The p53 family activates many of the same genes in response to DNA damage. Because p63 and p73 have structural differences from p53 and play distinct biological functions in development and metastasis, it is likely that they activate a unique transcriptional network. Therefore, we performed a genome-wide analysis using cells lacking the p53 family members after treatment with DNA damage. We identified over 100 genes involved in multiple pathways that were uniquely regulated by p63 or p73, and not p53. Further validation indicated that BRCA2, Rad51, and mre11 are direct transcriptional targets of p63 and p73. Additionally, cells deficient for p63 and p73 are impaired in DNA repair and p63+/−; p73+/− mice develop mammary tumors suggesting a novel mechanism whereby p63 and p73 suppress tumorigenesis.
p53 acts as a tumor suppressor gene by transcriptionally regulating a multitude of target genes in response to DNA damage [1]. Induction of these genes results in multiple cellular fates including apoptosis and cell cycle arrest. p63 and p73 share some of the same functions as p53; however, p63 and p73 are structurally more complex containing multiple isoforms [2], [3], [4]. The TA isoforms are structurally more like p53 and contain a transactivation domain while the ΔN isoforms lack this domain and are transcribed from an internal promoter unique to these isoforms [3], [5]. Based on the fact that the TA isoforms are more similar structurally to p53, the TA isoforms were hypothesized and shown to be the major isoforms that induce transcription and are thought to have tumor suppressive functions [3], [5], [6], [7], [8]. In contrast, the ΔN isoforms have been shown to act as dominant negatives against the TA isoforms of p63 and p73 and also against p53. Because of the ability of the ΔN isoforms to act as dominant negatives and their overexpression patterns in human tumors [2], [6], [9], [10], [11], these isoforms have been hypothesized to act as oncogenes [3], [5], [6]. Interestingly, recent data have revealed that the ΔN isoforms of p73 can induce apoptosis, cell cycle arrest and transactivate target genes, such as p21,14-3-3σ, and GADD45 [12]. Additional studies have demonstrated that expression of ΔNp73β at physiological levels can result in the suppression of cell growth in the presence or absence of p53 indicating that this isoform of p73 may act as a tumor suppressor gene [12]. Similarly, the ΔN isoforms of p63 have also been shown to have the ability to transactivate target genes [13]. In the case of p63, the ΔN isoforms are more highly expressed in epithelial tissues [14], and thus it is not be surprising that the ΔN isoforms transcriptionally regulate genes involved in the morphogenesis and differentiation of the epithelium. Given the structural complexity and expression of p63, p73, and their isoforms, the transcriptional targets of these genes are an area of growing research. We and others have shown previously that p63 and p73 can induce apoptosis in response to DNA damage [2], [8], [15]. Many of the target genes induced by p63 and p73 are shared with p53 [2], [8]. Additionally, we have shown that the p53 family of genes is interdependent on each other in the apoptotic response and in the suppression of tumorigenesis. p53+/−; p63+/− and p53+/−; p73+/− develop some of the same tumor types as p53+/− mice, but the phenotype of the tumors in the compound mutant mice is highly aggressive and metastatic indicative of cooperativity between family members [7], [15]. Mice heterozygous for combinations of the p53 family members develop a novel tumor spectrum compared to p53+/− mice indicative of functions of p63 and p73 independent of p53 [7]. These independent functions suggest that p63 and p73 may have unique transcriptional programs. To understand the transcriptional program of p63 and p73, we made use of MEFs deficient for each of the p53 family members individually and in combination and performed a genome wide analysis using cDNA microarray analysis to determine whether p63 and p73 transcriptionally regulate genes independently of p53 in response to DNA damage. Interestingly, we found that p63 and/or p73 transactivate sets of genes independent of p53. Among these sets of genes are those involved in homologous DNA repair, including Rad51, BRCA2, mre11 and Rad50. p63 and p73 were found to bind to these gene promoters by ChIP assay and to transactivate them as demonstrated by luciferase assay. Surprisingly, the ΔN isoforms of p63 and p73, which have been shown to be weak transactivators, transactivate the Rad51 and BRCA2 genes to high levels. In addition, p63−/−, p73−/− and p63−/−; p73−/− MEFs exhibited an impaired ability to repair their DNA and to survive in a clonogenic survival assay. Additionally, in vivo evidence from p63/p73 mutant mice supports this finding; p63+/−; p73+/− mice develop mammary adenocarcinomas at a high frequency [7]. Here, we show that these mammary tumors lose expression of p63, p73, BRCA2, and Rad51. Our findings indicate that p63 and p73 may suppress tumorigenesis by transcriptionally regulating critical genes in the DNA repair pathway.
