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MEF/ELF4 transactivation by E2F1 is inhibited by p53

MEF/ELF4 transactivation by E2F1 is inhibited by p53 76–88 Nucleic Acids Research, 2011, Vol. 39, No. 1 Published online 30 August 2010 doi:10.1093/nar/gkq762 MEF/ELF4 transactivation by E2F1 is inhibited by p53 1,2 1 1 1 Manabu Taura , Mary Ann Suico , Ryosuke Fukuda , Tomoaki Koga , 1 1 1 2 1, Tsuyoshi Shuto , Takashi Sato , Saori Morino-Koga , Seiji Okada and Hirofumi Kai * Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Global COE ‘Cell Fate Regulation Research and Education Unit’, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973 and Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan Received March 14, 2010; Revised July 4, 2010; Accepted August 10, 2010 INTRODUCTION ABSTRACT MEF/ELF4 is a member of the E-twenty -six (ETS) family Myeloid elf-1-like factor (MEF) or Elf4 is an of transcription factors, which function as transcriptional E-twenty-six (ETS)-related transcription factor with activators or repressors and regulate critical aspects of strong transcriptional activity that influences cellular differentiation, proliferation and transform- cellular senescence by affecting tumor suppressor ation (1). MEF was originally isolated from human p53. MEF downregulates p53 expression and megakaryocytic leukemia cell line, and is known to inhibits p53-mediated cellular senescence by tran- activate the expression of a variety of cytokine genes, such as interleukin (IL)-3 and IL-8 (2,3) and antibacterial scriptionally activating MDM2. However, whether peptides, such as lysozyme and human b-defensin and the p53 reciprocally opposes MEF remains unex- cytolytic molecule perforin (4–6). MEF expression and plored. Here, we show that MEF is modulated by activity are regulated by its post-translational modifica- p53 in human cells and mice tissues. MEF expres- tion, protein–protein interaction and by transcription. sion and promoter activity were suppressed by p53. MEF expression is highest at G1 phase; and at G1 to While we found that MEF promoter does not contain S-phase transition, MEF is phosphorylated by cyclinA– p53 response elements, intriguingly, it contains E2F skp2 cdk2 complex, ubiquitinated by SCF and degraded consensus sites. Subsequently, we determined that by proteasome (7,8). SUMOylation of MEF inhibits its E2F1 specifically binds to MEF promoter and transcriptional activity (9), whereas translocation of transactivates MEF. Nevertheless, E2F1 DNA MEF into promyelocytic leukemia (PML) nuclear bodies binding and transactivation of MEF promoter was induces interaction with PML and increases MEF tran- scriptional activator function (10,11). Sp1 was previously inhibited by p53 through the association between determined to positively influence the transcription of p53 and E2F1. Furthermore, we showed that activa- MEF (12). Epigenetic regulation, promoter methylation tion of p53 in doxorubicin-induced senescent cells and histone deacetylation mediate MEF gene silencing increased E2F1 and p53 interaction, diminished (our unpublished data) (13). E2F1 recruitment to MEF promoter and reduced Besides its function as an activator of cytokines and MEF expression. These observations suggest that innate immune molecules, MEF also impacts on cell-cycle p53 down-regulates MEF by associating with and progression by promoting the transition of cells from G inhibiting the binding activity of E2F1, a novel to S phase (7). The loss of MEF was shown to increase transcriptional activator of MEF. Together with tumor suppressor p53 protein and enhance hematopoietic previous findings, our present results indicate that stem cell (HSC) quiescence in murine embryonic fibro- a negative regulatory mechanism exists between blasts, implicating MEF in driving HSC from quiescence to G phase by opposing p53 function (14,15). A study p53 and MEF. *To whom correspondence should be addressed. Tel/Fax: +81 96 371 4405; Email: hirokai@gpo.kumamoto-u.ac.jp The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2011, Vol. 39, No. 1 77 previously demonstrated that MEF upregulates the tran- 37 C in a humidified atmosphere of 5% CO . Treatment scription of MDM2, the E3 ubiquitin ligase of p53, of cells with nutlin-3 or DMSO (control) was carried out thereby suppressing p53 protein stability that led to the for 24 h (HCT116 cells) or 48 h (HepG2 cells). Transient inhibition of p53-dependent oncogene-induced cellular transfections of plasmids were performed using Hilymax senescence (16). Considering that MEF contributes to (Dojindo Laboratories, Kumamoto, Japan) following the driving cell-cycle progression and that MEF suppresses manufacturer’s recommendation. Specifically, Hilymax p53, which is known for promoting cell-cycle arrest and diluted in Opti-MEM (Gibco) was mixed with total senescence, we hypothesized that p53, in turn, affects DNA in a ratio of 1:4 (DNA/Hilymax) and applied to MEF expression. Here, we present evidence that p53 subconfluent cells. Small-interfering RNA (siRNA) for downregulates MEF expression. p53 overexpression or ac- p53 or E2F1 was transfected into cells using Trans-IT tivation of endogenous p53 repressed MEF levels, whereas TKO (Mirus, Madison, WI, USA) according to the manu- in the absence of p53 in human epithelial cells and mice facturer’s instruction. p53 or E2F1 siRNA duplex (50 nM) tissues, higher MEF expression level was observed. By complexed with Trans-IT TKO (1:4; siRNA/TKO ratio) investigating the mechanism of this downregulation, we was transfected into 70% confluent cells. GL2-luc siRNA found that p53 inhibits the promoter-binding activity of duplex was used as control. The cells were harvested 48 h E2F1, which we also show here as a novel transcriptional after transfection. The siRNA oligonucleotide sequences activator of MEF. Exogenous addition of E2F1 are listed in Table 1. upregulated MEF expression and promoter activity; con- versely, E2F1 knockdown reduced MEF transcription. Real-time quantitative reverse transcriptase polymerase Furthermore, p53 inhibited the DNA binding of E2F1 chain reaction analysis to MEF promoter by associating with E2F1, which led Total RNA was isolated from cells and mice tissues using to the suppression of MEF levels. These findings TRIzol (Invitrogen, Carlsbard, CA, USA) according to the describe the direct positive regulation of MEF by E2F1 manufacturer’s instruction. Real-time quantitative reverse and the suppression of MEF by p53. transcriptase polymerase chain reaction (RT–PCR) analyses for human or mouse MEF and internal controls MATERIALS AND METHODS GAPDH or 18S ribosomal RNA (18S rRNA) were carried out with SYBR Green Master Mix (Applied Biosystems, Reagents and antibodies Carlsbard, CA, USA) following the manufacturer’s in- Nutlin-3 was from Alexis Biochemicals (San Diego, CA, structions. PCR amplifications were performed as USA). Doxorubicin was from Sigma-Aldrich Co. described previously (17). The Ct values for each gene amp- (St Louis, MO, USA). Antibody for MEF was obtained lification were normalized by subtracting the Ct value from Transgenic Inc. (Kumamoto, Japan). Mouse calculated for GAPDH or 18S rRNA. The normalized anti-p53 (DO-1), rabbit anti-E2F1 (C-20), mouse IgG gene expression values were expressed as the relative (sc-2025), rabbit IgG (sc-2027) and g-tubulin (sc-7396) quantity of MEF gene-specific messenger RNA (mRNA). antibodies were purchased from Santa Cruz The oligonucleotide primers used in the real-time quantita- Biotechnology (Santa Cruz, CA, USA). The horseradish tive PCR amplifications are shown in Table 2. peroxidase (HRP)-conjugated secondary antibodies used in this study were from Jackson ImmunoResearch Plasmids and luciferase assay Laboratories, Inc. (West Grove, PA, USA). The cloning of MEF promoter constructs (–849/+181; Cell culture, treatment and transfection 384/+181; 204/+181 bp) in luciferase reporter vector, +/+ pGL3 basic vector, (pGL3b; Promega Corp. Madison, Human colorectal cancer cell line, HCT116 p53 and WI, USA) was described previously (12). MEF promoter HCT116 p53 cells were kindly provided by Dr. B. constructs containing mutation/s in E2F binding sites Vogelstein from Johns Hopkins University. These cells (MT1, MT2 and MT1&2) were prepared using were maintained in Dulbecco’s modified Eagle’s QuikChange II XL site-directed mutagenesis kit medium/Ham’s F-12 (DMEM/F12) medium supple- (Stratagene. La Jolla, CA, USA) following the recom- mented with 10% (v/v) fetal bovine serum (FBS), mended protocol. The primers used for generating MEF 100 IU/ml penicillin and 100 mg/ml streptomycin. Human mutant promoters are listed in Table 3. The p53 expres- bronchial epithelial cells, 16HBE14o-, were donated by sion plasmid was cloned in pCDM8 expression vector as Dr. D. Gruenert from the California Pacific Medical described in (18). The E2F1 expression plasmid (Addgene Center (San Francisco, CA, USA). 