A Genomic Map of P53 Binding Sites Identifies Novel P53 Targets Involved in an Apoptotic Network

Research Article

A Genomic Map of p53 Binding Sites Identifies Novel p53 Targets Involved in an Apoptotic Network

Chaouki Miled, Marco Pontoglio, Serge Garbay, Moshe Yaniv, and Jonathan B. Weitzman

Unit of Gene Expression and Disease CNRS FRE2850, Department of Developmental Biology, Pasteur Institute, Paris, France

Abstract for p53-regulated genes that underlie its tumor-suppressive The transcriptional activity of the p53 protein is central to its function (2). role in tumor suppression. Identification of the complete The ability of p53 to inhibit cell proliferation in the G1 phase can be largely accounted for by its transcriptional regulation of the repertoire of p53-regulated genes is critical for dissecting the p21Cip1 gene encoding an inhibitor of cyclin-dependent kinases (4). complexity of the p53 network. Although several different However, the transcriptional program responsible for p53-mediated approaches have been used to characterize the p53 genetic apoptosis is much less clearly defined. The finding that mice program, we still lack a comprehensive molecular understand- lacking the p21Cip1 gene, unlike p53-null mice, do not develop ing of how p53 prevents cancer. Using a computational tumors implies that the apoptotic program plays an important role approach, we generated a genome-wide map of p53 binding in p53 tumor suppression. Several proapoptotic p53 target genes sites (p53BS) to identify novel p53 target genes. We show that have been identified, including BAX, DR5, PUMA, NOXA, FAS, PIDD, the presence of nearby p53BS can identify new proapoptotic and APAF1 (2). The products of some of these genes are involved in members of the Bcl-2 family. We show that p53 binds to p53BS the mitochondrial apoptotic pathway (e.g., Bax, Puma, and Noxa), identified in the BCL-G/BCL2L14 gene and that induction of whereas others are components of the death receptor–mediated this gene contributes to p53-mediated apoptosis. We found pathway (e.g., DR5, Fas, and PIDD). Studies of knockout mice have that p53 activates the COL18A1 gene encoding the precursor suggested that no single p53-regulated gene can alone account for the antiangiogenic factor endostatin. We also show that p53 for p53-induced cell death (5). Combinations of target genes are up-regulates the MAP4K4 gene and activates the c-Jun NH - 2 therefore probably responsible for p53-induced apoptosis in terminal kinase (JNK) pathway to drive apoptosis. Thus, different tissues and different cellular contexts. unbiased mapping of the genomic landscape of p53BS provides Despite the identification of a several dozen p53 target genes a systematic and complementary approach to identify novel using a variety of experimental approaches, the diversity of the p53- factors and connections in the p53 genetic network. Our study regulated transcriptional programs remain to be fully elucidated. illustrates how systematic genomic approaches can identify Defining new components of the p53 network remains a priority to binding sites that are functionally relevant for a p53 understand how p53 functions to prevent tumorigenesis. A number transcriptional program. The genetic link among p53, anti- of different approaches have been applied to the identification of angiogenic factors, and the JNK signaling pathway adds new p53-regulated target genes. In recent years, genomic approaches, dimensions to understanding p53 function in highly connected such as serial analysis of gene expression (SAGE) or microarray genetic networks. (Cancer Res 2005; 65(12): 5096-104) analysis (6–9), have identified p53-regulated genes involved in cell cycle arrest, apoptosis, and DNA repair (2). These data have been Introduction complemented by parallel approaches that exploit genomic-scale The p53 tumor suppressor gene is a key regulator of the cellular sequence information. Distinct bioinformatic approaches have response to stress and plays a critical role in preventing cancer been applied to explore the presence of sequences resembling to progression. The activation of p53 in response to DNA damage or the p53 consensus binding site and chromatin immunoprecipita- cellular stress leads to cell cycle arrest, apoptosis, or senescence, tion (ChIP) assays have begun to identify genomic loci where p53 depending on the cellular context (1, 2). The ability of the p53 to binds (10–12). function as a transcription factor is central to its role in tumor It is clear, however, that we are far from a complete suppression (3). Mutations in the p53 gene are associated with the understanding of how the p53-regulated genetic program accounts majority of human tumors. Such mutations often disrupt its for its major role in tumor suppression. It is only by defining the transcriptional function leading to loss of the growth arrest or complete repertoire of p53-regulated target genes that we can apoptotic transcriptional programs that limit the proliferation of appreciate its role in apoptotic responses, as well as other damaged cells. These observations have fired an intensive search anticancer functions such as inhibition of angiogenesis or metastasis. The task of prescribing regulatory functions to noncoding genomic sequences is in its infancy; predicting and validating the role of cis-regulatory motifs is the first step towards dissecting regulatory transcriptional networks (13). To this end, we Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). have generated a map of the genomic landscape of p53 binding C. Miled is currently at the Institut National de la Sante et de la Recherche sites (p53BS) by an unbiased, highly selective identification of Medicale, ‘‘Biology of the mononuclear phagocyte system’’ Necker Medical School, Necker-Enfants Malades Hospital, Paris, France. putative p53 cis-acting motifs using a computational approach. We Requests for reprints: Jonathan B. Weitzman, Unit of Gene Expression and show how this genomic p53BS map can be navigated to identify Disease CNRS FRE2850, Department of Developmental Biology, Pasteur Institute, novel components within the p53 genetic network. We have 25, rue du Docteur Roux, Paris, France, 75724 cedex 15. Phone: 33-1-4061-3480; Fax: 33-1-4061-3033; E-mail: [email protected]. identified new p53 target genes that shed light on how p53 I2005 American Association for Cancer Research. regulates angiogenesis and orchestrates the apoptotic program.