The p53 family members, p63 and p73, have previously been shown to share many of the same target genes as p53 [2], [8]. Additionally, both p63 and p73 have the ability to bind to the p53 consensus binding site. p63 and p73 also have biological activities independent of p53. Consequently, we were interested in determining whether p63 and p73 had unique transcriptional target genes. A cDNA microarray analysis was performed using E1A expressing MEFs deficient for each p53 family member individually (p53−/−, p63−/−, p73−/−) and in combination (p63−/−; p73−/−). These cells were treated with doxorubicin, a DNA damaging agent, to induce apoptosis in wild-type E1A MEFs. p53−/− and p63−/−; p73−/− E1A MEFs have previously been shown to be resistant to this treatment while the p63−/− and p73−/− E1A MEFS are partially resistant to apoptosis [15]. Microarray analysis revealed a large number of genes differentially expressed in the MEFs deficient for each p53 family member. Because we were interested in identifying genes that are transactivated by the p53 family members in response to DNA damage, genes that are down regulated in the absence of the p53 family members were further analyzed. After filtering and statistical analysis using SAM [16], 620 out of 15,488 genes were found to be down regulated in at least one of the single knockout E1A MEF lines compared to wild-type E1A MEFs in response to DNA damage. Eight-six of the 620 genes were down regulated in the p53−/−, p63−/−, and p73−/− E1A MEFs as illustrated by the Venn diagram (Figure 1 and Figure S1). There were also sets of genes that were uniquely regulated by each p53 family member; the p53−/−, p63−/−, and p73−/− MEFs each had 109,148, and 131 genes down regulated respectively. Lastly, there were sets of genes that were regulated by two family members only; forty-seven were down regulated in the absence of p53 and p63,41 in the absence of p53 and p73, and 58 in the absence of p63 and p73. The final list of differentially regulated genes was processed through multiple bioinformatic pipelines to identify biological pathways regulated by the p53 family members. Pathway analysis using the web-based KEGG, BioCarta, and GenMAPP databases indicated that the p53 family members regulate numerous pathways including: cell cycle, DNA-damage, p53 signaling, apoptosis, ribosomal proteins, metabolic pathways, and growth factor signaling (Table S1). The putative target genes identified by microarray analysis were analyzed for the presence of a p53 or p63 consensus binding sites using a computer based genome wide search and HMMER1 software [17]. The promoter sequences (defined as 5 kb upstream and downstream of the transcription start site excluding exons) from the 724 down regulated genes were queried, and 700 of these genes were found to have p53 family member motifs. Of these, 669 genes contained p53 family member motif sites with the ideal p53 spacer of 6 nucleotides between the two half sites. Scores were then given to each identified binding site corresponding to how well they matched with previously published p53 or p63 matrices (Table S2). Hierarchical clustering was then performed in the specified knock out MEFs relative to wild-type after DNA damage to highlight patterns between the downregulated genes in the p53−/−, p63−/− and p73−/− E1A MEFs (Figure 2 and Figure S1). Interestingly, many genes were differentially regulated in the various MEF lines (Figure 2) indicating that p63 and p73 have unique target genes. Also, many genes were found to be down-regulated in all mutant cell types supporting the hypothesis that all three transcription factors can transactivate some of the same gene targets (Figure 2). DNA damage triggers numerous cellular responses including an extensive DNA repair pathway involving numerous genes [18]. Microarray analysis revealed that the p53 family members regulate numerous genes involved in the DNA repair pathway. Many of these genes seemed to be uniquely regulated by p63 and/or p73. After DNA damage, loss of p63 or p73 prevents induction of Brca2 (Figure 2, cluster 4), an essential co-factor in Rad51-dependent DNA repair of double-stranded breaks, and Rad51 itself (Figure 2, cluster 3) [18]. Sequence analysis also indicates that p53/p63 response elements exist in both the promoter and intronic region of Brca2 and Rad51 (Table 1 and Table S2). Clustered with Rad51 are Dbf4, a regulator of Cdc7 and a prognostic determinant for melanoma development, and Gas6, which cooperates with the tyrosine receptor kinase Axl in tumor proliferation and cell survival (Figure 2, cluster 3). We also found additional genes that were uniquely down-regulated in p63−/−, p73−/−, and p63−/−; p73−/− MEFs. These hits indicate that p63 and p73 have roles independent of p53 in the DNA-damage response pathway. For example, expression of Rad50, which forms a complex with mre11 and Nebrin, is found to be down regulated in p63−/−; p73−/− MEFs relative to wild-type MEFs treated with doxorubicin. There are also p53 family response elements upstream of the transcription start site of Rad50 (Table S2). In addition to genes that are uniquely regulated by p63 and/or p73, genes controlled by all three p53 family members were identified. Mre11, a gene that functions in the repair of DNA double strand breaks, was found to be down-regulated in p53, p63, and p73 deficient E1A expressing MEFs (Figure 2, cluster 1). In addition, sequence analysis revealed multiple p53/p63 response elements (Table S2). Genes with similar expression profiles as mre11 include the growth factor signaling components Ghr and Sos1 as well as the apoptotic components Traf1 and Cathepsin D all of which contain p53 family member binding sites (Figure 2, cluster 1 & 6 & Table S2). Multiple genes involved in other biological processes, including tumor progression, metastasis and development were found to be differentially regulated in the various E1A MEF cells. For example, Mmp2, a gene shown to play a role in