Ei24, a Novel E2F Target Gene, Affects p53-independent Cell Death upon Ultraviolet C Irradiation*

Background: Rb loss deregulates E2F and thus induces the expressions of E2F target genes. Results: Ei24 is an E2F target up-regulated in Rb−/− MEFs and affects susceptibility of p53-deficient MEFs against UVC. Conclusion: E2F1 can provide a p53-independent modulation of cellular sensitivity against UVC via Ei24. Significance: EI24 may be a potential therapeutic target for treating p53-deficient tumors. The deficiency of retinoblastoma (Rb) gene deregulates E2F transcription factors and thus induces E2F target genes directly or p53 target genes indirectly via mouse p19Arf (or p14ARF in humans), an E2F target gene. Here, we identified that etoposide-induced 2.4 mRNA (Ei24)/p53-induced gene 8 (Pig8), a p53 target gene involved in apoptosis and autophagy, was up-regulated in Rb−/− mouse embryonic fibroblasts (MEFs). The Ei24 promoter was activated by E2F1 via multiple E2F-responsive elements, independently of the previously reported p53-responsive element. Chromatin immunoprecipitation assays revealed that E2F1 directly acts on the mouse Ei24 promoter. We observed that Ei24 expression was suppressed in p53−/− MEFs upon UVC irradiation, which was exacerbated in p53−/− E2f1−/− MEFs, supporting the positive role of E2F1 on Ei24 transcription. Furthermore, Ei24 knockdown sensitized p53−/− MEFs against UVC irradiation. Together, our data indicate that Ei24 is a novel E2F target gene contributing to the survival of p53-deficient cells upon UVC irradiation and thus may have a potential significance as a therapeutic target of certain chemotherapy for treating p53-deficient tumors.

The deficiency of retinoblastoma (Rb) gene deregulates E2F transcription factors and thus induces E2F target genes directly or p53 target genes indirectly via mouse p19 Arf (or p14 ARF in humans), an E2F target gene. Here, we identified that etoposideinduced 2.4 mRNA (Ei24)/p53-induced gene 8 (Pig8), a p53 target gene involved in apoptosis and autophagy, was up-regulated in Rb ؊/؊ mouse embryonic fibroblasts (MEFs). The Ei24 promoter was activated by E2F1 via multiple E2F-responsive elements, independently of the previously reported p53-responsive element. Chromatin immunoprecipitation assays revealed that E2F1 directly acts on the mouse Ei24 promoter. We observed that Ei24 expression was suppressed in p53 ؊/؊ MEFs upon UVC irradiation, which was exacerbated in p53 ؊/؊ E2f1 ؊/؊ MEFs, supporting the positive role of E2F1 on Ei24 transcription. Furthermore, Ei24 knockdown sensitized p53 ؊/؊ MEFs against UVC irradiation. Together, our data indicate that Ei24 is a novel E2F target gene contributing to the survival of p53-deficient cells upon UVC irradiation and thus may have a potential significance as a therapeutic target of certain chemotherapy for treating p53-deficient tumors.
The retinoblastoma gene (Rb) 4 was the first tumor suppressor gene to be identified in humans, and its loss of function is implicated in a wide variety of human cancers, including retinoblastomas (1). The Rb gene product (pRb) is a critical component of the cell cycle because it regulates the activity of E2F transcription factors, which induce the expression of genes required for cell cycle progression, including cyclins, cyclin-dependent kinases (CDKs), and proliferating cell nuclear antigen (2). When E2F is activated or deregulated, Rb-induced cell cycle arrest is compromised (3). Furthermore, deregulated E2F transcription factors can enforce quiescent cells to re-enter the cell cycle (4).
pRb/E2F is controlled by p53, which is a critical tumor suppressor known as the guardian of the genome (5). More than 50% of human cancers contain a mutation of p53 (6), and p53mediated tumor suppression is mainly dependent on its activity as a transcription factor. The mutations occur most frequently in the DNA-binding domain of the p53 protein (7). In fact, p53 can activate a plethora of genes that regulate cell cycle progress, apoptosis, and DNA repair under stressful conditions (8). p21 Waf1 is a well known p53 target gene that is involved in the regulation of the Rb/E2F pathway. p21 Waf1 can directly bind to and inhibit all cyclin-CDK complexes, whereas p16 Ink4a specifically inhibits cyclin D-associated complexes of CDK4 and CDK6 (9). The resulting hypophosphorylation of pRb blocks the activation of E2F transcription factors.
