Wild-type p53 and p73 negatively regulate expression of ... - Nature

Oncogene (2008) 27, 2583–2593

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ORIGINAL ARTICLE

Wild-type p53 and p73 negatively regulate expression of proliferation related genes MJ Scian1,4, EH Carchman1,4, L Mohanraj1,4, KER Stagliano1, MAE Anderson1, D Deb1, BM Crane1, T Kiyono2, B Windle3, SP Deb1 and S Deb1 1 Department of Biochemistry and Molecular Biology and the Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA; 2Virology Division, National Cancer Center Research Institute, Tokyo, Japan and 3Department of Medicinal Chemistry and the Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA

When normal cells come under stress, the wild-type (WT) p53 level increases resulting in the regulation of gene expression responsible for growth arrest or apoptosis. Here we show that elevated levels of WT p53 or its homologue, p73, inhibit expression of a number of cell cycle regulatory and growth promoting genes. Our analysis also identified a group of genes whose expression is differentially regulated by WT p53 and p73. We have infected p53-null H1299 human lung carcinoma cells with recombinant adenoviruses expressing WT p53, p73 or b-galactosidase, and have undertaken microarray hybridization analyses to identify genes whose expression profile is altered by p53 or p73. Quantitative real-time PCR verified the repression of E2F5, centromere protein A and E, minichromosome maintenance proteins (MCM)-2, -3, -5, -6 and -7 and human CDC25B after p53 expression. 5-Fluorouracil treatment of colon carcinoma HCT116 cells expressing WT p53 results in a reduction of the cyclin B2 protein level suggesting that DNA damage may indeed cause repression of these genes. Transient transcriptional assays verified that WT p53 repressed promoters of a number of these genes. Interestingly, a gain-of-function p53 mutant instead upregulated a number of these promoters in transient transfection. Using promoter deletion mutants of MCM-7 we have found that WT p53-mediated repression needs a minimal promoter that contains a single E2F site and surrounding sequences. However, a single E2F site cannot be significantly repressed by WT p53. Many of the genes identified are also repressed by p21. Thus, our work shows that WT p53 and p73 repress a number of growth-related genes and that in many instances this repression may be through the induction of p21. Oncogene (2008) 27, 2583–2593; doi:10.1038/sj.onc.1210898; published online 5 November 2007 Keywords: wild-type p53; transcriptional repression; cell cycle; DNA replication; microarray analysis

Correspondence: Dr S Deb, Department of Biochemistry and Molecular Biology and the Massey Cancer Center, Virginia Commonwealth University, Goodwin Research Laboratory, 401 College Street, Richmond, VA 23298, USA. E-mail: [email protected] 4 These authors contributed equally to this work Received 13 December 2006; revised 17 September 2007; accepted 1 October 2007; published online 5 November 2007

Introduction Elevated levels of wild-type (WT) p53 in response to stress situations lead to apoptosis or cell cycle arrest by modulating expression of genes involved in cellular growth regulation (el-Deiry, 1998; Prives and Hall, 1999; Maxwell and Davis, 2000; Vogelstein et al., 2000; Zhao et al., 2000; Kannan et al., 2001; Aldaz et al., 2002). Although most of the work has been focused on WT p53-mediated transactivation, p53 has also been shown to repress transcription of cellular and viral genes (Subler et al., 1992; el-Deiry, 1998; Kubicka et al., 1999; Lee et al., 1999; Murphy et al., 1999; Krause et al., 2000; Maxwell and Davis, 2000; Sun et al., 2000; Zhang et al., 2000; Kannan et al., 2001; Koumenis et al., 2001; Aldaz et al., 2002; Hoffman et al., 2002; Maeda et al., 2006; Moskovits et al., 2006). Additionally, WT p53 inhibits transcription of genes promoting cell survival and genes involved in G2/M transition (Murphy et al., 1996, 1999; Lee et al., 1999; Johnsen et al., 2000; Krause et al., 2000; Manni et al., 2001; Hoffman et al., 2002; Chae et al., 2005; Ho et al., 2005; Imbriano et al., 2005; Sengupta et al., 2005; Scoumanne and Chen, 2006; Spurgers et al., 2006; Van Bodegom et al., 2006). The ability of WT p53 to induce cell cycle arrest or apoptosis is predominantly related to the protein’s ability to bind DNA and act as a transcriptional activator (reviewed in el-Deiry (1998); Prives and Hall (1999); Vogelstein et al. (2000); Zhao et al. (2000)). Most tumorderived p53 mutants are defective in this sequence-specific DNA binding and therefore, lose their transactivation. Thus, these mutants are generally defective in protective cellular functions, such as p53-mediated apoptosis and cell cycle arrest. Interestingly, these mutants are also defective as repressors (Deb et al., 1992; Subler et al., 1992). In mice, mutation of residues G25 and S26 results in a mutant p53 with decreased transactivation and transrepression activities (Johnson et al., 2005). This mutant protein is also deficient in inducing cell cycle arrest (Chao et al., 2000). Using recombinant adenoviruses to express WT p53, p73 or b-galactosidase followed by microarray hybridization analyses, we have identified a number of genes repressed by WT p53 and p73 involved in cell growth and DNA replication. We have verified the repression for a number of these target genes by quantitative real-time