embryonic development and tumor metastasis, is also down regulated in the absence of p73 after doxorubicin treatment. Clustered with Mmp2 are many signaling components such as Grb2, Stat1, Map3k14, and Mapk8ip3- all of which have at least one p53 family member binding motif present near its promoter (Figure 2, cluster 5 and Table S2). Interestingly, brachyury, the developmental transcription factor, was identified as a putative p63 target gene (Figure 2, cluster 2). Given the identified roles of brachyury in limb development, cancer, and hematopoetic stem cells and the development phenotype of the p63−/− mouse, this putative target has important biological significance [19], [20], [21], [22]. We found brachyury to contain multiple p53 family response elements both upstream of its transcriptional start site and within the first intron (Table S2). Other p63 dependent genes that cluster with brachyury include Abr, the GAP for the small GTPase Rac, Socs3, involved in cytokine and apoptotic signaling, and the zinc-finger transcription factor Klf9 which is implicated in control of cell proliferation, cell differentiation, and cell fate (Figure 2, cluster 2). Strikingly, the results from the cDNA microarray indicate that genes in the DNA repair pathway are differentially regulated in MEFs lacking p63 and/or p73 after treatment with DNA damaging agents. To verify these putative transcriptional targets of p63 and p73, quantitative real time PCR was performed. The expression of mre11, BRCA2, Rad51, and Rad50 was examined in wild-type, p53−/−, p63−/−, p73−/− and p63−/−; p73−/− E1A MEFs before and after treatment with doxorubicin for 12 hours and 5 Gy of gamma radiation. Interestingly, mre11, BRCA2, Rad51, and Rad50 are all induced in wild-type E1A MEFs after these treatments (Figure 3). We measured the baseline levels of mRNA of mre11, BRCA2, Rad51, and Rad50 to determine levels of these transcripts prior to DNA damage (Figure S2). After treatment with doxorubicin or gamma radiation, levels of mre11 mRNA are not induced to wild-type levels in p63−/−and p63−/−; p73−/− E1A indicating that p63 may transcriptionally regulate this gene (Figure 3). Similarly, the levels of BRCA2 are significantly lower in p73−/− and p63−/−; p73−/− E1A MEFs than in wild-type or p53−/− E1A MEFs (Figure 3) after treatment with doxorubicin and gamma radiation. Likewise, the Rad51 gene is not induced to wild-type levels in p63−/−, p73−/−, and p63−/−; p73−/− E1A MEFs after treatment with DNA damaging agents (Figure 3), indicating again that p63 and p73 may be critical transcriptional activators of Rad51 after DNA damage. Lastly, Rad50 also showed a pattern indicative of transcriptional regulation by both p63 and p73. The mRNA levels of Rad50 are approximately 4-fold lower in p63−/−; p73−/− E1A MEFs than in wild-type E1A MEFs (Figure 3) after treatment with doxorubicin and gamma radiation. Taken together, these data indicate that mre11, BRCA2, Rad51, and Rad50 may be transcriptional targets of p63 and p73 in response to DNA damage. As previously reported, twenty percent of mice heterozygous for p63 and p73 (p63+/−; p73+/−) develop mammary adenocarcinomas [7] (Figure 4), and ninety percent of these tumors lose the wild-type allele of p63 and p73 [7]. Given that BRCA2 plays an important role in the pathogenesis of mammary adenocarcinoma, this made it a relevant biological target for p63 and p73 in mammary tumors. The protein levels of Rad51 was first examined by Western blot analysis using wild-type and p63−/−; p73−/− MEFs. Interestingly, the basal level of Rad51 is lower in p63−/−; p73−/− MEFs compared to wild-type MEFs (Figure 4A). The levels of Rad51 in p63−/−; p73−/− MEFs are not induced in response to gamma irradiation; however, a 2-fold increase in expression of Rad51 was detected in the wild-type MEFs after DNA damage. To determine whether this change in expression pattern of Rad51 was cell-type specific, we performed immunohistochemistry on mammary adenocarcinomas from p63+/−; p73+/− mice where LOH of p63 and p73 had occurred (n = 10) (Figure 4F–4I). Indeed, Rad51 as well as BRCA expression is detected in normal mammary glands (n = 10) of p63+/−; p73+/− mice (Figure 4B and 4D) and is lost in hyperplastic mammary glands (n = 4) and mammary adenocarcinomas (n = 6) in these mice (Figure 4C and 4E). Both the cDNA microarray and real-time RT-PCR data provide evidence that BRCA2, Rad51, and mre11 are transcriptionally regulated by p63 and p73 after DNA damage (Figure 3). Consequently, chromatin immunoprecipitation (ChIP) assay was performed to determine whether p63 and/or p73 could directly bind to the promoter region of these two genes. A subset of putative binding sites identified and summarized in Table 1 were assayed using ChIP. Sites chosen included those with the best scores for p53 and p63. Four putative binding sites were assayed for RAD51 (Table 1). RAD51-1 and 2 are located in intron 1, upstream of the start site, while RAD51-3 and 4 are found in intron 2, downstream of the start site. One putative element was assayed for BRCA2 in intron 2,133 nucleotides downstream of the start site (Table 1). Lastly, three putative p53 family response elements were queried for mre11: MRE11-1,2, and 3, located in intron 1, upstream of the start site (Table 1). ChIP analysis was performed using an antibody for p53, p63 or p73 in wild-type, p53−/−, p63−/−, and p73−/− E1A MEFs treated with doxorubicin for 12 hours (Figure 5). Interestingly, p73 was the only p53 family member that binds to the RAD51 promoter after DNA damage treatment. p73 was found to bind to RAD51-2 and 3 in intron 1 and intron 2 respectively. The primers used for this PCR reaction did not distinguish between the two sites; therefore, it is possible that p73 only binds to one of these sites. p63 and p73, but not p53, were found to bind to the response element in BRCA2 after DNA damage (Figure 5). Lastly, p63 was the only family member found bound to the mre-11 promoter at site mre11-3 within