16HBE14o- cells plasmid 10736) was purchased from Addgene. were cultured in Minimum Essential medium (MEM) sup- For luciferase assays, HCT116 and HEK293 cells plemented with 10% FBS and antibiotics, and grown in seeded onto 12-well plates were transfected with 0.2 mg fibronectin-coated dishes. Lung adenocarcinoma, A549, and human embryonic kidney, HEK293, were maintained of MEF-luc promoter construct, together with 20 ng of in DMEM containing 10% FBS and antibiotics. Human Renilla luciferase plasmid (phRG-TK; Promega), p53, hepatoma cells, HepG2, were maintained in MEM supple- E2F1 expression plasmids and/or pcDNA3.1 empty mented with 10% FBS and antibiotics. A549, HEK293 vector (control). Luciferase activity was determined and HepG2 cell lines were obtained from American using a Dual-Luciferase Reporter assay system Type Culture Collection. All cell lines were cultured at (Promega) as described previously (19). 78 Nucleic Acids Research, 2011, Vol. 39, No. 1 Table 1. siRNA oligonucleotide sequences Gene Sense Antisense 0 0 0 0 p53 siRNA 5 -GACUCCAGUGGUAAUCUACTT-3 5 -GUAGAUUACCACUGGAGUCTT-3 0 0 0 0 E2F1 siRNA 5 -AAGUCACGCUAUGAGACCUCATT-3 5 -UGAGGUCUCAUAGCGUGACUUTT-3 0 0 0 0 GL2 siRNA 5 -CGUACGCGGAAUACUUCGATT-3 5 -UCGAAGUAUUCCGCGUACGTT-3 Table 2. Primers used for real-time quantitative RT-PCR Gene Sense Antisense 0 0 0 0 Human MEF 5 -TGGAAGGCAGTTTTTTGCTGA-3 5 -GACTTCCGCGGTTGACATG-3 0 0 0 0 Human GAPDH 5 -CGGGAAGCTTGTGATCAATGG-3 5 -GGCAGTGATGGCATGGACTG-3 0 0 0 0 Human 18S rRNA 5 -CGGCTACCACATCCAAGGAA-3 5 -GCTGGAATTACCGCGGCT-3 0 0 0 0 Mouse MEF 5 -TCCTGGATGAGAAGCAGATCTTCA-3 5 -ATGGTGCTGCCTTTGCCATC-3 0 0 0 0 Mouse 18S rRNA 5 -GTAACCCGTTGAACCCCATT-3 5 -CCATCCAATCGGTAGTAGCG-3 Table 3. Primers used for the generation of mutant MEF promoter constructs Gene Sequence 0 0 MEF prom MT1_sense 5 -GACCGGGCGCCCGTGGATCCTTCCACTTCTC-3 0 0 MEF prom MT1_antisense 5 -GAGAAGTGGAAGGATCCACGGGCGCCCGGTC-3 0 0 MEF prom MT2_sense 5 -CTTGCCATTGGCGGCACCTAGGGTGGGAGAGC-3 0 0 MEF prom MT2_antisense 5 -GCTCTCCCACCCTAGGTGCCGCCAATGGCAAG-3 Table 4. Primers used for ChIP assay Gene Sense Antisense 0 0 0 0 MEF promoter 5 -CTCGAGCCTCCAACTTCCCATTGG-3 5 -CTCGAGGCTCAACTTCCACTTCTCC-3 0 0 0 0 CDC6 promoter 5 -AAAGGCTCTGTGACTACAGCCA-3 5 -GATCCTTCTCACGTCTCTCACA-3 0 0 0 0 GAPDH promoter 5 -AAAAGCGGGGAGAAAGTAGG-3 5 -CTAGCCTCCCGGGTTTCTCT-3 and E2F1, we used nuclear extracts of control, transfected Chromatin immunoprecipitation assay or treated cells. After blocking, the membranes were probed with the appropriate antibodies, and blots were To determine E2F1 binding to MEF promoter, nuclear visualized using SuperSignal (PIERCE, Rockford, IL, extracts from HCT116 and HEK293 cells were used for USA). chromatin immunoprecipitation (ChIP) assay following the protocol described previously (20). Two micrograms Senescence induction and senescence-associated of anti-E2F1 antibody or normal rabbit IgG was b-galactosidase staining incubated with pre-cleared chromatin. Samples were analyzed by PCR using LA Taq Polymerase (TaKaRa) To induce DNA damage-associated cellular senescence, +/+ / according to the recommended protocol. The primers HCT116 p53 and p53 cells were treated with used (Table 4) recognize a fragment of the human MEF 100 nM doxorubicin (dox) for 24 h and re-incubated in promoter, CDC6 promoter or GAPDH promoter. normal medium for 3 days (for PCR analysis) or 5 days (for staining and protein analyses) according to the Immunoprecipitation and western blotting protocol reported previously (21). Cells were fixed with 2% formaldehyde/0.2% glutaraldehyde and stained for To analyze the interaction between p53 and E2F1, trans- senescence-associated b-galactosidase (SA-b-gal) activity fected or doxorubicin-treated HCT116 cells were lysed using 5-bromo-4-chloro-3-indolyl b-D-galactoside (X-gal) with nuclear extraction buffer as previously described in at pH 6.0 as described before (22). SA-b-gal-positive cells ref. 5. Nuclear extracts were incubated for 12 hr at 4 C were detected by bright-field microscopy. with 2 mg anti-p53 or anti-E2F1 antibodies or control IgG immobilized in protein G Sepharose beads (Amersham Animals Bioscience, Sweden). Immunoprecipitates were washed, eluted and subjected to immunoblotting, following essen- The p53 knockout (p53 ) mice were kindly provided by tially our protocol reported previously (20). Blots of IP Dr. Shin Aizawa from RIKEN (Kobe, Japan). The samples or input fraction were probed with anti-p53 p53-deficient mice were produced through an ordinary and anti-E2F1 antibodies or anti-g-tubulin antibody (for knockout (KO) strategy for p53 gene in C57BL/6 mice input fraction). For western blotting analysis of MEF, p53 as described earlier (23). RNA isolates from different Nucleic Acids Research, 2011, Vol. 39, No. 1 79 tissues of 12-week-old p53 mice and age-matched p53 suppresses MEF promoter activity controls were used for Q-PCR analyses. The mice used The effect of p53 on MEF expression was observed at the in this study were housed in a vivarium in accordance mRNA level; thus, we focused on MEF promoter to as- with the guidelines of the animal facility center of certain how p53 downregulates MEF. To establish the Kumamoto University. The animals were fed with chow influence of p53 on MEF promoter activity, we ad libitum. All experiments were performed according to co-transfected in HCT116 p53 cells increasing the protocols approved by the Animal Welfare Committee amounts of p53 expression plasmid and MEF promoter of Kumamoto University (#A19-115). (–849/+181 bp). We found that increased levels of p53 caused a concomitant decline in MEF promoter activity Statistical analysis (Figure 3A). Next, to identify the region of MEF For statistical analysis, the data were analyzed by promoter that is important for its response to p53, we Student’s t-test or by one-way analysis of variance used three promoter constructs with varying lengths, (ANOVA) with Tukey–Kramer multiple comparison test labeled: 800 bp (–849/+181), 400 bp (–384/+181) and (JMP software, SAS Institute, NC, USA) as indicated in 200 bp (–204/+181) (Figure 3B). These constructs and each figure legend. A P-value of <0.05 is considered stat- p53 expression plasmid were co-transfected in p53 istically significant. cells. Reporter assays revealed that activity for all three constructs was downregulated by p53, suggesting that p53 suppresses MEF transcriptional activity by affecting at RESULTS least the 200 bp proximal region of MEF promoter p53 negatively regulates MEF expression in human cell (Figure 3B). However, in silico analysis of MEF lines and mice tissues promoter did not reveal any p53 response element within the 800 bp region upstream from the transcription MEF has been implicated in the suppression of p53 ex- start site. Intriguingly, we found E2F consensus binding pression (16), but it is unknown whether this regulation is sites in this promoter region (Figure 3C). Because it was reciprocal. To address this question, we first compared the previously demonstrated that p53 physically interacts with +/+ / basal level of MEF mRNA in HCT116 p53 and p53 and inhibits the transcriptional activity of E2F1 (24), we cells. Interestingly, quantification of mRNA showed that hypothesized that p53 indirectly suppresses MEF tran- +/+ the amount of MEF in HCT116 p53 cells was 5-fold / scription by affecting E2F1, which could be a novel tran- lower than in p53 cells (Figure 1A). Conventional PCR scriptional activator of MEF. To assess this possibility, we analysis also