -127/+51, and pBAX -76/+51 were generated by PCR1 amplification of the appropriate fragments from the original pBAX luciferase reporter plasmid (8). Upstream primers were engineered with the NheI restriction site. Downstream primers contained the HindIII restriction site. Following PCR, products were digested with both NheI and HindIII and cloned into pGL3-E1bTATA which was also double-digested with NheI and HindIII, removing the adenovirus E1b minimal promoter. To construct pBAXDelta -126/-77, PCR amplification of the original pBAX was used to generate two fragments corresponding to -315 to -127 and to -76 to +51 from the start site of transcription. The -315 to -127 fragment was engineered to contain the NheI restriction site on the upstream side and the SacI restriction site on the downstream side. The -76 to +51 fragment was engineered to contain the SacI site upstream and the HindIII site downstream. Following PCR amplification each fragment was double-digested with the appropriate restriction enzymes (NheI and SacI or SacI and HindIII). A three-way ligation with the two PCR-generated fragments and pGL3-E1bTATA, double-digested with NheI and HindIII, then was performed, replacing the BAX sequence from -126 to -77 with the SacI restriction site. To construct pBAXDelta -113/-104, PCR amplification of the original pBAX was used to generate two fragments corresponding to -315 to -114 and to -103 to +51 from the start site of transcription. The -315 to -114 fragment was engineered to contain the NheI restriction site on the upstream side and the NcoI restriction site on the downstream side. The -103 to +51 fragment was engineered to contain the NcoI site upstream and the HindIII site downstream. Following PCR amplification each fragment was double-digested with the appropriate restriction enzymes (NheI and NcoI or NcoI and HindIII). A three-way ligation with the two PCR-generated fragments and pGL3-E1bTATA, double-digested with NheI and HindIII, then was performed, replacing the BAX sequence from -113 to -104 with the NcoI restriction site. The generation of pBAXDelta -103/-93, pBAXDelta -92/-83, and pBAXDelta -113/-93 was accomplished as above with pBAXDelta -113/-104 but using PCR-generated fragments corresponding to -315 to -104 and -92 to +51, -315 to -93 and -82 to +51, and -315 to -114 and -92 to +51, respectively. The expression plasmid pCMV-p53wt, originally referred to as pC53-SN3 (35), encodes the wild-type human p53 protein under the control of the cytomegalovirus promoter. The expression plasmid pPacSp1 contains the 2.1-kilobase pair XhoI restriction fragment of Sp1 cloned downstream of the Actin 5C promoter (36). pPacU was generated by removing the 2.1-kilobase pair XhoI fragment from pPacSp1.

Transfections-- Saos-2 cells were transfected using LipofectAMINE Plus Reagent (Life Technologies, Inc.). 2 × 105 cells were seeded into 35-mm plates. Cells were transfected 24 h later according to the manufacturer's instructions. Cellular lysates were prepared 24 h post-transfection, and total protein concentration was determined by protein assay (Bio-Rad), and luciferase assays were quantitated using a commercially available kit (Promega) and a TD-20e Luminometer (Turner). Drosophila SL2 cells were transfected using Cellfectin (Life Technologies, Inc.). 60-mm dishes were seeded with 2 × 106 cells in Schneider's Drosophila media containing 10% heat-inactivated fetal bovine serum but no penicillin or streptomycin. The DNA to be transfected was added to 500 µl of serum-free media containing 8 µl of Cellfectin reagent, mixed gently, and incubated at room temperature for 20 min. This mixture then was added directly to the cells. 48 h post-transfection cells were lysed by sonication (6 × 20 s pulse). Total protein and luciferase activity was determined as above.

HeLa Cell Nuclear Extraction-- Unless otherwise stated, all procedures were conducted at 4 °C. HeLa S3 cells were obtained as a packed cell pellet from the National Cell Culture Center (Minneapolis, MN). Cell pellets were resuspended in 5 volumes of Buffer A (10 mM HEPES, pH 7.6, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) and incubated on ice for 10 min. Cells then were centrifuged at 500 × g for 12 min. The supernatant was removed, and the pellet was resuspended in two packed cell volumes of Buffer A. Cells were homogenized 10 times in a Dounce homogenizer with pestle A (tight). The resulting solution was centrifuged at 430 × g for 10 min to pellet the nuclei. The supernatant was decanted, and the pellet was recentrifuged at 24,000 × g for 20 min. The supernatant again was removed. The pellet was resuspended in 3 ml of Buffer C (20 mM HEPES, pH 7.6, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT) per 109 cells. The solution was homogenized 10 times with pestle B (loose). The resulting solution was transferred to a beaker and stirred for 30 min on ice. The solution then was centrifuged at 24,000 × g for 30 min. The resulting nuclear extract was dialyzed against Buffer D (20 mM HEPES, pH 7.6, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT) for 5 h. The extract was clarified by centrifugation at 24,000 × g for 20 min. Nuclear extracts were aliquoted, frozen in a dry ice/ethanol bath, and stored at -70 °C.