In addition to their roles in cell cycle progression, E2F genes are important for apoptosis. In fact, diverse proapoptotic genes are directly regulated by E2F transcription factors (2), whose physiological significance has been demonstrated using mouse models deficient for E2F transcription factors. E2f1-deficient mice exhibit defects in apoptosis and are rather prone to tumorigenesis (10,11). E2F2 and E2F3 are required for S phase entry induced by c-Myc (12). These phenotypes are supported by E2F-mediated regulation of proapoptotic genes including Bax/Bad, Apaf1, caspase-3, and caspase-9 (2).
Etoposide-induced 2.4 mRNA (Ei24), also known as p53-induced gene 8 (Pig8), was first identified as an up-regulated transcript in etoposide-treated NIH3T3 cells (13) and was later characterized as a p53 target gene (14). Ei24 was also identified as a prerequisite gene for executing autophagy (15). Because its ectopic expression induces cell death and suppresses cellular growth, Ei24 has been regarded as a tumor suppressor gene with a proapoptotic function (14). In support of this characterization, Ei24 is located on chromosome 11q24, which is fre-quently lost in neoplastic lesions, including breast cancers (16). Furthermore, because Ei24 is a Bcl2-binding protein localized on the endoplasmic reticulum with potential roles in preventing the spreading of tumors (17), it is believed to be a critical downstream regulator of p53 in tumor suppression. We recently reported that Ei24 stabilizes protein kinase C␣ (PKC␣) and that its deficiency attenuates PKC␣ signaling and skin carcinogenesis in mice (18), suggesting that the physiological role of EI24 is complex and may manifest differently according to tissue-and/or in a stress-specific manner.
Even though a large number of genes have been identified as E2F target genes, the role of pRb/E2F has not been fully elucidated. Recently, we analyzed the alteration in the gene expression pattern induced by Rb deficiency (19). Among the deregulated genes, the induction of Ei24 was observed in Rb Ϫ/Ϫ mouse embryonic fibroblasts (MEFs). In the present study, we characterized Ei24 as an E2F target gene, suggesting that Ei24 may act as a key mediator in the downstream pathways of both pRb/E2F and p53.

EXPERIMENTAL PROCEDURES
Cell Culture, Transfection, and Reporter Gene Assay-MEFs deficient for Rb, p53, and/or E2f1 were prepared as described previously (20). In addition to MEFs, NIH3T3 fibroblasts and H1299 cells were grown in DMEM containing 10% fetal bovine serum (Sigma). Reporter gene assays were conducted using NIH3T3 and H1299 cells. Cells were transiently transfected using WelFect-EX plus reagent (WelGENE) or Lipofectamine 2000 reagent (Invitrogen). Firefly and Renilla luciferase activities were measured 24 h after transfection using Dual-Luciferase Reporter Assay System (Promega) and a luminometer (Turner Designs).
