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• W2077230345 abstract "Article1 August 2002free access Ets1 is required for p53 transcriptional activity in UV-induced apoptosis in embryonic stem cells Dakang Xu Dakang Xu Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author Trevor J. Wilson Trevor J. Wilson Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author David Chan David Chan Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author Elisabetta De Luca Elisabetta De Luca Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author Jiong Zhou Jiong Zhou Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author Paul J. Hertzog Corresponding Author Paul J. Hertzog Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author Ismail Kola Ismail Kola Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Present address: 7245-24-110, Pharmacia and Upjohn, 301 Henrietta Street, Kalamazoo, MI, 39007 USA Search for more papers by this author Dakang Xu Dakang Xu Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author Trevor J. Wilson Trevor J. Wilson Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author David Chan David Chan Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author Elisabetta De Luca Elisabetta De Luca Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author Jiong Zhou Jiong Zhou Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author Paul J. Hertzog Corresponding Author Paul J. Hertzog Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Search for more papers by this author Ismail Kola Ismail Kola Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia Present address: 7245-24-110, Pharmacia and Upjohn, 301 Henrietta Street, Kalamazoo, MI, 39007 USA Search for more papers by this author Author Information Dakang Xu1, Trevor J. Wilson1, David Chan1, Elisabetta De Luca1, Jiong Zhou1, Paul J. Hertzog 1 and Ismail Kola1,2 1Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, 246 Clayton, Clayton, Victoria, 3168 Australia 2Present address: 7245-24-110, Pharmacia and Upjohn, 301 Henrietta Street, Kalamazoo, MI, 39007 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4081-4093https://doi.org/10.1093/emboj/cdf413 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Embryonic stem (ES) cells contain a p53-dependent apoptosis mechanism to avoid the continued proliferation and differentiation of damaged cells. We show that mouse ES cells lacking Ets1 are deficient in their ability to undergo UV-induced apoptosis, similar to p53 null ES cells. In Ets1−/− ES cells, UV induction of the p53 regulated genes mdm2, perp, cyclin G and bax was decreased both at mRNA and protein levels. While p53 protein levels were unaltered in Ets1−/− cells, its ability to transactivate genes such as mdm2 and cyclin G was reduced. Furthermore, electrophoretic mobility shift assays and immunoprecipitations demonstrated that the presence of Ets1 was necessary for a CBP/p53 complex to be formed. Chromatin immunoprecipitations demonstrated that Ets1 was required for the formation of a stable p53–DNA complex under physiological conditions and activation of histone acetyltransferase activity. These data demonstrate that Ets1 is an essential component of a UV-responsive p53 transcriptional activation complex in ES cells and suggests that Ets1 may contribute to the specificity of p53-dependent gene transactivation in distinct cellular compartments. Introduction Pre-implantation embryos are hypersensitive to DNA damage and can rapidly undergo apoptosis without cell-cycle arrest (Aladjem et al., 1998; Heyer et al., 2000). Indeed, apoptosis is observed in the inner cell mass of up to 75% of pre-implantation embryos (Hardy et al., 1989). The pro-apoptotic protein p53 plays a critical role in the cellular stress response and appears to be involved in these early embryonic checkpoint controls. Significantly, the absence of p53 resulted in a high rate of embryonic malformations (Corbet et al., 1999; Heyer et al., 2000). This suggests that mechanisms exist to avoid the expansion of potentially abnormal embryonic cells, which could contribute to developmental abnormalities. Murine embryonic stem (ES) cells, which are derived from the inner cell mass of pre-implantation embryos, have provided an important resource to investigate these early embryonic checkpoints. Exposure of these cells to DNA-damaging agents, such as UV irradiation, have demonstrated that p53 levels rapidly increase, together with associated transcriptional activity (Ko and Prives, 1996; Levine, 1997; Corbet et al., 1999). The factors which contribute to this activation of p53 could thus have significant roles in