showed lower expression of MEF in HCT116 +/+ / first investigated the effect of E2F1 on MEF expression p53 cells than in p53 cells (Supplementary Figure and promoter activity. S1A). Consistent with these observations, transfection of p53 plasmid in HCT116 p53 cells downregulated MEF E2F1 is a novel MEF transcriptional activator mRNA level (Figure 1B and Supplementary Figure S1B). Introduction of E2F1 in HCT116 p53 cells resulted in Treatment with nutlin-3, a specific activator of p53, dose-dependent increase of MEF mRNA level as resulted in a drop in MEF mRNA expression level in +/+ / determined by Q-PCR (Figure 4A). Similarly, the p53 cells but not in p53 cells (Figure 1C). relative amount of MEF mRNA was upregulated by Treatment of HepG2 cells with nutlin-3 also decreased overexpression of E2F1 in other cell lines tested MEF mRNA level (Figure 1D). Conversely, knockdown (Figure 4B–E). In contrast to E2F1 overexpression, of p53 by siRNA in A549, 16HBE14o- and HEK293 siRNA targeting E2F1 induced a drop in basal MEF cells up–regulated the mRNA expression of MEF (Figure 1E–G). These observations implied that p53 mRNA level in human epithelial cells (Figure 4F–H). suppresses MEF transcription. To assess whether this The upregulation of MEF mRNA by E2F1 translated to decrease also occurs at the protein level, we examined an increase in MEF protein level as determined by western +/+ MEF expression in nuclear extracts of HCT116 p53 blotting of nuclear extracts of cells transfected with E2F1 and p53 cells. Basal MEF protein level in HCT116 (Figure 4I and J). +/+ / p53 cells was lower than in p53 cells (Figure 1H). To analyze the effect of E2F1 on MEF transactivation, Exogenous addition of p53 in HCT116 p53 cells we co-transfected increasing amounts of E2F1 with MEF decreased the expression of MEF (Figure 1I). Moreover, promoter (–849/+181 bp) in HCT116 p53 cells. E2F1 activation of endogenous p53 by nutlin-3, reduced MEF clearly stimulated MEF transcriptional activity in a +/+ expression in HCT116 p53 cells but had no effect on dose-dependent manner (Figure 5A). To define the MEF in p53 cells (Figure 1J). Knockdown of p53 by region of MEF promoter that is controlled by E2F1, we si-RNA in A549 and 16HBE14o- cells increased MEF assessed the effect of E2F1 on three different lengths of protein expression (Figure 1K and L). In addition, by MEF promoter construct [as used above (–800 bp, using tissues of p53 mice, we confirmed the effect of 400 bp and 200 bp)]. These constructs were transfected p53 on MEF mRNA expression in vivo. Consistent with in HCT116 p53 cells together with E2F1 expression the results in human cell lines, MEF mRNA level was vector. Reporter assays revealed that the minimal region higher in various tissues of p53-deficient mice than in of MEF that is activated by E2F1 is at 200 bp upstream those of p53 wild-type mice (Figure 2A–F). These data from transcription start site (Figure 5B). The 200 bp collectively indicated that p53 downregulates MEF proximal region contains two E2F binding sites expression. (Figure 3C). Mutation of the E2F site nearest to the 80 Nucleic Acids Research, 2011, Vol. 39, No. 1 +/+ Figure 1. p53 downregulates MEF expression in human cell lines. (A) Relative amount of MEF mRNA was examined in HCT116 p53 and / / p53 cells. (B) HCT116 p53 cells were transfected with control pcDNA3.1 empty vector or p53 plasmid. Forty-eight hours post-transfection, +/+ / MEF mRNA level was analyzed. (C) HCT116 p53 and p53 cells were untreated or treated with nutlin-3 for 24 h. Total RNA was isolated for analysis. (D) HepG2 cells were treated with 20 mM nutlin-3 for 48 h then total RNA was recovered. (E–G) A549, 16HBE14o- and HEK293 cells were transfected with si-GL2 (control) or si-p53 as described in ‘Materials and Methods’ section. Total RNA was isolated and analyzed for MEF mRNA. For (A–G) MEF mRNA level, assessed by quantitative RT-PCR, was normalized to GAPDH or 18S rRNA, which served as internal controls. Values are mean ± SD of triplicate measurements. *P< 0.05; **P< 0.01 versus control cells, assessed by Student’s t-test or ANOVA with Tukey– +/+ / Kramer test (for (C)). (H) Endogenous MEF and p53 protein expressions in nuclear extracts of HCT116 p53 and p53 cells were examined by +/+ / / western blotting. (I) HCT116 p53 and p53 cells were transfected with control pcDNA3.1 empty vector, or p53 plasmid for p53 cells. +/+ / Forty-eight hours post-transfection, nuclear extracts were isolated. (J) HCT116 p53 and p53 cells were untreated or treated with Nutlin-3 (5 mM) for 24 h, then the nuclear extracts were isolated. (K and L) A549 and 16HBE cells were transfected with si-GL2 (control) or si-p53. Forty-eight hours after transfection, nuclear extracts were isolated. For (H–L), protein levels of MEF and p53 were analyzed by western blotting. g-tubulin was used as internal control in these experiments. start site (MT1) abrogated the basal transcriptional of both sites (MT1&2) diminished the basal promoter activity of MEF compared to wild-type promoter while activity of MEF in comparison with wild-type to a level mutation of the second E2F site (MT2) did not suppress similar to that of MT1 (Figure 5C), indicating that E2F the basal transactivation of MEF (Figure 5C). Mutation binding site 1 in the 200 bp proximal region is important Nucleic Acids Research, 2011, Vol. 39, No. 1 81 / +/+ / Figure 2. MEF mRNA level is upregulated in p53 mice tissues. (A–F) Total RNA isolated from the indicated tissues of p53 and p53 mice was analyzed for the expression of MEF by real-time quantitative RT–PCR. MEF mRNA level was normalized to mouse 18S rRNA (internal control). Results represent mean ± SD (n = 3). *P< 0.05, **P< 0.01 against p53 wild-type mice assessed by Student’s t-test. for efficient up-regulation of MEF basal transcription. by increasing amounts of co-transfected p53 (Figure 6A The residual activity that was not suppressed by the and B). To verify the physical interaction between p53 and mutation of E2F site could be attributed to the effect of E2F1, we performed immunoprecipitation (IP) using Sp1, which we previously showed as a basal transcription- nuclear extracts of HCT116 p53 transfected with p53 al activator of MEF that binds to 91/–82 and 63/– and E2F1. Consistent with previous reports (24,26), we 54 bp in MEF promoter (12). To establish the association confirmed that p53 associates with E2F1 (Figure 6C). of E2F1 on MEF promoter, we analyzed E2F1 recruit- To assess the functional consequence of this interaction, ment to MEF promoter by ChIP assay of nuclear lysates we studied its effect on E2F1 binding on MEF promoter. / / from HCT116 p53 cells transfected with E2F1 or As expected, addition of p53 in HCT116 p53 cells sub- pcDNA3.1 using ChIP primers that recognize MEF stantially lessened the steady-state association of E2F1 on promoter region at 204/+181 bp. Immunoprecipitation MEF promoter region as detected by ChIP analysis of with E2F1 antibody but not with control IgG and subse- nuclear extracts (Figure 6D, left). Similar effect of p53 quent PCR reactions revealed the recruitment of endogen- was also observed on CDC6 promoter (Figure 6D, ous and overexpressed E2F1 to the promoter region of right). Because the expression level of E2F1 is relatively MEF (Figure 5D). In addition, we verified the binding similar in cells with or without p53 (Figure 6E), we ruled of endogenous E2F1 to MEF promoter in HEK293 cells out the possibility that the lack of E2F1 binding to pro- (Figure 5E). Taken together, we substantiated that E2F1 moters in the presence of p53 was due to reduced level of is a novel MEF transcriptional activator that binds to E2F E2F1. Collectively, these data indicated that p53 consensus site in MEF proximal promoter region. downregulates MEF transcription by diminishing the recruitment of E2F1 to MEF promoter. p53 inhibits E2F1 binding to MEF promoter Cellular senescence-induced p53 activation downregulates It has been demonstrated previously that the association MEF expression via inhibition of E2F1 binding to MEF of p53 with E2F1 blocks the ability of E2F1 to bind to promoter DNA and transactivate gene expression (25). Having shown that E2F1 binds and activates