Transcriptional repression by p53 MJ Scian et al

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QPCR. While our manuscript was in preparation Spurgers et al. (2006) has reported similar findings. In vivo transcriptional analyses indicate that the promoters of many of these genes are inhibited after expression of WT p53. QPCR data shows that various genes repressed by WT p53 and p73 are also repressed by p21 suggesting that WT p53-mediated repression occurs through the induction of p21. We have also investigated repression of the MCM-7 gene and have found that this repression may occur through E2F, although other sequences flanking the E2F site are also required.

Results p53 and p73 have highly similar gene expression response profiles The p53 homologue, p73, has structural and functional similarities to WT p53. For example, p73 can induce transcription of genes normally induced by WT p53 is also stabilized by genotoxic stress resulting in apoptosis or cell cycle arrest (Kaghad et al., 1997; Di Como et al., 1999). Since p73 can substitute for WT p53 as a transcriptional activator, it is expected that p73 may also substitute for WT p53 as a transcriptional repressor (Fontemaggi et al., 2002). To examine this possibility, overexpression of p73 and WTp53 were compared for their effects on gene expression in the lung carcinoma cell line, H1299. The gene expression profile of H1299 and 21PT cells infected with a recombinant adenovirus expressing WT p53 was compared to that of cells infected with a recombinant adenovirus expressing b-galactosidase in order to identify genes modulated by WT p53. Additionally, a H1299derived WT p53 inducible cell line (HIp53, Flatt et al., 2000) was used to complement the gene expression results obtained using the adenoviral system. For the p73 gene expression response profile, H1299 cells were infected with an adenovirus expressing p73a and p73b and compared to cells infected with one expressing b-galactosidase. Gene expression was assessed by microarray hybridization analyses using the Affymetrix U95Av2 arrays and analyzed as described in Materials and methods. We wanted to identify any statistically significant association between p53 and p73, from significant yet subtle effects on genes due to biological effects or a low sensitivity of the microarray for some genes. An enrichment process for differentially expressed genes for p53 versus control and p73 versus control was used followed by identification of overlapping genes between p53 and p73 and between known biological processes and the genes common to p53 and p73. A 10-fold enrichment of differentially expressed genes, selecting for the lowest P-values, generated lists of genes with a falsediscovery rate from 0.34 to 0.37 for the differentially expressed genes of p53 and p73. While this provided lists of genes insufficient for study by themselves individually, substantially higher confidence was added with the subsequent association analyses. There were 435 genes in common between the p53 response genes and the p73b response genes, with most Oncogene

of these genes overlapping those for the p73a response genes. The P-value for the p53-p73b association is 10144, and thus these overlapping genes reflect a substantial and significant similarity in gene expression effects between p53 and p73. The p53-p73 response genes were further analyzed by association analysis with Gene Ontology (GO) biological processes to identify subsets of genes of particular interest. The overrepresentation of genes from known biological processes could shed light on the types of genes regulated by both p73 and p53. Supplementary Table S1 shows the GO biological processes with statistically significant associations with the p53-p73a and p53-p73b response genes. An FDR of 0.10 was used for identifying these pathways and functions. The pathways and functions represented for the p53p73a and the p53-p73b response genes are similar, and any difference may merely reflect differences in expression between the two forms of p73. Cell cycle-related processes are prominent and consistent with the known effects on the cell cycle by p53. The processes of cell cycle (Table 1), mitosis and cytokinesis are all highly interrelated, and some of these genes identified in this analysis were studied further. Of the 435 genes in common between p53 and p73b, there were 43 genes that were inversely correlated, either induced by p53 and repressed by p73 or vice versa (Supplementary Table S2). Association analysis with GO biological processes yielded no significant association and thus any biological significance of these genes will await investigation at a later date. We have used QPCR to verify the data from microarray analyses. RNA was isolated and cDNA synthesized from WT p53- (or b-galactosidase-) expressing H1299 cells. Standard curves were generated for target genes and used for the relative quantitation of gene expression. The relative mRNA levels in b-galactosidaseexpressing cells was set to 1 and compared to mRNA levels in cells after expression of WT p53. We have confirmed WT p53-mediated repression of E2F-5, CENP-A, CENP-E, hCDC25B and MCM-6 by QPCR (Figure 1). In some cases we have also tested related genes, although our microarray analyses might not have directly identified them; for example, array analyses have identified MCM-6 and -7 as transcriptional targets of p53, we have also checked other MCM genes. Transcription of MCM-2, -3, and -5, was also inhibited after expression of WT p53 (Figure 1); similar is the situation for CENP-E. The table inset shows the fold inhibition calculated for each of the genes tested. Similar results were obtained after induction of WT p53 in the HIp53 inducible cell line system (Supplementary Figure S1). The apparent reason for the difference in the level of repression observed is most likely due to the method of WT p53 expression attainable by the two methodsrecombinant adenovirus (expressing WT p53) infection generates a much higher level of WT p53, leading to a higher level of repression. To test whether p53-inhibited genes are repressed because of accumulated p53, we used protein synthesis inhibitor cycloheximide (CHX) that has been shown to inhibit p53 accumulation and p53-dependent transcription


