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Molecular Cancer Research 1:1048-1057 (2003)
© 2003 American Association for Cancer Research

Cell Cycle, Cell Death, and Senescene

Multiple Response Elements and Differential p53 Binding Control Perp Expression During Apoptosis1

Elizabeth E. Reczek1, Elsa R. Flores1, Alice S. Tsay1, Laura D. Attardi2 and Tyler Jacks1,3

1 Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA;
2 Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA; and
3 Howard Hughes Medical Institute, Chevy Chase, MD

Requests for reprints: Tyler Jacks, Center for Cancer Research, Massachusetts Institute of Technology, Room E17-110, 77 Massachusetts Avenue, Cambridge, MA 02139. Phone: (617) 253-0262; Fax: (617) 253-9863. E-mail: tjacks{at}mit.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The p53 tumor suppressor gene responds to cellular stress by activating either cell cycle arrest or apoptosis. A growing number of target genes involved in each of these pathways have been identified. However, the mechanism by which the apoptosis versus arrest decision is made remains to be elucidated. Perp is a proapoptotic target gene of p53 expressed to high levels in apoptotic cells compared with those undergoing cell cycle arrest. This pattern of expression is unusual among p53 target genes, many of which are induced to similar levels during arrest and apoptosis. Here, we describe the regulation of the Perp gene by p53 through at least three response elements in the Perp promoter and first intron. These sites are occupied in vivo in E1A-expressing mouse embryo fibroblasts undergoing apoptosis but not cell cycle arrest, in contrast to the p21 5' response element, which is occupied during both. The apoptosis-deficient p53 point mutant, p53V143A, displays a selective deficit in binding to the Perp elements, demonstrating that p53 can distinguish between Perp and p21 at the level of DNA binding. These results provide mechanistic insight into the selective expression of Perp during apoptosis and may provide a useful model for studying the p53-dependent cell cycle arrest versus apoptosis decision.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The p53 tumor suppressor gene is a critical component of the anticancer defense mechanism (1). At the cellular level, p53 is a central regulator of stress response pathways (2), becoming active in response to a variety of conditions, including abnormal proliferation, hypoxia, DNA damage, nucleotide depletion, and disruption of the mitotic spindle (3–9). Increased levels of active p53 can have many outcomes, including cell cycle arrest, apoptosis, senescence, and differentiation, depending on numerous cell intrinsic and extrinsic factors (10). Following some forms of cellular stress, p53 can induce reversible cell cycle arrest and appears to participate in damage repair mechanisms (11, 12). The ability of p53 to direct damaged or dangerous cells to undergo apoptosis is also critical to its tumor suppressive function (13). However, the mechanisms that determine whether cells will undergo p53-dependent cell cycle arrest or apoptosis following a given stress stimulus remain to be elucidated.

The p53 protein is a transcriptional activator with a transactivation domain at its NH2 terminus (14), a regulatory region and tetramerization domain at its COOH terminus (15, 16), and a central sequence-specific DNA binding domain (17, 18). The majority of p53 mutations found in human cancers reside within the DNA binding domain, suggesting that sequence-specific binding and transactivation of specific target genes is crucial for the tumor suppressive properties of p53 (17). p53 is capable of inducing expression of several genes containing one or more copies of a p53 binding element, located either within the promoter region (19–24) or in the introns of the responsive gene (5, 25–29). el-Deiry et al. (30) defined a consensus sequence for p53 binding consisting of two copies of a 10-bp motif, 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3', separated by 0–13 bp, and most p53-responsive genes identified to date contain a sequence roughly corresponding to this consensus. Transactivation by p53 is required for both arrest and apoptotic activities of p53 (31–34). However, a subset of p53 point mutants shows a selective defect in the transactivation of apoptosis-related target genes and induction of an apoptotic response, suggesting that the apoptosis- and arrest-inducing functions of p53 can be separated (35–41). Additionally, the p53 family members, p63 and p73, have been shown to be required for p53-dependent apoptosis but not cell cycle arrest, and loss of these proteins specifically affects transactivation of apoptosis-specific target genes (42). Other proteins such as ASPP1/2 and JMY have been found to selectively induce transactivation of apoptosis-related p53 target genes and to stimulate p53-dependent apoptosis but not cell cycle arrest (43, 44). Both apoptotic and arrest functions of p53 are likely to be tightly regulated through these and other factors to prevent their inappropriate activation.