intron 1,171 nucleotides upstream of the start site. The same binding pattern in the ChIP assay was obtained with other DNA damaging agents, such as gamma radiation (data not shown). The ChIP results clearly demonstrate that p63 and/or p73 can bind to the promoters of these genes; however to gain a clear indication of which isoforms of p63 and p73 transactivate Rad51, BRCA2, and mre11, luciferase assays were performed with TA and ΔN isoforms of p63 and p73. Regions shown to bind by ChIP assay were used to construct firefly luciferase reporters. pGL3-Rad51-1 was designed by cloning intron 1 containing RAD-51-1 and 2 (Table 1) in to the pGL3 basic vector and pGL3-Rad51-2 containing the elements, RAD51-3 and 4, was cloned in to the pGL3 basic vector. These constructs were transfected in to p63−/−; p73−/− MEFs along with a renilla luciferase gene and one of the following isoforms of p63 or p73: TAp63α, TAp63γ, TAp73α, TAp73β, ΔNp63γ, ΔNp73α, and ΔNp73β. Interestingly, both ΔNp63α and ΔNp73β are the isoforms that transactivate the Rad51 reporter gene to appreciable levels. ΔNp63α transactivates pGL3-Rad51-1 11 fold and ΔNp73β transactivates this reporter 6 fold (Figure 6A). These isoforms more modestly transactivate the pGL3-Rad51-2 reporter indicating that the p63/p73 element resides in intron 1 (Figure 6A and 6B). Surprisingly, the TA isoforms did not transactivate the reporter gene. The p63/p73 family members also transactivate this reporter gene. ΔNp63α and ΔNp73β together can transactivate the Rad51-1 reporter 19 fold (Figure 6A and 6B). Additionally, the other ΔN isoforms that modestly transactivate this reporter alone can transactivate this reporter to higher levels. For example, ΔNp63α along with ΔNp73α can transactivate this reporter gene 9. 8 fold, demonstrating additive effects between these family members. Similar to the experiments for RAD51, the BRCA2 region within intron 1 found to be bound by both p63 and p73 was cloned in to the pGL3 basic vector. Dual-luciferase reporter assay was performed in p63−/−; p73−/− MEFs as described above. Strikingly, the isoform with the highest ability to transactivate this reporter was ΔNp73β with a 4 fold induction. Additionally, ΔNp63α and ΔNp73β can transactivate the reporter 6 fold and other combinations of ΔN isoforms also show increases in transactivation of this reporter (Figure 6C). The ability of p63 and p73 to transactivate the mre11 gene was also tested by luciferase assay. The region shown to bind to p63 by ChIP analysis was cloned in to the pGL3 basic vector to generate pGL3-Mre11. This reporter was induced 3. 8 fold by ΔNp63α and ΔNp73β together (Figure 6D). pPERP-luc, which has previously been shown to be responsive to TAp63γ was used as a positive control for these experiments [23], [24]. To determine whether p53 could transactivate these reporters, p53 was transfected with each reporter and luciferase activity was measured. p53 did not induce any of the reporters assayed (Figure 6A–6D). In addition, we performed luciferase assays using the Rad51-1 and BRCA2 reporters in MEFs lacking p53, p53−/−; p73−/− (Figure 6E and 6F) and p53−/−; p63−/− (data not shown). These experiments yielded similar results as those shown in Figure 6A and 6C. Taken together, these data indicate that the trasactivation of Rad51, BRCA2, and mre11 is p53-independent. Rad51 and BRCA2 are both involved in homologous recombination (HR) DNA repair, one of the major pathways for repair of double strand breaks (DSBs). Cells lacking genes involved in HR, like BRCA2 and Rad51, have been shown to have an impaired ability to repair their DNA [18], [25], [26], [27]. Consequently, we hypothesized that cells lacking p63 and/or p73, which have low levels of these two proteins, may have a defect in repairing DSBs in damaged DNA. To test this hypothesis, wild-type, p53−/−, p63−/−, p73−/−, and p63−/−; p73−/− primary and E1A MEFs were treated with 5 Gy gamma-radiation or doxorubicin to generate DSBs. A comet assay was then performed to determine the DSB repair capacity in these cells. Comet assay, or single cell gel electrophoresis, is a commonly applied approach for detecting DNA damage in a single cell. The unwound, relaxed DNA migrates out of the cell during electrophoresis and forms a “tail” [28]. Therefore, cells that have damaged DNA appear as comets with tails containing fragmented and nicked DNA, while normal cells do not. The degree of DNA damage is represented using the parameter known as tail moment defined as the product of the tail length and the portion of total DNA in the tail. MEFs lacking the p53 family members were treated with DNA damage and incubated for a total of 16 hours allowing the homologous recombination repair to take place. Cells were and harvested at 0 (untreated), 1, and 16 hours for the Comet assay. In all cases, p63−/−, p73−/−, and p63−/−; p73−/− MEFs were found to have the largest tail moment after DNA damage (Figure 7A–7D). The tail moment after DNA damage was significantly higher for p63−/−, p73−/−, and p63−/−; p73−/− primary and E1A MEFs (18. 8) compared wild-type samples (p <0. 0001). This result indicates that p63 and p73 play a critical role in DNA repair. Because loss of p63 and p73 impair DSB repair by regulating Rad51, BRCA2, and mre11, it is likely that loss of p63 and p73 results in poor cell survival due to the inability to repair damaged chromosomal DNA. To determine whether loss of p63 and p73 results in a decrease in cell survival, a clonogenic survival assay was performed using both primary MEFs and E1A expressing MEFs after treatment with 1,2 and 3 Gy of gamma radiation and 0. 34,0. 5, and 1. 0 µM doxorubicin. After 12 hours, cells were replated and assayed for the ability to form colonies. p63−/−; p73−/− E1A MEFs and primary MEFs have an impaired ability to form colonies after gamma radiation indicative of defects in DNA repair (Figure 7E and 7F). A similar result was seen after treatment with doxorubicin in these cells (Figure 7G and 7H).