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This study has unraveled a link between p53 and the c-Jun NH2- Reverse transcription-PCR and short interfering RNA assay. We terminal kinase (JNK) signaling pathway suggesting additional isolated cytoplasmic mRNA using RNeasy (Qiagen, Courtaboeuf, France) complexity and synergic interactions between key pathways and prepared cDNA using SuperScript II reverse transcriptase (Invitrogen, regulating cell fate. Cergy-Pontoise, France) following the manufacturers’ instructions. Reverse transcription-PCR analysis was done using Taq polymerase (Promega, Charbonnie`res, France) and a Perkin-Elmer Thermal Cycler machine. Materials and Methods Quantitative PCR analysis was done using SYBR PCR Mix (Applied Screening for genomic p53BS. We aligned the sequences of 21 well- Biosystems, Courtaboeuf, France) and the Abiprism 7700 machine (Applied characterized p53BS to construct the p53 position weight matrix (PWM). Biosystems). In all quantitative PCR calculations, the amount of nucleic acid These sites come from 19 p53 target genes. When aligning these material was standardized using oligonucleotide primers for 18S RNA genes. sequences, we tested both potential orientations (either DNA strand) to All quantification data are presented as the standardized values, mean F SD maximize the number of matrix positions where a given nucleotide of triplicates. frequency is zero. Each aligned sequence was taken on the DNA strand Short interfering RNA (siRNA) oligonucleotides were designed accord- that maximized the number of matrix positions, where a given nucleotide ing to the criteria established by Reynolds et al. (19) and manufactured frequency is zero. This optimization resulted in three alternative possible by Proligo (Paris, France; see Supplementary Table S3). p53-Saos-2 cells matrices with the same number of zeros. We reasoned that zero frequency were transfected with 20 pmol siRNA using the Lipofectamine 2000 re- values indicate positions where a particular nucleotide is deleterious for agent (Invitrogen) according to manufacturer’s instructions. Cells were p53 binding, and we assigned these with a negative weight to counter- incubated with doxycycline 24 hours post-transfection and apoptosis was select sequences with nonrepresented nucleotides. We incorporated the measured 48 hours later by flow cytometry (Beckman Dickinson, Le Pont negative weight value À177 (corresponding to the difference between the de Claix, France) analysis using CellQuest software. Apoptosis measure- maximum and minimum scores of sequences with only permitted ments were also done by trypan blue exclusion and manual counting. The sequence variations) at 26 ‘‘invariant’’ zero positions and an intermediate results are presented as mean F SD of triplicates. For siRNA MAP4K4 counterweight values (À89) at two positions that varied between the three experiments, p53-dependent apoptosis was calculated as apoptosis in matrix possibilities. Scores were calculated as the sum of nucleotide presence of dox À apoptosis in absence of dox. frequencies at each matrix position for 20 nucleotide windows throughout Promoter analysis assays. To clone the p53BS-containing sequences of the human genome (14). The minimal threshold of 211, corresponded to p53 target genes, we amplified DNA fragments and cloned them into a the lowest score of p53BS in the training set sequences (p53DINP1 Luciferase reporter plasmid (pGL2, Promega). A 1,430-bp fragment from the promoter). We calculated the distances from the most upstream COL18A1 promoter was amplified with two oligonucleotides 5V-CTCGAGG- annotated transcription start site to the nearest p53BS (upstream or TACCAAGGAGGCCTGGATCCCTG-3V and 5V-AAGCTTGAGCTCTCCCT- downstream of the transcriptional start site) for the entire set of CACCTCCAAGCAC-3Vand digested with KpnI and SacI enzymes before annotated human genes (22,883 genes). The program used for the binding cloning. A 1,558-bp fragment from the BCL-G gene, containing the intronic site search was written in C and run on a Linux system on which we have p53BS, was amplified with two oligonucleotides 5V-CTCGAGGTAC- installed a mirror of the Ensembl database. The complete p53BS data set CAGTGTTCCCAGAGGACACG-3V and 5V-GATCTGAGCTCGGTCAGAAT- is available on request. TAGGTGCGGC-3V and digested with KpnI and SacI enzymes before Cells and protein analysis. p53-Saos-2 cells containing a doxycycline- cloning. Reporter plasmids, or an empty vector control, were transfected inducible p53 allele (ref. 15; kindly provided by Karen Vousden, Scotland, (using Lipofectamine 2000, Invitrogen) into p53-Saos-2 cells and cells were United Kingdom) were cultured in DMEM containing 10% FCS. p53 was treated with or without doxycycline for 48 hours. Luciferase expression induced by treating cells with 2.5 Ag/mL doxycycline for 24 hours (unless levels were monitored by quantitative PCR and standardized using 18s otherwise indicated). HCT116 cells lacking p53 (kindly provided by Bert quantification. Vogelstein, Baltimore, MD) were grown in McCoy’s medium containing 10% FCS and p53 was induced in control cells by treatment with 25 mmol/L Results cisplatin. Primary mice embryonic fibroblasts from wild-type or Trp53À/À animals were isolated and characterized as described previously (16). Constructing a genomic map of potential p53BS. Many Fluorescence immunohistochemistry and immunoblot analysis of protein groups have used genome-wide approaches to identify p53- expression were done as previously described (16, 17). We used antibodies regulated gene targets. Invariably, these studies have begun with specific for p53, p21, Mdm2, h-tubulin (all from Santa Cruz Biotechnology, measurement of gene expression levels in cells with or without p53, through Tebu-Bio, Le Perray en Yvelines, France), endostatin, and phospho- or following stimulation with genotoxic agents. The results of such c-Jun (17). transcriptome analyses generally fail to discriminate between Chromatin immunoprecipitation. p53-Saos-2 cells were treated with direct and indirect regulatory programs. In some cases, genes that A 2.5 g/mL doxycycline for 24 hours. Cells were fixed with formaldehyde are regulated by p53 have subsequently been shown to be located (final concentration, 1%), incubated for 10 minutes at room temperature, near sequences that resemble the p53 consensus motif 5V- and the reaction was stopped by adding glycine to a final concentration of V 0.125 mol/L. Nuclear extracts were centrifuged at 10,000 Â g,4jC for 10 RRRCWWGYYY-3 (R = G or A, W = T or A, Y = C or T; ref. 20). minutes and resuspended in sonication buffer containing 50 mmol/L We sought to identify novel, direct p53 target genes by a reverse HEPES (pH 7.9), 140 mmol/l NaCl, 1 mmol/L EDTA, 1% Triton X-100, 0.1% approach, beginning with a genome-wide assessment of potential Na-deoxycholate, 0.1% SDS, and protease inhibitor cocktail. Chromatin p53BS and subsequently examining the regulation of nearby aliquots were sonicated in ice water with a Cosmobio/Bioruptor sonicator genetic elements. and centrifuged at 15,000 rpm at 4jC for 20 minutes. An aliquot was The construction of useful transcription factor binding site saved for the input sample. After centrifugation, the soluble chromatin (TFBS) maps requires the ability to predict true binding sites in was subjected to immunoprecipitation as previously described (18). For genomic DNA sequence with high accuracy. Computer programs immunoprecipitation, we used the anti-p53 polyclonal FL393 antibody exist that search for TFBS by scanning for sequences with (Santa Cruz Biotechnology). Primers were designed using PrimerExpress similarity to consensus binding site motifs, sometimes allowing software (see Supplementary Table S2). The fold enrichment of DNA bound was calculated by comparing output and input measurements by one or two positions to differ from the consensus. PWMs offer a quantitative PCR analysis with respect to uninduced cells. All quantifica- superior approach for identifying binding sites as they take into tion data are presented as the standardized values, mean F SD of account the nucleotide frequency at each position in previously triplicates. defined sites. This approach is particularly adapted to p53 as its www.aacrjournals.org 5097 Cancer Res 2005; 65: (12). June 15, 2005