2585 Table 1 A selection of genes common to p53 and p73b responses associated with cell cycle Gene name

Genbank accession no

Gene symbol

Ubiquitin-conjugating enzyme E2C TPX2, microtubule-associated protein homologue (Xenopus laevis) Putative lymphocyte G0/G1 switch gene MCM7 minichromosome maintenance deficient 7 MCM6 minichromosome maintenance deficient 6 CDC28 protein kinase regulatory subunit 2 CDC20 cell division cycle 20 homologue (Saccharomyces cerevisiae) Cyclin B1 Cyclin A2 Serine/threonine kinase 6 RAN, member RAS oncogene family Cell division cycle 25B Kinesin family member 22 V-myc myelocytomatosis viral oncogene homologue (avian) Cyclin-dependent kinase 2 Microtubule-associated protein, RP/EB family, member 2 Cyclin-dependent kinase inhibitor 3 G-2 and S-phase expressed 1 Kinesin family member 23 Deoxythymidylate kinase (thymidylate kinase) Cyclin F Cell division cycle 25C Cyclin-dependent kinase inhibitor 1A (p21, Cip1) Tumor protein p53 (Li-Fraumeni syndrome) Jagged 2 Stratifin V-Ha-ras Harvey rat sarcoma viral oncogene homologue Growth arrest-specific 2 like 1 Latent transforming growth factor beta binding protein 2 Septin 9 Neural precursor cell expressed, developmentally downregulated 9

U73379 NM_012112 NM_015714 D55716 NM_005915 NM_001827 NM_001255 M25753 X51688 NM_003600 NM_006325 S78187 AB017430 M13929 M68520 NM_014268 L25876 NM_016426 NM_004856 L16991 Z36714 M34065 U03106 X02469 NM_002226 NM_006142 NM_005343 NM_006478 NM_000428 NM_006640 NM_006403

UBE2C TPX2 G0S2 MCM7 MCM6 CKS2 CDC20 CCNB1 CCNA2 STK6 RAN CDC25B KIF22 MYC CDK2 MAPRE2 CDKN3 GTSE1 KIF23 DTYMK CCNF CDC25C CDKN1A TP53 JAG2 SFN HRAS GAS2L1 LTBP2 SEPT8 NEDD9

Fold change for p53

Fold change for p73 beta

6.04 5.53 5.26 4.69 4.52 4.28 3.32 3.20 2.71 2.32 1.92 1.73 1.72 1.66 1.63 1.43 1.36 1.34 1.29 1.25 1.22 1.15 32.26 19.82 6.40 4.13 3.14 2.12 1.47 1.24 1.08

4.37 3.85 4.67 2.59 2.94 3.29 2.61 2.66 2.03 1.82 1.73 1.58 1.53 1.40 1.43 1.40 1.32 1.39 1.12 1.28 1.14 1.24 5.17 1.21 14.67 63.49 2.48 1.63 1.63 2.76 1.08

A negative fold change indicates repression while a positive fold change indicates induction.

(Woo et al. 2002). We pretreated the HIp53 culture with 10 mg ml1 CHX for 30 min following the procedure described by Kho et al. (2004) prior to p53 induction and harvested the cells after 24 h. As expected, CHX treatment significantly abrogated p21 induction (Supplementary Figure S1) and impacted expression of cyclin B2. In the presence of CHX, we observed some downregulation of cyclin B2 expression in cells treated with Ponasterone A. QPCR analysis revealed an upregulation of 1.8-fold p53 expression (leading to a 2.5-fold induction of p21) in the presence of CHX compared to 11.7-fold in its absence by Ponasterone A. This suggests that residual WT p53 is responsible for downregulation of cyclin B2 even in the presence of CHX. In the absence of CHX, we see almost complete inhibition of cyclin B2 expression at 24 h. Thus, cyclin B2 may be considered as a p53-regulated gene. We have also investigated by QPCR analysis whether p73b could inhibit any of the genes repressed by WT p53. As was seen with our microarray data, p73b was able to inhibit most of the genes tested by QPCR (Supplementary Figure S2). QPCR data show that p73b expression is lower after infection of cells with the adenovirus expressing p73b compared to the p53 expression level in cells infected with WT p53 adenovirus (data not shown). This indicates that the relative lower repressive capacity of p73 may be a reflection of its lower expression level.

Promoters and cloned upstream sequences of a number of genes identified are repressed in transient transcriptional assays We cloned (or obtained from other sources) a number of promoters (or presumptive promoters) corresponding to a representative set of identified genes. DNA fragments generated by genomic PCR were cloned into pGL3luciferase (Promega, Madison, WI, USA), using sequence information from NCBI. Saos-2 p53-null osteosarcoma cells were cotransfected with promoter-luciferase constructs and increasing amounts of an expression plasmid for WT p53 (or vector alone) as indicated in the figure (Figure 2). Cells were harvested and luciferase assays carried out as described (Deb et al., 2001, 2002). All the promoters tested were significantly inhibited by WT p53 (Figures 2b and c). These results are significant since similar amounts of WT p53 can transactivate 17–30 folds a construct containing the p21 promoter (Figure 2a). Mutant p53, instead upregulated the promoters of MCM-3, -5 and -7, showing gain-of-function activity (Figure 2d). This is in line with our earlier data showing that mutant p53 could upregulate the MCM-6 promoter (Scian et al., 2005). Many WT p53 repressed genes are repressed through p21 induction Studies have shown that WT p53 transcriptional regulation may be affected by p21 (Taylor et al. 2001; Oncogene


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Relative mRNA level

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MCM-7

Figure 1 QPCR after adenoviral expression of WT p53. mRNA was extracted from cells infected with a WT p53-expressing recombinant adenovirus (or b-galactosidase-expressing adenovirus as a control). cDNA was prepared 16–24 h postinfection and analyzed by QPCR using gene-specific primers for various genes identified through microarray analysis as described in Materials and methods. The degree of expression was quantitated using a relative standard curve and normalized to an internal control (BMV RNA) corresponding to the cDNA batch as described (Deb et al., 2002). PCR reactions were carried out in triplicate. The average normalized mRNA levels for each gene were calculated and graphed. Fold change, **Pp0.001 and *Pp0.01. The table inset shows the relative fold repression observed.

Lohr et al., 2003; Shats et al., 2004). Therefore, we tested whether transcription of genes identified in this study can also be inhibited by expression of p21. H1299 cells were infected with a recombinant adenovirus expressing p21 or b-galactosidase as control (Prabhu et al., 1996). RNA was extracted 16 h postinfection. mRNA levels in cells expressing p21 were determined for the genes of interest and compared to levels in the control cells infected with an adenovirus expressing b-galactosidase. For all genes examined, p21 overexpression resulted in repression, although this inhibition was not as pronounced as that observed with WT p53 expression (Supplementary Figure S3). QPCR data show that p21 expression is lower after infection of cells with the adenovirus expressing p21 compared to those cells infected with WT p53 (Supplementary Figure S3). The table inset compares the fold inhibition observed after infection of cells with WT p53 or p21-expressing adenoviruses. Thus, many of the genes repressed by WT p53 detected by our microarray may be repressed via p21. Treatment of HCT116 cells with 5-fluorouracil causes p53 stabilization and a reduction of the cyclin B2 level We tested whether 5-fluorouracil (5-FU) treatment of the WT p53-expressing colon carcinoma cell line HCT116 stabilizes p53 and whether that leads to reduction of cyclin B2 expression. Following the method Oncogene

described by Kho et al. (2004), we treated HCT116 cells with 5-FU and allowed to incubate for 24 h. As shown in Figure 3, western blot analysis shows p53 stabilization while QPCR analysis indicates that cyclin B2 expression was inhibited by the treatment in HCT116 (p53 þ / þ , p21 þ / þ ) cells, but not in HCT116 (p53/, p21 þ / þ ) cells. Interestingly, in case of HCT116 (p53 þ / þ , p21/) cells cyclin B2 expression became higher because of the treatment; perhaps this indicates that in the absence of p21 accumulated WT p53 induces a G2/M arrest leading to an increase in the cyclin B2 level. Thus, WT p53 downregulates the expression of cyclin B2 in a p53 dependent way. Our data is in agreement with that shown by Kho et al. (2004), who also demonstrated downregulation of MCM-3, -6 and -7 among others. Basile et al. (2006) also showed that cyclin B2 is a WT p53 target. Promoter deletion analysis We examined whether there is a requirement for a specific sequence motif for WT p53-mediated repression of the MCM-7 promoter, and tested a number of MCM7 promoter deletions (Figure 4a). Figure 4b shows that the MCM-7 promoter sequences defined between the þ 53 and 186 positions are efficiently repressed suggesting that the minimal sequence requirement for the repression may be found within this region. Further deletion abolishes this repression. We repeated this