p53 transactivates a vast network of genes in response to DNA damage [1]. While p63 and p73 can also transactivate known p53 target genes to varying degrees, they play roles in distinct biological functions including development and metastasis and likely have unique transcriptional targets. The advantage of the system employed here is the use of isogenic primary cells with the deletion of a single p53 family member. Here, we used early passage MEFs lacking the p53 family members individually or both p63 and p73 in combination and expressing E1A, which sensitizes them to undergo apoptosis after DNA damage to identify changes in gene expression in this process. We identified sets of genes that are regulated by individual and multiple p53 family members indicating unique and overlapping functions for this family of genes in response to DNA damage. Six hundred twenty out of 15,488 genes queried were regulated by a p53 family member. Genes identified played a role in multiple processes including apoptosis and DNA repair. In addition to engaging pathways predicted to be induced by DNA damage, genes involved in other processes like development and metastasis were also induced. These are biologically significant given the reported developmental, tumor, and metastatic phenotypes of the p63/p73 mutant mice [7], [20], [22], [29]. Lastly, the majority of the targets identified had binding sites that closely fit the p53 and p63 consensus binding site [14], [30], [31] indicating that they may be bona fide direct transcriptional targets of these family members. Indeed, we verified that Rad51, BRCA2, and mre11, genes involved in DNA repair, are direct transcriptional targets of p63 and p73. Given the high prevalence of mammary adenocarcinoma in mice mutant for p63 and p73 (p63+/−; p73+/−), a group of genes of interest are those involved in DNA repair. These genes were induced in wild-type cells and down regulated in the absence of p63 or p73. The mechanism for the tumor suppressive activity of p63 and p73 is not completely understood [6], [7], [32]. Regulation of DNA repair genes by p63 and p73 has not been demonstrated previously and could be a pathway employed by these genes in tumor suppression. Both Rad51 and BRCA2 were found to be direct transcriptional targets of p63 and p73 indicating that these mechanisms may be triggered during tumorigenesis. Interestingly, Rad51 has been shown previously to be repressed by p53 through a site found upstream of the start site [33]. Here, we show that ΔNp63 and ΔNp73 transactivate Rad51 through a distinct element in intron 1 indicating that there is an intricate and complex regulation of this gene by the p53 family and is likely a critical target in tumor suppression by this family. We also showed that transcriptional regulation of Rad51, BRCA2, and Rad51 by p63 and p73 is p53-independent/ It was surprising that the ΔN isoforms of p63 and p73 were more potent transactivators of Rad51, BRCA2, and mre11 than the TA isoforms. The TA isoforms have an acidic N-terminal domain necessary for transactivation [2], [3], and many studies have shown previously that the TA isoforms are more potent transactivators than the ΔN isoforms [2], [8]. Furthermore, the ΔN isoforms are better known for the dominant negative activities that they impose on the TA isoforms of p63 and p73 and p53. Interestingly, a number of recent studies have shown that the ΔN isoforms are capable of transactivating target genes due to a proline-rich transactivation domain that exists in these isoforms [12], [13]. In addition, the ΔN isoforms of p63 are more highly expressed than TAp63 in certain tissues including the skin [14] making the ΔNp63 isoforms likely candidates for gene regulation in these tissues. Taken together, our results indicate that the roles of the ΔN isoforms are more complex than previously appreciated. We have shown previously that E1A expressing MEFs deficient for p63 and p73 are resistant to apoptosis [15]. Paradoxically, we found that p63−/−; p73−/− primary and E1A MEFs are radiosensitive in long-term clonogenic assays. This finding coupled with the inability of p63/p73 deficient cells to repair DNA as shown by Comet assay indicate that p63 and p73 play a critical role in DNA repair. This new finding does not preclude that p63/p73 deficient cells are resistant to apoptosis after acute exposure to DNA damage. These data demonstrate that surviving p63−/−; p73−/− cells are unable to proliferate and establish a colony after DNA damage. This is likely due to defects in the DNA repair mechanisms. Using a genome wide analysis, these studies have revealed novel transcriptional targets of the p53 family members. We have also identified a novel mechanism of the regulation of the DNA repair pathway by p63 and p73. Given the high incidence of mammary adenocarcinoma in p63/p73 mutant mice, these studies have unveiled a potential mechanism for p63 and p73 as tumor suppressor genes. In addition, our studies have revealed further complexity by indicating that the primary transactivators of these DNA repair genes are the ΔN isoforms of p63 and p73. These isoforms have previously been thought to act as oncogenes. More recent data have challenged this notion as these isoforms can also transactivate genes involved in apoptosis and the expression of these isoforms does not provide a growth advantage [12]. These studies provide further evidence that the ΔN isoforms may have some anti-tumor functions such as the ability to engage DNA repair pathways. Future studies using isoform specific knock out mice should yield important insights in to how each of these isoforms contributes to tumor suppression and shed light on the interactions of the complex p53 family.