Electrophoretic Mobility Shift Assay-- Production of baculovirus-infected Sf9 cell extracts and purification of recombinant human p53 protein were done as described previously (34). Purified p53 protein, extract from Sf9 cells expressing recombinant human Sp1 protein, or HeLa cell nuclear extract was incubated with 3 ng of radiolabeled double-stranded oligonucleotide and antibody (Sp1 PEP-2X, p300 N-15X, and CBP 451X, Santa Cruz Biotechnology), where appropriate, in a total volume of 30 µl of DNA binding buffer (20 mM HEPES, pH 7.5, 83 mM NaCl, 0.1 mM EDTA, 12% glycerol, 2 mM MgCl2, 2 mM spermidine, 0.7 mM DTT, and 17 µg/ml poly(dI-dC)) for 20 min at room temperature. Samples were loaded on a native 4% acrylamide gel in 0.5× TBE and electrophoresed at 4 °C at 225 V for 2 h. The gel was dried and exposed to Kodak XAR film using an intensifying screen at -70 °C. Phosphorimaging and densitometry data were collected with a Personal Molecular Imager FX and a GS-710 Calibrated Imaging Densitometer (Bio-Rad), and analyzed with Quantity One software (Bio-Rad).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

All Three Potential p53 Half-sites Are Required for the p53-dependent Transcriptional Activation of the Human BAX Promoter-- Previously it was demonstrated that in isolation the p53 response element from the human BAX promoter required sequences from three adjacent half-sites to confer p53-dependent transcriptional activation on a minimal promoter (37). To confirm the requirement of all three half-sites in the context of the BAX promoter, luciferase reporter plasmids with various deletions in the BAX promoter, both in and around the p53 response element, were cotransfected with either pCMV or a wild-type p53 expression vector into the p53-negative osteosarcoma Saos-2 cell line (Fig. 1). The previously characterized p53 response element of the BAX promoter is contained within the sequence from -113 to -83 from the start site of transcription. There was no significant difference between the p53-dependent transactivation of either a reporter construct lacking sequences 5' to the p53 response element (pBAX -127/+51) or the full-length promoter construct (pBAX -315/+51)(Fig. 1A). Deletion of a larger fragment, including the p53 response element (pBAX -76/+51), produced a reporter construct that was unresponsive to wild-type p53 (Fig. 1A). Furthermore, targeted deletion of the promoter region containing the p53 response element (pBAXDelta -126/-77) also produced a reporter plasmid that was unresponsive to wild-type p53 (Fig. 1A). These results show that -113 to -83 is the only region, within the 366-base pair promoter fragment investigated, that affects the ability of p53 to activate transcription.


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Fig. 1.   All three potential p53 half-sites are required for the p53-dependent transcriptional activation of the human BAX promoter. A and B, Saos-2 cells were transfected as described under "Experimental Procedures" with 1 µg of the indicated pBAX reporter constructs in the presence of either 10 ng of pCMV (white bars) or 10 ng of pCMV-p53wt (gray bars). 24 h post-transfection cells were lysed and assayed for total protein and luciferase activity as described under "Experimental Procedures." The indicated values are the average of three independent experiments each performed in duplicate. Error bars correspond to one S.D. The numbers above each bar indicate the fold activation for each reporter construct observed with pCMV-p53wt as compared with pCMV. A, the previously identified p53 response element is indicated by the dark gray box at -113 to -83. B, the three potential p53 half-sites are represented by the light gray (-113 to -104), white (-102 to -93), and dark gray (-92 to -83) boxes.

The region from -113 to -83 contains three potential p53 half-sites (represented in Fig. 1B as the light gray, white, and dark gray boxes). The role of each of these half-sites in the p53-dependent activation of the BAX promoter was examined. Removal of the first half-site from -113 to -104 (pBAXDelta -113/-104) significantly reduced the ability of p53 to activate transcription through this promoter (Fig. 1B, compare 63-fold with pBAX -315/+51 to 7-fold with pBAXDelta -113/-104), whereas removal of the second (pBAXDelta -103/-93) or the third half-site (pBAXDelta -92/-83) completely abolished the ability of p53 to activate transcriptionally the promoter (Fig. 1B). Consistent with the above results, removal of the first and second half-sites in combination (pBAXDelta -113/-93) also abolished the ability of p53 to activate transcriptionally the promoter (Fig. 1B). These results demonstrate that, as was observed with the isolated response element (37), p53 requires sequences from all three potential half-sites to mediate transcriptional activation of the BAX promoter.

The First Two Potential p53 Half-sites Constitute a Bona Fide p53 Response Element-- Each of the three potential p53 half-sites located in the BAX promoter from -113 to -83 closely resembles the consensus sequence of 5'-RRRCWWGYYY-3' (represented in Fig. 2 by the light gray, white, and dark gray boxes). The first, located at -113 to -104, deviates from the consensus at 2 bases (-113 and -104). The second half-site matches the consensus sequence at all 20 base pairs and is located at -102 to -93. The third half-site is located at -92 to -83 and deviates from the consensus at three bases (-84, -85, and -88)(see Fig. 2). These three half-sites can combine in different ways to produce a total of three possible p53 complete binding sites (half-sites 1 and 2, 2 and 3, and 1 and 3). Previous studies demonstrated that in electrophoretic mobility shift assays (EMSA), double-stranded oligonucleotides representing both -113 to -93 (half-sites 1 and 2) and -102 to -83 (half-sites 2 and 3) are capable of binding p53 in a sequence-specific manner with similar affinities (37). When cloned upstream of the adenovirus E1b minimal promoter in the pTATA luciferase reporter plasmid, however, the combination of the first and second half-sites (-113 to -93) is unable to mediate p53-dependent transcriptional activation (37). To examine further the ability of p53 to interact with this sequence in cells, the -113 to -93 sequence was multimerized (as three copies) and cloned into the pTATA luciferase reporter plasmid. This reporter plasmid was cotransfected with either pCMV or a wild-type p53 expression vector in the Saos-2 cell line (Fig. 2). These three copies of this p53-binding site were capable of mediating a significant degree of activation in response to p53 (Fig. 2, compare 4-fold with pTATA-113/-93 to 142-fold with pTATA(-113/-93)3), demonstrating that the sequence from -113 to -93 is indeed a bona fide p53 response element capable of both binding p53 in a sequence-specific manner in vitro and mediating p53-dependent transcriptional activation in cells. Confirming previous results, p53 was able to activate transcription through the second and third half-sites (-102 to -83), but this activation was significantly reduced as compared with that mediated by all three half-sites combined (Fig. 2, compare 44-fold with pTATA-102/-83 and 153-fold with pTATA-113/-83). To test the ability of half-sites one and three to mediate p53-dependent transcriptional activation, a synthetic oligonucleotide corresponding to -113 to -83 of the BAX promoter, with -102 to -93 scrambled to remove any contribution of the second half-site, was cloned into the pTATA reporter plasmid. This construct failed to be activated by p53 (Fig. 2, pTATA-113/-83(sc -102/-93)). The third half-site in isolation (-92 to -83) also failed to mediate p53-dependent transcriptional activation (Fig. 2, pTATA-92/-83).


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Fig. 2.   The first two potential p53 half-sites constitute a bona fide p53 response element. Saos-2 cells were transfected as described under "Experimental Procedures" with 1 µg of the indicated pTATA reporter constructs in the presence of either 10 ng of pCMV (white bars) or 10 ng of pCMV-p53wt (gray bars). 24 h post-transfection cells were lysed and assayed for total protein and luciferase activity as described under "Experimental Procedures." The indicated values are the average of three independent experiments each performed in duplicate. Error bars correspond to 1 S.D. The numbers above each bar indicate the fold activation for each reporter construct observed with pCMV-p53wt as compared with pCMV. The sequence of the BAX promoter from -113 to -83 is given at the top of the figure. Potential p53 quarter-sites are indicated by the solid bars above and below the sequence. Bases that deviate from the p53 DNA-binding consensus sequence are indicated by asterisks. The three potential half-sites are indicated by the brackets labeled 1-3, respectively, and are represented graphically as the light gray, white, and dark gray boxes, respectively. The vertical arrow above the BAX sequence indicates the 1-base pair insert between the first and second half-sites.

Sp1 Binds with Sequence Specificity to and Activates Transcription through the p53 Response Element from the Human BAX Promoter-- We previously reported the identification of a nuclear factor, termed Binder of BAX 1 (BoB1), that interacts with sequence specificity with the same region of the human BAX promoter that is required for p53-dependent transcriptional activation (37). These previous studies demonstrated that this factor binds to sequences within the region of -102 to -83. Analysis of this region using a MatInspector search of the TRANSFAC data base (38, 39) showed that it contains a sequence that potentially could bind the transcription factor Sp1. To test this, a synthetic oligonucleotide corresponding to -102 to -83 of the BAX promoter was used as a radiolabeled probe in an EMSA with HeLa cell nuclear extract (Fig. 3). As reported previously for Saos-2 (37), HeLa cell nuclear extract contains a factor that demonstrated marked sequence specificity for the labeled BAX probe. This factor was successfully competed by increasing amounts of unlabeled probe (Fig. 3, lanes 7-9) as well as by increasing amounts of oligonucleotide corresponding to the DNA-binding consensus sequence of Sp1 (Fig. 3, lanes 13-15). This binding was specific, as an oligonucleotide corresponding to the 5' p53 response element from the human p21 promoter failed to compete for binding (Fig. 3, lanes 10-12). In addition, this factor was successfully bound by an anti-Sp1 antibody, as demonstrated by a "supershifted" complex (Fig. 3, lanes 2 and 3), whereas a control anti-p300 antibody failed to bind the factor (Fig. 3, lanes 4 and 5). Together, these data demonstrate that Sp1 can bind a portion of the p53 response element from the human BAX promoter in a sequence-specific manner.


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Fig. 3.   Sp1 binds with sequence specificity to the p53 response element from the human BAX promoter. An electrophoretic mobility shift assay was performed using an oligonucleotide corresponding to the -102/-83 sequence from the human BAX promoter as radiolabeled probe. 8 µg of HeLa cell nuclear extract was incubated with 3 ng of the probe alone (lanes 1 and 6), in the presence of 2 or 4 µl of anti-Sp1 antibody (Ab) (lanes 2 and 3, respectively), 2 or 4 µl of anti-p300 antibody (lanes 4 and 5, respectively), a 100-, 200-, or 300-fold molar excess of either the unlabeled BAX -102/-83 oligonucleotide (lanes 7-9) or p21 5' oligonucleotide (lanes 10-12), or a 10-, 20-, or 30-fold molar excess of the unlabeled Sp1 consensus oligonucleotide (lanes 13-15). The arrows indicate the positions of the Sp1-DNA complex and the supershifted complex containing antibody, Sp1, and DNA.

To delineate further the sequences important for Sp1 binding, oligonucleotides were synthesized that replaced portions of the BAX sequence with corresponding sequence from the p21 5' p53 response element. The sequence from -102 to -83 in the BAX promoter contains two p53 half-sites (-102 to -93 and -92 to -83), and the p21 5' element also consists of two p53 half-sites. Hybrid oligonucleotides were synthesized in which the first of the two half-sites in the BAX element was combined with the second half-site of the p21 5' element and vice versa. The oligonucleotide corresponding to -102 to -83 of the BAX promoter again was used as a radiolabeled probe with HeLa nuclear extract in an EMSA (Fig. 4). Competitions, using unlabeled probe as well as the oligonucleotides corresponding to the p21 5' element and the two hybrid elements, were conducted. Sp1 bound the radiolabeled probe (Fig. 4, lane 1) and was recognized by an anti-Sp1 antibody (Fig. 4, lane 2) but not by a control anti-CBP antibody (Fig. 4, lane 3). Both unlabeled probe and the Sp1 DNA-binding consensus site oligonucleotide effectively competed for Sp1 binding (Fig. 4, lanes 4-5 and 12-13, respectively), whereas the p21 5' element did not (Fig. 4, lanes 10-11). Consistent with the notion that Sp1 binds DNA through GC box regions, the hybrid oligonucleotide in which the first half-site is derived from the p21 sequence and the second half-site from the BAX sequence (-92 to -83, 5'-GGGCGTGGGC-3') effectively competed for Sp1 binding (Fig. 4, lanes 8-9), whereas the other hybrid oligonucleotide that replaces this GC-rich region with sequence from the p21 5' element demonstrated a significantly reduced affinity for Sp1 binding (Fig. 4, lanes 6-7). These data indicate that Sp1 binds to sequence within -92 to -83 of the BAX promoter.


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Fig. 4.   The Sp1-binding site is localized to a GC-rich region of the p53 response element. A, an electrophoretic mobility shift assay was performed using an oligonucleotide corresponding to the -102/-83 sequence from the human BAX promoter as radiolabeled probe. 8 µg of HeLa cell nuclear extract was incubated with 3 ng of the probe alone (lane 1), in the presence of 4 µl of anti-Sp1 antibody (Ab) (lane 2), 4 µl of anti-CBP antibody (lane 3), a 100- or 200-fold molar excess of either the unlabeled BAX -102/-83 oligonucleotide (lanes 4 and 5), p21 5' oligonucleotide (lanes 10 and 11), BAX/p21 5' hybrid oligonucleotide (lanes 6 and 7) or p21 5'/BAX hybrid oligonucleotide (lanes 8 and 9), or a 10- or 20-fold molar excess of the unlabeled Sp1 consensus oligonucleotide (lanes 12 and 13). The arrows indicate the positions of the Sp1-DNA and the supershifted antibody-Sp1-DNA complexes. B, the sequences of the human BAX promoter from -102 to -83 from the start site of transcription (gray boxes) and the human p21 promoter from -2281 to -2262 from the start site of transcription (white boxes; corresponding to the p21 5' oligonucleotide) are shown. Each sequence is divided into two with the first half indicated as A and the second half indicated as B. Oligonucleotides in A are represented graphically according to this color and letter scheme. For example, the BAX/p21 5' hybrid oligonucleotide that corresponds to the first half of the BAX sequence followed by the second half of the p21 sequence is indicated by a gray box labeled A followed by a white box labeled B.

To determine whether or not Sp1 can interact with this element in cells, a pTATA luciferase reporter plasmid containing -113 to -77 of the human BAX promoter was cotransfected with increasing amounts of an Sp1 expression vector into the Sp1-deficient Drosophila SL2 cell line (Fig. 5). Expression of Sp1 successfully activated transcription of this reporter and yet failed to activate transcription of a control plasmid containing the 5' p53 response element of the p21 promoter (Fig. 5). Consistent with the in vitro EMSA results, this confirms that Sp1 is capable of activating transcription through the p53 response element of the human BAX promoter.


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Fig. 5.   Sp1 can activate transcription through the p53 response element of the human BAX promoter. Drosophila SL2 cells were transfected as described under "Experimental Procedures" with 2 µg of the indicated pTATA reporter constructs in the presence of 0, 300, 600, or 900 ng of pPacSp1. Appropriate amounts of the vector pPacU were added to each transfection mixture to maintain a constant level of total plasmid DNA of 2.9 µg/sample. 48 h post-transfection cells were lysed and assayed for total protein and luciferase activity as described under "Experimental Procedures." The indicated values are the average of four independent experiments expressed as the fold activation for each reporter plasmid with pPacSp1 as compared with pPacU. Error bars correspond to 1 S.D.

The Ability of Sp1 to Bind the p53 Response Element of the BAX Promoter in Vitro Correlates with the Ability of p53 to Activate Transcription through This Element in Cells-- To explore the significance of the Sp1-binding site to the ability of p53 to activate transcription through the BAX promoter, nucleotide substitutions were identified that differentially affected the ability of p53 to activate transcription through its response element in the BAX promoter (-113 to -83). Two mutated forms of the p53 response element from the BAX promoter, in which the indicated guanine bases were replaced with adenines (Fig. 6A, GG-92/-91AA and GG-85/-84AA), were cloned into the pTATA luciferase reporter plasmid. In cotransfection assays with a wild-type p53 expression vector in the Saos-2 cell line, substitution of bases -92 and -91 completely abolished the ability of p53 to activate transcription through this element (Fig. 6A, compare -113/-83 to GG-92/-91AA), whereas substitution of bases -85 and -84 did not (Fig. 6A). As observed in Figs. 1 and 2, removal of the third potential half-site (-92 to -83) inhibited the ability of p53 to mediate transcriptional activation through this element (Fig. 6A, compare -113/-83 and -113/-93), demonstrating the requirement for this Sp1-binding sequence in the p53-dependent transcriptional activation of this element.


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Fig. 6.   A mutant element that fails to bind Sp1 in vitro also fails to confer p53-dependent transcriptional activation in cells. A, Saos-2 cells were transfected as described under "Experimental Procedures" with 1 µg of the indicated pTATA reporter constructs in the presence of either 10 ng of pCMV (white bars) or 10 ng of pCMV-p53wt (gray bars). 24 h post-transfection cells were lysed and assayed for total protein and luciferase activity as described under "Experimental Procedures." The indicated values are the average of five independent experiments each performed in duplicate. Error bars correspond to 1 S.D. The GC-rich region that binds Sp1 is shown by the boxed sequence. Bases in the wild-type sequence that were mutated are shown in gray with the corresponding mutations indicated above. B, an electrophoretic mobility shift assay was performed using an oligonucleotide corresponding to the -113/-77 sequence from the human BAX promoter as radiolabeled probe. 50 ng of purified p53 was incubated with 3 ng of the probe alone (lane 1) or in the presence of a 500-, 1000-, or 1500-fold molar excess of either the unlabeled BAX -113/-83 oligonucleotide (lanes 2-4), the GG-92/-91AA oligonucleotide (lanes 5-7), or the GG-85/-84AA oligonucleotide (lanes 8-10). The arrow indicates the position of the p53-DNA complex. The vertical bar between lanes 4 and 5 represents the removal of irrelevant lanes from the gel. C, bands were quantitated by densitometry. D, an electrophoretic mobility shift assay was performed using an oligonucleotide corresponding to the -113/-77 sequence from the human BAX promoter as radiolabeled probe. Extract from Sf9 cells expressing human recombinant Sp1 protein was incubated with 3 ng of the probe alone (lane 2), in the presence of anti-Sp1 antibody (lane 1), or in the presence of a 50-, 100-, or 200-fold molar excess of either the unlabeled BAX -113/-83 oligonucleotide (lanes 3-5), the GG-92/-91AA oligonucleotide (lanes 6-8), or the GG-85/-84AA oligonucleotide (lanes 9-11). The arrow indicates the position of the Sp1-DNA complex, and the asterisk indicates the position of the supershifted anti-Sp1-DNA complex. The vertical bar between lanes 5 and 6 represents the removal of irrelevant lanes from the gel. E, bands were quantitated by densitometry.

Both of these mutant sequences were assayed for their ability to bind purified p53 in an EMSA. An oligonucleotide corresponding to -113 to -77 of the BAX promoter was used as a radiolabeled probe with purified p53 in an EMSA (Fig. 6B). Competitions were performed with increasing amounts of an oligonucleotide corresponding to -113 to -83 of the BAX promoter and the two mutant oligonucleotides. When compared with the wild-type oligonucleotide, both mutant oligonucleotides displayed a slightly decreased affinity for p53 (Fig. 6B, compare lanes 2-4 with lanes 5-7 and 8-10; Fig. 6C). Compared with one another, however, both mutant oligonucleotides demonstrated a comparable affinity for p53 (Fig. 6, A and B), suggesting that the differences in p53-dependent transcriptional activation observed in Fig. 6A are not due to differences in the affinity of p53 for the two sequences. In contrast, the abilities of the two mutant sequences to bind Sp1 differed (Fig. 6, D and E). An oligonucleotide corresponding to -113 to -77 of the BAX promoter was used as radiolabeled probe with extract from Sf9 cells expressing recombinant human Sp1 protein in an EMSA (Fig. 6D). Sp1 bound the probe and was recognized by an anti-Sp1 antibody (Fig. 6D, lanes 1-2). Sp1 binding was successfully competed by unlabeled BAX -113/-83 oligonucleotide as well as by the GG-85/-84AA mutated oligonucleotide (Fig. 6D, lanes 3-4 and 9-11, respectively; Fig. 6E). The GG-92/-91AA mutant, however, demonstrated a significant decrease in affinity for Sp1 (Fig. 6D, compare lanes 3-5 and 9-11 to lanes 6-8; Fig. 6E).

The results with the GG-85/-84AA mutant presented in Fig. 6 suggest that not all of the bases contained within the third potential half-site of the p53 response element are required for p53-dependent transcriptional activation. To identify the minimal sequence elements required to mediate p53-dependent transactivation, a series of oligonucleotides was synthesized in which each of the 10 bases of the third potential half-site (-92 to -83) was individually replaced. These mutant oligonucleotides then were cloned into the pTATA luciferase reporter plasmid and tested for their responsiveness to p53 in a cotransfection assay in the Saos-2 cell line (Fig. 7). Consistent with the results in Fig. 6A, substitution of the bases at either -85 or -84 did not inhibit the ability of p53 to activate transcription through this element (Fig. 7, G-85T and G-84T). Furthermore, substitution of -86 and -83 also failed to affect significantly the ability of p53 to activate transcription (Fig. 7, compare -113/-83 to G-86T and C-83A). Substitution of the base at -87, however, significantly reduced the ability of p53 to activate transcription through this element (Fig. 7, compare -113/-83 to T-87G). Together, these results suggest that the minimal response element consists of sequence from -113 to -87, with -86 to -83 being dispensable for p53-dependent transactivation.


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Fig. 7.   Mutational analysis shows that the BAX promoter sequence from -86 to -83 is not required for p53-dependent transcriptional activation. Saos-2 cells were transfected as described under "Experimental Procedures" with 1 µg of the indicated pTATA reporter constructs in the presence of either 10 ng of pCMV (white bars) or 10 ng of pCMV-p53wt (gray bars). 24 h post-transfection cells were lysed and assayed for total protein and luciferase activity as described under "Experimental Procedures." The indicated values are the average of three independent experiments each performed in duplicate. Error bars correspond to 1 S.D. The GC-rich region that binds Sp1 is shown by the boxed sequence. Bases in the wild-type sequence that were mutated are shown in gray with the corresponding mutations indicated above.

To confirm that the bases from -86 to -83 are not required for p53-dependent transcriptional activation, two additional mutant oligonucleotides were synthesized. The first mutant was generated by replacing all 10 nucleotides from -92 to -83 (Fig. 8A, sc-92/-83). The 4 bases from -86 to -83 were substituted as indicated to generate the second mutant oligonucleotide (Fig. 8A, sc-86/-83). Each oligonucleotide was cloned into the pTATA vector and tested for its responsiveness to p53 in a cotransfection assay (Fig. 8A). As observed with the reporter plasmid in which the sequence from -92 to -83 is removed entirely (pTATA-113/-93), the first mutant, in which all 10 bases of the third potential half-site (-92 to -83) are replaced, showed little to no response to p53 (Fig. 8A, compare pTATA-113/-93 to pTATA-113/-83 and pTATAsc-92/-83). In contrast, the second mutant, in which only the last 4 bases of the element (-86 to -83) are replaced, was efficiently activated by p53 (Fig. 8A, compare 312-fold with pTATA-113/-83 to 323-fold with pTATAsc-86/-83). This result demonstrates that the minimal p53 response element in the BAX promoter consists of sequence from -113 to -87. In an EMSA both mutants displayed a decreased affinity for p53 as compared with the wild-type sequence (Fig. 8B, compare lanes 2-4 to lanes 8-10 and 11-13; Fig. 8C). When compared with each other, there was no significant difference in the affinity of p53 for the two mutant sequences (Fig. 8B, compare lanes 8-10 to 11-13; Fig. 8C). This suggests that the differences in transcriptional activation observed in Fig. 8A cannot be explained by differences in p53 affinities. Furthermore, the oligonucleotide corresponding to -113 to -93 displayed a similar affinity for p53 as the two mutant oligonucleotides (Fig. 8B, compare lanes 5-7 to lanes 8-10 and 11-13; Fig. 8C) consistent with the idea that, in the case of the two mutants, p53 is interacting with the first and the second half-sites only. The sc -86/-83 mutant oligonucleotide efficiently competed for Sp1 binding in an EMSA (Fig. 8D, compare lanes 2-4 to lanes 8-10; Fig. 8E), whereas the ability of the sc -92/-83 mutant to bind Sp1 was significantly reduced compared with the wild-type sequence (Fig. 8D, compare lanes 2-4 to lanes 5-7; Fig. 8E), further strengthening the correlation between Sp1 binding in vitro and p53 activation in cells.


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Fig. 8.   The minimal element from the BAX promoter that confers p53-dependent transcriptional activation consists of a single p53-binding site and an adjacent Sp1-binding site. A, Saos-2 cells were transfected as described under "Experimental Procedures" with 1 µg of the indicated pTATA reporter constructs in the presence of either 10 ng of pCMV (white bars) or 10 ng of pCMV-p53wt (gray bars). 24 h post-transfection cells were lysed and assayed for total protein and luciferase activity as described under "Experimental Procedures." The indicated values are the average of three independent experiments each performed in duplicate. Error bars correspond to 1 S.D. The numbers above each bar indicate the fold activation for each reporter construct observed with pCMV-p53wt as compared with pCMV. The GC-rich region that binds Sp1 is shown by the boxed sequence. Bases in the wild-type sequence that were mutated are shown in gray with the corresponding mutations indicated above. B, an electrophoretic mobility shift assay was performed using an oligonucleotide corresponding to the -113/-83 sequence from the human BAX promoter as radiolabeled probe. 50 ng of purified p53 was incubated with 3 ng of the probe alone (lane 1) or in the presence of a 500-, 1000-, or 1500-fold molar excess of either the unlabeled BAX -113/-83 oligonucleotide (lanes 2-4), the BAX -113/-93 oligonucleotide, the BAX sc -92/-83 oligonucleotide (lanes 5-7), or the BAX sc -86/-83 oligonucleotide (lanes 8-10). The arrow indicates the position of the p53-DNA complex. C, bands were quantitated by phosphorimaging. D, an electrophoretic mobility shift assay was performed using an oligonucleotide corresponding to the -113/-83 sequence from the human BAX promoter as radiolabeled probe. Extract from Sf9 cells expressing human recombinant Sp1 protein was incubated with 3 ng of the probe alone (lane 1) or in the presence of a 10-, 50-, or 100-fold molar excess of either the unlabeled BAX -113/-83 oligonucleotide (lanes 2-4), the BAX sc -92/-83 oligonucleotide (lanes 5-7), or the BAX sc -86/-83 oligonucleotide (lanes 8-10). The arrow indicates the position of the Sp1-DNA complex. E, bands were quantitated by phosphorimaging.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The data presented in this report demonstrate that the minimum p53 response element in the BAX promoter consists of the sequence from -113 to -87 from the start site of transcription. This sequence contains a p53-binding site (-113 to -93) that can function as a bona fide response element as demonstrated by its ability when multimerized to confer p53-dependent transcriptional activation on a minimal promoter (Fig. 2). Immediately adjacent to this p53-binding site are 6 base pairs that are GC-rich in nature (-92 to -87: 5'-GGGCGT-3'). These 6 bases are required for p53-dependent transcriptional activation as deletion or mutation of this region in the context of either the promoter or the isolated response element completely abrogates the ability of p53 to activate transcription through this sequence (Figs. 1B, 2, 6A, and 8A). The addition of these bases to the -113/-93 sequence appears to have little effect on the affinity of p53 for this sequence (Fig. 8, B and C), consistent with a model in which these 6 bases function to recruit a co-activator as opposed to simply enhancing p53 binding. Furthermore, these 6 base pairs mediate sequence-specific binding to the Sp1 transcription factor (Figs. 3, 4, 6D, and 8D), and a positive correlation is seen between the ability of Sp1 to bind this element in vitro and the ability of p53 to mediate transcriptional activation through its response element in cells (Figs. 6 and 8). In addition, the results with electrophoretic mobility shift assays with the GG-92/-91AA mutant oligonucleotide (Fig. 6B) are not consistent with the published p53 DNA-binding consensus sequence of (RRRCWWGYYY)2 (18, 19). This consensus allows for a purine in the first three positions of each half-site. The GG-92/-91AA mutant contains a conservative substitution of purines (adenines) for purines (guanines) and, as such, does not represent a substantive change in terms of the p53 DNA-binding consensus sequence. This substitution, however, did produce a significant decrease in the ability of p53 to bind to this oligonucleotide in vitro (Fig. 6B), suggesting that, in these limited circumstances, the p53 DNA-binding sequence involves greater specificity than implied by the consensus.

Previous studies have suggested a connection between p53 and Sp1. The two proteins physically interact under certain circumstances (40-42), and, transcriptionally, p53 and Sp1 have been shown to function in a cooperative manner in some settings and an antagonistic manner in others (41, 43, 44). In addition to p53, Sp1 has been found to synergize with other transcription factors, including YY1 and SREBP (45-47). Studies with the Sp family of transcription factors, however, are complicated by the fact that there are at least 16 mammalian members of this family. Due to marked conservation in the DNA-binding domain, many of these family members have similar if not identical in vitro DNA binding characteristics (48, 49). Originally, this led to the misclassification of many GC boxes solely as Sp1-binding sites because of the ubiquitous nature of Sp1 and the fact that it was the first family member cloned. Given this, the possibility exists that the true in vivo cofactor required for the p53-dependent transactivation of the BAX promoter is an Sp1-related family member that is obscured in in vitro assays by the sheer abundance of Sp1 in nuclear extracts from tissue culture cells. Consistent with this, antibodies used in a supershift EMSA identified other Sp family members as minor components of the Sp1-DNA complex.2 Furthermore, cotransfection assays in the Sp1-deficient Drosophila SL2 cell line failed to demonstrate cooperation between Sp1 and p53 in transcriptionally activating the p53 response element of the BAX promoter.2 The Drosophila assays, however, are difficult to interpret as the ability of p53 alone to activate transcription through a control plasmid was significantly impaired in the SL2 cell line. Complicating interpretation of the results in the Drosophila system is the recent identification of a Drosophila p53 homolog (50, 51) that may affect the ability of transiently expressed human p53 to function properly in this system.

Regardless of whether the cofactor required for the p53-dependent transactivation of the BAX promoter is Sp1 or a related family member, the requirement of this cooperating protein suggests a model for the observed cell type- and tumor type-specific regulation of the BAX gene by wild-type p53 (Fig. 9). In this model, cells that are permissive to p53-dependent up-regulation of the BAX gene express both p53 and the cofactor, and these proteins function together to activate transcriptionally the gene. In those cells that fail to show p53-dependent BAX expression, one can propose three possible mechanisms to explain the apparent failure of wild-type p53 to activate the BAX gene (Fig. 9). First, the required cofactor may be absent, either due to mutation or due to cell type-specific limitations on its expression. Second, this factor may be inactivated by post-translational modification. Finally, another factor that cannot cooperate with p53 may compete with the cofactor for binding to its site in the BAX promoter. Data with the Sp family of transcription factors support each of these possibilities. Although several of the Sp family members, like Sp1, are ubiquitously expressed, other members of the family display high degrees of tissue specificity (48, 49). Even the ubiquitously expressed family members fluctuate in levels under particular cellular conditions (52-55). Sp1 mRNA, for example, varies up to 100-fold depending on the cell type and developmental stage of the mouse (56). Consistent with a model of post-translational modification, certain Sp family members, including Sp1 and EKLF, are phosphorylated, glycosylated, and acetylated (57-59). Finally, given the high level of conservation in the DNA-binding domain of the Sp family of transcription factors, it is not surprising that DNA binding competition can be observed between various members of this family. In certain cases, including Sp1/Sp3, BTEB1/AP-2rep, and BKLF/EKLF, this competition has ramifications on gene expression (60-62). In each case, transcriptional activation by one family member is repressed by the other member by competing for the same DNA-binding site. The data in this report, in combination with the previous studies of the Sp family of transcription factors, support a model in which the regulation of a required cofactor controls cell type-specific p53-dependent expression of the BAX gene.


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Fig. 9.   Model for the cell type-specific regulation of the BAX promoter by the tumor suppressor protein p53. A, in cells that are permissive to p53-dependent transcriptional activation of the BAX gene, p53 and the required cofactor cooperate to mediate activation. In cells that do not support the p53-BAX pathway, three possible mechanisms may explain the apparent failure of wild-type p53 to activate the BAX gene. B, the cofactor may be absent due to mutation or to cell type-specific limitations on its expression. C, the cofactor may be inactivated by post-translational modification as follows: P, phosphorylation; G, glycosylation; or A, acetylation. D, another factor that cannot cooperate with p53 may compete with the required cofactor for binding to its site in the BAX promoter. The p53-binding site (-113 to -93) is represented by the black box. The Sp1-binding site (-93 to -87) is represented by the white box. p53, the required cofactor, and the inhibitory factor are represented by the gray circle, the dotted oval, and the cross-hatched triangle, respectively.

The ability of the proapoptotic BAX to function as a tumor suppressor protein has been substantiated by several studies. In certain mouse models, BAX has been shown to be an important mediator of p53-dependent apoptosis and a suppressor of oncogenic transformation, with loss of BAX leading to accelerated rates of tumor growth, increased tumor numbers, larger tumor mass, and decreased survival rates (63, 64). A significant correlation between decreased BAX expression and both a corresponding resistance to apoptotic stimuli, as well as a shorter survival period also have been observed in a number of human tumor types, including breast, ovarian, pancreatic, colorectal, and non-Hodgkin's lymphoma (65-69). In addition, in colon and gastric cancers of the microsatellite mutator phenotype mutational inactivation of the BAX gene has been shown to confer a strong survival advantage during tumor clonal evolution (70). Complimenting these data are observations showing that overexpression of the BAX protein in certain tumor cell lines both sensitizes these cells to chemotherapy- and radiation-induced apoptosis and reduces their ability to form tumors in SCID mice (71-73). Together, these results strongly support a tumor suppressor role for the BAX protein.

An important regulator of the BAX gene is the tumor suppressor protein p53. Several reports have demonstrated the significance of the p53-BAX pathway in tumor suppression. Both the identification of tumor-derived p53 mutants that selectively fail to activate transcription through the BAX promoter and subsequently fail to induce apoptosis (29-32) as well as the TgT121 transgenic studies that demonstrate that BAX is an obligatory downstream effector of p53 in the suppression of choroid plexus tumor growth (33) suggest that the ability of p53 to activate transcription through the BAX promoter is important to the tumor suppressor function of p53. Furthermore, the resistance of certain tumor cell lines to radiation therapy is associated with a failure of wild-type p53 to induce BAX expression (28, 74), and certain human tumors have been identified that are genetically wild-type for both p53 and BAX and yet fail to express significant levels of BAX protein (75). Thus, a complete understanding of the transcriptional regulation of the BAX gene by the tumor suppressor p53 may provide important information concerning both the molecular origins of cancer as well as the development of tumor resistance to certain cancer treatments.

    ACKNOWLEDGEMENTS

We thank Bert Volgelstein (The Johns Hopkins University) for the wild-type p53 expression plasmid; John Reed (Burnham Institute) for the original pBAX reporter construct; Robert Tjian and Andres Naar (University of California at Berkeley) for the pPacSp1 expression plasmid and the recombinant baculovirus expressing human Sp1; and Ze'ev Ronai (Mount Sinai) for the recombinant baculovirus expressing His-tagged human p53. We thank Ron Magnusson for help with the recombinant baculovirus infections. We also thank Lois Resnick-Silverman of the Manfredi laboratory for help and support. The technical assistance of Andrea DaCosta is gratefully acknowledged.

    FOOTNOTES

* This work was supported by NCI Grant CA69161 from the National Institutes of Health and the Breast Cancer Program of the United States Army Medical Research and Materiel Command Grants DAMD-17-97-1-7336, DAMD-17-97-1-7337, and DAMD-17-99-1-9308).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Cancer Center, Box 1130, Mount Sinai School of Medicine, New York, NY 10029. Tel.: 212-659-5495; Fax: 212-849-2446.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc. M011643200

2 E. C. Thornborrow and J. J. Manfredi, unpublished data.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assays.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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