ensuring the normal development of healthy embryos. Although the mechanisms that regulate p53 transcriptional activity are not precisely defined, this complex process is known to include post-translational events such as phosphorylation and acetylation (Meek, 1998; Sakaguchi et al., 1998). Phosphorylation of specific residues alters the stability of p53 and its ability to form complexes with DNA and/or other proteins (Kapoor and Lozano, 1998; Lu et al., 1998; Unger et al., 1999). For example, phosphorylation of N-terminal serine residues is believed to be important for the interaction of p53 with the transcriptional co-activators p300/CREB-binding protein (CBP) and p300/CBP-associated factor (PCAF; Lambert et al., 1998; Sakaguchi et al., 1998). Furthermore, p300/CBP binding and subsequent acetylation of p53 has also been shown to enhance sequence-specific p53–DNA binding (Gu and Roeder, 1997). These complex regulatory events, which are just beginning to be defined, result in p53-dependent transactivation of a number of genes involved in the apoptotic process. These include MDM2, GADD45, cyclin G, bax, p21 and PERP (El-Deiry et al., 1993; Perry et al., 1993; Ko and Prives, 1996; Matsuyama et al., 1998; Attardi et al., 2000). Production of these proteins activates a signaling cascade that results in cell death via DNA cleavage and nuclear fragmentation characteristic of apoptosis. There is considerable circumstantial evidence to link p53 and the transcription factor Ets1. Both p53 and Ets1 induce apoptosis, are regulated by Ras pathway phosphorylation, bind to a comparable region of CBP and have similar expression patterns during development (Schmid et al., 1991; Kola et al., 1993; Maroulakou et al., 1994). Indeed, the p42 Ets1 splice variant has been shown to induce apoptosis in human colon cancer cells (Li et al., 1999) and Ets1 is involved in the cellular response to stress (Seth et al., 1999; Lelievre et al., 2001). Ets1 transcriptional activity is also enhanced following phosphorylation by the Ras pathway (Wasylyk et al., 1997; for a review, see Watson et al., 2002). Furthermore, the CH3 domain of CBP binds Ets1, is the same domain that has been shown to bind p53 (Yang et al., 1998), and is involved in the regulation of genes controlling apoptosis and the cell cycle (Avantaggiati et al., 1997). Based upon this evidence, we propose that Ets1 and p53 may compete and/or complex with CBP and other proteins to modulate transcriptional activity of genes in p53-dependent signaling pathways. In the absence of direct data linking Ets1 and p53, we generated Ets1 null mouse ES cells by homologous recombination and determined that they have a reduced apoptotic response following exposure to UV. This was strikingly similar to that shown for p53 null ES cells (Corbet et al., 1999). Ets1 null ES cells also had significantly lower levels of mdm2, perp, cyclin G and bax mRNA, which are all defined p53 target genes. Significantly, Ets1 was found to be part of a p53 and CBP-containing complex bound to a p53 consensus binding site in electrophoretic mobility shift assays (EMSA) and was necessary for the assembly of CBP to this UV-induced p53 complex. Results Generation of Ets1 null mouse ES cells Ets1-deficient ES cells were generated by homologous recombination using the loxP-CRE system. The vector detailed in Figure 1 was used to generate a targeted allele where exons 3–6 and a neomycin resistance gene were floxed. This strategy was designed such that specific recombination by CRE recombinase removed the floxed exons (and neomycin), which would result in a frameshift and premature stop codon (should mRNA still be synthesized). Heterozygous targeted ES clones were obtained from screening G418 and gancyclovir-resistant colonies (Figure 1C and D). Correctly targeted clones were identified with a 5′ external probe, which detected 5.5 kb wild-type and 3.8 kb targeted bands on EcoRV-digested ES cell DNA (Figure 1C). These clones were also confirmed using a 3′ internal probe (Figure 1D). Clones with two targeted alleles were generated by culture in 2.0 mg/ml G418 and identified by loss of the wild-type band (5.5 kb) using a larger 5′ probe on EcoRV-digested DNA (e.g. clones 2 and 17; Figure 1E). Two independent double-targeted Ets1 ES cell clones (2 and 17) were transiently transfected with a PGK-CRE recombinase vector. Both targeted clones were able to generate sub-clones with appropriately spliced Ets1 alleles (2–15 and 17–34, respectively), which gave the expected 13 kb EcoRV band on a Southern blot (Figure 1E). To verify that these ES cell clones were truly Ets1 null, RNA was extracted and the absence of Ets1 expression was confirmed by northern blot analysis (Figure 1F; data not shown). These targeted clones (2 and 17) and their CRE-recombined derivatives (2–15 and 17–34) were used in subsequent assays. Figure 1.Targeted mutagenesis of the Ets1 gene. (A) The wild-type Ets1 locus and targeting vector showing the targeting strategy and restriction enzyme sites used for screening. The construct was designed such that exons 3–6 and a neomycin cassette were floxed for subsequent excision by CRE recombinase. 