Mouse embryo fibroblasts (MEFs) undergo p53-dependent G1 arrest in response to treatment with DNA damaging agents (5). Expression of the adenoviral E1A oncoprotein induces hyperproliferation and sensitizes wild-type MEFs to undergo p53-dependent apoptosis in response to DNA damage (45, 46), as does expression of c-myc or E2F-1 (47–51). The mechanism by which E1A and other oncoproteins can modulate the response of the cell to DNA damage is not fully understood. However, the transactivation function of p53 is crucial for the apoptotic response in E1A-MEFs, suggesting that transcriptional targets of p53 are likely to be involved (32). We previously reported the cloning of the p53 target gene Perp, which is expressed to higher levels in apoptotic E1A-MEFs than in MEFs undergoing G1 arrest (52). This pattern of expression is unusual for p53 target genes, many of which are induced to similar levels during both cell cycle arrest and apoptosis in this system. Thus, understanding Perp regulation could provide insight into the mechanisms through which p53 controls these cellular fates. We described previously the identification of two matches to the consensus p53 binding site in the murine Perp promoter (52). Here, we describe the identification of two additional p53 response elements in the Perp gene, one in the promoter region and one in intron 1. We demonstrate that at least three of these elements are functional p53 binding sites and can direct a p53 response in reporter assays, supporting the conclusion that Perp is a direct transcriptional target of p53. Further, we show that these sites can be distinguished from p53 response elements derived from cell cycle arrest related target genes in several ways, including their affinity for wild-type p53, their ability to bind to and mediate transactivation by apoptosis-deficient point mutants of p53, and their occupation by p53 in vivo under conditions of arrest and apoptosis.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Four Putative p53 Binding Sites Are Present Within the Promoter and First Intron of the Murine Perp Gene
We previously identified two sites (A and C) with significant similarity to the p53 binding site consensus within the Perp promoter region (Fig. 1, A and B) and demonstrated that the 4-kb upstream region of the murine Perp gene could direct a p53-dependent response (52). Further analysis of the murine Perp genomic sequence from -3850 to +11975 revealed the presence of two additional putative sites, one within the promoter region (site B) and one within the first intron (site D; Fig. 1, A and B). Sites A and C each contain a short spacer region between the two half-sites, while sites B and D consist of two half-sites immediately adjacent to one another. Site B contains a third half-site 4 bases upstream. Of the four sites identified, only the intronic site (site D) was conserved in the human Perp sequence obtained from the Human Genome Project public database (Fig. 1A). The mouse and human sequences at site D were identical at 14 of 20 residues, and four of six changes were conservative such that the mouse and human sites match the consensus at 18 of 20 and 19 of 20 bases, respectively. Several potential sites were found by sequence analysis of the human promoter region, but these sites have not been tested for function (data not shown).



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  		FIGURE 1. Cloning and sequencing of the murine Perp gene reveals four putative p53 binding sites. A. The murine Perp genomic locus. Black boxes, exons. Filled circles, matches to the p53 binding site consensus. Positions are labeled with respect to the transcriptional start site. The alignment between human and mouse site D is shown. Solid lines, perfect matches. Dotted lines, mismatches where both residues are consistent with the consensus. Asterisks, mismatches to the consensus sequence. B. The sequences of the four putative p53 binding sites from the Perp locus. The p53 binding site consensus and the 5' site from the murine p21 locus are shown for comparison. Bold letters, four core nucleotides of each half-site. Underlined sequences, mismatches to the consensus sequence. Half-site sequences separated from one another and from spacer nucleotides are indicated by a space.

 
Radiolabeled oligonucleotide probes representing these four sites were tested for the ability to bind p53 in electrophoretic mobility shift assays (EMSA). As shown in Fig. 2, only oligonucleotide probes corresponding to sites B and D were capable of binding to p53 in extracts from doxorubicin-treated E1A-MEFs. Binding could be competed by a 50-fold excess of an unlabeled consensus oligonucleotide (lanes 7 and 15; Fig. 2) but not by unlabeled mutant oligonucleotide (lanes 8 and 16; Fig. 2), demonstrating the specificity of the interaction. Binding was not observed in the absence of the activating antibody, monoclonal antibody 421 (lanes 5 and 13; Fig. 2), consistent with previous reports (53–56).



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  		FIGURE 2. Perp sites B and D are capable of sequence-specific binding to p53. EMSAs were performed using oligonucleotide probes corresponding to the four Perp sites. Lysates from wild-type E1A-MEFs treated with 0.2 µg/ml doxorubicin for 12 h were incubated with the labeled probes in the presence or absence of p53 antibody 421. A 50-fold molar excess of unlabeled wild-type probe (Wt comp; lanes 3, 7, 11, and 15) or mutant probe (Mut comp; lanes 4, 8, 12, and 16) was added to demonstrate specificity of binding. Asterisk, position of a nonspecific band in lanes 5, 6, 7, and 8. Lower panel, free probe.

 
Sites B and D Are Required for Full p53-Dependent Transactivation
Because sites B and D were capable of binding to p53 in vitro, we next tested the ability of sites A–D to direct p53-dependent expression of Perp in a luciferase reporter assay. The E1A-MEF system, in which Perp was originally identified, was not suitable for these assays because the process of transfection resulted in high levels of apoptosis. Therefore, we chose the HIp53 cell line (57), a derivative of the p53-deficient H1299 human lung carcinoma cell line (58) expressing His-tagged, wild-type, human p53 that can be induced by addition of the ecdysone analog, Ponasterone A (PonA). It should be noted that induction of p53 in HIp53 cells results in G1 arrest, and we were unable to sensitize these cells to apoptosis with irradiation or additional chemical treatments. Therefore, while we do observe endogenous Perp expression on p53 induction in these cells (data not shown) and reporters tested were strongly and reproducibly induced, this system is not ideal for distinguishing between arrest- and apoptosis-specific regulation of Perp. However, it has been very useful for assessing the contribution of different putative p53 binding sites to p53-dependent transactivation more generally. To address apoptosis-specific regulation, we have relied on chromatin immunoprecipitation (ChIP) experiments in wild-type and E1A-expressing MEFs.

We described previously the construction of the pPERPluc1 vector by insertion of ~4-kb upstream genomic fragment of the Perp gene into the pGL3Basic vector (Promega, Madison, WI; 52). While this reporter was strongly induced in p53-/- MEFs when cotransfected with a p53 expression vector (52), we observed minimal induction of pPERPluc1 expression in the HIp53 system (Fig. 3A). This difference is likely due to the modest level of p53 induced by PonA treatment in this system compared with levels achieved through overexpression of p53 by transient transfection (data not shown). In contrast, the pPERPlucFL reporter, containing an 8-kb Perp genomic fragment extending from -3850 to +4150, showed 9-fold induction with PonA treatment comparable with the induction of the murine p21 (59) and rat Cyclin G (60) reporters (Fig. 3A).



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  		FIGURE 3. Perp sites B and D are required for full transactivation by p53. Left, Schematics of the relevant luciferase reporter constructs (A and B). Black boxes, exons. Filled circles, putative p53 binding sites. Gray boxes, binding sites from p21 and Cyclin G. X, point-mutated sites. SA, insertion of a splice acceptor sequence from Perp exon 2. Positions are designated in reference to the transcriptional start site. Reporters were cotransfected with the pRL-null Renilla expression vector into HIp53 cells, treated with PonA or ethanol (EtOH) control 12 h post-transfection, and assayed for firefly and Renilla luciferase activities 12 h later. Firefly activity was normalized to Renilla activity. Fold induction is graphed relative to ethanol control. Columns, averages of at least three separate experiments each performed with six replicates; bars, 1 SD. Differences in levels of induction of pPERPlucPS between A and B may reflect differences in batches of PonA or HIp53 responsiveness to PonA treatment in experiments performed several months apart.

 
Because site D was capable of binding p53 in gel shift assays (Fig. 2), we inserted a 1.5-kb fragment of intron 1 (from +2375 to ~+3875) containing site D downstream of the luciferase cassette in the pPERPluc1 vector to generate pPERPlucPS. pPERPlucPS showed a 5-fold induction in HIp53 cells after PonA treatment (Fig. 3A), suggesting that a p53-responsive element was present within this fragment. However, the response of pPERPlucPS was weaker than seen with pPERPlucFL. Thus, an additional regulatory site(s) may be present in the upstream portion of intron 1, or the endogenous spacing of the elements within the Perp locus may be important for full functionality. Point mutation of the central cytosine and guanine residues of each half-site of site D to thymidine residues reduced the response of the pPERPlucFL vector by approximately two-thirds (pPERPlucFL-mutD; Fig. 3A) and completely abrogated the response of the pPERPlucPS vector (pPERPlucPS-mutD; Fig. 3A) to PonA treatment. Insertion of an oligonucleotide corresponding to site D into the unresponsive pPERPluc1 vector at a cloning site downstream of the luciferase cassette (pPERPluc1+D) was sufficient to restore the level of induction to ~70% of that seen with the pPERPlucPS vector; a mutant oligonucleotide (pPERPluc1+mutD) had no effect (Fig. 3A). Together, these data strongly implicate site D in the response of Perp to p53 but suggest that additional sites or endogenous spacing might be required for full induction of Perp in response to p53.

We next examined the ability of sites within the promoter region of Perp to enhance the p53 response mediated by site D. To this end, a series of deletions was made in the upstream region of the pPERPlucPS reporter (Fig. 3B). Deleting the region from -2180 to -650, which includes sites A and B, decreased the response by ~30% (pPERPlucPS-650). A deletion from -650 to -50 bp, including sites A–C, further decreased expression following p53 induction (pPERPlucPS-50). These data suggest that a p53 response element is present in the region from -2180 to -650 and an additional site may lie in the region from -650 to -50. To determine whether site A, B, or C might mediate the observed response, we point mutated these sites individually and in combination. Point mutation of sites A and C together (pPERPlucPS-mutAC) or site C alone (pPERPlucPS-650-mutC) had no effect on the ability of the reporter to respond to p53 in this system (Fig. 3B). However, point mutation of site B in the pPERPlucPS reporter (pPERPlucPS-mutB) decreased the response to PonA treatment by ~30% (Fig. 3A). These data suggest that site D is responsible for the majority of the response of Perp to p53 in this system but that site B might augment or modulate that response. Because the HIp53 cell line fails to undergo apoptosis, however, we cannot rule out the possibility that sites A and C might function under apoptotic conditions.

p53 Is Present at the Perp Locus in Apoptotic but not Cell Cycle Arrested MEFs
Having established the ability of sites B and D to mediate transactivation by p53, we performed ChIP for p53 on wild-type MEFs, wild-type E1A-MEFs, and p53-/- E1A-MEFs in the presence and absence of DNA damage to examine the functionality of the four putative response elements in vivo under conditions of apoptosis and cell cycle arrest (Fig. 4). Twelve hours after doxorubicin treatment, 80% of wild-type MEFs are in G1 arrest, while only 60% of E1A-MEFs are viable at this time point. In these cells, we examined the 5' p53 response element from promoter of the p21 gene, which is induced under conditions favoring both cell cycle arrest and apoptosis, in addition to the four putative sites from the Perp locus. Due to the proximity of Perp sites A and B, these sites could not be distinguished in this assay.



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  		FIGURE 4. Differential binding of p53 at the Perp and p21 sites in vivo in apoptotic versus cell cycle arrested cells. ChIP analysis for the presence of p53 at the 5' p21 response element and Perp sites A/B (A&B), C, and D either untreated (U) or following treatment of wild-type MEFs (WT), wild-type E1A-MEFs (E1A WT), or p53-/- E1A-MEFs (E1A p53-/-) with doxorubicin (dox) for 12 h. Twenty percent of total input chromatin (IN) and no antibody (NA) controls are shown. Perp sites A&B could not be separated in this assay due to their proximity to one another in the Perp promoter. PCR reactions were run on a 2% gel and stained with ethidium bromide to visualize amplified fragments.

 
Association of p53 with the p21 promoter was strongly stimulated in both wild-type MEFs and wild-type E1A-MEFs following doxorubicin treatment (Fig. 4). In contrast, association of p53 with the Perp sites was greatly induced by doxorubicin treatment in wild-type E1A-MEFs but not in wild-type MEFs without E1A. These data correlate well with the observed expression patterns of p21 and Perp in cell cycle arrested and apoptotic MEFs (52) and provide the first mechanistic insight into the differential regulation of Perp under these conditions. Interestingly, while site C failed to bind p53 in gel shift experiments (Fig. 2) and point mutation of site C had no effect on the ability of p53 to transactivate Perp luciferase reporters in the HIp53 system (Fig. 3B), we observed a strong association of p53 with Perp site C in vivo. This discrepancy may suggest the presence of a nonconsensus response element within the amplicon used to detect binding to site C or may indicate that additional sequences surrounding the minimal consensus element used for EMSA analysis are required. Alternatively, p53 binding to site C in vivo may require association of other factors that are not present in the in vitro gel shift conditions or in the context of the reporter assay.