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Cancer Research binding site is relatively long (two palindromic decamers) and flexibility. Identification of these ‘‘gapped’’ sites requires a modified significant degeneracy has been observed. To identify potential approach (see Discussion). Several design features make our p53BS in the human genome, we constructed a PWM (see Materials approach particularly selective: (i) the careful choice of biochem- and Methods for details) that models the binding specificities of ically validated sequences to construct the PWM, (ii) the p53 by aligning the sequences of experimentally validated p53BS optimization of alignment to maximize the number of forbidden from the cis-regulatory regions of well-characterized p53 target positions and the introduction of a negative weight to eliminate genes (Fig. 1A). We focused our search on well-studied p53BS that false positives, (iii) the genome-wide selection of motifs on either comprise the minimal 20-nucleotide p53 recognition site identified strand orientation above a nonarbitrary threshold (211, by a nonbiased alignment algorithm. We aligned these sequences corresponding to the lowest scoring true p53BS), and (iv) the to maximize the number of matrix positions where a given unbiased search for both upstream and downstream sites. nucleotide frequency is zero and introduced negative values at 28 We identified 25,044 potential p53BS in the human genome (i.e., positions to counterselect sequences with nonrepresented nucleo- one per 110 kb). This number increases significantly if sequences tides. This strategy reduces the number of false positives and that allow for a variable gap between the decamers are also enhances selectivity. We chose to begin with a strict criterion for selected (see Discussion). This number is greater than previous p53BS selection in order to focus on sites with a strong predictive estimates of the number of p53BS (10). Our genome-wide map of potential. A number of p53-regulated genes contain a p53BS with p53BS provides a panoramic view of p53 binding. We anchored our a gap of 1 to 14 nucleotides between the core decamer binding physical p53BS map to the genetic landscape by assessing the sites, illustrating that p53 binding tolerates an additional level of relationship between p53BS and the annotated transcription start