5′ and 3′ probes, which were used to discriminate between alleles, are also shown. LoxP sites are indicated by filled triangles. (B) Schematic representation of the wild-type and modified Ets1 locus: (1) wild-type locus; (2) targeted locus with appropriate insertion of loxP sites flanking exons 3–6; (3) double-targeted Ets1 locus generated by High G418 selection; and (4) double-knockout ES cells generated by CRE-mediated excision. (C) EcoRV-digested DNA probed with the 1.5 kb genomic fragment external to the targeting construct. WT and clone 270 show the 5.5 kb WT allele only, whereas clone 269 shows both the WT and 3.8 kb targeted band. (D) Confirmation of targeting of clone 269. Genomic DNA from wild-type and Ets1-targeted clone 269 probed with the 1.7 kb 3′ internal genomic fragment. Only clone 269 shows the expected 2.5 and 5.7 kb targeted bands (HindIII and BglII digested DNA, respectively) in addition to the expected WT bands. (E) Double-targeted ES cell clones generated by High G418 resistance (17, 2) and subsequent removal of the Ets1 exons 3–6 by CRE recombinase (17–15, 2–34). The CRE spliced and unspliced targeted alleles are indicated by the 13 and 1.7/3.8 kb bands, respectively, using the 5.5 kb 5′ probe. (F) Northern blotting showing absence of Ets1 mRNA expression in Ets1-targeted and spliced ES cell clones using a murine Ets1 cDNA probe. Poly(A)+ mRNA (3 μg) of each was used and reprobed for GAPDH to demonstrate equal loading. These clones were subsequently considered Ets1−/−. Download figure Download PowerPoint Ets1−/− ES cells have decreased p53 mRNA levels Ets1 and Ets2 have been shown to activate p53 promoter constructs in vitro and have high affinity for an element within the promoter containing palindromic Ets-binding sites (Venanzoni et al., 1996). We therefore analysed the level of p53 mRNA in feeder cell-depleted wild-type and Ets1−/− ES cells by northern blot analysis (Figure 2A). Interestingly, Ets1−/− ES cells expressed less p53 mRNA than that observed in wild-type ES cells and our targeted cells prior to CRE-mediated Ets1 inactivation. Similar results were observed for a second independently derived Ets1−/− clone (data not shown). Thus the absence of Ets1 was associated with a reduction in p53 mRNA levels in these ES cells. Figure 2.Reduced expression of p53 mRNA in Ets1−/− ES cells is not due to altered morphology or differentiation status. (A) Northern blotting showing reduced expression of p53 in Ets1−/− ES cells compared with wild-type (WT), double-targeted (Ets1loxP) ES cells. The same blot was used to determine the that relative levels of Oct4 mRNA were unaltered and GAPDH was used to demonstrate equal loading. (B) Photomicrographs of wild-type (1 and 4), Ets1loxP (2 and 5) and Ets1−/− (3 and 6) ES cells. The upper panel is a phase-contrast image whereas the lower panel is labeling with anti-SSEA1–FITC, which is expressed only in undifferentiated ES, indicating that these cultures contain very few differentiated cells. Bar corresponds to 100 μm. Download figure Download PowerPoint Morphology and differentiation status of Ets1−/− ES cells Since p53 has been reported to be down-regulated after ES cell differentiation (Louis et al., 1988), we examined the morphology and differentiation status of the ES cell clones. The double-targeted ES cell lines (before CRE, and are Ets1+/+) had a normal phenotype and were indistinguishable from wild-type cells in all assays. Ets1−/− ES cells demonstrated morphology similar to control cells (Figure 2B). We have also determined that these ES cells have similar expression of SSEA1 [immunohistology and flow cytometry (>95%)], alkaline phosphatase (histology) and Oct4 [northern blot and flow cytometry (>95%)] (Figure 2; data not shown). Each of these markers are only expressed in undifferentiated ES cells and not in differentiated derivatives (Wobus et al., 1984; Scholer et al., 1989). These data indicate that the differentiation status of control and Ets1 null ES cells was similar. ES cells lacking Ets1 are less susceptible to UV-induced apoptosis Since there was a lower level of p53 mRNA in