A p53 Point Mutant Deficient in Induction of Apoptosis Fails to Transactivate Perp
We also examined the ability of an apoptosis-deficient, temperature-sensitive p53 mutant (p53V143A) to transactivate the pPERPlucFL reporter. The V143A mutation lies within the DNA binding domain of p53 and shows a temperature-sensitive phenotype with respect to transactivation of some target genes, including GADD45, Cyclin G, p21, and mdm2 (35). However, p53V143A fails to transactivate target genes involved in apoptosis, including Bax and IGF-BP3, and is specifically defective in inducing apoptosis at the permissive temperature (35). Consistent with previous reports, in our experiments, the V143A mutant was unable to transactivate any of the target gene reporter constructs tested in transient cotransfection assays in H1299 cells at 37°C. At 32°C, p53V143A transactivated the Cyclin G and mdm2 reporters but still failed to induce expression of the pPERPlucFL and IGF-BP3 box B reporters (Fig. 5A). These data place Perp in the apoptotic category of p53 target genes.



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  		FIGURE 5. The p53V143A point mutant can discriminate between Perp and arrest-related target genes. A. Relative transactivation of target genes by wild-type p53 (wt) and p53V143A (V143A) at 37°C and 32°C in transiently transfected H1299 cells. Cells were cotransfected with reporter, the relevant expression vector, and the pRL-null Renilla luciferase vector and transferred 3 h after transfection to 32°C or left at 37°C. Data are normalized to activity in the empty vector condition and are plotted as a percent induction by wild-type p53 at each temperature at 24 h. Columns, averages of three separate experiments performed with four replicates per condition; bars, 1 SD. B. EMSA showing a selective defect in the ability of p53V143A to bind probes corresponding to Perp sites B and D compared with the 5' p21 probe. H1299 cells were transfected with the appropriate expression vector and were transferred 3 h after transfection to 32°C or left at 37°C. Cells were lysed 24 h after temperature shift. Forty micrograms of whole cell lysate were used per well. Probes were counted in a scintillation counter and equal counts of all probes were loaded per lane. Lower panel, free probe. C, Western blot for p53 demonstrating approximately equal expression of wild-type and mutant p53 at each temperature. Both wild-type and mutant p53 showed significantly higher expression at 37°C compared with 32°C.

 
To determine whether the observed differences in transactivation between these reporters were mediated by the p53 response elements themselves or required additional sequences, we examined the ability of wild-type p53 and p53V143A to bind sites B and D from the Perp gene and the 5' p21 site using gel shift analysis. Consistent with previous reports, p53V143A expressed at 37°C bound much more weakly to all sites examined (Fig. 5B) despite being strongly expressed at levels comparable with wild-type p53 at this temperature (Fig. 5C). At 32°C, p53V143A was capable of binding to all sites. However, p53V143A binding to Perp sites B and D was sharply lower than the binding of wild-type p53 to those sites at the same temperature (Fig. 5B; 18- and 11-fold, respectively), consistent with the failure of the point mutant to transactivate pPERPlucFL at 32°C (Fig. 5A). This difference was not due to differences in the expression of wild-type and mutant p53, as levels of both proteins were comparable at 32°C (Fig. 5C). In contrast, binding of p53V143A to the 5' p21 probe was robust and was only 2.3-fold lower than wild-type at this temperature (Fig. 5B), correlating with the ability of p53V143A to transactivate the p21 reporter at 32°C (Ref. 35, and data not shown). Thus, the deficiency in binding of the p53V143A mutant at 32°C compared with the wild-type protein was more pronounced with respect to Perp sites B and D than with respect to the 5' p21 site, demonstrating that the minimal response element contains sufficient information to allow p53V143A to distinguish between Perp and p21.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Given the well-established role of p53 in tumor suppression and the apoptotic response of tumor cells to chemotherapeutic agents, a clear understanding of its many functions will be crucial for rational design of improved treatments. One key outstanding question is the mechanism by which p53 distinguishes between classes of target genes to induce cell cycle arrest or apoptosis when appropriate. In this report, we have dissected the transcriptional control elements of the Perp gene to gain insight into this process. As described above, the regulation of the Perp promoter by p53 appears to involve a complex interplay between multiple response elements. The data presented here demonstrate the existence of two novel p53 response elements within the murine Perp gene, sites B and D, in addition to sites A and C, which we described previously (52). In addition, we show that Perp can be distinguished from p21 at the level of DNA binding in vivo and in vitro.

Several models have been proposed to explain the different cellular outcomes downstream of p53 activation. In one model, p53 responds to cellular insults in a consistent manner, and the ultimate fate of the cell is determined by the presence of other p53-independent survival or death signals (61). While other factors are undoubtedly involved in the decision, our data argue against such a limited role for p53. We observed differential association of p53 with the Perp promoter in ChIP analysis of apoptotic cells compared with cell cycle arrested cells (Fig. 4), suggesting that the activity of p53 itself toward apoptotic targets is stimulated under conditions favoring an apoptotic response. In addition, the temperature-sensitive p53V143A mutant showed a selective defect in binding to Perp gel shift probes compared with the 5' p21 probe at the permissive temperature, which was reflected in the inability of p53V143A to transactivate Perp and other apoptotic target genes but not cell cycle targets at this temperature (Fig. 5). These data agree with previous observations that the cell cycle arrest and apoptotic functions of p53 can be uncoupled through specific point mutations and that loss of apoptotic function is correlated with the inability to transactivate a specific subset of target genes implicated in apoptosis (35, 38, 41). While there is no question that p53-independent factors can influence the eventual cellular outcome, p53 itself can clearly play a direct role as well. Therefore, a second model, in which p53 plays an active role in determining the outcome following a given insult (61), appears to be more consistent with our observations.

Although the mechanisms behind this role of p53 remain largely unclear, the ability of p53 to distinguish between arrest and apoptosis targets requires, at least, that intrinsic differences exist in the regulatory regions of these genes, either within the response element itself or in surrounding sequences. The ability of p53 to distinguish between short oligonucleotide probes corresponding to Perp and p21 sites (Fig. 5B) suggests that the minimal response element, in the absence of surrounding sequences, contains such information. Our understanding of specific sequence requirements for functional p53 response elements within the accepted consensus sequence put forth by el-Deiry et al. in 1992 is still rudimentary, but several groups have begun making progress on this front. Recently, Qian et al. (62) compared binding sites from 57 known and potential target genes of p53 and noted the presence of spacer regions between half-sites and three or more mismatches relative to the consensus in apoptotic targets, while response elements from cell cycle related target genes generally lacked spacers and had two or fewer mismatches. Inga et al. (63) showed that p53 response elements from various target genes could vary by as much as 1000-fold in their ability to direct p53-dependent transactivation in yeast and that the presence of a CATG at the core of one or both half-sites correlated strongly with a high transactivation capacity.

Based on these predictions, the four p53 binding sites we identified at the Perp locus would be expected to display a range of affinities for p53. Sites B and D each contain two mismatches (excluding the third half-site in site B) and lack spacers, suggesting that these would be relatively strong sites. In addition, site D contains a CATG core at the 3' half-site, consistent with it being a particularly strong site. In fact, we observed robust binding to both sites B and D in gel shifts (Figs. 2 and 5B), and a strong requirement for site D for full induction by p53 in reporter assays (Fig. 3). In addition, there was clear occupancy of site D by p53 in ChIP analysis in E1A-MEFs even in the absence of DNA damage (Fig. 4). The requirement for site B in reporter assays was somewhat weaker.

In contrast, sites A and C contain two and four mismatches, respectively, and each has a spacer region, suggesting that these would be relatively weak sites. As predicted, these sites failed to bind p53 in gel shift experiments (Fig. 2) and were dispensable for induction of Perp reporters by p53 (Fig. 3). However, site C carries a CATG motif at the core of its 3' half-site, and p53 was found to associate strongly with site C in ChIP analysis in E1A-MEFs following DNA damage (Fig. 4). Thus, perhaps in vivo, the presence of a CATG motif can compensate to some degree for the presence of mismatches and a spacer. Finally, the 5' p21 site, to which the Perp sites were compared, contains no spacer, has two mismatches, and has a CATG motif at the core of each half-site (Fig. 1B). This site bound very strongly to p53 in gel shift assays (Fig. 5B). It was required for transactivation of p21 reporters by p53 (Fig. 2A; Ref. 20) and is occupied by p53 during both apoptosis and arrest conditions in ChIP analysis (Fig. 4). These data are consistent with the hypothesis that intrinsic differences in response elements may be partially responsible for determining their affinity for p53. However, the presence of multiple sites with varying characteristics within the Perp promoter suggests that a simple model in which cell cycle arrest related targets contain high-affinity sites and proapoptotic targets contain low-affinity sites is insufficient to explain the differential regulation of proarrest and proapoptotic target genes. Instead, the regulation of Perp expression, and perhaps that of other target genes, may be achieved through a complex interaction of p53 with multiple sites of varying affinity as well as with other cofactors.

Several groups have recently identified cofactors such as ASPP1/2 and JMY that bind p53 and can selectively stimulate transactivation of apoptosis-related genes without affecting expression of arrest-specific genes (43, 44). Additionally, we have shown previously that the p53 family members, p63 and p73, are required for binding of p53 to response elements in apoptotic targets and induction of apoptosis but not for cell cycle arrest (42). In that report, p63 was shown to be associated with the Perp promoter in E1A-MEFs following DNA damage and loss of both p63 and p73 prevented association of p53 with the Perp promoter following DNA damage. In contrast, p63 and p73 were not required for binding of p53 to the p21 and mdm2 promoters. These data show that p53 family members play an important role in the transactivation of apoptotic targets of p53 and suggest the intriguing possibility that p63, and perhaps p73, might bind to the Perp promoter following DNA damage and recruit p53 to the otherwise weak response elements at the Perp locus, thereby activating Perp to stimulate an apoptotic response.

The regulation of the apoptosis versus cell cycle arrest decision by p53 is clearly complex and likely involves an intricate interplay between numerous factors to produce the appropriate cellular response under each set of conditions. Moreover, the large number of apoptosis-related targets of p53 identified to date in various systems may imply that the specific mechanisms differ depending on cell type and specific death stimulus. The results presented here confirm the role of Perp as a direct transcriptional target of p53 involved in the apoptosis response. They demonstrate that interaction of p53 with the Perp locus occurs through at least three p53 response elements of varying affinities and is regulated at the level of p53 DNA binding. Understanding how these sites function and the mechanisms by which Perp expression is enhanced under apoptotic conditions will likely provide valuable insight into the regulation of the critical cell death decision.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cloning and Sequence Analysis of Perp Genomic DNA
A 200-bp RsaI fragment of the Perp cDNA was used to probe a murine 129/SvJae genomic {lambda}FIX II phage library. Isolated clones were sequenced and intron-exon boundaries were determined from the cDNA sequence. All putative boundaries contain consensus splice donors and acceptors. Perp genomic DNA sequence was scanned for sequences matching the p53 consensus sequence (30). The human Perp sequence was obtained from the Sanger Center Web site (http://www.sanger.ac.uk/HGP/Chr6/) and was used for comparative sequence analysis with the murine Perp sequence. Regions of high conservation were identified using several Internet-based alignment programs (Bayesian Phylogenetic Footprint Homepage, http://bayesweb.wadsworth.org/cgi-bin/bayes_align12.pl, and PipMaker, http://bio.cse.psu.edu/pipmaker) and the VectorNTI alignment program (Informax, Frederick, MD.).

Cell Culture
p53-inducible HIp53 cells (57) were selected in 600 µg/ml G418 and 400 µg/ml Zeocin. Wild-type and p53-/- MEFs were isolated as described. To generate E1A-expressing MEFs, wild-type and p53-/- MEFs were infected with retroviral E1A as described previously (64).

EMSA
EMSAs were performed using the p53 NuShift Plus kit from Active Motif, Carlsbad, CA. Antibody 421 (Ab-1, Oncogene Science, Cambridge, MA) at 1 µg/µl was used in place of the supplied polyclonal anti-p53 antibody. Cells were plated at 2 x 106 cells per 15-cm dish 24 h before treatment. After 12–24 h of treatment, cells were rinsed once in PBS, scraped from the plate, pelleted, and lysed in 100 µl cell lysis buffer [100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1% NP40] plus protease inhibitors (Complete Mini, Roche, Basel, Switzerland) for 30 min at 4°C. Total protein concentrations were determined using BCA Protein Assay (Pierce Chemical Co., Rockford, IL). Total protein used per binding reaction varied from 40 to 100 ng depending on the experiment but was constant for all lanes in a given experiment. Probes were end labeled with T4 PNK and purified in G-25 Microspin columns (Amersham Pharmacia Biotech, Piscataway, NJ). All probes were in excess and equal counts were used per reaction. Binding reactions were run on a 4% polyacrylamide gel. Oligonucleotide probes were Perp site A: 5'-AGAGGCGCTAGTCCACACAGACTAGATTCCTC-3', Perp site B: 5'-TATTTGCTTGGTCTCACATTCTAGCCCAGGCTTGCCCAGA-3', Perp site C: 5'-TGGAGCTCAAGTGTAGCCTTAGCCATGCTCCTTA-3', Perp site D: 5'-CTTCAGATCCTGGGCAAGCTCTGGCATGTCAGGGTGGAGGG-3', p21 5' site: 5'-GAACATGTCTTGACATGTTC-3', wild-type competitor: 5'-AGCTGGACATGCCCGGGCATGTCC-3', and mutant competitor: 5'-AGCTGGATCGCCCCGGGCATGTCC-3'.

Plasmids
Wild-type p53 and p53V143A expression vectors were described previously (65, 66). All Perp luciferase reporters were constructed in the pGL3Basic vector. The pPERPluc1 reporter was described previously (52). The pPERPlucFL vector was constructed by insertion of a splice acceptor upstream of the luciferase cassette in pGL3Basic and insertion of a BamHI Perp genomic fragment (from -3850 to +4150) into the polylinker region. pPERPlucPS was derived from pPERPluc1 by blunt insertion of a PmeI-SphI fragment of intron 1 (from +2377 to +3870) into the downstream cloning site in the pGL3Basic backbone. pPERPlucPS-mutD and pPERPlucPS-mutB vectors were derived from pPERPlucPS through site-directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA). Oligonucleotides used in site-directed mutagenesis were Perp site A: 5'-CCTTAAGAGGCGTTATTCCACACAGATTATATTCCTCCATATG-3', Perp site B: 5'-GGTCTCACATTTTATCCCAGGTTTTCCCAGAATTAAC-3', Perp site C: 5'-GTGCTTGGAGCTTAATTGTAGCCTTAGCTATTCTCCTTACACTG-3', and Perp site D: 5'-CTTCAGATCCTGGGTAATCTCTGGTATTTCAGGGTGGAGGG-3'. The pPERPlucFL-mutD vector was derived from pPERPlucFL by substitution of the point-mutated PmeI-SphI intron 1 fragment for the wild-type fragment. pPERPluc1+D and pPERPluc1+mutD were constructed using the QuikChange kit to insert an oligonucleotide containing wild-type or mutant site D downstream of the luciferase coding region. The wild-type and mutant p21 luciferase reporters were derived from p21 chloramphenicol acetyltransferase reporters (59). The Bax, Cyclin G, mdm2, and IGF-BP3 reporters were described previously (60). Deletion constructs were derived from pPERPlucPS vector using standard cloning methods. The pRL-null Renilla luciferase expression vector (Promega) was used in all luciferase assays.

DNA Transfection and Transactivation Assays
Transient transfections were performed using Fugene6 (Roche) at a 1:1.5 or 1:3 DNA/Fugene6 ratio according to manufacturer's instructions. Luciferase assays were performed using the Dual Luciferase System (Promega). Cells were plated 18–24 h before transfection at 5000 cells/well of a 96-well plate. Fifty nanograms of reporter, 50 ng of pRL-null, and 100 ng of wild-type or mutant p53 were transfected per well. HIp53 cells were treated with 10 µM PonA or an equal volume of ethanol 6–12 h post-transfection. Cells were lysed 12 h post-treatment for HIp53 or 24 h post-transfection for cotransfection experiments with p53V143A in 20 µl/well 1x Passive Lysis Buffer (Promega) by shaking at room temperature for 15 min. The 96-well plates were read on a LB96V plate-reading luminometer (Berthold, OakRidge, TN) with 45 µl injection volumes for each reagent and a 2-s delay followed by a 10-s integration time. Firefly luciferase readings were divided by Renilla luciferase readings as a control for variations in cell number and transfection efficiency.

ChIP Assay
After treatment with doxorubicin for 12 h, wild-type MEFs with or without E1A and p53-/- E1A-MEFs were cross-linked with 1% formaldehyde. ChIP was carried out as described previously (42). Primers for the 5' site of the p21 promoter and Perp sites A/B and C were as described previously (42). Primers for Perp site D were 5'-TGAATGTTTGGCTTATATTTGTGGAG-3' and 5'-CCTTCTTTCAGTGCATACCTCATCCC-3'. Annealing temperature was 60°C. PCR products were electrophoresed on a 1.2% agarose gel and were detected by ethidium bromide staining.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank M. Oren for Cyclin G, mdm2, and IGF-BP3 reporters, J. Pietenpol for the HIp53 cell line, K. Macleod for p21 chloramphenicol acetyltransferase reporters, and B. Vogelstein for pCMV-wild-type p53 and pCMV-p53V143A expression vectors.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Anna Fuller Fund (E. E. R.), Leukemia and Lymphoma Society of America (E. R. F.), Damon Runyon Cancer Research Foundation (L. D. A.), and NIH and Howard Hughes Medical Institute (T. J.). Back

Received August 21, 2003; revised November 12, 2003; accepted November 14, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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