The Laboratory of Genetics at The National Institute on Aging (NIA) cloned approximately 15,000 unique cDNAs into the NotI/SalI site of Ampicillin-resistant pSPORT1 vector (Life Technologies). Average insert size of the clones is 1. 5 kb (0. 5–3 kb). Inserts were amplified for microarray printing following a modified version of the protocol described previously [34]. In 96 well format, bacterial stocks were grown overnight in 2X YT medium (100 µg/ml ampicillin) with agitation. Ten microliters of the overnight bacterial culture was added to 90 µl ddH2O in PCR plates (MJ Research) and denatured at 95° C for 10 minutes. Following denaturation, plates were centrifuged for 10 minutes. To perform PCR, 5 µl of supernatant from each well was used as template in a 100 µl reaction with 3. 5 units of AmpliTaq DNA polymerase (Applied Biosystems), forward primer (5′–CCAGTCACGACGTTGTAAAACGAC-3′) reverse primer (5′-GTGTGGAATTGTGAGCGGATAACAA-3′), and deoxynucleotide triphosphates (dNTPs). Amplification was carried out in thermocyclers with a program that contained an initial denaturation step at 95°C for 2 minutes followed by 38 cycles of 30 s at 94°C, 45 s at 65°C, and 3 minutes at 72°C, and a final extension of 5 minutes at 72°C. The amplified inserts were then purified using Montage PCR96 cleanup Filter Plates (Millipore) on a BIO-TEK Precision 2000 Automated Microplate Pipetting System to a purified volume of 100 µl. Thirty-five microliters of each purified PCR product was added to a 384-well plate, and desiccated using a large Savant Speed-vac apparatus, then reconstituted in 7 µl of 3X SSC/1. 5 M betaine to a mean concentration of 600 ng/µl. The microarrays were fabricated at the MIT BioMicro Center using Corning GAPS II Gamma Amino Propyl Silane slides. cDNA clones were printed using a BioRobotics Microgrid 600 TAS Arrayer with a 32-pin print head and quill pin microfluidic liquid transfer technology. All procedures involving mice were approved by the IACUC at U. T. M. D. Anderson Cancer Center and M. I. T. E1A-expressing mouse embryonic fibroblasts (MEFs) (wild-type, p53−/−, p63−/−, p73−/−, and p63−/−; p73−/−) were generated as described previously [15] from passage 1 primary MEFs. 3×106 E1A MEFs were plated on each of 6–15 cm dishes. Twenty-four hours after plating, the cells were treated with 0. 34 µM doxorubicin. Twelve hours after treatment, total RNA (150–300 µg) was extracted from treated and untreated E1A MEFs using the RNAeasy Midi Kit (Qiagen). For each microarray hybridization, 100 µg of total RNA prepared from the reference or experimental cells were labeled by incorporating Cy3- or Cy5-labeled dUTP (NEN) using oligo d (T) (MWG) and Superscript II reverse transcriptase (Invitrogen). The resulting probes were purified using the Qiaquick PCR purification Kit (Qiagen) and recovered in a volume of 30 µl ddH20. The printed slides were rehydrated, UV cross-linked, and blocked to reduce background using succinic anhydride (Sigma), 1-methyl 2-pyrrolidinone and sodium borate. Each slide was incubated in 60 µl total volume of hybridization solution containing Cy3- and Cy5-labeled target (one probe is the reference invariant target and the other is the experimental target), 1 µg of Mouse Cot-1 DNA (Invitrogen), 0. 1 units of poly-A40–60 (Amersham Pharmacia), and 10. 1 µg of Salmon Testes DNA (Sigma), 25% Formamide, 5X SSC, 0. 1% SDS under a 22×40-mm lifterslip (Erie Scientific Company) at 42°C for 16 hours exactly. The slide was placed in a sealed hybridization chamber (Corning) containing two side wells with a total of 20 µl 3X SSC for humidification in a light-sealed water bath. After exactly 16 hours of hybridization, the slide was washed in 500 ml of 1X SSC, 0. 03% SDS for 5 minutes after the lifterslips are gently removed in the wash solution. Then, the slides were washed for 5 minutes in 0. 1X SSC, 0. 01% SDS followed by 0. 1X SSC. Slides were centrifuged in a speed-vac to dry. Each slide was scanned using an arrayWoRx Auto Biochip Reader that employs white light, polychromatic filter-wheel/CCD camera (Applied Precision) at wavelengths corresponding to each analog' s emmision wavelength (595 and 685 nm for Cy3 and Cy5, respectively). RNA from each sample was hybridized to four independent cDNA microarrays. For 2 replicates, the invariant target was labeled with Cy3 and the experimental target was labeled with Cy5. For the other 2 replicates for each sample, the invariant target was labeled with Cy5 and the experimental target was labeled with Cy3. The invariant reference target RNA used was extracted from untreated wild type- E1A MEFs. These cells were chosen as a source of reference target RNA because this species of RNA robustly hybridized to a large percentage of genes, and it is relevant to the experimental design. Total RNA was extracted from the E1A MEFs of the genotypes described above using the RNeasy Midi and Rnase-free Dnase kits (Qiagen). RNA was quantified and tested for quality on the Agilent 2100 Bioanalyzer (Agilent Technologies). To generate cDNA, RNA (2 µg) from each E1A MEF line treated with 0. 