Constructs-pBabe-HA-ER and pBabe-HA-ER-E2F1 were generous gifts from Dr. Kristian Helin (Biotech Research and Innovation Center, University of Copenhagen). pSG5L-HA-E2F1 (Addgene plasmid 10736) was routinely used as an effector for the luciferase and chromatin immunoprecipitation (ChIP) assays. pcDNA3-HA-E2F1, -E2F2, and -E2F3 were generous gifts from Dr. Joseph R. Nevins (Duke University) and were used to evaluate the effect of individual E2F members on the activation of the Ei24 promoter. pcDNA3-FLAG-p53 was a generous gift from Dr. Jaewhan Song (Yonsei University) and was used to validate the activation of the human EI24 promoter. pRL-SV40 (Promega) was used as an internal control for the Dual-Luciferase Reporter Assay System (Promega). The mouse Ei24 promoter was cloned from 129 mouse genome and subcloned into pGL3 Basic vector (Promega). Site-directed mutagenesis was done using the following primers: 5Ј-GTGT-TATAAGCCCCGGCTCTCC-3Ј for BS1, 5Ј-GGTATATGA-GCAGGCGAACCGAG-3Ј for BS2, and 5Ј-CCGCGGCGG-TAGGGTGTATTGGTAGGTGCAGGCCGC-3Ј for BS3. The human EI24 promoter region (Ϫ2093/ϩ477) was amplified by PCR from human EI24 BAC clone (clone ID: RP11-57M13) and was subcloned into pGL3 Basic vector. The reporter gene constructs were verified by direct sequencing. Lentiviral constructs encoding mouse Ei24-specific shRNAs were purchased from Sigma.
Stable Cell Lines and Treatments-Retroviruses were generated with pBabe-HA-ER and pBabe-HA-ER-E2F1, as described previously, and were used for the infection (21). After selection with puromycin (Sigma), infected NIH3T3 cells were cloned and used after the expressions of HA-ER and HA-ER-E2F1 were confirmed. Cells were pretreated with cycloheximide (10 g/ml, Sigma) for 1 h and then co-treated with 30 nM, 300 nM, or 3 M 4-hydroxytamoxifen (4-OHT, Sigma) dissolved in ethanol for 24 h. Infectious lentiviral particles were produced as described previously (18) and were used to infect p53 Ϫ/Ϫ MEFs. Infected cells were selected by treatment with puromycin (Sigma), and polyclonal cell populations were used for the following experiments. UVC irradiation and the following cell death assays, done by measuring fluorescein isothiocyanate (FITC)-labeled annexin V (annexin V-FITC) staining and propidium iodide permeability were conducted as described previously (20).

Up-regulation of Ei24 in Rb Ϫ/Ϫ MEFs-To identify novel
Rb/E2f-regulated genes, we previously analyzed the gene expression profile of Rb Ϫ/Ϫ MEFs (19). Among the deregulated genes, we observed the highly up-regulated expression of Ei24, which has been identified as a p53 target gene (14). Northern blot and quantitative RT-PCR analyses confirmed the significant increase of Ei24 mRNA in Rb Ϫ/Ϫ MEFs (Fig. 1, A and B). Consistent with this finding, known E2F target genes including apoptosis protease-activating factor 1 (Apaf1) (23) and Stathmin (22) were also up-regulated in Rb Ϫ/Ϫ MEFs (Fig. 1B). These data indicate that Ei24 expression can be regulated by Rb/E2F.
Because deregulated E2F1 directly up-regulates p19 Arf , Rb deficiency alters the expressions of diverse p53 target genes (24). To examine whether Ei24 transcription is directly induced by E2F1, stable cell lines expressing the hemagglutinin (HA)tagged ligand-binding domain of the estrogen receptor (ER) and estrogen receptor-fused E2F1 (ER-E2F1) were established Ei24 as an E2F Target Gene using Ink4a/Arf-deficient NIH3T3 as a parental cell line (Fig.  1C). Because 4-OHT binding causes the translocation of the ER into the nucleus, the activity of ER-E2F1 can be conditionally activated by 4-OHT (25). These cell lines were pretreated with cycloheximide (CHX) to prevent de novo protein synthesis and then co-treated with 4-OHT for 24 h to activate the transcriptional activity of ER-E2F1 (Fig. 1, D and E). The data revealed that the Ei24 transcript level was considerably up-regulated in cells expressing ER-E2F1 but not in cells expressing ER only (Fig. 1D). Consistently, Stathmin, a known E2F target gene, showed a comparable expression pattern (Fig. 1D). In Fig. 1E, the effect of CHX treatment was confirmed by PKC␣ protein levels as a control on Western blot assays (18). Because de novo protein synthesis was blocked, these results indicate that Ei24 should be transcriptionally regulated by E2F1.
Ei24 Is a Novel E2F Target Gene-To identify potential ciselements responsive to E2F transcription factors, the proximal region of the Ei24 promoter was analyzed with the TFBIND software (26). Three putative E2F-responsive elements (RE1, 2, and 3) and two cryptic REs with moderate similarity to the consensus were identified in the proximal region of mouse and human EI24 promoters ( Fig. 3A and data not shown). Based on this analysis, the corresponding regions of the Ei24 promoters were cloned from human and mouse genomes, and a series of reporter genes was constructed. Similar to mouse Ei24 (14), human EI24 promoter was strongly activated by p53 ( Fig. 2A). Because Rb deficiency up-regulated Ei24 expression in MEFs (Fig. 1A), and as pRb mainly regulates E2F1-3 (2), we examined the effect of these E2F transcription factors on the activation of the Ei24 promoter. Although E2F1-3 comparably activated the synthetic E2F reporter construct, they had variable effects on the activation of the Ei24 promoter (Figs. 2B and 3B). E2F1 showed an ϳ4-fold higher luciferase activity compared with basal activity, whereas E2F2 and E2F3 increased the activity by ϳ2-fold (Figs. 2B and 3B). These results suggest that, among the different E2Fs, deregulation of E2F1 has a major effect on the transcriptional activation of Ei24 in Rb Ϫ/Ϫ MEFs.
To discriminate the relative potential of the putative E2Fresponsive elements on the Ei24 promoter, they were deleted or mutated. The longest promoter (Ϫ396 ϳ ϩ453) was strongly activated by E2F1 in NIH3T3 cells (Fig. 3C). The deletion of two cryptic REs had no effect on the E2F1-mediated activation of reporter genes, but the responsiveness of the Ei24 promoterluciferase reporter to E2F1 was almost completely blocked by deleting RE1, 2, and 3 (Fig. 3C). In addition to the deletion, site-directed mutations in RE1 and RE2 significantly decreased E2F1-induced reporter gene activity but did not completely abolish the reporter gene activation by E2F1 (Fig. 3D, upper  panel). An RE3 mutation had minimal effect when RE1 and 2 were intact (Fig. 3D, lower panel). However, the combined mutations of RE1-3 effectively suppressed the reporter gene activation by E2F1 (Fig. 3D, lower panel). These results imply that RE1, 2, and 3 are important for E2F-mediated activation of the Ei24 promoter.
Based on the E2F1-dependent activation of the Ei24 promoter, we examined whether E2F1 binds directly to the Ei24 promoter. An Ei24 reporter gene containing wild-type E2Fresponsive elements was transiently transfected into H1299 cells with or without an E2F1 expression construct, and ChIP assays were conducted with an E2F1-specific antibody (Fig. 3E,  left panel). Although the signals from the input were similar, a more prominent signal was detected in the cells transfected with the E2F1-expressing construct (Fig. 3E, left panel). As  E2F1 is functional in H1299 cells (27), a relatively weak signal may also be detected without E2F1 overexpression (Fig. 3E, left  panel). To verify this result, the ChIP assay was done in an endogenous condition using wild-type and E2f1 Ϫ/Ϫ MEFs with the same E2F1-specific antibody. The promoter region of the Ei24 promoter containing RE1-3 was specifically immunoprecipitated from the wild-type MEFs, but no signal was detected from the E2f1 Ϫ/Ϫ MEFs (Fig. 3E, right panel), suggesting the direct binding of E2F1 on the Ei24 promoter. Taken together, these results imply that Ei24 is a novel transcriptional target of E2F1.