Ets1−/− ES cells and Ets1 has been reported to be proapoptotic, we examined UV-induced apoptosis in these cells by propidium iodide staining. There was no significant difference in the rates of proliferation or apoptosis between untreated wild-type, Ets1loxP and two independent Ets1−/− ES cell lines (15 and 34; Figure 3; data not shown). However, when we exposed these cells to various doses of UV irradiation, which is effective at inducing p53-mediated apoptosis in ES cells (Sabapathy et al., 1997; Corbet et al., 1999), the number of apoptotic cells was significantly reduced in the Ets1−/− ES cells (Figure 3). The proportion of cells undergoing apoptosis in wild-type ES cells (and the parental Ets1 double-targeted ES cells) increased with increasing doses of UV irradiation with ∼60% of Ets1+/+ ES cells apoptosing after 40 J/m2 UV (Figure 3A and B). In contrast, only 30% apoptosis was observed in Ets1−/− ES cells exposed to the same dose of UV (Figure 3A and B). Time-course analysis after 40 J/m2 UV revealed that there was initially no significant difference in proportion of apoptotic cells, as all genotypes had a relatively small fraction of apoptotic cells 4 h post-irradiation (Figure 3C), and by 9 h, 20–30% of cells from both genotypes were apoptotic. After this initial period of cell death, the percentage of apoptotic Ets1−/− cells did not significantly increase, whereas the fraction of wild-type and Ets1loxP cells continued to increase for up to 12 h. To confirm that this was truly apoptosis, we also examined these cells for morphological features characteristic of apoptosis by Hoechst staining (Figure 3D–I) and the characteristic fragmentation of DNA (Figure 3J). Untreated wild-type and Ets1−/− ES cells showed very similar morphological features (Figure 3D–F); however, after 40 J/m2 of UV treatment, the wild-type and targeted (Figure 3G and H) but not Ets1−/− cells (Figure 3I) demonstrated nuclear condensation and fragmentation. Similarly, agarose gel electrophoresis of DNA extracted from wild-type cells 12 h after UV irradiation (40 J/m2) showed DNA laddering, which was not observed in UV-treated Ets1−/− cells (Figure 3J). Figure 3.Ets1−/− ES cells are resistant to UV irradiation-induced apoptosis. (A) Flow cytometric analysis of fixed wild-type, Ets1-targeted and two independent Ets1 null ES cell clones stained with propidium iodide 12 h after UV irradiation (40 J/m2). Percentage of cells with a hypodiploid (sub-2N) DNA content indicative of apoptosis are shown. (B) Graphical representation of the percentage of cells undergoing apoptosis after various doses of UV irradiation. Data is shown as mean ± SD of three independent experiments. (C) Percentage of cells undergoing apoptosis at different time points after irradiation with 40 J/m2 of UV. Data is shown as mean ± SD of three independent experiments. (D–I) Immunofluorescent images of Hoechst 33342-stained WT (D and G), Ets1loxP (E and H) and Ets1−/− (F and I) ES cells, with and without UV irradiation. Significant nuclear condensation and fragmentation is not observed in untreated cells (D–F) or UV-irradiated Ets1 null cells (I). However, a significant number of WT and Ets1-targeted ES cells showed nuclear fragmentation after UV irradiation (G and H). Bar corresponds to 20 μm. (J) Ethidium bromide staining of DNA from WT, Ets1loxP and Ets1−/− ES cells after 40 J/m2 UV irradiation analysed by agarose gel electrophoresis, demonstrating bands of DNA fragmentation in WT and targeted but not Ets1−/− cells. Lane 1 shows pUC19/HpaII marker. Download figure Download PowerPoint Addition of exogenous Ets1, but not exogenous p53, restores sensitivity to UV irradiation-induced apoptosis in Ets1−/− ES cells To verify that the resistance to apoptosis in Ets1 null ES cells was only due to the Ets1 status, we transfected Ets1−/− cells with FLAG-tagged Ets1 or vector alone constructs (pEFBOS 1 FLAG) as a control. Transfected cells were shown to express FLAG-Ets1 protein by αFlag western blotting (Figure 4A). These ES cell lines were exposed to 40 J/m2 UV and examined for apoptosis by propidium iodide staining and DNA fragmentation. There was no significant difference in the apoptosis rates between untreated Ets1−/− ES cells expressing FLAG-Ets1 or FLAG alone (Figure 4C), indicating these Ets1 expression alone does not activate apoptosis. However, after UV treatment, Ets1−/− cells transfected with FLAG-Ets1 showed increased sensitivity to apoptosis, ∼57% cell death at 12 h after UV irradiation, similar to that observed for wild-type cells (Figure 4C). In contrast, cells transfected with FLAG alone showed only 27% apoptosis (Figure 4C). These observations demonstrate that the inability of our ES cells lacking Ets1 to undergoing UV-induced apoptosis is due to their Ets1 status only, since exogenous Ets1 can restore the normal response. Figure 4.Addition of exogenous Ets1, but not exogenous p53, restores sensitivity to UV irradiation-induced apoptosis in Ets1−/− ES cells. Western blots of ES cell lysates after transfection with FLAG-Ets1 (A) and exogenous p53 (B), which demonstrate expression of FLAG-Ets1 and increased expression of p53, respectively. (C) FACS analysis of propidium iodide-stained wild-type and Ets1−/− ES cells expressing exogenous CMV-p53 or EF1α-FLAG/Ets1 after UV treatment. These demonstrate characteristic apoptosis after UV treatment of ES cells, which express endogenous or exogenous Ets1, regardless of p53 status. Download figure Download PowerPoint Since we determined that p53 mRNA levels were also decreased in Ets1−/− ES cells and a similar reduced sensitivity to UV-induced apoptosis has been observed in ES cells lacking p53 (Corbet et al., 1999), we tested whether addition of p53 would reconstitute the apoptotic response. Ets1−/− and wild-type ES cells were transfected with a CMV-p53 construct that has previously been shown to induce cell-cycle arrest and apoptosis in human and murine cells (Wills et al., 1994; Linke et al., 1996). CMV-p53 transfectants did show increased levels of p53 protein by western blotting; however, unlike p53 mRNA levels (Figure 2A), there was no significant difference in endogenous p53 protein levels between Ets1−/− and wild-type cells (Figure 4B). Furthermore, high levels of exogenous p53 did not induce apoptosis or cell-cycle arrest, and the ability of Ets1−/− ES cells to undergo apoptosis was not restored even after UV treatment, as determined by propidium iodide staining (Figure 4C) and DNA laddering analysis (data not shown). For example, in Ets1−/− cells expressing increased levels of p53, only 35% of cells were apoptotic compared with 27% with endogenous p53 only. There was also no significant difference in the proportion of Ets1+/+ cells undergoing apoptosis after increasing p53 levels by transfection of exogenous p53. Thus the decreased sensitivity of Ets1−/− ES cells to UV-induced apoptosis appears to be due to the lack of Ets1, and not related to levels of p53 protein. The expression of p53-dependent UV-activated genes is reduced in cells lacking Ets1 Although UV-induced ES cell apoptosis has been shown to be p53 dependent (Sabapathy et al., 1997; Corbet et al., 1999), our data demonstrate that UV-induced apoptosis is Ets1 dependent and is reduced in the absence of Ets1. Furthermore, it has previously been shown that Ets1 can be proapoptotic via a p53-independent mechanism (Li et al., 1999, 2000), thus we examined the expression of a number of p53-dependent and -independent apoptosis-regulating genes, including perp, mdm2, cyclin G, bax (p53 dependent), Bcl-2 and Bcl-XL (p53 independent; Figure 5). Northern blot analysis of wild type and two Ets1−/− ES cell clones demonstrated that there was no significant difference between the non-induced levels of the p53-dependent genes perp, mdm2, cyclin G and bax (Figure 5A). How ever, these genes were strongly induced by UV irradiation in wild-type and Ets1-targeted ES cells, but were poorly induced by UV treatment in Ets1−/− ES cells (Figure 5A). These data correlated with western blot analysis, which demonstrated that mdm2 and Bax proteins were induced by UV in Ets1+/+ but not Ets1−/− ES cells (Figure 5B). In contrast, levels of the p53-independent proteins Bcl-2 and Bcl-XL were not induced by UV irradiation in wild-type or Ets1−/− cells (Figure 5B). Interestingly, western blot analysis of p53 protein indicated that p53 protein was expressed and induced by UV similarly in Ets1+/+ and Ets1−/− ES cells. Figure 5.The expression of p53 transactivated genes is reduced in cells lacking Ets1 following UV irradiation. (A) Results of a typical northern blot experiment measuring the expression of p53-regulated genes in wild-type (wt), Ets1-targeted (Ets1loxP) and Ets1−/− ES cells before and after UV irradiation (40 J/m2). Cells were analysed and RNA isolated at times indicated after UV treatment. Probes specific for mouse perp, mdm2, cyclin G and bax were used for northern blot analyses with GAPDH as a loading control. Data from five independent experiments of two independent Ets1−/− clones were quantified using a Fuji Image Reader VI.3E and expressed as mean ± SD, relative to a GAPDH loading control. (B) Expression of p53-regulated proteins in wild-type and Ets1−/− ES cells. Cells were isolated at the indicated time points (0, 1, 2, 4 and 8 h) post-UV irradiation (40 J/m2). Antibodies specific for mouse p53, mdm2, Bax, Bcl-2, Bcl-XL and β-tubulin (loading control) were used for western blot analyses. Analysis was performed at least three times on two independent Ets1−/− clones and data from a representative experiment is shown. Download figure Download PowerPoint These data suggest that Ets1 may regulate perp, mdm2, cyclin G and bax, which are defined p53 target genes, either independently of p53 or via post-translational regulation of p53 transactivational activity. p53 transactivational activity induced by UV requires Ets1 p53 transactivational activity is regulated, at least in part, by post-translational modification such as phosphorylation, acetylation and interaction with other proteins (Meek, 1998; Sakaguchi et al., 1998). In the absence of Ets1, the levels of p53-regulated genes were altered although p53 protein levels were unchanged, thus we investigated whether the transactivational activity of p53 was altered in the absence of Ets1. Mdm2 and cyclin G promoter constructs were used to determine whether the alterations observed in mRNA and protein levels were due to alterations in transcription. Consistent with our other data, when these constructs were introduced into Ets1+/+, Ets1loxP or two Ets1−/− ES cell clones, they were more efficiently activated by UV in wild-type rather than Ets1−/− cells (Figure 6). To demonstrate that this action was mediated by p53, constructs with mutations in the p53REI site of the mdm2 promoter and p53REII site of cyclin G promoter, which have been shown to remove p53 responsiveness (Zauberman et al., 1995a, b; Yardley et al., 1998), were similarly introduced into these cell lines. These mutations removed all responsiveness of these promoters to UV irradiation regardless of the Ets1 status (Figure 6). Indeed, the response of the promoters containing a p53RE site mutation were transactivated less in Ets1+/+ cells than the wild-type promoters in Ets1−/− ES cells. In contrast, the mdm2 promoter containing mutations in one of the Ets-binding elements was able to respond to UV treatment of wild-type ES cells at a reduced level (Figure 6A), similar to that observed for the wild-type mdm2 promoter in Ets1 null cells. The Ets-binding element mutation did not alter the response in Ets1−/− ES cells. Figure 6.UV enhances p53 transcriptional activity in wild-type but not Ets1−/− ES cells. (A) Wild-type and Ets1−/− ES cells were transfected with wild-type mdm2 promoter-luciferase constructs or those containing mutations in the defined p53- or Ets-binding sites (mdm2, mdm2Δp53I and mdm2ΔEtsA, respectively). Cells were exposed to UV irradiation 24 h after transfection and harvested at 36 h. Luciferase activity was determined relative to a β-gal control. Each assay was performed at least three times in triplicate. Data is shown as the mean of independent experiments ± SD. (B) As above, except the mouse cyclin G promoter and p53 element mutant cyclin GΔp53II promoter-reporter vectors were transfected into wild-type and Ets1−/− ES cells and exposed to UV irradiation. Mean of at least three independent experiments is shown ± SD. Download figure Download PowerPoint These data demonstrate that the p53-binding element of these promoters is required for the UV response, but the presence of the transcription factor Ets1 and the Ets(A)-binding site of the mdm2 promoter is necessary for optimal transactivation. The p53–CBP complex induced by UV irradiation contains and requires Ets1 Our data indicate that the reduced induction of p53-dependent genes after UV irradiation in Ets1−/− ES cells was transcriptionally mediated; however, the mechanism by which Ets1 contributed to the transactivation of these genes was unclear. Thus we determined whether the ability of p53 to bind DNA was altered in these cells. Nuclear extracts were isolated from UV-irradiated ES cells and EMSA performed with a p53-binding element consensus sequence that has previously been shown to bind p53 after UV exposure (Figure 7; El-Deiry et al., 1992; Sabapathy et al., 1997). These data demonstrated that p53-binding activity was induced in both wild-type and Ets1 null cells at 5 h after UV treatment (Figure 7A, lanes 3 and 4, and lanes 8 and 9). Specificity of the p53–DNA complex is demonstrated both by competition with unlabelled oligonucleotide (Figur" @default.
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• W2077230345 title "Ets1 is required for p53 transcriptional activity in UV-induced apoptosis in embryonic stem cells" @default.
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