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Figure 2 Promoter analysis. The presumptive promoters for CENP-E, MCM-3, murine CDC25B (mCDC25B) and human CDC25B (hCDC25B) were cloned from genomic DNA using information obtained in the NCBI database (Materials and methods). The MCM7, p21 and mCDC25A promoter constructs have been previously described (Deb et al., 2001; Korner et al., 2001; Suzuki et al., 1998). Promoters were studied using reporter luciferase constructs and p53-null human osteosarcoma Saos-2 cells. Cells were transfected with 200 ng of promoter-luciferase reporter construct and 5, 10 or 50 ng of an expression plasmid for WT p53 (or an equivalent amount of empty vector as control). Saos-2 cells were transfected with (a) p21.luc and WT p53 expression plasmid (or expression vector alone), (b) MCM-7.luc, mCDC25B.luc or hCDC25B.luc and WT p53 expression plasmid (or expression vector alone), (c) CENP-E.luc or MCM3.luc and WT p53 expression plasmid (or expression vector alone) and (d) MCM-3.luc, MCM-5.luc or MCM-7.luc and p53-D281G expression plasmid (or expression vector alone). Cell extracts were prepared 48 h after transfection and luciferase activity measured. The vector reading was arbitrarily set to 100 and the relative luciferase activity (RLA) calculated for remaining samples. Fold change, **Pp0.001 and *Pp0.01, when not indicated, P-value >0.01. Oncogene


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experiment using HIp53 cells where WT p53 closed under inducible promoter. We have also used a pair of genotypically matched HCT116-derived cell lines lacking p21 but expressing endogenous p53 (Bunz et al., 1998; Flatt et al., 2000). As shown in Figures 4c and d, deletion of sequences located between residues 186 and 85 resulted in a loss of inhibition. These data suggest that the sequences required for WT p53 repression of the MCM-7 promoter are located within this region (186 to 85).

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WT p53-mediated repression of MCM-7 requires the E2F site CDC2 gene transcription is inhibited by WT p53 (Taylor et al., 2001). Interestingly, this promoter contains a p130/ E2F-4 binding site. Data presented above, suggest that WT p53 may inhibit the MCM-7 promoter through sequences located between residues 186 and 85, which has a putative E2F-binding site (Figure 4a). Therefore, we tested whether this site can interact with E2F factors and whether WT p53 interferes with this interaction. Equal amounts of protein from WT p53-induced and un-induced nuclear extracts were incubated in the

12h

presence of a radiolabeled probe containing the putative E2F site. Electrophoretic mobility shift assays (EMSAs) were carried out as described in Materials and methods (Figure 5). The arrow indicates the suspected E2F containing complexes. The shift observed can be abolished by the addition of nonradiolabeled specific competitor (SC) to the reaction (Figure 5, lanes 4 and 5). On the other hand, a nonspecific competitor (NSC) does not decrease the amount of protein-DNA complexes formed (Figure 5, lanes 6 and 7). Induction of p53 decreases the amount of protein-DNA complexes formed (compare lane 2 with 3). We have performed chromatin immunoprecipitation (ChIP) assays to determine whether E2F sites are occupied by E2F1 in vivo and whether expression of WT p53 impacts the occupancy. For this experiment we have used HIp53 cells where WT p53 is induced by treatment with Ponasterone A. The ChIP assays were carried out as described by Scian et al. (2004a) using induced and un-induced cells and, E2F-1-agarose to pull down E2F-1-specific complexes. QPCR was carried out to determine the presence of MCM-7 promoter sequences using primers specific for the MCM-7 upstream sequences. As shown in Figure 5b, there is significant

p53 HCT116p53+/+

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30.00 20.00 10.00 0.00 p53+/+

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Figure 3 Treatment of HCT116 cells with 5-flurouracil cause p53 stabilization and a reduction of cyclin B2 expression. HCT116 cells were treated with 375 mM 5-FU (Kho et al., 2004) for the indicated length of time after which cells were harvested, cell extracts prepared as described (Scian et al., 2005) and analyzed by western blot analysis using antibodies directed against human b-actin and p53. From another batch of cell treated identically RNA was isolated after 24 h, and QPCR was done as described in Materials and methods to assay for cyclin B2 and GAPDH. The data indicate p53 stabilization results in subsequent reduction of the cyclin B2 level.

Figure 4 MCM-7 promoter inhibition by WT p53 expression. (a) MCM-7 promoter deletions used in this study. The white boxes represent E2F-binding sites; the gray box represents a c-myc binding site. (b) Promoter deletions of the MCM-7 promoter were cotransfected into Saos-2 cells in the presence of a WT p53 expression plasmid (or empty expression plasmid as control). Cell extracts were prepared and luciferase readings taken 48 h after transfection as described in Materials and methods. The vector reading was arbitrarily set to 100 and the relative luciferase activity (RLA) calculated for remaining samples. (c) HIp53 cells were transfected with 200 ng of the various MCM-7 promoter deletions in the presence or absence of Ponasterone A (100 mM final concentration). Addition of Ponasterone A to the medium results in the conditional induction of p53 in this cell line. Cell extracts were prepared 24 h after transfection and luciferase activity measured. Luciferase units in the absence (Pon A) and presence of p53 ( þ Pon A) are graphed. The vector reading was arbitrarily set to 100 and RLA calculated for remaining samples. (d) HCT116 p21/ or p21 þ / þ cells were transfected with 200 ng of the various MCM-7 promoter deletions. Cell extracts were prepared 24 h after transfection and the luciferase activity measured. The vector reading was arbitrarily set to 100 and RLA calculated for remaining samples. Fold change, **Pp0.001 and *Pp0.01, when not indicated, P-value >0.01. Oncogene


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reduction in the occupancy by E2F-1 on the MCM-7 promoter after induction of WT p53. This is consistent with the transcriptional repression observed.

vector pTATA-luc (Altschmied and Duschl, 1997) as described in Materials and methods and tested the effect of WT p53 on their promoter function. Surprisingly, as shown in Supplementary Figure S4, WT p53 failed to repress the promoter activity of the plasmid containing the WT E2F site, although it repressed activity from the promoter containing the mutated E2F site, which had very low promoter function as evident by low luciferase activity

E2F site alone is not enough to be repressed by WT p53 We tested whether the E2F site present on the MCM-7 promoter is enough to confer repression by WT p53 by cloning the E2F site in its WT and mutated form in the

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2590 E2F-WT: 5’-ctagcgtcacgtgggggggggcgacgtttcgcgccaatttcga-3’ 3’-cagtgcccccccccccgacgtaaagcgcggtaaaagcttcga-5’

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Figure 5 (a) Electrophoretic mobility shift assays. Nuclear extracts were prepared from exponentially growing cells of the HIp53 cell line grown in the presence ( þ ) or absence () of 100 mM final concentration of Ponasterone A for 24 h. Radiolabeled probes containing the putative E2F binding site from the MCM-7 promoter were used. Competition studies were done using unlabeled E2F-binding site containing probe as a specific competitor (E2F-WT, lanes 4 and 5) or a probe containing an unrelated sequence as a nonspecific competitor (E2F-MT, lanes 6 and 7) at 40 molar excess. The single arrow indicates the suspected DNA complexes containing E2F. Decreased E2F binding is observed in the presence of WT p53 (compare lanes 2 and 3). Equal amounts of protein were added to each lane. Oligonucleotide sequences are shown on the figure. (b) Chromatin immunoprecipitation (ChIP) assays. Exponentially growing cells of HIp53 cell line (un-induced or induced to express WT p53) were crosslinked, and harvested as described in Materials and methods. p53 was immunoprecipitated using the specified antibody (DO1). Normal IgG was used in the control immunoprecipitation. The presence of the MCM-7 promoter in the immunoprecipitate was detected using PCR-specific primers as described in Materials and methods. The graph shows the induced/un-induced ratio of the signals obtained for the specified antibody immunoprecipitation experiments. This ratio has been given an arbitrary number of 1 for induced cells. The average fold difference for the signals and standard deviations were calculated for both experiments. Oncogene

(not shown). Thus, these results suggest that the E2F site alone is not enough for WT p53-mediated repression and that the associated sequences containing c-myc binding site are also necessary. The repression observed with mutated E2F construct is not altogether surprising as we have seen similar results with synthetic promoters containing a single TATA box (Deb et al., 1992).

Discussion It is now clear that p53 can integrate signals from various pathways in the cell in response to cellular stress. Since p53 and p73 have structural and functional similarities, we have undertaken a comparative gene expression analyses to identify genes whose transcriptional activity is affected by expression of WT p53 and/or p73 (Supplementary Tables 1, S1, S2). From these analyses, we have chosen to mainly focus our attention on those genes downregulated by p53 and p73. We have confirmed the transcriptional inhibition of various target genes after expression of WT p53 by QPCR using two different methods of p53 expression (Figure 1 and Supplementary Figure S1a). In vivo transient transcriptional assays suggest that this repression occurs at the promoter level (Figure 2). Interestingly, although the majority of the upregulated genes appear to be involved in cell cycle, apoptosis and various metabolic pathways, genes inhibited by WT p53 mostly include genes involved in the various aspects of cell cycle suggesting an interrelationship between WT p53-mediated repression and WT p53-mediated cell cycle arrest. Since WT p53 and p73 expression leads to cell cycle arrest or apoptosis, it is not absolutely clear from our work whether the genes that we have seen to be affected by overexpression of WT p53 and p73 are a direct effect or a result from cell cycle arrest or apoptosis. Any effect that causes a global change in the state of a cell, such as cell cycle changes, can potentially result in gene expression changes because of the change in cell cycle. Those genes affected are nonetheless important in understanding the p53 pathway. To argue that there are gene expression changes that are more immediate, one has to look at a time before complex cell states have established. Our 16–24 h time-point after p53 expressing recombinant adenovirus infection is conceivably short enough, when considering time to get p53 protein levels up. Therefore, the genes identified should be primarily immediate effects, and not the effect of cell cycle changes. However, to address this point more thoroughly, one needs to do a time course, along with p53 and cell cycle analysis. We have identified a region between positions 186 and 85 on the MCM-7 promoter that is responsive to WT p53 expression. Deletion of this sequence abolished the ability of WT p53 to repress this gene (Figure 4) suggesting that the transrepression of this promoter may have some sequence specificity. An analysis of these DNA sequences reveals a putative E2F DNA binding site within this region; however, no WT p53 consensus


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binding sites were found in the same region (data not shown). ChIP assays indicated that the E2F-MCM-7 promoter interaction is quantitatively reduced in the presence of p53 (Figure 5) showing p53 impacts E2F binding to the promoter. Our transient transcriptional analyses also suggest that only one E2F site is not enough to be a target of WT p53-mediated repression (Supplementary Figure S4). In agreement with earlier data (Lohr et al., 2003; Shats et al., 2004), we have also found that expression of the CDK inhibitor p21 inhibited transcription of all the genes tested (Supplementary Figure S3). As an inhibitor of cyclins, p21 prevents phosphorylation of the retinoblastoma (Rb) protein, which results in accumulation of hypophosphorylated Rb (Bartek and Lukas, 2001). This protein form in turn binds to E2F factors and converts them from transcriptional activators into transcriptional repressors. The story is complicated by the presence of a c-myc binding site in the MCM-7 promoter (gray box, Figure 4a). A report by Ho et al. (2005) showed that p53-mediated cell cycle arrest required the inhibition of c-myc. This inhibition was not dependent on p21 activation and involved the recruitment of the corepressor mSin3A. Consistent with this report we have identified c-myc as a transcriptional target of p53, suggesting that transcriptional repression of MCM-7, and perhaps other genes, may occur after WT p53mediated repression of c-myc. Finally, we have also explored the ability of p73a and p73b to modulate expression of genes normally regulated by WT p53. Expression analysis show that in most cases, p73 can replace WT p53 and affect expression of genes modulated by WT p53 (Table 1) (Kovalev et al., 1998; Tannapfel et al., 2000; Ichimiya et al., 2001; Senoo et al., 2001; Melino et al., 2002; Urist and Prives, 2002; Moll and Petrenko, 2003; Stanelle et al., 2003; Flores et al., 2005). However, there is a subgroup of genes differentially modulated after expression of p53 or p73 (Supplementary Tables S2a and b), showing that p73 has its own set of transcriptional target genes. Functional grouping of genes activated by WT p53 but differentially modulated by p73 shows that most of these genes are involved in various metabolic and energy pathways; whereas the group of genes repressed by WT p53 but unaffected or activated by p73 include genes involved in various phases of the cell cycle (Supplementary Table S2 and data not shown). The significance of this is unknown and remains to be investigated. Several mechanisms have been proposed to explain WT p53-mediated repression (Ho and Benchimol, 2003). First, transrepression can occur through interference with the functions of transcriptional activators through binding of p53 to the transcription factor itself or direct binding of p53 to the target DNA thus preventing another transcription factor from binding. Such a mechanism has been described for the a-fetoprotein (AFP), polymerase d catalytic subunit (POLD1) and survivin gene promoters (Ho and Benchimol, 2003). WT p53 can also interfere with the basal transcriptional machinery without an apparent need to act through gene-specific activators. Instead, WT p53 may interact with TATA-binding protein and certain

TAFs leading to a decrease in transcription (Ho and Benchimol, 2003). Repression may also occur through remodeling of the chromatin accomplished by recruitment of histone deacetylases. Repression of the Map4, a-tubulin, survivin and cyclin B2 among other genes occur through this mechanism (Ho and Benchimol, 2003; Imbriano et al., 2005; Basile et al., 2006). Our studies do not exclude any of these possibilities but suggest p53-mediated repression occurring through upregulation of p21. However, we do gain new insights into the various inhibitory functions of WT p53. Additionally, we provide further evidence to suggest that p73 can act as a transcriptional repressor of p53modulated genes suggesting that at least one of the mechanisms through which WT p53 represses gene expression is shared with p73.

Materials and methods Cell culture H1299, Saos-2 and 21PT p53-null cells were cultured as described (Scian et al., 2005). The HIp53, WT p53 inducible cell line, a kind gift of Dr Jennifer Pietenpol, was maintained as previously described (Flatt et al., 2000). WT p53 in this cell line was induced for 24 h with 100 mM final concentration of Ponasterone A. HCT116 (p53 þ / þ , p21 þ / þ ), (p53/, p21 þ / þ ) and (p53/, p21 þ / þ ) cells were maintained in McKoy’s 5A media with 10% Fetal Bovine Serum, and are kind gifts from Dr Bert Vogelstein. Adenovirus preparation, infection and RNA isolation The viruses were prepared as described (Valerie et al., 1999). The p21-expressing adenovirus was a kind gift from Dr Wafik el-Diery (Prabhu et al., 1996). Virus infection and RNA preparation were done as described (Deb et al., 2001) and as discussed in Supplementary Information. DNA microarray hybridization, data management and analysis All microarray hybridization analyses were performed in duplicate using Affymetrix U95Av2 (HG-U95Av2) chips by Virginia Commonwealth University’s Institutional Nucleic Acid Research facility. The general procedures for microarray hybridization and analysis are described elsewhere (Thibault et al., 2001). H1299 cells infected with a WT p53-expressing adenovirus (or b-galactosidase control) and HIp53 (() or ( þ ) Ponasterone A) cells were analyzed. Array hybridizations have been done at least two times with two batches of RNA. Data normalization was performed using the robust microarray average method and RMAExpress software (Irizarry et al., 2003). Differentially expressed genes were identified using a ttest with the false discovery rate estimated by the Benjamini and Hochberg method to correct for multiple testing (Benjamini and Hochberg, 1995). Association analysis was performed using the hypergeometric distribution and false discovery rate estimated by the Benjamini and Hochberg method. GO annotation was obtained from the Stanford Source (http:// smd-www.stanford.edu/cgi-bin/source/sourceSearch). Confirmation of analysis was obtained using the David tools (http://david.abcc.ncifcrf.gov/tools.jsp) (Dennis et al., 2003). Quantitative real-time PCR (QPCR) QPCR was performed as previously described (Deb et al., 2002; Stagliano et al., 2003; Scian et al., 2004a). Total RNA Oncogene


2592 was isolated from cells infected with recombinant adenovirus. The primer sets used are given in Supplementary Information section. Cloning of the presumptive promoters Cloning of the presumptive gene promoters was done using available genomic sequences in the NCBI database, as discussed in Supplementary Information. Transfections and luciferase assays Transfections and luciferase assays were carried as described (Scian et al., 2004b) and discussed in Supplementary Information. Electrophoretic mobility shift assays Exponentially growing HIp53 cells were incubated in the presence or absence of Ponasterone A for 24 h. Nuclear extracts were prepared following the method described by Yang et al. (2000), and EMSA was carried out as described (Scian et al. 2005), and as elaborated in Supplementary Information. Chromatin immunoprecipitation ChIPs were done as described (Scian et al., 2004a), and is detailed in Supplementary Information briefly.

Treatment of HCT116 cells with 5-fluorouracil and QPCR analysis HCT116 (p53 þ / þ , p21 þ / þ ), (p53/, p21/) and (p53 þ / þ , p21/) colon carcinoma-derived cells were treated with 375 mM 5-FU (Sigma) as suggested (Kho et al., 2004) and were incubated for different time intervals. Cells were then harvested and RNA prepared as described (Scian et al., 2005). QPCR was performed to check for expression cyclin B2 and p21with specific primers (see Supplementary Information). GAPDH acted as an internal control. In the western analysis to test whether p53 (detected by monoclonal antibody PAb1801) was induced b-actin was used as a loading control. Acknowledgements We thank Arnold Levine, Bert Vogelstein and Vimla Band for providing us with cells and plasmids. This work was supported by grants from Philip Morris USA Inc and by Philip Morris International (04-I176-01) and NIH (CA70712) to Sumitra Deb and from NIH (CA74172) to Swati Palit Deb. Katherine Stagliano was supported by Susan G Komen Breast Cancer Foundation (DISS0201749) and Mariano Scian by a predoctoral fellowship from NCI (F31-CA97520).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc). Oncogene