Ei24 Expression and Its Physiological Effect upon UVC Irradiation-Because E2F1 is important in the response to DNA damage (28), the expression of Ei24 might be altered by genotoxic stresses independent of p53. To test this possibility, we employed p53 Ϫ/Ϫ and p53 Ϫ/Ϫ E2f1 Ϫ/Ϫ MEFs to test the p53-independent role of E2F1 in regulating Ei24 expression upon genotoxic stresses. Adriamycin, etoposide, and mitomycin C did not induce significant expressional changes between these genotypes (data not shown), but UVC irradiation significantly reduced Ei24 expression in both genotypes (e.g. mock versus 10 J/m 2 , p Ͻ 0.005 for p53 Ϫ/Ϫ and p Ͻ 0.05 for p53 Ϫ/Ϫ E2f1 Ϫ/Ϫ ) (Fig. 4). Notably, the reduction in expression was more severe in the p53 Ϫ/Ϫ E2f1 Ϫ/Ϫ MEFs than in the p53 Ϫ/Ϫ MEFs (Fig. 4). Consistently, Stathmin, an E2F target gene, showed a similar pattern of a reduction in expression in a timedependent experiment (Fig. 4B). These results indicate that UVC irradiation suppresses Ei24 expression upon p53 deficiency and that E2F1 mitigates the negative effect of UVC irradiation on Ei24 expression in MEFs.
Because UVC irradiation is a potent inducer of both p53-dependent and p53-independent cell death, we examined whether Ei24 knockdown might affect UVC-induced cell death in p53 Ϫ/Ϫ MEFs. Ei24 expression was suppressed with lentiviruses encoding Ei24-specific short hairpin RNAs (shRNAs) in p53 Ϫ/Ϫ MEFs (Fig. 5A). When these MEFs were irradiated with 20 J/m 2 UVC, annexin V-FITC-and/or propidium iodidestained cells were prominently detected at 24 and 48 h (Fig. 5B). Notably, Ei24 knockdown significantly increased UVC-induced cell death (Fig. 5, B and C). In contrast to previous reports showing the proapoptotic function of Ei24, these results provide evidence that EI24 may have a protective role against UVC-induced cell death in a p53-deficient genetic background.

DISCUSSION
In the present study, we identified Ei24, a p53 target gene, as an up-regulated gene in Rb Ϫ/Ϫ MEFs, and we subsequently characterized it as an E2F1 target gene. p53 and pRb/E2F are FIGURE 3. A, schematic representation of the proximal Ei24 promoter. The sequences for putative E2F-responsive elements (RE1, 2, and 3) and a consensus sequence for E2F-responsive elements (cons) are denoted. B, differential effects of E2F transcription factors on the Ei24 promoter (Ϫ292/ϩ453). The Ei24 reporter gene (Ei24-Luc) containing RE1-3 and p53 RE without the cryptic E2F REs was used. C, effect of Ei24 promoter deletions on their responsiveness to E2F1. D, effect of site-directed mutagenesis on E2F1-dependent activation of the reporter genes. Upper panel, promoter region of Ei24, Ϫ292/ϩ453. Each mutant construct is denoted as M1, M2, and M3 for the site-directed mutation of RE1, RE2, and both, respectively. Lower panel, the promoter region of Ei24 encompassing Ϫ300/Ϫ116. It does not contain p53 RE. M4, M5, and M6 indicate the mutant constructs with site-directed mutation on RE3 only, commonly on RE1 and RE2, and on all three REs, respectively. ƒ, site-directed mutation ruining the E2F consensus sequence. White bars, basal promoter activity; black bars, E2Factivated reporter gene activity. White boxes, previously reported p53-responsive elements; gray boxes, cryptic E2F-responsive elements; black boxes, putative E2F-responsive elements. The average of a triplicate experiment Ϯ S.D. (error bars) is shown. E, ChIP analysis showing the association of the Ei24 promoter with E2F1. The Ei24 reporter gene containing wild-type RE1-3 (Ϫ300/Ϫ116) were transiently transfected into p53-deficient H1299 cells with or without E2F1 expression construct, and ChIP analysis was done (left panel) or endogenous interaction of E2f1 with the Ei24 promoter was monitored using wild-type and E2f1 Ϫ/Ϫ MEFs (right panel). E2F1-associated chromatin fractions were immunoprecipitated with an E2F1-specific antibody detecting both human and mouse E2F1 proteins.