34 µM doxorubicin was used for random hexanucleotide- primed cDNA synthesis. Each 40 µl reaction contained 1X buffer, 10 µM DTT, 1 µg random hexamer, 2 µl of Superscript II (Invitrogen), 0. 5 mM each of all four dNTPs, and 80 units of RNase inhibitor (Promega). Using heating blocks, reactions were incubated at 42°C for 1 hour, 70°C for 15 minutes, 37°C for 20 minutes, and 95°C for 2 minutes. RNase H (2 units) (Invitrogen) was added to each reaction following the 70°C incubation. Afterwards, each reaction was diluted with ddH2O to a final working volume of 200 µl. cDNAs (2 µl) were added to 25-µl reaction mixtures containing 12. 5 µl of 2X SYBR Green master mix (Applied Biosystems), and 40 nm of gene-specific primers. Primers were designed using Primer Express software (Applied Biosystems). Assays were performed in triplicate with an ABI Prism 7000 Sequence Detector (Applied Biosystems). All data were normalized to an internal standard (18 S ribosomal RNA; TaqMan Ribosomal RNA Control Reagents VIC Probe: Protocol: Rev C, Applied Biosystems) or GAPDH. ChIP Assay was performed as described previously, E1A MEFs (wild-type, p53−/−, p63−/−, p73−/−, and p63−/−; p73−/−) were untreated or treated with 0. 34 µM doxorubicin for 12 hours, which are the same conditions used for the array and real time PCR. Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products). Immunprecipitated complexes were recovered by Staphylococcus A cells, treated with proteinase K, and DNA was purified. PCR was performed for putative p53 family binding elements. Putative p53 family member binding sites were identified by scanning 1000 bp of the 5′ UTR, exon 1, intron 1, exon 2 and intron 2 for the consensus p53 binding site [31]. These sites are summarized in Table 1. Sequences for primers used are available upon request. To generate the pGL3-Rad51 luciferase reporter, DNA was amplified from a BAC clone containing the Rad51 gene (RP23-15121, CHORI BACPAC resources) using primers designed containing the p73 binding site shown by ChIP and 5′ NheI and 3′ XhoI cloning restriction enzyme sites: forward primer (5′- ACTAGCTAGCAGCAGGGCGACCAACCGAC-3′) and reverse primer (5′-CCGCTCGAGTGGCCCTCCCTATCCACAGG-3′). To construct the pGL3-BRCA2 luciferase reporter, the DNA fragment containing the p63/p73 binding site shown by ChIP was amplified from C57/B6 genomic DNA by PCR using the following primers with 5′ XhoI and 3′ BglII cloning restriction enzyme sites: forward primer (5′-CCGCTCGAGAGAGGGATCCGGCGCGTC-3′) and reverse primer (5′-GGAAGATCTGGTCTAAGCTCTGTTGCTCCTG-3′. To generate the pGL3-Mre11 luciferase reporter, DNA was amplified from a BAC clone containing the mre11a gene (RP23-149D5, CHORI BACPAC resources) using primers designed containing the p63 binding site shown by ChIP and 5′ XhoI and 3′ BglII cloning restriction enzyme sites: forward primer (5′- CCGCTCGAGACAGAGAGAACCTCACCGAGAAC -3′) and reverse primer (5′-GGAAGATCTCTGTACCAGGTTCCTCTCCAAG-3′). The resulting amplified DNA fragments were gel-purified (Wizard Prep Kit, Promega) after restriction enzyme digestion and then ligated to pGL3-basic vector (Promega) between the respective cloning sites. 6×105 wild-type and p63−/−; p73−/− MEFs were plated on 6 cm dishes. Twelve hours after plating, the MEFs were irradiated with 5 Gy of gamma-irradiation and then harvested at 10 minutes, 30 minutes, 1,2, and 4 hours. The MEFs were lysed on ice in lysis buffer (100 mM Tris, 100 mM NaCl, 1% Nonidet P40, protease inhibitor cocktail (Roche) ). Thirty micrograms of each lysate was subjected to electrophoresis on a 10% SDS PAGE for Rad51 and transferred to PVDF membrane. Rad51 was detected using the anti-Rad51 antibody (clone 51RAD01, Neomarkers), and BRCA2 was detected using the anti-BRCA2 antibody (clone H-300, Santa Cruz). Slides were dewaxed in xylene and rehydrated in a graded series of ethanol following standard protocols [7]. Slides were incubated with primary antibodies for p63 (4A4, Santa Cruz), p73 (IMG-259A, Imgenex), Rad51 (clone 51RAD01, Neomarkers), or BRCA2 (clone H-300), Santa Cruz). at a dilution of 1∶100 for 18 hours at 4 deg C. Detection was performed using the Vectastain kit (Vector Labs) followed by the VIP kit or DAB kit (Vector Labs) and counterstained with methyl green (Vector Labs). Ten normal mammary glands and ten mammary adenocarcinomas were stained with each antibody. p63−/−; p73−/−, p53−/−; p73−/− or p53−/−; p63−/− MEFs were plated on 6-well plates (3. 