Figure 1. Mapping potential p53BS in the human genome. A, we constructed a p53 PWM by aligning p53BS from well-characterized target genes. The matrix values reflect the nucleotide frequency at each position. We introduced negative weights to counterselect sequences with nonrepresented nucleotides (boxes). The score for each 20-nucleotide window is the sum of the frequency of bases at each matrix position. The name of each target gene, the p53BS sequence, and the matrix scores (right column). Two sites were used for the MDM2 and GDF15 genes. B, histogram plot of the distance of the nearest identified potential p53BS for every annotated gene in the human genome (top) or for the known targets that were used to construct the PWM (bottom). The gene frequency is plotted against the log of the distance in nucleotides. C-D, we measured the distance to the nearest p53BS for genes that have been reported to be induced (n) or noninduced (5) by p53 expression in microarray experiments (C) in human lung cancer cells (7) or (D) colon carcinoma cells (9).

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Figure 2. The BCL2L14/BCL-G gene is a proapoptotic p53 target. A, we analyzed the distance to the nearest potential p53BS for proapoptotic and antiapoptotic members of the Bcl-2-like gene family. Bcl-2-like genes that have been reported to be p53 targets (bold). B-C, we detected upregulation of BCL2L14/BCL-G and BBC3/PUMA mRNA upon induction of p53. p53-Saos-2 cells, containing an inducible p53 allele, were treated with doxycycline for 24 hours and the levels of gene expression were measured (B) by reverse transcription-PCR analysis or (C) by quantitative PCR using gene-specific primers. The levels of transcripts for p53, BAX, CDKN1A, b-actin (ACTB) were monitored as controls. D, a genomic fragment containing the intronic p53BS from the BCL2L14/BCL-G gene was cloned into a Luciferase reporter plasmid, transfected into p53-Saos-2 cells, and Luciferase levels were monitored 48 hours later in the presence (+) or absence (À) of doxycycline (dox). An empty vector construct was used as a negative control.

sites for the entire set of human genes (22,883 annotated genes). of the expression of several Bcl-2-like genes, including BAX, and Whereas 79% of the matrix genes have a p53BS within 4 kb of the BH3-only members BBC3/PUMA and PMAIP1/NOXA (2, 15, 21, 22). start site, only 8% of the whole-genome set harbor such close sites, Our analysis predicted that the BCL2L14/BCL-G gene might also be demonstrating enrichment for nearby p53BS in p53 target genes a p53-regulated target as it has a p53BS within the first intron. Bcl-G (Fig. 1B). Approximately 9% of the potential p53BS lie within 10 kb has been previously shown to be proapoptotic in COS-7 cells, but upstream of an annotated gene (Supplementary Table S1). To little is known about its transcriptional regulation (23). We found explore this map of genomic p53BS, we first analyzed the distance that BCL2L14/BCL-G mRNA levels were up-regulated upon to the nearest p53BS for genes reported to be induced or repressed activation of a tetracycline-inducible p53 allele in p53-Soas-2 cells by p53 in microarray experiments (7, 9). Kannan et al. examined (ref. 15; Fig. 2B-C). These results suggest that the presence of the gene expression in human lung cancer cells expressing a p53BS was sufficient to identify BCL-G as a p53 target gene. To temperature-sensitive p53 (7). We found that 28% of induced investigate this further, we cloned a 1.5-kb portion of the BCL2L14/ genes have a potential p53BS within 6 kb, representing a 5-fold BCL-G promoter containing the putative regulatory p53BS in enrichment compared with uninduced genes (Fig. 1C). Our analysis a reporter Luciferase plasmid. This plasmid was transfected into of gene profiling data representing p53 induction in human colon p53-Soas-2 cells and the expression of Luciferase monitored after carcinoma cells from Zhao et al. (9) also found enrichment for induction of p53 with doxycycline. Induction of p53 resulted in a p53BS near p53-induced genes compared with uninduced genes 4-fold activation of Luciferase expression, demonstrating that (Fig. 1D). We did not observe any enrichment for p53BS in the the BCL-G gene is p53-regulated (Fig. 2D). subset of genes that were repressed by p53 expression (7, 9). Thus, To show that the p53 protein actually binds to the predicted p53BS genes that were shown experimentally to be p53-regulated are in the BCL-G gene, we did ChIP experiments (Fig. 3A-B). The enriched for nearby p53BS compared with noninduced or promoters of the CDKN1A and GAPDH genes were used as positive repressed genes. and negative controls in ChIP analysis (see below). We found that the The BH3-only BCL-G gene is a proapoptotic p53 target gene. intronic BCL-G site bound to p53 in ChIP experiments, whereas A well-characterized function of p53 is the induction of apoptosis, a more distant upstream site showed no binding (Fig. 3A). Our but the exact mechanisms are not fully understood (1, 2). Among the algorithm also identified two p53BS in the BBC3/PUMA gene list of proapoptotic genes regulated by p53 are several members of (Fig. 3B). One of these, the most 5V, was missed in previous the Bcl-2-like family (15, 21, 22). The Bcl-2-like family can be divided characterization of this promoter (15, 22). We found that both sites into two groups: antiapoptotic proteins such as Bcl-2 itself and bind p53 in ChIP experiments (Fig. 3B). Thus, the up-regulation of proapoptotic proteins such as the BH3-only members. Our genomic these proapoptotic Bcl-2 family genes upon p53 induction is p53BS map allowed us to assess for the first time the relationship associated with binding to the p53BS that we have identified within between the entire family of Bcl-2-like genes and the genomic the BCL-G and PUMA genes. To investigate whether induction of landscape of p53BS. Strikingly, we found that putative p53BS were BCL2L14/BCL-G is critical for p53-induced cell death, we did RNA closer to genes encoding proapoptotic Bcl-2-like proteins than to interference (RNAi) experiments to knockdown the gene (Fig. 3C-D). antiapoptotic genes (Fig. 2A). p53 has been described as a regulator Transfection with siRNA oligonucleotides for the BCL-G transcript www.aacrjournals.org 5099 Cancer Res 2005; 65: (12). June 15, 2005

Figure 3. p53 binds to sites in the BCL-G gene that plays a role in p53-mediated apoptosis. We identified potential p53BS in (A) the BCL2L14/BCL-G and (B) the BBC3/PUMA genes. Sequence of the p53 sites and matrix scores. We showed that p53 binds to a p53BS in the BCL2L14/BCL-G gene (A) and two sites in the BBC3/ PUMA gene (B) using ChIP analysis experiments. We did ChIP experiments with extracts from p53-Soas-2 cells treated with (+) or without (À) doxycycline (dox) and determined the binding to the promoters by site-specific, quantitative PCR analysis. The promoters of the GAPDH and CDKN1A genes were used as negative and positive controls, respectively (see data in Fig. 4). C, we treated p53-Saos-2 cells with siRNA duplexes against BCL2L14/ BCL-G or a control gene (Luciferase) before induction of p53 with doxycycline. We measured mRNA levels of BCL2L14/BCL-G to show that the RNAi had effectively reduced expression. D, we measured the levels of apoptosis by flow cytometry after 48 hours in the presence or absence of doxycycline and siRNA against BCL2L14/BCL-G or a control gene.

led to a 90% reduction in expression levels compared with siRNA the COL18A1 promoter into a Luciferase reporter plasmid and found reagents for a control gene (Fig. 3C). We observed a significant that it was p53-responsive when transfected into p53-Saos-2 cells reduction in apoptosis (f40-50%) when p53 was induced in p53- (Fig. 4C). Furthermore, ChIP experiments showed that p53 bound Saos-2 cells transfected with BCL-G siRNA oligonucleotides, very efficiently to the p53BS cluster in the COL18A1 promoter whereas control siRNA reagents had no effect on p53-induced (Fig. 4D). In control experiments, we observed a 4-fold enrichment of apoptosis (Fig. 3D). Hence, we identified BCL2L14/BCL-G as a p53 bound to the CDKN1A promoter that encodes the p21Cip1 downstream effector in the p53 apoptotic network by analyzing the protein, whereas there was no p53 binding to the GAPDH promoter, p53BS repertoire for an entire family of apoptosis regulators. This which is not p53-regulated (Fig. 4D). Furthermore, immunostaining shows that our algorithm is particularly efficient for identifying and Western blotting showed that p53 induction leads to an increase functional p53BS. Furthermore, the creation of a genome-wide in endostatin polypeptide levels (Fig. 4E). Thus, we have identified a p53BS map allows an evaluation of an entire annotated gene functionally relevant cluster of p53BS that regulate expression of a family in a way that has not been examined previously. gene with antiangiogenic function. A p53BS cluster identifies the endostatin-containing colla- p53 regulation of the c-Jun NH2-terminal kinase signaling gen XVIII gene as a p53 target. It is well established that pathway contributes to apoptosis. The only gene in the human clustering of TFBS contributes to gene regulation (24). The genome that contained a nearby p53BS cluster downstream of the genomic p53BS map allowed us to search for genes associated promoter is the MAP4K4 gene (Fig. 5A), which encodes an upstream with higher concentrations of p53BS. We examined clustering of activator of the JNK signaling pathway (27). JNK signaling has been p53BS in our genomic map by assessing the number of p53BS in a implicated in the cellular response to stress and apoptosis (28, 29). window around the promoter of all human genes. Using a 6-kb The MAP4K4 gene contains six potential p53BS in the first intron window, only two genes had a cluster of three sites upstream of the (Fig. 5A). We showed that four of these sites bind p53 in ChIP gene and only one gene had a cluster of sites downstream of the experiments (Fig. 5B). The induction of p53 in p53-Saos-2 cells led start site. It has been previously shown that the statistical to a reproducible 2-fold up-regulation of the MAP4K4 mRNA significance of such clusters increases with their rarity (25). (Fig. 5C). p53 induction was also accompanied by an increase in One of the genes associated with an upstream cluster is the gene phospho-c-Jun protein levels in these cells (Fig. 5D). This was more encoding collagen XVIII (Fig. 4A). This gene is particularly marked in immunofluorescence experiments, probably because interesting as the collagen XVIII polypeptide is cleaved to release the p53 protein is not equally induced in all cells of the Saos-2-p53 the antiangiogenic peptide endostatin (26). p53 can negatively cell line (Fig. 6A). To test whether endogenous p53 also regulated regulate tumor angiogenesis, but the mechanisms remain unclear. the JNK pathway, we examined c-Jun phosphorylation in HCT116 We found that the COL18A1 gene was indeed up-regulated upon cells with or without p53 after treatment with cisplatin. This induction of p53 in p53-Saos-2 cells (Fig. 4B) or in U2OS cells treated experiment showed that the induction of JNK signaling is also p53- with camptothecin (data not shown). We cloned a 1.4-kb fragment of dependent in these cells (Fig. 5D).