Ei24 as an E2F Target Gene
closely related in modulating diverse cellular physiologies. When Rb is deficient, deregulated E2F up-regulates the expression of p19 Arf , an important tumor suppressor gene alternatively encoded by Ink4a/Arf locus (24,29,30). p19 Arf then executes a critical role in regulating the p53 activity by compromising the MDM2-mediated degradation of p53 (31)(32)(33). This molecular interaction allows diverse p53 and E2F target genes to be simultaneously induced in Rb Ϫ/Ϫ MEFs. Besides the Ink4a/Arf locus, cross-talk between p53 and pRb/E2F is also achieved by sharing target genes or by regulating distinct genes of a common physiological process. For example, Apaf1 is directly up-regulated by both p53 and E2F1 (23). Bax, a p53 target gene, and Apaf1, a common target of E2F and p53, are critical components for transducing intrinsic apoptotic signals and cooperate with E2F target genes including caspases-3 and 7-9, to properly execute apoptosis (34). Along these lines, considering its expressional regulation, EI24 may participate in cellular processes that are commonly governed by both p53 and E2F.
The potential significance of Ei24 has been highlighted as a p53 target gene for apoptosis and growth suppression (14) and as an autophagy gene (15). As a lethal stress, UVC irradiation actively induces cell death through both p53-dependent and independent pathways (35,36). Our results indicate that Ei24 expression is suppressed by UVC irradiation (Fig. 3), presumably by the activation of pRb and/or its homologues, p107 and p130 (37). However, because this phenomenon was accelerated by E2f1 deficiency, E2F1 should have a positive role in regulating Ei24 expression upon UVC irradiation. Previous studies have described Ei24 as a proapoptotic gene, and thus its putative role as a tumor suppressor is expected (14,17). In fact, etoposide induces Ei24 expression (13), and Ei24 knockdown confers cellular resistance to etoposide-induced cell death in NIH3T3 cells (38). Although these lines of evidence suggest the possible role of EI24 as a tumor suppressor, we recently reported that Ei24-heterozygous knock-out mice manifested attenuated skin tumorigenesis phenotypes upon 7,12-dimethylbenz[␣]anthracene (DMBA)/ 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment (18). Consistently, Ei24 knockdown sensitizes p53 Ϫ/Ϫ MEFs to UVC irradiation (Fig. 5), raising the possibility that EI24 might exert a protective effect under a certain stress independently of p53. These results also suggest that the physiological role of EI24 in cell death is complicated and may be context-and/or environment-dependent. Meanwhile, its physiological role can be deduced from the function of EI24 as an autophagy gene (15).   OCTOBER 25, 2013 • VOLUME 288 • NUMBER 43 Autophagy is also regulated by both p53 and E2F1 (39). Because UVC irradiation seriously damages cells, the damaged organelles, including mitochondria, should be removed by autophagy. If they are not properly eliminated by autophagy, lethal materials produced and released by the damaged mitochondria (e.g. reactive oxygen species or proapoptotic molecules) may make Ei24-silenced cells susceptible to UVC irradiation.

Ei24 as an E2F Target Gene
As Ei24 knockdown increased the sensitivity of p53 Ϫ/Ϫ MEFs against UVC irradiation (Fig. 5), EI24 might be critical for determining cellular survival under a certain stress independently of p53. In other words, it is highly possible that EI24 is a critical factor modulating the survival of p53-deficient cells in human diseases, e.g. cancers. Therefore, EI24 can be considered as a potential therapeutic target in p53-deficient tumors.
Taken together, these results imply that EI24 is a critical mediator/effector required for the crosstalk between p53 and pRb/E2F pathways dedicated to tumor suppression. Therefore, understanding and identifying the specific conditions that require EI24 may provide important clues for the prevention and treatment of neoplastic diseases in humans, and it may be helpful to scrutinize the molecular mechanisms of EI24 action and to evaluate the extent of mutations and expression of Ei24 gene in diverse human cancers for proper cancer treatments.