5×105 cells per well). Twelve hours after plating, the MEFs were transiently transfected using Fugene HD (Roche) with 2. 5 µg of the following Firefly luciferase reporter plasmids (pGL3-Rad51-1, pGL3-Rad51-2, pGL3-BRCA2) or pPERP-luc [24], 1 µg of Renilla luciferase plasmid (transfection control), and 2. 5 µg of empty vector (pcDNA3) or plasmids encoding the p63/p73 isoforms (TAp63α, TAp63γ, ΔNp63γ, TAp73α, TAp73β, ΔNp73α and ΔNp73β) or p53/ In experiments where 2 isoforms of p63 and p73 were assayed simultaneously, 1. 25 µg of each isoform was used. After 24 hr, cells were harvested and luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega) and a Veritas microplate luminometer (Turner BioSystems). The relative luciferase activity was determined by dividing the Firefly luciferase value with the Renilla luciferase value and the fold increase in relative luciferase activity was determined by dividing the relative luciferase value induced by p63 and p73 isoforms with that induced by the pcDNA3 control vector. Each experiment was performed in triplicate. E1A MEFs or primary MEFs were plated in 6-well plates (1×106 cells per well) of the following genotypes (wild-type, p53−/−, p63−/−, p73−/−, and p63−/−; p73−/−) [15]. Twelve hours later, MEFs were irradiated with 1,2, and 3 Gy of gamma radiation or 0. 34,0. 5, and 1 µM doxorubicin. After 12 hr, 1200 cells were plated on 10 cm dishes. After 12 days of incubation, the cells were stained with clonogenic reagent (0. 25% of 1,9-dimethyl-methylene blue in 50% ethanol). Surviving colonies were counted, and the survival rate was calculated as the ratio of the surviving colonies after DNA damage treatment over the number of colonies for each genotype before treatment. Each experiment was performed in triplicate on three independent MEF lines for each indicated genotype. Wild-type, p53−/−, p63−/−, p73−/−, and p63−/−; p73−/− primary and E1A MEFs were plated on 6-well dishes (1. 6×105 cells per well). Twelve hours after plating, MEFs were irradiated with 5 Gy of gamma radiation. Cells were harvested 0,1, and 16 hours later for Comet Assay (Trevigen) according to the manufacturer' s protocol specific for DSB detection. Briefly, cells were suspended in PBS at a density of 3×105 cell/mL. Twenty microliters of each cell suspension was mixed with 200 µL of melted low melting point agarose (LMA) and 75 µL of this mixture was placed onto the Trevigen CometSlide for electrophoresis. Subsequent to electrophoresis, samples were visualized with SYBR Green I and fluorescence microscopy. Twenty pictures were taken for each sample and at least 135 cells per experiment were examined for comet tails using CometScore software (TriTek Corporation). Three independent MEF lines for each genotype were assayed in triplicate. Student' s t test was used for statistical analysis. All experiments were performed at least in triplicate. Data are represented as the mean ± SEM. Statistics for qRT-PCR, luciferase, clonogenic, and comet assays was performed using Student' s t test for comparison between two groups. A p value of 0. 05 was considered significant.
p63 and p73 have been identified as important suppressors of tumorigenesis and metastasis. Although they are structurally similar to p53, they have many functions that are unique including roles in development and metastasis. Here we show, using a genome-wide analysis of cells lacking p63 and p73 individually and in combination, that p63 and p73 regulate many unique target genes involved in multiple cellular processes. Interestingly, one of these pathways is DNA repair. Further validation of differentially expressed target genes in this pathway, revealed that p63 and p73 transcriptionally regulate BRCA2, Rad51, and mre11 providing a novel mechanism for the action of p63 and p73 in tumor suppression. These findings have important therapeutic implications for cancer patients with alterations in the p63/p73 pathway.
genetics and genomics/animal genetics
genetics and genomics/cancer genetics
genetics and genomics/bioinformatics
p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair