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7 November 2002, Volume 21, Number 51, Pages 7901-7911
Table of contents    Previous  Article  Next   [PDF]
Oncogenomics
Groups of p53 target genes involved in specific p53 downstream effects cluster into different classes of DNA binding sites
Hua Qian1,a, Ting Wang1, Louie Naumovski2, Charles D Lopez3 and Rainer K Brachmann1,b

1Division of Oncology, Department of Medicine, Washington University School of Medicine, 660 S Euclid Avenue, Box 8069, St Louis, Missouri, MO 63110, USA

2Division of Hematology, Oncology and Stem Cell Transplantation, Stanford University, 269 Campus Drive, CCSR Room 1215, Stanford, California, CA 94305, USA

3Division of Hematology and Medical Oncology, Department of Medicine, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Baird Hall Room 3030, Portland, Oregon, OR 97201, USA

Correspondence to: R K Brachmann, E-mail: rbrachma@uci.edu

aCurrent address: Department of Surgery, Xin Hua Hospital, Shanghai Second Medical University, 1665 Kong Jiang Road, Shanghai, 200092, PR China

bCurrent address: Division of Hematology/Oncology, Department of Medicine, University of California at Irvine, Medical Sciences I, C250, Irvine, California, CA 92697, USA

Abstract

The tumor suppressor protein p53, once activated, can cause either cell cycle arrest or apoptosis through transactivation of target genes with p53 DNA binding sites (DBS). To investigate the role of p53 DBS in the regulation of this profound, yet poorly understood decision of life versus death, we systematically studied all known and potential p53 DBS. We analysed the DBS separated from surrounding promoter regions in yeast and mammalian assays with and without DNA damage. p53 efficiently utilized the DBS of MDM2 and of genes connected to cell cycle arrest, DNA repair and the death receptor pathway of apoptosis. However, p53 was unable to utilize two-thirds of the isolated DBS, a subset that included almost all DBS of apoptosis-related genes. Neither ASPP2, a p53-interacting protein reported to specifically stimulate p53 transcriptional activity on apoptosis-related promoters, nor DNA damage resulted in p53 utilization of isolated DBS of apoptosis-related genes. Thus, a major regulation of p53 activity occurs at the level of p53 DBS themselves by posing additional requirements for the successful utilization of apoptosis-related DBS.

Oncogene (2002) 21, 7901-7911. doi:10.1038/sj.onc.1205974

Keywords

apoptosis; cell cycle; DNA binding site; DNA repair; p53

Introduction

p53 exerts its tumor suppressor function, in part, as a transcription factor that induces effector genes once activated by upstream signals, such as DNA damage. Activated p53 results in DNA repair, cell cycle arrest and/or apoptosis (Prives and Hall, 1999; Vogelstein et al., 2000). Direct target genes of p53 have one or more p53 DNA binding site(s) (DBS) very similar to the p53 consensus DBS that consists of two copies of the 10 base pair (bp) motif 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' separated by 0-13 bp (el-Deiry et al., 1992; Funk et al., 1992). However, only one p53 DBS, Type IV Collagenase, is a perfect match for the p53 consensus DBS. There are over 60 genes with known or potential p53 DBS (see Tables 1 and 2). The biological activities of many are consistent with established p53 effects. For example, the p21 and 14-3-3sigma genes are involved in p53-mediated G1 and G2 cell cycle arrest, respectively. The GADD45, p53R2 and PCNA genes have roles in DNA repair, and the growing list of apoptosis-related genes includes Bax, Noxa, p53AIP1, PERP and PIG3. Genes that are part of the death receptor pathway of apoptosis are also represented, such as Fas, KILLER/DR5 and Pidd (Bates and Vousden, 1999; Hengartner, 2000; Ko and Prives, 1996; Levine, 1997; Prives and Hall, 1999; Rich et al., 2000; Sionov and Haupt, 1999; Vogelstein et al., 2000; Vousden, 2000). In addition, p53 induces MDM2, a target gene that creates a negative feedback loop with p53 to maintain low p53 protein levels in unstressed cells (Haupt et al., 1997; Kubbutat et al., 1997; Prives, 1998). The role of many other known or potential target genes in the p53 context remains poorly defined. For example, p53 may drive the transcription of growth-promoting genes, such as c-fos, c-met, Epidermal Growth Factor Receptor and Transforming Growth Factor-alpha (Elkeles et al., 1999; Ludes-Meyers et al., 1996; Seol et al., 1999; Shin et al., 1995).

It is also still unclear why some cells respond to p53 activation with cell cycle arrest, while others undergo apoptosis. Cell type, amount of DNA damage, the presence of survival factors and the inappropriate activity of oncogenes appear to be important, but what exact underlying mechanisms govern the profound decision of survival versus death after p53 activation remains unknown (Bates and Vousden, 1999; Sionov and Haupt, 1999; Vousden, 2000). Several models have been put forth. In the 'p53 dumb' model, activated p53 always leads to the same downstream signals, including upregulated cell cycle arrest and apoptotis genes, and these signals are then modulated by other pathways. In the 'p53 smart' model, increased p53 protein levels, specific post-translational p53 modifications and/or the recruitment of specific coactivators determine the expression levels of cell cycle arrest and apoptotis genes (Vousden, 2000). ASPP1 and ASPP2 have been characterized as p53 coactivators that specifically stimulate the apoptotic function of p53. They appear to do so by enhancing the DNA binding and transcriptional activity of p53 at apoptosis-related promoters (Lane, 2001; Samuels-Lev et al., 2001). Thus, the activity of ASPP proteins would fit into the 'p53 smart' model (Vousden, 2000). In addition, genetic approaches in mice and chromatin immunoprecipitation (ChIP) analysis have recently shown that p53 requires its family members p73 and p63 to be present in order to bind to the promoters of and transactivate apoptosis genes (Flores et al., 2002).

If a cell's response to activated p53 is indeed determined at the level of promoters, then p53 DBS themselves may be important components of the regulatory mechanisms by providing more or less perfect binding sites for p53. Differences between p53 DBS have been previously noted. For example, p53 cancer mutants were found to have differential DNA binding properties and transcriptional activities when tested with several p53 DBS (Campomenosi et al., 2001; di Como and Prives, 1998; Friedlander et al., 1996). Several studies suggested that wild-type p53 may adopt more than one conformation to utilize certain DBS (Thornborrow and Manfredi, 1999; 2001). Lastly, Szak et al. (2001) have recently demonstrated by gel shift assays that the PIG3 DBS (as originally reported by Polyak et al., 1997) is a low-affinity p53 DBS, as compared to the p21, 5' site, and MDM2 DBS.

However, the question whether p53 DBS play a broader role in determining a cell's response to activated p53 has never been stringently and comprehensively addressed. Therefore, we studied isolated p53 DBS of all known and putative p53 downstream target genes systematically in yeast and mammalian assays with and without DNA damage. Strikingly, two thirds of p53 DBS were unable to direct p53-mediated transcription, and almost all DBS from apoptosis-related genes were included in this set. To the contrary, the DBS of MDM2 and of genes connected to cell cycle arrest, DNA repair and the death receptor pathway of apoptosis were efficiently used by p53. Based on our findings, we propose rules for p53 DBS that successfully predict the behavior of 62 out of 67 DBS in our assays. The results suggest that p53 DBS themselves are an important part of the regulatory mechanisms that guide a cell on the path to either cell cycle arrest or apoptosis.

Results

To determine the ability of p53 to utilize isolated DBS in yeast, we generated URA3 reporter gene plasmids for 67 known and potential p53 DBS representing 57 known and potential p53 downstream target genes (see Tables 1 and 2). The reporter plasmids were identical except for the DBS itself; 5 bp of flanking region were added, if the information was available (see Figure 1a, Tables 1 and 2). p53 transcriptional activity was evaluated through the Ura-phenotype of yeast strains at 30 and 37°C. Unexpectedly, only 23 of 67 DBS (34%) were utilized by p53 in the yeast assay (Table 2) representing 22 of 57 p53 target genes (39%). Strikingly, all p53 target genes related to cell cycle arrest and DNA repair (Ko and Prives, 1996; Levine, 1997; Vogelstein et al., 2000) had at least one DBS that was functional in yeast (Figure 1b,c; for simplicity, hereafter referred to as cell-cycle-like DBS). The only exceptions were BTG/PC3/Tob and RB. All known p53-dependent genes that are part of the death receptor pathway of apoptosis (Bates and Vousden, 1999; Hengartner, 2000; Rich et al., 2000; Sionov and Haupt, 1999; Vousden, 2000) also had DBS that were functional in yeast (Figure 1d; Fas (APO-1/CD95), KILLER/DR5 and Pidd). In contrast, all other DBS of genes that are part of apoptotic pathways (apoptosis-like DBS) did not function in yeast, except Noxa and p53AIP1 (Figure 1d) (Bates and Vousden, 1999; Hengartner, 2000; Rich et al., 2000; Sionov and Haupt, 1999; Vousden, 2000). Several other genes with varying biological activities also had DBS that were utilized by p53 in yeast, including MDM2, the p53 target gene that is part of a negative feedback loop with p53 (see Figure 1b-d, and Table 2) (Haupt et al., 1997; Kubbutat et al., 1997; Prives, 1998).

We next determined the ability of transfected p53 to utilize the DBS for cell cycle arrest, DNA repair and apoptosis genes in the p53-negative H1299 cell line. The DBS were cloned into a mammalian luciferase reporter plasmid using the same strategy to ensure that all DBS were tested under identical conditions (Figure 1a). We found the pattern of DBS utilization by p53 to be essentially the same in H1299 cells as in yeast (Figure 2a,b). The two exceptions were the PTGF-beta, SBS01 and the Bax DBS (negative in yeast, but activity above background in H1299 cells). Of note, the MDM2 DBS resulted in 13-fold more activity than the p21, 5' site, DBS (used as a reference of 100% throughout the figures). We investigated whether higher p53 protein levels could lead to utilization of apoptosis-like DBS in H1299 cells, but the results were essentially unchanged as compared to Figure 2a,b (Figure 2c).

We considered the possibility that transiently overexpressed p53 may be sufficient to utilize cell-cycle-, but not apoptosis-like DBS. We therefore decided to evaluate the utilization of DBS by endogenous wild-type p53 in the absence and presence of DNA damage. We chose A549 cells with endogenous wild-type p53 because they show markedly increased p53 protein levels and an up to fivefold increase in p53 transcriptional activity after doxorubicin-induced DNA damage (Wang et al., 2001). In A549 cells without DNA damage, the pattern for cell-cycle-like DBS was similar to H1299 cells. Additionally, all DBS that were positive in yeast led to a 2-5-fold increase in p53 transcriptional activity after DNA damage. Consistent with the H1299 experiments, MDM2 was the DBS most efficiently utilized by p53 (Figure 3a). In contrast, apoptosis-like DBS, except Noxa and p53AIP1, were unable to direct p53-mediated transcription, with or without DNA damage (Figure 3b). The Bax DBS was also negative, suggesting cell-dependent differences in the utilization of the Bax DBS or that p53 overexpression in H1299 cells caused the observed moderate amount of transcriptional activity (compare Figures 2b to 3b).

These results suggested that DNA-damage-induced post-translational modifications of p53 were unable to compensate for the underutilized apoptosis-like DBS (Figure 6I). In theory, A549 cells may lack certain pathways upstream of p53, or they may be devoid of an essential p53 cofactor that needs to interact with post-translationally modified p53 for the efficient utilization of apoptosis-like DBS (Figure 6II). To find a condition in which p53 utilizes apoptosis-like DBS, we evaluated representative DBS in HepG2, MCF7, U2OS (all with endogenous wild-type p53), SW480 (mutated endogenous p53 plus transfected wild-type p53) and H1299 (p53-negative plus transfected wild-type p53) cells with and without DNA damaging treatments known to induce apoptosis in these cell lines. None of the tested conditions resulted in the utilization of apoptosis-like DBS by p53 (Figure 3c-f, and data not shown). This may have been due to the fact that all tested cell lines have genetic alterations that result in the inability of p53 to utilize apoptosis-like DBS. More likely, apoptosis-like DBS alone may not be sufficient to allow essential p53 cofactors to aid p53 (see Figures 4 and 5).

Removal of the carboxy-terminal 30 amino acids (residues 364-393) of p53 leads to its activation under some conditions (Hupp et al., 1992; Ko and Prives, 1996). With the p21, 5' site, DBS, p53Delta364-393 showed higher activity than wild-type p53. However, improved utilization of PERP, -218, as well as DBS for Cathepsin D, ei24/PIG8, IGF-BP3, MCG10 and PIG3, was not observed (Figure 4a, and data not shown).

We reasoned that cis elements adjacent to p53 DBS that allow cofactors to colocalize and interact with p53 (Figure 6III) might be required for the utilization of apoptosis-like DBS. In yeast, no differences were observed between the isolated DBS and reporter constructs for the p21, 5' site, Bax and PIG3 DBS that contained 100 bp up- and downstream of the p53 DBS itself (Figure 4b). In A549 cells, the p21, 5' site, DBS with addition of promoter fragments showed enhanced p53 transcriptional activity with or without DNA damage (Figure 4c). In contrast, adding 200 bp of native promoter sequence around the Bax and PIG3 DBS did not result in an enhanced usage of these DBS by p53 (Figure 4c). This suggested that cis elements in close proximity to a p53 DBS enhance p53 activity in the case of a cell-cycle- (p21, 5' site), but not apoptosis-like DBS (Bax and PIG3). We then compared full-length promoters for p21, Bax and PIG3 to p53 transcriptional activity with the isolated p21, 5' site, DBS. The presence of additional promoter elements resulted in p53 transcriptional activity that was at least equivalent to the p21, 5' site, DBS for all tested promoters (Figure 4d).

ASPP1 and ASPP2 have been characterized as p53 coactivators that specifically enhance p53 transcriptional activity at apoptosis-related promoters. The behavior of both ASPP proteins appeared to be very similar (Samuels-Lev et al., 2001). We therefore evaluated whether ASPP2, expressed in A549 cells with endogenous wild-type p53, facilitates p53 transcriptional activity with apoptosis-like DBS lacking surrounding promoter sequences. Although ASPP2 enhanced p53 transcriptional activity with cell-cycle-like DBS, it was unable to promote the utilization of apoptosis-like DBS by p53 (Figure 5a). Providing 100 bp of additional cis elements up- and downstream of the p21, 5' site, Bax and PIG3 DBS did not change these findings (Figure 5b).

Mouse knock-out models have recently shown that the p53 family members p73 and p63 are required for p53-mediated transactivation of apoptosis genes. ChIP assays further established that p63 proteins are present at the promoters of apoptosis-related target genes in order to facilitate p53-mediated transactivation (Flores et al., 2002). We tested two of the numerous p73 and p63 family members for their ability to assist p53 in utilizing isolated apoptosis-like DBS. In H1299 cells with transiently transfected p53, the Bax DBS results in some p53 transcriptional activity (see Figures 2b and 3f). In this setting, p73alpha and p73beta were able to enhance p53 transcriptional activity to at least the level of the p21, 5' site, DBS (Figure 5c,d). gamma-irradiation in this assay system did not lead to enhanced p53 transcriptional activity (see Figure 3f) suggesting that essential cofactors for p53 may become limiting under the conditions of p53 overexpression. For two apoptosis-like DBS that had been negative under all tested conditions, an increase in p53 transcriptional activity was not observed (IGF-BP3, Box A and PIG3, Figure 5c,d).

Discussion

We have performed a comprehensive and stringent study of known and potential p53 DBS that have been identified in the promoters, introns or exons of 57 known and potential p53 downstream target genes. To our surprise, a substantial number of reported p53 DBS (66%) were not utilized by p53 in both yeast and mammalian assays. This included DBS for almost all genes that are part of the mitochondrial pathway of apoptosis. Equally remarkable was the fact that almost all genes related to cell cycle arrest, DNA repair or the death receptor pathway of apoptosis were represented in the collection of positive DBS.

The question arises how seemingly equivalent DBS, all conforming to the p53 consensus DBS (el-Deiry et al., 1992) except for very few base pair changes, can behave so differently. A closer look, however, reveals striking patterns that lead us to postulate the following rules for p53 DBS. Cell-cycle-like DBS that are utilized by p53 do not have sequences interspersed between the two half-sites of the p53 DBS (22 of 24) and have two or fewer mismatches with the p53 consensus DBS (22 of 24). Apoptosis-like DBS that are not utilized by p53 have interspersed sequences between the two half-sites of the p53 DBS (29 of 43) and/or three or more mismatches (32 of 43). All p53 DBS, except for GPX, p53AIP1, PAI-1, Pidd and RGC, conform to these rules (see Tables 1 and 2).

The negative effect of interspersed sequences on p53-mediated transcription has been previously recognized (Tokino et al., 1994). Such additional sequences and increasing numbers of mismatches with the p53 consensus DBS likely result in low affinity DBS, as has been demonstrated by gel shift assays in a comparison of the p21, 5' site, MDM2 and PIG3 DBS (Szak et al., 2001). These and our results suggest a hierarchy of p53 DBS: the MDM2 DBS that is used by p53 in a relatively inactive state, the collection of cell-cycle-like DBS that are utilized by a more activated pool of p53 and lastly apoptosis-like DBS that have additional requirements for usage by p53.

It should be noted that recently an additional class of p53 DBS has been identified by Contente et al. (2002). Detailed studies of the PIG3 promoter showed that the previously reported PIG3 DBS for p53 (Polyak et al., 1997; used in our study) was not required for p53-mediated transactivation of luciferase reporter constructs. Rather, p53 depended on and interacted with repeats of a pentanucleotide motif that represented a microsatellite sequence. The pentanucleotide repeats have limited homology with the consensus DBS reported by el-Deiry et al. (1992), and thus would fall into our class of apoptosis-like DBS as well. However, the lack of conformity to the consensus p53 DBS may be compensated for by the repeat of the pentanucleotide sequence of up to 17 times. The report of this intriguing new class of p53 DBS appeared too late to be incorporated into the experiments of our studies.

We attempted to further define the 'rules of engagement' between p53 and its DBS through activation of p53 by DNA damage. For cell-cycle-like DBS, we demonstrated that no additional promoter regions were required for enhanced p53 transcriptional activity after DNA damage. This may simply reflect increased p53 protein levels, but is also consistent with p53 being activated by post-translational modifications (Figure 6I) that, in addition, may lead to the recruitment of p53 cofactors (Figure 6II). In contrast, we were unable to identify conditions in which p53 could utilize apoptosis-like DBS, demonstrating that increased p53 protein levels, post-translational modifications of p53 and/or cofactors that function in close proximity to a p53 DBS (100 bp) are not sufficient to drive transcription of apoptosis-related genes (Figure 6I-III). Whole promoters for Bax and PIG3 resulted in p53 transcriptional activity that was at least as high as for the isolated p21, 5' site, DBS. This strongly suggests that p53 can utilize apoptosis-like DBS only in the context of additional promoter elements that are likely involved in recruiting essential p53 cofactors.

ASPP1 and ASPP2 have been characterized as p53 coactivators that are specific for apoptosis-related genes and that lead to p53-mediated apoptosis, but not cell cycle arrest (Lane, 2001; Samuels-Lev et al., 2001). Using A549 cells and reporter plasmids containing isolated DBS or DBS flanked by 100 bp of 5' and 3' promoter sequence, we found that ASPP2 stimulated p53 transcriptional activity with cell-cycle-like p53 DBS (p21, 5' site, and KILLER/DR5), but not apoptosis-like DBS (Bax; ei24/PIG8; IGF-BP3, Box A, and PIG3). DNA damage further enhanced ASPP2 stimulation of p53 transcriptional activity with cell-cycle-like DBS, but did not lead to p53 utilization of apoptosis-like DBS. These results are opposite to what the model of Samuels-Lev et al. (2001) would predict and may be different because our study focused on isolated p53 DBS.

The findings for p53, ASPP2 and isolated p53 DBS lead us to propose two hypotheses. Since ASPP2 has been characterized as a p53 coactivator that is specific for apoptosis-related genes (Samuels-Lev et al., 2001), its activity must be very tightly regulated. Cells must have a specific inhibitory mechanism to prevent ASPP2-directed p53 stimulation at cell cycle arrest promoters (since cell-cycle-like DBS alone with ASPP2 are sufficient to result in p53 stimulation). Likewise, cells must have complex mechanisms to specifically enhance p53 activity at apoptosis-related promoters (since apoptosis-like DBS alone with ASPP2 were unable to do so). Such regulatory mechanisms most likely rely on additional, more distant cis elements that were not addressed in our study. Since ASPP proteins do not appear to bind DNA directly, these cis elements may recruit regulatory proteins that then interact with the amino-terminus of ASPP proteins, as suggested by Samuels-Lev et al. (2001). Our model system will facilitate the systematic identification and examination of these cis elements that are important for regulating ASPP function.

The recent finding that the p53-related families of p73 and p63 proteins are necessary for the appropriate transactivation of apoptosis genes by p53 (Flores et al., 2002) has raised an intriguing question. What is the exact interplay of these three proteins? Flores et al. (2002) established by ChIP analysis that p63 did engage with the promoters of established p53-dependent apoptosis genes for successful p53-mediated transactivation. Yet, at present it is not known whether all involved factors bind to the promoters directly at all times, what the specific promoter elements are and which p73 and/or p63 family members assist in the response of p53 to DNA damage. This has led to the proposal of two models by Urist and Prives (2002). In the 'Dynamic Exchange Model', p53 and family members rely on a single 'p53 Family Response Element'; the family members would reside in a stabilized complex, but only one of them could be bound to the response element at any given time. In the 'Dual Site Stabilization Model', at least two 'p53 Family Response Elements' are required to allow the simultaneous binding of p53 and p73 and/or p63 family members, thus resulting in the recruitment of other required cofactors. Our results for three apoptosis-like DBS suggest that both models might be correct. In the case of the Bax DBS, fairly low transcriptional activity of p53 could be enhanced by p73alpha and/or p73beta. In contrast, p73alpha and/or p73beta were not sufficient to enhance p53 transcriptional activity with either the PIG3 or IGF-BP3, Box A DBS. Additional studies are needed to establish which of the at least eight p73 and nine p63 family members (Yang et al., 2002) are involved in p53-mediated activation of apoptosis genes. Our system lends itself to dissecting the complexity of the family members and is an excellent tool to identify the promoter elements that are sufficient to result in p53 transcriptional activity.

The comprehensive and systematic approach of our study required assay systems that were relatively easy to manipulate. Clearly, the regulation of p53 transcriptional activity in normal cells is much more complex, as it involves full-length promoters and occurs in the context of chromatin. However, the complexity will only be fully understood when every single regulatory component has been appropriately identified and characterized. Our classification of p53 DBS is an important step forward in systematically dissecting the mechanisms that determine cellular outcomes after p53 activation. The clear differences between p53 DBS strongly argue that p53 DBS themselves are important determinants of a cell's decision to undergo either cell cycle arrest or apoptosis. Our rules for p53 DBS will help to assign p53 target genes to different p53 downstream pathways and identify and characterize the specific requirements for activating these pathways.

Materials and methods

p53 assays in S cerevisiae

For URA3 reporter plasmids, annealed oligonucleotides with suitable linkers representing p53 DBS plus 5 bp up- and downstream (if the information was available, see Table 1) were cloned into the PstI-EcoRI sites of a modified pMV252 (Vidal et al., 1996) that had the -368 to -170 EcoRI fragment replaced with a PstI-EcoRI linker (Figure 1a). After sequence-confirmation, the NotI-SPO13-promoter-URA3-gene-XhoI cassettes were cloned into pRS414 (Sikorski and Hieter, 1989). For constructs with 100 bp up- and downstream of the DBS, the promoter regions were amplified from human genomic DNA for p21 or reporter constructs for Bax and PIG3 (Miyashita and Reed, 1995; Polyak et al., 1997) with Pfx (Invitrogen, CA, USA) and primers with linkers that allowed in vivo homologous recombination of the promoter fragment into the PstI-EcoRI gap of the URA3 reporter plasmid. Plasmids were then rescued from yeast and sequence-verified. p53 yeast assays were performed as described (Brachmann et al., 1996; 1998; Vidal et al., 1996).

p53 assays in mammalian cells

For luciferase reporter plasmids, the SacII-EcoRI fragments of yeast reporter plasmids were cloned into a modified pp53-TA-Luc (Clontech Laboratories, CA, USA) that had the KpnI-BglII fragment upstream of the minimal promoter replaced with a SacII-EcoRI linker (Figure 1a). For experiments in H1299 cells, the p53 expression plasmids pCMV-p53 (50-100 ng; Clontech Laboratories, CA, USA) and pC53-SN3 (25-500 ng; Baker et al., 1990) were used. To obtain a matching set of expression plasmids for p53 and p53Delta364-393, the natural open reading frame (ORF) of p53 in pCMV-p53 was replaced by p53 ORFs with silent nucleic acid substitutions (T Wang and RK Brachmann, unpublished data) encoding for wild-type p53 or p53 lacking codons 364-393 (50 ng each per reporter gene experiment). To induce DNA damage, A549, HepG2 and SW480 cells were exposed to doxorubicin (SIGMA, MO, USA) at 0.6 mug/ml for 16-24 h prior to lysis, and H1299 and MCF7 cells to 6000 rad of gamma-irradiation 7 h prior to lysis. Reporter gene assays were performed as described (Brachmann et al., 1998), except that a Renilla luciferase construct (Promega, WI, USA) was used to normalize results. For ASPP2-related experiments, a Cep4-based plasmid expressing full-length ASPP2 (500 ng) was used. G Melino provided mammalian expression plasmids for HA-tagged p73alpha and p73beta (50 ng per experiment; de Laurenzi et al., 1998). The luciferase reporter constructs for whole promoters were provided by WS El-Deiry for p21 (based on el-Deiry et al., 1993), M Oren for Bax (based on Miyashita and Reed, 1995) and B Vogelstein for PIG3 (Polyak et al., 1997). Standard procedures and the DO-1 antibody (Santa Cruz Biotechnology, CA, USA) were used for anti-p53 immunoblotting.

Acknowledgements

We thank WS El-Deiry, H McLeod, G Melino, M Oren and B Vogelstein for providing reagents and Nataya Boonmark for constructing the Cep4-ASPP2 plasmid. This work was supported in part by grants from the James S McDonnell Foundation and National Institutes of Health grants CA81511 (RK Brachmann) and CA76316 (L Naumouski).

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Figures

Figure 1 Systematic analysis of p53 DNA binding sites (DBS) in yeast reporter gene assays. (a) Two reporter gene systems were used in this study. For yeast reporter constructs, 67 p53 DBS were cloned into the SPO13 promoter upstream of the URA3 reporter gene. Promoter fragments containing the DBS were then moved into a mammalian luciferase reporter construct. PTA denotes the TATA box of the herpes simplex virus thymidine kinase promoter. (b,c) All cell-cycle-arrest- and DNA-repair-related genes had at least one DBS that was utilized by p53 in the yeast assay (called cell-cycle-like DBS for simplicity), resulting in a Ura+ phenotype, except BTG/PC3/Tob and RB. Three independent transformants are shown per DBS. (d) DBS of genes that are part of the death receptor pathway also conferred a Ura+ phenotype, while the remainder of DBS of genes connected to apoptosis (apoptosis-like DBS) were not utilized by p53. Results for the MDM2 DBS are shown in (b) through (d) for comparison, since this DBS conferred the highest p53 transcriptional activity in mammalian assays

Figure 2 Systematic analysis of p53 DBS in mammalian reporter gene assays using H1299 cells. Results that were essentially superimposable on the p53 yeast assay were obtained in mammalian reporter gene assays in p53-negative H1299 cells transiently transfected with 50 ng of the p53 expression plasmid pCMV-p53. (a) Cell-cycle-like DBS conferred p53 transcriptional activity similar to the reference DBS p21, 5' site (always shown as 100%). The DBS of MDM2, a gene that is important for a negative feedback loop with p53, led to markedly enhanced luciferase activity. (b) Apoptosis-like DBS were not utilized by p53, while DBS of death-receptor-related genes were. The control for (a) and (b) is the SPO13 promoter without a p53 DBS; all DBS constructs were also tested in the absence of p53 with results similar to the shown control. (c) Increased levels of p53 did not lead to increased utilization of apoptosis-related DBS by p53. Controls are the specific DBS constructs without p53. The expression plasmid pC53-SN3 was used because of higher p53 expression levels than pCMV-p53. Immunoblotting was performed with anti-p53 antibody DO-1 after the cell lysates of the shown reporter gene assay had been adjusted to control for Renilla luciferase activity. The reduced transcriptional read-out for the p21, 5' site, DBS with 500 ng as compared to 50 ng p53 expression plasmid is likely due to increased p53-mediated apoptosis that the assay system does not completely control for

Figure 3 Systematic analysis of p53 DBS in A549 cells with and without DNA damage and evaluation of apoptosis-like DBS under various assay conditions. Experiments were performed as in Figure 2a-c, except that many of the reporter gene assays relied on endogenous p53 in the absence or presence of DNA damage. (a) In A549 cells, cell-cycle-like DBS were again positive (14-3-3sigma, BDS-2; PTGF-beta; GADD45 and PCNA to a lesser extent than in H1299 cells), and all positive DBS showed significantly enhanced p53 transcriptional activity after exposure of the cells to doxorubicin at 0.6 mug/ml for 16 h. The MDM2 DBS conferred markedly enhanced activity compared to the others. (b) Apoptosis-like DBS were again negative and showed no stimulation of p53 transcriptional activity after DNA damage. As in yeast and H1299 cells, exceptions were Noxa and p53AIP1, as well as genes of the death receptor pathway. The Bax DBS (approximately 30% activity in p53-negative H1299 cells with transiently transfected, overexpressed p53) was negative in A549 cells, suggesting that the cell context is important for this DBS or that large amounts of p53 can lead to some utilization of the Bax DBS. (c-f) Apoptosis-like DBS were also negative in several other cell lines tested with and without DNA damage (Doxorubicin or gamma-irradiation). Similar to H1299 cells, the Bax DBS led to transcriptional activity above background in SW480 cells (with mutated p53) that were transfected with p53

Figure 4 Search for conditions that allow p53 to utilize apoptosis-like DBS. (a) Removal of the last 30 amino acids of p53, a potential negative autoregulatory domain (p53Delta364-393), led to a doubling in luciferase activity with the p21, 5' site, DBS, a rather small increase, considering the significantly higher protein levels for p53Delta364-393. However, it did not lead to p53 activity with the apoptosis-like DBS PERP, -218, as well as several others (data not shown). (b) The addition of 100 bp up- and downstream of the p53 DBS did not change the yeast phenotypes for p21, 5' site, Bax and PIG3. (c) These results were confirmed in mammalian reporter gene assays in A549 cells. The addition of p21 promoter elements enhanced p53 transcriptional activity, but also abolished the increase in p53 activity after DNA damage that was seen with the isolated DBS. (d) The evaluation of whole promoters for p21, Bax and PIG3 led to p53 transcriptional activity that was at least as high as for the isolated p21, 5' site, DBS

Figure 5 Evaluation of the p53-interacting protein ASPP2 and p73 family members with p53 and isolated p53 DBS. (a) ASPP2 enhanced p53 transcriptional activity with isolated cell-cycle-like DBS, in the absence and presence of DNA damage, but was unable to aid p53 in utilizing apoptosis-related DBS. (b) These results remained unchanged when p53 DBS were evaluated with additional 100 bp up- and downstream of the DBS. (c,d) p73alpha and/or p73beta were able to increase p53 transcriptional activity in the case of the Bax DBS that shows approximately 30% activity of the p21, 5' site, DBS in H1299 cells with overexpressed p53. However, the two p73 family members were unable to increase p53 transcriptional activity with the IGF-BP3, Box A and the PIG3 DBS

Figure 6 'Rules of engagement' for p53 and its DBS. Inactive p53 is unable to utilize DBS, with the likely exception of the MDM2 DBS that showed markedly enhanced activity in our mammalian assays. This fits nicely with the role of the MDM2 gene in a negative feedback loop that maintains relatively inactive p53 at low protein levels in unstressed cells. The MDM2 DBS actually contains two p53 DBS; this in itself, however, does not explain our findings, since the negative c-fos DBS does so as well (see Table 1). Partially or fully activated p53 is sufficient to utilize cell-cycle-like DBS (I). This is illustrated in our mammalian reporter gene assays where p53 shows transcriptional activity with cell-cycle-like DBS which can be further enhanced by DNA damage. Our data is also consistent with the requirement for additional cofactors that bind to post-translationally modified p53 in the context of a DBS alone in order to augment p53 activity (II). In contrast, activated p53 is unable to utilize apoptosis-like DBS alone (I and II). Additional cis elements surrounding apoptosis-like DBS (100 bp up- and downstream) do not change this situation (III), suggesting that the 'rules of engagement' for p53 and apoptosis-like DBS are much more complex than for p53 and cell-cycle-like DBS. ASPP proteins have been characterized as p53 coactivators that specifically enhance p53 transcriptional activity at apoptosis-related promoters. Our results suggest that this coactivator role must require complex regulatory mechanisms and rely on additional cofactors and cis elements. The p73 and p63 families of p53-related proteins have recently been shown to be required for p53 to activate apoptosis-related target genes. Our data for p73alpha and p73beta suggest that negative apoptosis-like DBS are not sufficient to allow for p73-mediated enhancement of p53 transcriptional activity. However, a broader evaluation of apoptosis-like DBS and the at least 17 p73 and p63 family members is needed

Tables

Table 1 Sequences of known and potential p53 DNA binding sites

Table 2 Phenotypes for 67 p53 yeast assay

Received 22 April 2002; revised 12 August 2002; accepted 13 August 2002
7 November 2002, Volume 21, Number 51, Pages 7901-7911
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