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We tested genetically whether JNK activation by genotoxic stress weighting to eliminate ‘‘false-positive’’ motifs. Our search is not is p53-dependent by examining phospho-c-Jun staining in mouse exhaustive, as we chose to focus our initial search on p53BS that fibroblasts from embryos with or without functional p53. c-Jun contain a minimal 20-nucleotide recognition site without allowing phosphorylation was induced by genotoxic stress in a p53- for a variable gap of 1 to 14 nucleotides between the palindromic dependent manner, whereas the tumor necrosis factor-a–induced decamers, or the contribution of adjacent nucleotides. Our signal was p53-independent (Fig. 6B). Finally, to show that the JNK approach can, however, be easily extended to search for p53BS signaling pathway is important for p53-mediated apoptosis, we with a gap between the two palindromic motifs (20) and to map blocked components of the pathway using pharmacologic or p53BS (or other TFBS) that are conserved between species. An genetic approaches. We tested whether pharmacologic inhibition of initial survey of p53 PWM with variable gaps indicates that each JNK activity could affect p53-induced apoptosis. We found that the matrix adds a large number of potential p53BS (Supplementary specific JNK inhibitor SP600125 reduced p53-induced cell death in Table S1). Combining the maps for these additional PWM offers p53-Soas-2 cells by around 30% to 40% (Fig. 6C). We also used RNAi potential to identify novel p53 targets that have been missed by the assays to knockdown expression of the MAP4K4 gene and tested gap-less PWM. It is noteworthy that analysis of clustering for a the effect on p53-induced cell death. siRNA against MAP4K4 lead to combined p53BS data set indicates that the COL18A1 gene shows a significant reduction in p53-dependent apoptosis, whereas enrichment for additional p53BS with gaps. For example, a 6-kb control siRNA reagents had no effect (Fig. 6D). These results window around the start site contains eight upstream p53BS and suggest that the JNK signaling pathway contributes to p53- four intronic p53BS when all gapped motifs are included. Thus, the regulated apoptosis, and that at least one way that p53 regulates combined p53BS map will provide a resource for future mining of this pathway is via induction of the MAP4K4 gene. p53 binding sites and their relationship with adjacent genes. Although bioinformatics approaches have been applied to p53 Discussion transcriptional regulation in the past (11, 12), this is the first time We have shown that a genome-wide approach to map p53BS that genome-wide in silico prediction of p53 target genes has led to motifs can identify novel bona fide p53-regulated genes. Several the identification of specific genes whose biological function has design features contribute to the predictive nature of our been experimentally shown to contribute to p53-induced apopto- bioinformatics approach. PWMs offer more powerful selection sis. Previous studies have tended to analyze restricted gene sets than conventional searches for consensus binding sites and and rarely showed that the potential sites bound to p53 in vivo or selectivity was enhanced by the choice of characterized sites to that the genes identified were functionally relevant for p53 construct our PWM, the high score threshold, and negative biological outcomes (11, 12). Furthermore, our analysis identified

Figure 4. p53 induces COL18A1 expression via a cluster of upstream p53BS. A, we identified a cluster of four p53BS upstream of the COL18A1 gene. The position, sequence, and PWM score of the sites are indicated. B, we analyzed the expression of COL18A1 mRNA in p53-Soas-2 cells treated with doxycycline for 24 hours by quantitative PCR analysis. C, we cloned the COL18A1 promoter containing the p53BS cluster and tested expression of the Luciferase reporter gene upon transfection into p53-Saos-2 cells and treated with (+) or without (À) doxycycline (dox) for 48 hours. An empty vector plasmid was used as a negative control. D, we did ChIP experiments to show that p53 binds to the COL18A1 promoter. A single pair of primers was used to encompass the four clustered p53BS. p53-Soas-2 cells were treated with (+) or without (À) doxycycline for 24 hours before ChIP analysis with anti-p53 specific antibodies. Analysis of p53 binding to the GADPH or CDKN1A promoters served as negative and positive controls, respectively. ChIP experiments were done using quantitative PCR. E, we detected expression of the endostatin polypeptide using specific antibodies in p53-Soas-2 cells treated with doxycycline by immunoblot (left)or immunohistochemistry (right) experiments. p53 induction was detected with antibodies against p53 protein and nuclei were detected by 4V,6-diamidino-2-phenylindole (DAPI) staining.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Cancer Research novel p53 target genes that have been missed in previous genomic The fact that our p53BS map is truly genome-wide allowed us studies, emphasizing the need to develop multiple parallel to assess the relationship between all known human transcripts approaches if we are to understand the full complexity of the and the local distribution of p53BS. Our analysis emphasizes p53 genetic network. Notably, the COL18A1 gene that we have the importance of mapping both upstream binding sites in the identified here is located on chromosome 21. Previous mapping of promoter region, as well as intronic p53BS within the body of TFBS along chromosome 21 by large-scale ChIP experiments (10) the gene. Indeed, many more p53BS are found within genes than in failed to detect the cluster of p53BS that we have shown here can the 10-kb upstream of genes (Supplementary Table S1). For bind p53 in our ChIP assays (Fig. 4). Again, this result emphasizes example, we identified and characterized functionally relevant the sensitivities of different experimental systems and supports the p53BS in the first intron of BCL-G and MAP4K4 genes. These need for multiple, complementary approaches for a comprehensive observations underscore the importance of genome-wide searches definition of the entire repertoire of p53-regulated genes. The rather than those restricted to small portions of cloned promoters. generation of TFBS maps for other factors (e.g., E2F, Myc, and AP-1) Genomic TFBS maps also allow a broad analysis of the could be superimposed on our p53BS map to provide insights into regulation of large gene families. Our novel observation that coregulated transcriptional programs in response to different stress p53BS lie closer to a proapoptotic subset of the Bcl-2-like genes led conditions. Furthermore, we show how TFBS mapping data can be to the identification of BCL2L14/BCL-G as a new proapoptotic p53 combined with microarray data (Fig. 1) to focus on up-regulated target. This is the first time this approach has been taken to predict genes that are enriched for local binding sites and are therefore target genes. Interestingly, the BCL-G gene maps to a tumor more likely to represent direct transcriptional targets. The data suppressor locus at chromosome 12p12 (30). Thus, p53 regulates from our p53BS map allows one potentially to distinguish between multiple members of the BH3-only family of death-inducing direct and indirect targets suggested from microarray or SAGE proteins in its apoptotic program (5). Inhibition of BCL-G analysis. The ability to identify easily the nearest upstream or expression by RNAi significantly reduced, but did not completely downstream p53BS from a characterized or annotated gene abolish, p53-induced cell death (Fig. 3). Similar results were accelerates the next steps in functional characterization (e.g., ChIP reported when the related PUMA gene was inhibited using and promoter assays). We have shown how the analysis of p53BS antisense oligonucleotides (15). These observations support the map information can be effective in predicting functional targets idea that multiple avenues contribute to p53-induced apoptosis for network analysis. This approach aids the integration of large and no single effector gene can solely account for the p53-regulated data sets of genomic sequence and gene expression data to dissect cell death program. Study of other gene families is likely to lead to functionally relevant transcriptional programs. The same PWM and the identification of additional targets. Thus, the identification of tools can be easily applied to genomic sequences from other the entire repertoire of p53-regulated genes is essential to species, allowing an assessment of the p53 regulation of understand the mode of action of p53 and the combination of orthologous genes and aiding in the interpretation of phylogenetic multiple complementary approaches is vital. footprinting approaches to identify functionally relevant noncoding Our search for higher local densities of p53BS led to the genomic regions. identification of functionally relevant p53BS clusters in the COL18A

Figure 5. p53 regulates JNK pathway activation via induction of the MAP4K4 gene. A, we identified a cluster of potential p53BS in the first intron of the MAP4K4 gene. The position, sequence, and PWM score of the sites. B, we did ChIP experiments to show that p53 binds to sites within the MAP4K4 gene. p53-Soas-2 cells were treated with (+) or without (À) doxycycline (dox) for 24 hours before ChIP analysis with anti-p53 specific antibodies. Precipitated DNA was examined by quantitative PCR analysis. C, we analyzed the expression of MAP4K4 mRNA in p53-Soas-2 cells treated with doxycycline for 24 hours by quantitative reverse transcription-PCR analysis. D, immunoblot analysis with an anti-phospho-Jun (P-Jun) antibody revealed activation of the JNK pathway upon p53 induction in p53-Saos-2 cells (left), or upon cisplatin treatment of HCT116 cells with or without p53 (right).

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Figure 6. p53 activation of the JNK pathway contributes to apoptosis. We detected phospho-c-Jun (P-Jun) immunostaining (indicative of JNK activation) in p53-Saos-2 cells treated with (A) doxycycline (dox)or(B) in mouse embryonic fibroblasts (MEF) in which the p53 pathway was activated by treatment with etoposide for 18 hours. We failed to observe a phospho-c-Jun signal in MEFs lacking the Trp53 gene (p53À/À) or in untreated cells. Tumor necrosis factor-a (TNF-a, 10 ng/mL) induced JNK signaling (detected by a positive signal with the anti-phospho-c-Jun antibody) independently of the p53 genotype. C, we showed that p53-induced apoptosis is dependent on JNK signaling, as the SP600125 inhibitor (50 Amol/L) reduced p53-dependent apoptosis. We show phase-contrast micrographs of treated p53-Saos-2 cells and flow cytometry quantification of apoptosis. Columns, mean of triplicates of a representative experiment; bars, FSD. D, siRNA against MAP4K4 inhibits p53-dependent apoptosis. p53-Saos-2 cells were treated with siRNA reagents for MAP4K4 or a control gene, and the extent of p53-dependent apoptosis was monitored by flow cytometry after doxycycline treatment.

and MAP4K4 genes. We suggest that in addition to cell autonomous a wide variety of cell types in response to different stimuli (29). JNK effects of p53 on growth arrest and apoptosis, p53 may regulate activation has been shown to result in the phosphorylation of signaling pathways and extracellular components that affect several proapoptotic proteins in the Bcl-2 family (35). As BH3-only neighboring cells. p53 was previously linked to the transcriptional encoding genes are also p53 target genes, this should lead to a control of angiogenic regulators, such as thrombospondin and synergistic regulation of cell fate. Once again, the activation of the VEGF (31, 32). However, these proangiogenic genes are down- JNK/MAP4K4 signaling pathway and the concomitant induction of regulated by p53 via an unknown mechanism. Endostatin proapoptotic JNK substrates provides insights into the logic represents the first antiangiogenic factor that is directly regulated underlying the circuitry of the p53 network. by p53. It will be interesting to test whether the antiangiogenic The induction of MAP4K4 and endostatin links p53 to a wider function of p53 is mediated by endostatin in vivo. Interestingly, the genetic network affecting diverse cell signaling events. The recent enzyme that cleaves collagen XVIII to generate endostatin, MMP2, reports that BCL2L14/BCL-G (30), components of the JNK pathway is also encoded by a p53 target gene (33). The observation that (36–38), and endostatin (26, 34) play different roles in tumor p53 regulates both MMP2 and its substrate collagen XVIII, suppression suggest that these novel targets may be related to p53’s underlies the logic behind p53 genetic programs to ensure effective critical anticancer function. These p53 targets contribute to an antitumor function. Furthermore, endostatin has recently been interconnected genetic network that reinforces the apoptotic linked to regulation of a complex genetic network associated with program initiated by p53 in response to stress. apoptosis and proliferation (34). The MAP4K4 gene represents a rare example of transcriptional Acknowledgments regulation of an upstream kinase involved in JNK signaling. Notably, Received 11/29/2004; revised 3/15/2005; accepted 4/9/2005. we did not observe a clear correlation between the extent of Grant support: Association for International Cancer Research grant AICR #02-347, transcriptional induction and the number of p53BS or the efficiency the European Community grant CEE HPRNCT 2002-00256, the Pasteur-Weizmann Foundation, the Association pour la Recherche sur le Cancer (ARC, France), and the of p53 binding. We envisage that the modest transcriptional Ligue Nationale Contre le Cancer (Ile de France). induction of the upstream kinase is amplified by downstream The costs of publication of this article were defrayed in part by the payment of page signaling cascades. Indeed, we observed robust p53-dependent JNK charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. activation (c-Jun phosphorylation) in HCT116 cells. The JNK We thank Samia Ben Hassine for experimental assistance and members of the signaling pathway has been implicated in apoptotic regulation in Yaniv laboratory for helpful discussion.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. A Genomic Map of p53 Binding Sites Identifies Novel p53 Targets Involved in an Apoptotic Network

Chaouki Miled, Marco Pontoglio, Serge Garbay, et al.

Cancer Res 2005;65:5096-5104.

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