MEF promoter, We next asked whether the regulation of MEF by p53 via we next assessed whether p53 abrogates these effects. E2F1 occurs under physiologically relevant setting. Given Congruent with the above observations, E2F1 significant- that MEF has been linked to p53 in the context of cellular ly stimulated MEF transcriptional activity when E2F1 senescence (16) and considering our data above, we looked was co-transfected with MEF promoter (–204/+181 bp) into the possibility that senescence-activated p53 can in HCT116 p53 and HEK293 cells (Figure 6A and suppress the expression of MEF. We induced cellular sen- B). However, this positive regulation was titrated away escence by treating cells with DNA-damaging reagent 82 Nucleic Acids Research, 2011, Vol. 39, No. 1 Figure 3. p53 suppresses MEF promoter activity. (A) HCT116 p53 cells were transiently transfected with MEF (–849/+181 bp) promoter con- struct or pGL3b vector (0.2 mg) and the indicated amount of p53 plasmid or pcDNA3.1 empty vector. Luciferase activity was determined 48 h after transfection of plasmids and is expressed as fold activation over the pGL3b vector. Values are the mean ± SE of triplicate platings. ***P< 0.001 versus pcDNA3.1, determined by ANOVA with Tukey–Kramer test. n.s, not significant. (B) HCT116 p53 cells were transiently transfected with the indicated MEF promoter constructs (0.2 mg) and p53 plasmid (0.1 mg) or pcDNA3.1 empty vector (as control). Luciferase activity was determined 48 h after transfection of plasmids and is expressed as fold activation over the pcDNA3.1 vector (con) in each promoter construct. Values are mean ± SE of triplicate platings. **P< 0.01; ***P< 0.001 versus control, assessed by Student’s t-test. (C) The –849 bp 5 -flanking region of MEF. Nucleotide sequence of 5 -flanking region of human MEF gene is shown. The site indicated (+1) denotes the start site of the first exon. The predicted binding sites for E2F1 are marked on the sequence. Nucleic Acids Research, 2011, Vol. 39, No. 1 83 Figure 4. E2F1 upregulates MEF expression in human cell lines. (A–E) Cells were transiently transfected with pcDNA3.1 vector (con) or the indicated amount of E2F1 (A) or 1.0 mg E2F1 (B–E). Forty-eight hours after transfection, total RNA was extracted and analyzed for MEF mRNA expression. (F–H) si-GL2 or si-E2F1 (50 nM) was transfected into the indicated cell lines and MEF mRNA expression was assessed 48 h post-transfection. For (A–H) MEF mRNA level, determined by quantitative RT–PCR, was normalized to GAPDH or 18S rRNA (internal control) and expressed as relative amount of mRNA. Values are mean ± SD of triplicate measurements. *P< 0.05; **P< 0.01; ***P< 0.001 versus control, assessed by ANOVA with Tukey–Kramer (A) or Student’s t-test (B–H). (I–J) MEF and E2F1 protein expressions were examined by western blotting in nuclear extracts of HCT116 p53 (I) or 16HBE14o- cells (J) transiently transfected with E2F1 or pcDNA3.1 empty vector. g-tubulin was used as internal control. +/+ doxorubicin (dox). Treatment with moderate dose p53 cells was also downregulated upon senescence (100 nM) of dox for 24 h and additional incubation for induction (Figure 7C). Next, we assessed the associ- 5 days was reported to induce cellular senescence in ation between E2F1 and p53 during cellular senescence HCT116 wild-type cells (21). Intense SA-b-gal staining by performing IP analysis. IP using antibody specific +/+ was observed in HCT116 p53 cells while faint to E2F1 and probing with p53 antibody revealed that staining was seen in p53 cells (Figure 7A), consistent cellular senescence augmented the physical interaction with the results obtained by Chang et al. (21). between E2F1 and p53 (Figure 7D). Notably, we Interestingly, under senescent condition, concomitant detected by ChIP assay that steady-state binding of with an enhanced level of p53, MEF protein expression E2F1 to MEF promoter was abolished in HCT116 +/+ +/+ was reduced compared with control in HCT116 p53 p53 senescent cells (Figure 7E). Taken together, these cells (Figure 7B). On the other hand, dox treatment did results suggested that under cellular senescence condition, not suppress MEF in HCT116 p53 cells (Figure 7B), activated p53 downregulates MEF expression by arguing for p53-dependency of MEF downregulation associating with E2F1 and inhibiting E2F1 binding to during senescence. MEF mRNA expression in HCT116 MEF promoter. 84 Nucleic Acids Research, 2011, Vol. 39, No. 1 Figure 5. E2F1 increases MEF promoter activity by binding to E2F site. (A) HCT116 p53 cells were transiently transfected with MEF (–849/ +181 bp) promoter construct or pGL3b vector (0.2 mg) and the indicated amounts of E2F1 plasmid or pcDNA3.1 empty vector. Luciferase activity was determined 48 h after transfection and is expressed as fold activation over the pGL3b vector. Values are the mean ± SE of triplicate platings. yyy / P< 0.001 versus pGL3b; ***P< 0.001 versus pcDNA3.1, determined by ANOVA with Tukey–Kramer. (B) HCT116 p53 cells were transiently transfected with the indicated MEF promoter constructs (0.2 mg) and E2F1 plasmid or pcDNA3.1 vector (0.1 mg). Luciferase activity was determined 48 h after transfection and is expressed as fold activation over the pcDNA3.1 vector in each promoter construct. Values are mean ± SE of triplicate platings. ***P< 0.001 versus control, assessed by Student’s t-test. (C) HCT116 p53 cells were transfected with 0.2 mg pGL3b vector or MEF (– 200/+181 bp) promoter wild-type (WT), MT1, MT2 or MT1&2. (Left) MT1, MT2 represent MEF (–200/+181 bp) promoter containing mutated E2F site. Luciferase activity was determined 48 h after transfection and is expressed as fold activation over pGL3b vector. Values are mean ± SE of triplicate platings. ***P< 0.001 versus WT, assessed by ANOVA with Tukey–Kramer test. (D) E2F1 binding on MEF promoter was determined by ChIP assay using nuclear extracts of HCT116 p53 cells transiently transfected with E2F1 or pcDNA3.1 vector (1.0 mg). CDC6 promoter was used as positive control and GAPDH promoter was used as negative control for E2F1 binding. Upper panel illustrates the MEF promoter region (– 204 bp/+181 bp) in which binding was assessed. (E) Nuclear extract from HEK293 cells was used to assess the endogenous binding of E2F1 on MEF promoter. CDC6 and GAPDH promoters were used as positive and negative controls, respectively. DISCUSSION in MEF promoter while p53 antagonizes this positive interaction by associating with E2F1 and reducing its We have identified a dual, opposing transcriptional regu- lation mechanism of MEF by E2F1 and p53. E2F1 acti- DNA-binding activity. The extensive crosstalk between vates MEF transcription by binding to E2F consensus site p53 and E2F pathways is well documented (27). It was Nucleic Acids Research, 2011, Vol. 39, No. 1 85 Figure 6. p53 inhibits E2F1 binding to MEF promoter and reduces E2F1-induced MEF promoter activation. (A and B) HCT116 p53 and HEK293 cells were transiently transfected with MEF (–204/+181 bp) promoter construct or pGL3b vector (0.2 mg), pcDNA3.1 or E2F1 (0.1 mg) and the indicated amounts of p53 plasmid. Luciferase activity was determined 48 h after transfection and is expressed as fold activation over the pGL3b yyy ### vector. Values are the mean ± SE of triplicate platings. P< 0.001 versus pGL3b; P< 0.001 versus pcDNA3.1; *P< 0.05; ***P< 0.001 versus E2F1, determined by ANOVA with Tukey–Kramer. (C) HCT116 p53 cells were transiently transfected with p53 and E2F1 plasmids. Nuclear extracts were analyzed for p53-E2F1 association by IP using antibody specific to p53, E2F1 or control mouse IgG. Immunoprecipitates were loaded onto SDS–PAGE gel, blotted and probed with E2F1 or p53 antibodies. (D) HCT116 p53 cells were transiently transfected with E2F1 and p53 plasmid or pcDNA3.1 empty vector. Nuclear extracts were isolated and used for ChIP assay to determine E2F1 binding on MEF promoter and CDC6 promoter in the presence or absence of p53. (E) Endogenous protein level of E2F1 was examined in nuclear extracts of HCT116 +/+ / p53 and p53 cells. g-Tubulin was used as internal control. described earlier by O’Connor et al. (24) that E2F1 and gene that regulates the early steps of DNA replication p53 reciprocally inhibit each other’s transcriptional (28–30), raising the possibility that p53 interferes with activity through their physical association, probably the transcription of genes other than MEF that are forming a protein–protein complex and thereby prevent- targeted by E2F1. Being two pivotal regulators of cell ing DNA binding. E2F1 and p53 are also known to proliferation, the functional interaction between E2F1 control genes that influence cell cycle progression and p53 most likely affects cell fate. (24,27). Indeed, we found here that their interaction Sashida et al. (16) has demonstrated that MEF de- (Figures 6C and 7D) affects the regulation of MEF, a creases p53 protein stability by inducing the transcription molecule that is involved in the transition of cells from of MDM2, and MEF also downregulates Rb, an endogen- G1 to S phase (7) and in driving quiescent hematopoietic ous inhibitor of E2F1. Because the loss of MEF substan- stem cells to G1 phase (14). We also noted that exogenous tially enhanced the senescent phenotype of murine addition of p53 slightly lessened the recruitment of E2F1 embryonic fibroblasts and activated the p53 function, it to the promoter of CDC6 (Figure 6D), an E2F1 target was evident that MEF inhibits cellular senescence by 86 Nucleic Acids Research, 2011, Vol. 39, No. 1 +/+ / Figure 7. p53 suppresses MEF expression during cellular senescence. (A and B) HCT116 p53 and p53 cells were treated with doxorubicin (Dox) for 24 h and incubated for 5 days to induce cellular senescence. Cells were fixed and stained with X-gal to detect SA-b-gal activity. Stained cells were photographed at phase contrast with 20-fold magnification (Scale bar, 100 mm) (A). The nuclear extracts from Dox-treated or untreated cells +/+ were subjected to immunoblotting using the indicated antibodies. g-tubulin was used as internal control (B). (C) HCT116 p53 cells were untreated or treated with Dox for 24 h and incubated for 3 days. Total RNA was recovered and MEF mRNA expression was examined by Q-PCR. Amount of MEF mRNA was normalized to GAPDH and expressed as relative amount compared to control cells. Values are mean ± SD of triplicate platings. +/+ **P< 0.01, determined by Student’s t-test. (D) Cellular senescence was induced in HCT116 p53 cells by doxorubicin treatment similar to (A). p53 binding to E2F1 was examined by immunoprecipitation of nuclear extracts. (E) E2F1 binding on MEF promoter was examined by ChIP assay using +/+ nuclear extracts of control or Dox-induced senescent HCT116 p53 cells. (F) Negative regulatory mechanism between p53 and MEF. (Left) DNA damage stimulates p53 that enhances its binding to E2F1. This leads to reduced recruitment of E2F1 to the MEF promoter and the suppression of MEF transcription. (Right) MEF upregulates MDM2 (described in ref. 16), which inhibits p53. suppressing p53 pathway (16) (Figure 7F, right diagram). combined with our data here showing that MEF mRNA However, whether this occurs at steady state or induced and protein levels were suppressed by p53 implies that a by pathogenic/pathological condition is still unclear (16). negative regulatory mechanism exists between MEF and The previous study showing that MEF downregulates p53 p53 pathways. We propose that during DNA damage, Nucleic Acids Research, 2011, Vol. 39, No. 1 87 p53 is activated and interacts with E2F1, which minimizes now shown that p53 reciprocally opposes MEF transactivation. E2F1 recruitment to MEF promoter (Figure 7F, left diagram). The lessening of MEF levels may partly contrib- ute to the modulation of cell cycle progression. We SUPPLEMENTARY DATA showed here that p53 affects MEF transcription indirectly, but the possibility that p53 can also directly affect MEF at Supplementary Data are available at NAR online. the post-translational level cannot be fully ruled out. Until now, the regulation of MEF protein stability mediated by cyclin A is the only known molecular event ACKNOWLEDGEMENTS by which MEF is controlled especially during cell cycle +/+ / HCT116 p53 and HCT116 p53 cell lines were (7). Our data here indicated that MEF transcription is provided by Dr. Bert Vogelstein (Johns Hopkins also influenced by E2F1, an activator E2F family University). 16HBE14o- cells were donated by member that affects the cell cycle (31,32). Thus, mechan- Dr. Dieter C. Gruenert (California Pacific Medical isms of controlling MEF exist at both transcriptional and Center). Wild-type p53 plasmid was supplied by post-translational levels. Because E2F1 binds to MEF Dr. Hideyuki Saya (Keio University). p53 mice were promoter and mutation of E2F binding site inhibited made available by Dr. Shin Aizawa (Laboratory for basal MEF promoter activity, MEF may be considered Animal Resources and Genetic Engineering, Center for a target of E2F1 transcription factor. While it is possible Developmental Biology, RIKEN). that E2F2 and E2F3, which are also trans-activating E2Fs, can affect MEF, we found that specific silencing of E2F1 efficiently suppressed endogenous MEF mRNA FUNDING level, suggesting a considerable specificity of E2F1 where transactivation is concerned. However, although we The Ministry of Education, Science, Sports and Culture observed that MEF transcription was reduced by p53 at (MEXT) of Japan (Grant # 19390045); and the Global the organismal level (Figure 2), the effect of E2F1 on COE Program (Cell Fate Regulation Research and MEF in vivo still awaits verification. As far as we know, Education Unit), MEXT, Japan. Funding for open MEF is the first member of the Ets transcription factor access charge: The Ministry of Education, Science, family to be identified as a direct E2F target gene despite Sports and Culture of Japan. the fact that Ets transcription factors are notable for their Conflict of interest statement. None declared. roles in cellular growth and proliferation (1). It might not be surprising that future research efforts will unveil direct molecular links between members of E2F and Ets families. REFERENCES Interestingly, it was reported that p53 downregulates Ets1 and Ets2 at transcriptional level, although the molecular 1. Sharrocks,A.D. (2001) The ETS-domain transcription factor family. Nat. Rev. Mol. Cell Biol., 2, 827–837. mechanism for this downregulation has not been 2. Miyazaki,Y., Sun,X., Uchida,H., Zhang,J. and Nimer,S. (1996) elucidated (33). As our transcription factor search MEF, a novel transcription factor with an Elf-1 like DNA yielded a few E2F consensus sites in their promoter binding domain but distinct transcriptional activating properties. regions (data not shown), E2F might participate in the Oncogene, 13, 1721–1729. 3. Hedvat,C.V., Yao,J., Sokolic,R.A. and Nimer,S.D. 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MEF/ELF4 transactivation by E2F1 is inhibited by p53

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Abstract

76–88 Nucleic Acids Research, 2011, Vol. 39, No. 1 Published online 30 August 2010 doi:10.1093/nar/gkq762 MEF/ELF4 transactivation by E2F1 is inhibited by p53 1,2 1 1 1 Manabu Taura , Mary Ann Suico , Ryosuke Fukuda , Tomoaki Koga , 1 1 1 2 1, Tsuyoshi Shuto , Takashi Sato , Saori Morino-Koga , Seiji Okada and Hirofumi Kai * Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Global COE ‘Cell Fate Regulation Research and Education Unit’, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973 and Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan Received March 14, 2010; Revised July 4, 2010; Accepted August 10, 2010 INTRODUCTION ABSTRACT MEF/ELF4 is a member of the E-twenty -six (ETS) family Myeloid elf-1-like factor (MEF) or Elf4 is an of transcription factors, which function as transcriptional E-twenty-six (ETS)-related transcription factor with activators or repressors and regulate critical aspects of strong transcriptional activity that influences cellular differentiation, proliferation and transform- cellular senescence by affecting tumor suppressor ation (1). MEF was originally isolated from human p53. MEF downregulates p53 expression and megakaryocytic leukemia cell line, and is known to inhibits p53-mediated cellular senescence by tran- activate the expression of a variety of cytokine genes, such as interleukin (IL)-3 and IL-8 (2,3) and antibacterial scriptionally activating MDM2. However, whether peptides, such as lysozyme and human b-defensin and the p53 reciprocally opposes MEF remains unex- cytolytic molecule perforin (4–6). MEF expression and plored. Here, we show that MEF is modulated by activity are regulated by its post-translational modifica- p53 in human cells and mice tissues. MEF expres- tion, protein–protein interaction and by transcription. sion and promoter activity were suppressed by p53. MEF expression is highest at G1 phase; and at G1 to While we found that MEF promoter does not contain S-phase transition, MEF is phosphorylated by cyclinA– p53 response elements, intriguingly, it contains E2F skp2 cdk2 complex, ubiquitinated by SCF and degraded consensus sites. Subsequently, we determined that by proteasome (7,8). SUMOylation of MEF inhibits its E2F1 specifically binds to MEF promoter and transcriptional activity (9), whereas translocation of transactivates MEF. Nevertheless, E2F1 DNA MEF into promyelocytic leukemia (PML) nuclear bodies binding and transactivation of MEF promoter was induces interaction with PML and increases MEF tran- scriptional activator function (10,11). Sp1 was previously inhibited by p53 through the association between determined to positively influence the transcription of p53 and E2F1. Furthermore, we showed that activa- MEF (12). Epigenetic regulation, promoter methylation tion of p53 in doxorubicin-induced senescent cells and histone deacetylation mediate MEF gene silencing increased E2F1 and p53 interaction, diminished (our unpublished data) (13). E2F1 recruitment to MEF promoter and reduced Besides its function as an activator of cytokines and MEF expression. These observations suggest that innate immune molecules, MEF also impacts on cell-cycle p53 down-regulates MEF by associating with and progression by promoting the transition of cells from G inhibiting the binding activity of E2F1, a novel to S phase (7). The loss of MEF was shown to increase transcriptional activator of MEF. Together with tumor suppressor p53 protein and enhance hematopoietic previous findings, our present results indicate that stem cell (HSC) quiescence in murine embryonic fibro- a negative regulatory mechanism exists between blasts, implicating MEF in driving HSC from quiescence to G phase by opposing p53 function (14,15). A study p53 and MEF. *To whom correspondence should be addressed. Tel/Fax: +81 96 371 4405; Email: hirokai@gpo.kumamoto-u.ac.jp The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2011, Vol. 39, No. 1 77 previously demonstrated that MEF upregulates the tran- 37 C in a humidified atmosphere of 5% CO . Treatment scription of MDM2, the E3 ubiquitin ligase of p53, of cells with nutlin-3 or DMSO (control) was carried out thereby suppressing p53 protein stability that led to the for 24 h (HCT116 cells) or 48 h (HepG2 cells). Transient inhibition of p53-dependent oncogene-induced cellular transfections of plasmids were performed using Hilymax senescence (16). Considering that MEF contributes to (Dojindo Laboratories, Kumamoto, Japan) following the driving cell-cycle progression and that MEF suppresses manufacturer’s recommendation. Specifically, Hilymax p53, which is known for promoting cell-cycle arrest and diluted in Opti-MEM (Gibco) was mixed with total senescence, we hypothesized that p53, in turn, affects DNA in a ratio of 1:4 (DNA/Hilymax) and applied to MEF expression. Here, we present evidence that p53 subconfluent cells. Small-interfering RNA (siRNA) for downregulates MEF expression. p53 overexpression or ac- p53 or E2F1 was transfected into cells using Trans-IT tivation of endogenous p53 repressed MEF levels, whereas TKO (Mirus, Madison, WI, USA) according to the manu- in the absence of p53 in human epithelial cells and mice facturer’s instruction. p53 or E2F1 siRNA duplex (50 nM) tissues, higher MEF expression level was observed. By complexed with Trans-IT TKO (1:4; siRNA/TKO ratio) investigating the mechanism of this downregulation, we was transfected into 70% confluent cells. GL2-luc siRNA found that p53 inhibits the promoter-binding activity of duplex was used as control. The cells were harvested 48 h E2F1, which we also show here as a novel transcriptional after transfection. The siRNA oligonucleotide sequences activator of MEF. Exogenous addition of E2F1 are listed in Table 1. upregulated MEF expression and promoter activity; con- versely, E2F1 knockdown reduced MEF transcription. Real-time quantitative reverse transcriptase polymerase Furthermore, p53 inhibited the DNA binding of E2F1 chain reaction analysis to MEF promoter by associating with E2F1, which led Total RNA was isolated from cells and mice tissues using to the suppression of MEF levels. These findings TRIzol (Invitrogen, Carlsbard, CA, USA) according to the describe the direct positive regulation of MEF by E2F1 manufacturer’s instruction. Real-time quantitative reverse and the suppression of MEF by p53. transcriptase polymerase chain reaction (RT–PCR) analyses for human or mouse MEF and internal controls MATERIALS AND METHODS GAPDH or 18S ribosomal RNA (18S rRNA) were carried out with SYBR Green Master Mix (Applied Biosystems, Reagents and antibodies Carlsbard, CA, USA) following the manufacturer’s in- Nutlin-3 was from Alexis Biochemicals (San Diego, CA, structions. PCR amplifications were performed as USA). Doxorubicin was from Sigma-Aldrich Co. described previously (17). The Ct values for each gene amp- (St Louis, MO, USA). Antibody for MEF was obtained lification were normalized by subtracting the Ct value from Transgenic Inc. (Kumamoto, Japan). Mouse calculated for GAPDH or 18S rRNA. The normalized anti-p53 (DO-1), rabbit anti-E2F1 (C-20), mouse IgG gene expression values were expressed as the relative (sc-2025), rabbit IgG (sc-2027) and g-tubulin (sc-7396) quantity of MEF gene-specific messenger RNA (mRNA). antibodies were purchased from Santa Cruz The oligonucleotide primers used in the real-time quantita- Biotechnology (Santa Cruz, CA, USA). The horseradish tive PCR amplifications are shown in Table 2. peroxidase (HRP)-conjugated secondary antibodies used in this study were from Jackson ImmunoResearch Plasmids and luciferase assay Laboratories, Inc. (West Grove, PA, USA). The cloning of MEF promoter constructs (–849/+181; Cell culture, treatment and transfection 384/+181; 204/+181 bp) in luciferase reporter vector, +/+ pGL3 basic vector, (pGL3b; Promega Corp. Madison, Human colorectal cancer cell line, HCT116 p53 and WI, USA) was described previously (12). MEF promoter HCT116 p53 cells were kindly provided by Dr. B. constructs containing mutation/s in E2F binding sites Vogelstein from Johns Hopkins University. These cells (MT1, MT2 and MT1&2) were prepared using were maintained in Dulbecco’s modified Eagle’s QuikChange II XL site-directed mutagenesis kit medium/Ham’s F-12 (DMEM/F12) medium supple- (Stratagene. La Jolla, CA, USA) following the recom- mented with 10% (v/v) fetal bovine serum (FBS), mended protocol. The primers used for generating MEF 100 IU/ml penicillin and 100 mg/ml streptomycin. Human mutant promoters are listed in Table 3. The p53 expres- bronchial epithelial cells, 16HBE14o-, were donated by sion plasmid was cloned in pCDM8 expression vector as Dr. D. Gruenert from the California Pacific Medical described in (18). The E2F1 expression plasmid (Addgene Center (San Francisco, CA, USA). 16HBE14o- cells plasmid 10736) was purchased from Addgene. were cultured in Minimum Essential medium (MEM) sup- For luciferase assays, HCT116 and HEK293 cells plemented with 10% FBS and antibiotics, and grown in seeded onto 12-well plates were transfected with 0.2 mg fibronectin-coated dishes. Lung adenocarcinoma, A549, and human embryonic kidney, HEK293, were maintained of MEF-luc promoter construct, together with 20 ng of in DMEM containing 10% FBS and antibiotics. Human Renilla luciferase plasmid (phRG-TK; Promega), p53, hepatoma cells, HepG2, were maintained in MEM supple- E2F1 expression plasmids and/or pcDNA3.1 empty mented with 10% FBS and antibiotics. A549, HEK293 vector (control). Luciferase activity was determined and HepG2 cell lines were obtained from American using a Dual-Luciferase Reporter assay system Type Culture Collection. All cell lines were cultured at (Promega) as described previously (19). 78 Nucleic Acids Research, 2011, Vol. 39, No. 1 Table 1. siRNA oligonucleotide sequences Gene Sense Antisense 0 0 0 0 p53 siRNA 5 -GACUCCAGUGGUAAUCUACTT-3 5 -GUAGAUUACCACUGGAGUCTT-3 0 0 0 0 E2F1 siRNA 5 -AAGUCACGCUAUGAGACCUCATT-3 5 -UGAGGUCUCAUAGCGUGACUUTT-3 0 0 0 0 GL2 siRNA 5 -CGUACGCGGAAUACUUCGATT-3 5 -UCGAAGUAUUCCGCGUACGTT-3 Table 2. Primers used for real-time quantitative RT-PCR Gene Sense Antisense 0 0 0 0 Human MEF 5 -TGGAAGGCAGTTTTTTGCTGA-3 5 -GACTTCCGCGGTTGACATG-3 0 0 0 0 Human GAPDH 5 -CGGGAAGCTTGTGATCAATGG-3 5 -GGCAGTGATGGCATGGACTG-3 0 0 0 0 Human 18S rRNA 5 -CGGCTACCACATCCAAGGAA-3 5 -GCTGGAATTACCGCGGCT-3 0 0 0 0 Mouse MEF 5 -TCCTGGATGAGAAGCAGATCTTCA-3 5 -ATGGTGCTGCCTTTGCCATC-3 0 0 0 0 Mouse 18S rRNA 5 -GTAACCCGTTGAACCCCATT-3 5 -CCATCCAATCGGTAGTAGCG-3 Table 3. Primers used for the generation of mutant MEF promoter constructs Gene Sequence 0 0 MEF prom MT1_sense 5 -GACCGGGCGCCCGTGGATCCTTCCACTTCTC-3 0 0 MEF prom MT1_antisense 5 -GAGAAGTGGAAGGATCCACGGGCGCCCGGTC-3 0 0 MEF prom MT2_sense 5 -CTTGCCATTGGCGGCACCTAGGGTGGGAGAGC-3 0 0 MEF prom MT2_antisense 5 -GCTCTCCCACCCTAGGTGCCGCCAATGGCAAG-3 Table 4. Primers used for ChIP assay Gene Sense Antisense 0 0 0 0 MEF promoter 5 -CTCGAGCCTCCAACTTCCCATTGG-3 5 -CTCGAGGCTCAACTTCCACTTCTCC-3 0 0 0 0 CDC6 promoter 5 -AAAGGCTCTGTGACTACAGCCA-3 5 -GATCCTTCTCACGTCTCTCACA-3 0 0 0 0 GAPDH promoter 5 -AAAAGCGGGGAGAAAGTAGG-3 5 -CTAGCCTCCCGGGTTTCTCT-3 and E2F1, we used nuclear extracts of control, transfected Chromatin immunoprecipitation assay or treated cells. After blocking, the membranes were probed with the appropriate antibodies, and blots were To determine E2F1 binding to MEF promoter, nuclear visualized using SuperSignal (PIERCE, Rockford, IL, extracts from HCT116 and HEK293 cells were used for USA). chromatin immunoprecipitation (ChIP) assay following the protocol described previously (20). Two micrograms Senescence induction and senescence-associated of anti-E2F1 antibody or normal rabbit IgG was b-galactosidase staining incubated with pre-cleared chromatin. Samples were analyzed by PCR using LA Taq Polymerase (TaKaRa) To induce DNA damage-associated cellular senescence, +/+ / according to the recommended protocol. The primers HCT116 p53 and p53 cells were treated with used (Table 4) recognize a fragment of the human MEF 100 nM doxorubicin (dox) for 24 h and re-incubated in promoter, CDC6 promoter or GAPDH promoter. normal medium for 3 days (for PCR analysis) or 5 days (for staining and protein analyses) according to the Immunoprecipitation and western blotting protocol reported previously (21). Cells were fixed with 2% formaldehyde/0.2% glutaraldehyde and stained for To analyze the interaction between p53 and E2F1, trans- senescence-associated b-galactosidase (SA-b-gal) activity fected or doxorubicin-treated HCT116 cells were lysed using 5-bromo-4-chloro-3-indolyl b-D-galactoside (X-gal) with nuclear extraction buffer as previously described in at pH 6.0 as described before (22). SA-b-gal-positive cells ref. 5. Nuclear extracts were incubated for 12 hr at 4 C were detected by bright-field microscopy. with 2 mg anti-p53 or anti-E2F1 antibodies or control IgG immobilized in protein G Sepharose beads (Amersham Animals Bioscience, Sweden). Immunoprecipitates were washed, eluted and subjected to immunoblotting, following essen- The p53 knockout (p53 ) mice were kindly provided by tially our protocol reported previously (20). Blots of IP Dr. Shin Aizawa from RIKEN (Kobe, Japan). The samples or input fraction were probed with anti-p53 p53-deficient mice were produced through an ordinary and anti-E2F1 antibodies or anti-g-tubulin antibody (for knockout (KO) strategy for p53 gene in C57BL/6 mice input fraction). For western blotting analysis of MEF, p53 as described earlier (23). RNA isolates from different Nucleic Acids Research, 2011, Vol. 39, No. 1 79 tissues of 12-week-old p53 mice and age-matched p53 suppresses MEF promoter activity controls were used for Q-PCR analyses. The mice used The effect of p53 on MEF expression was observed at the in this study were housed in a vivarium in accordance mRNA level; thus, we focused on MEF promoter to as- with the guidelines of the animal facility center of certain how p53 downregulates MEF. To establish the Kumamoto University. The animals were fed with chow influence of p53 on MEF promoter activity, we ad libitum. All experiments were performed according to co-transfected in HCT116 p53 cells increasing the protocols approved by the Animal Welfare Committee amounts of p53 expression plasmid and MEF promoter of Kumamoto University (#A19-115). (–849/+181 bp). We found that increased levels of p53 caused a concomitant decline in MEF promoter activity Statistical analysis (Figure 3A). Next, to identify the region of MEF For statistical analysis, the data were analyzed by promoter that is important for its response to p53, we Student’s t-test or by one-way analysis of variance used three promoter constructs with varying lengths, (ANOVA) with Tukey–Kramer multiple comparison test labeled: 800 bp (–849/+181), 400 bp (–384/+181) and (JMP software, SAS Institute, NC, USA) as indicated in 200 bp (–204/+181) (Figure 3B). These constructs and each figure legend. A P-value of <0.05 is considered stat- p53 expression plasmid were co-transfected in p53 istically significant. cells. Reporter assays revealed that activity for all three constructs was downregulated by p53, suggesting that p53 suppresses MEF transcriptional activity by affecting at RESULTS least the 200 bp proximal region of MEF promoter p53 negatively regulates MEF expression in human cell (Figure 3B). However, in silico analysis of MEF lines and mice tissues promoter did not reveal any p53 response element within the 800 bp region upstream from the transcription MEF has been implicated in the suppression of p53 ex- start site. Intriguingly, we found E2F consensus binding pression (16), but it is unknown whether this regulation is sites in this promoter region (Figure 3C). Because it was reciprocal. To address this question, we first compared the previously demonstrated that p53 physically interacts with +/+ / basal level of MEF mRNA in HCT116 p53 and p53 and inhibits the transcriptional activity of E2F1 (24), we cells. Interestingly, quantification of mRNA showed that hypothesized that p53 indirectly suppresses MEF tran- +/+ the amount of MEF in HCT116 p53 cells was 5-fold / scription by affecting E2F1, which could be a novel tran- lower than in p53 cells (Figure 1A). Conventional PCR scriptional activator of MEF. To assess this possibility, we analysis also showed lower expression of MEF in HCT116 +/+ / first investigated the effect of E2F1 on MEF expression p53 cells than in p53 cells (Supplementary Figure and promoter activity. S1A). Consistent with these observations, transfection of p53 plasmid in HCT116 p53 cells downregulated MEF E2F1 is a novel MEF transcriptional activator mRNA level (Figure 1B and Supplementary Figure S1B). Introduction of E2F1 in HCT116 p53 cells resulted in Treatment with nutlin-3, a specific activator of p53, dose-dependent increase of MEF mRNA level as resulted in a drop in MEF mRNA expression level in +/+ / determined by Q-PCR (Figure 4A). Similarly, the p53 cells but not in p53 cells (Figure 1C). relative amount of MEF mRNA was upregulated by Treatment of HepG2 cells with nutlin-3 also decreased overexpression of E2F1 in other cell lines tested MEF mRNA level (Figure 1D). Conversely, knockdown (Figure 4B–E). In contrast to E2F1 overexpression, of p53 by siRNA in A549, 16HBE14o- and HEK293 siRNA targeting E2F1 induced a drop in basal MEF cells up–regulated the mRNA expression of MEF (Figure 1E–G). These observations implied that p53 mRNA level in human epithelial cells (Figure 4F–H). suppresses MEF transcription. To assess whether this The upregulation of MEF mRNA by E2F1 translated to decrease also occurs at the protein level, we examined an increase in MEF protein level as determined by western +/+ MEF expression in nuclear extracts of HCT116 p53 blotting of nuclear extracts of cells transfected with E2F1 and p53 cells. Basal MEF protein level in HCT116 (Figure 4I and J). +/+ / p53 cells was lower than in p53 cells (Figure 1H). To analyze the effect of E2F1 on MEF transactivation, Exogenous addition of p53 in HCT116 p53 cells we co-transfected increasing amounts of E2F1 with MEF decreased the expression of MEF (Figure 1I). Moreover, promoter (–849/+181 bp) in HCT116 p53 cells. E2F1 activation of endogenous p53 by nutlin-3, reduced MEF clearly stimulated MEF transcriptional activity in a +/+ expression in HCT116 p53 cells but had no effect on dose-dependent manner (Figure 5A). To define the MEF in p53 cells (Figure 1J). Knockdown of p53 by region of MEF promoter that is controlled by E2F1, we si-RNA in A549 and 16HBE14o- cells increased MEF assessed the effect of E2F1 on three different lengths of protein expression (Figure 1K and L). In addition, by MEF promoter construct [as used above (–800 bp, using tissues of p53 mice, we confirmed the effect of 400 bp and 200 bp)]. These constructs were transfected p53 on MEF mRNA expression in vivo. Consistent with in HCT116 p53 cells together with E2F1 expression the results in human cell lines, MEF mRNA level was vector. Reporter assays revealed that the minimal region higher in various tissues of p53-deficient mice than in of MEF that is activated by E2F1 is at 200 bp upstream those of p53 wild-type mice (Figure 2A–F). These data from transcription start site (Figure 5B). The 200 bp collectively indicated that p53 downregulates MEF proximal region contains two E2F binding sites expression. (Figure 3C). Mutation of the E2F site nearest to the 80 Nucleic Acids Research, 2011, Vol. 39, No. 1 +/+ Figure 1. p53 downregulates MEF expression in human cell lines. (A) Relative amount of MEF mRNA was examined in HCT116 p53 and / / p53 cells. (B) HCT116 p53 cells were transfected with control pcDNA3.1 empty vector or p53 plasmid. Forty-eight hours post-transfection, +/+ / MEF mRNA level was analyzed. (C) HCT116 p53 and p53 cells were untreated or treated with nutlin-3 for 24 h. Total RNA was isolated for analysis. (D) HepG2 cells were treated with 20 mM nutlin-3 for 48 h then total RNA was recovered. (E–G) A549, 16HBE14o- and HEK293 cells were transfected with si-GL2 (control) or si-p53 as described in ‘Materials and Methods’ section. Total RNA was isolated and analyzed for MEF mRNA. For (A–G) MEF mRNA level, assessed by quantitative RT-PCR, was normalized to GAPDH or 18S rRNA, which served as internal controls. Values are mean ± SD of triplicate measurements. *P< 0.05; **P< 0.01 versus control cells, assessed by Student’s t-test or ANOVA with Tukey– +/+ / Kramer test (for (C)). (H) Endogenous MEF and p53 protein expressions in nuclear extracts of HCT116 p53 and p53 cells were examined by +/+ / / western blotting. (I) HCT116 p53 and p53 cells were transfected with control pcDNA3.1 empty vector, or p53 plasmid for p53 cells. +/+ / Forty-eight hours post-transfection, nuclear extracts were isolated. (J) HCT116 p53 and p53 cells were untreated or treated with Nutlin-3 (5 mM) for 24 h, then the nuclear extracts were isolated. (K and L) A549 and 16HBE cells were transfected with si-GL2 (control) or si-p53. Forty-eight hours after transfection, nuclear extracts were isolated. For (H–L), protein levels of MEF and p53 were analyzed by western blotting. g-tubulin was used as internal control in these experiments. start site (MT1) abrogated the basal transcriptional of both sites (MT1&2) diminished the basal promoter activity of MEF compared to wild-type promoter while activity of MEF in comparison with wild-type to a level mutation of the second E2F site (MT2) did not suppress similar to that of MT1 (Figure 5C), indicating that E2F the basal transactivation of MEF (Figure 5C). Mutation binding site 1 in the 200 bp proximal region is important Nucleic Acids Research, 2011, Vol. 39, No. 1 81 / +/+ / Figure 2. MEF mRNA level is upregulated in p53 mice tissues. (A–F) Total RNA isolated from the indicated tissues of p53 and p53 mice was analyzed for the expression of MEF by real-time quantitative RT–PCR. MEF mRNA level was normalized to mouse 18S rRNA (internal control). Results represent mean ± SD (n = 3). *P< 0.05, **P< 0.01 against p53 wild-type mice assessed by Student’s t-test. for efficient up-regulation of MEF basal transcription. by increasing amounts of co-transfected p53 (Figure 6A The residual activity that was not suppressed by the and B). To verify the physical interaction between p53 and mutation of E2F site could be attributed to the effect of E2F1, we performed immunoprecipitation (IP) using Sp1, which we previously showed as a basal transcription- nuclear extracts of HCT116 p53 transfected with p53 al activator of MEF that binds to 91/–82 and 63/– and E2F1. Consistent with previous reports (24,26), we 54 bp in MEF promoter (12). To establish the association confirmed that p53 associates with E2F1 (Figure 6C). of E2F1 on MEF promoter, we analyzed E2F1 recruit- To assess the functional consequence of this interaction, ment to MEF promoter by ChIP assay of nuclear lysates we studied its effect on E2F1 binding on MEF promoter. / / from HCT116 p53 cells transfected with E2F1 or As expected, addition of p53 in HCT116 p53 cells sub- pcDNA3.1 using ChIP primers that recognize MEF stantially lessened the steady-state association of E2F1 on promoter region at 204/+181 bp. Immunoprecipitation MEF promoter region as detected by ChIP analysis of with E2F1 antibody but not with control IgG and subse- nuclear extracts (Figure 6D, left). Similar effect of p53 quent PCR reactions revealed the recruitment of endogen- was also observed on CDC6 promoter (Figure 6D, ous and overexpressed E2F1 to the promoter region of right). Because the expression level of E2F1 is relatively MEF (Figure 5D). In addition, we verified the binding similar in cells with or without p53 (Figure 6E), we ruled of endogenous E2F1 to MEF promoter in HEK293 cells out the possibility that the lack of E2F1 binding to pro- (Figure 5E). Taken together, we substantiated that E2F1 moters in the presence of p53 was due to reduced level of is a novel MEF transcriptional activator that binds to E2F E2F1. Collectively, these data indicated that p53 consensus site in MEF proximal promoter region. downregulates MEF transcription by diminishing the recruitment of E2F1 to MEF promoter. p53 inhibits E2F1 binding to MEF promoter Cellular senescence-induced p53 activation downregulates It has been demonstrated previously that the association MEF expression via inhibition of E2F1 binding to MEF of p53 with E2F1 blocks the ability of E2F1 to bind to promoter DNA and transactivate gene expression (25). Having shown that E2F1 binds and activates MEF promoter, We next asked whether the regulation of MEF by p53 via we next assessed whether p53 abrogates these effects. E2F1 occurs under physiologically relevant setting. Given Congruent with the above observations, E2F1 significant- that MEF has been linked to p53 in the context of cellular ly stimulated MEF transcriptional activity when E2F1 senescence (16) and considering our data above, we looked was co-transfected with MEF promoter (–204/+181 bp) into the possibility that senescence-activated p53 can in HCT116 p53 and HEK293 cells (Figure 6A and suppress the expression of MEF. We induced cellular sen- B). However, this positive regulation was titrated away escence by treating cells with DNA-damaging reagent 82 Nucleic Acids Research, 2011, Vol. 39, No. 1 Figure 3. p53 suppresses MEF promoter activity. (A) HCT116 p53 cells were transiently transfected with MEF (–849/+181 bp) promoter con- struct or pGL3b vector (0.2 mg) and the indicated amount of p53 plasmid or pcDNA3.1 empty vector. Luciferase activity was determined 48 h after transfection of plasmids and is expressed as fold activation over the pGL3b vector. Values are the mean ± SE of triplicate platings. ***P< 0.001 versus pcDNA3.1, determined by ANOVA with Tukey–Kramer test. n.s, not significant. (B) HCT116 p53 cells were transiently transfected with the indicated MEF promoter constructs (0.2 mg) and p53 plasmid (0.1 mg) or pcDNA3.1 empty vector (as control). Luciferase activity was determined 48 h after transfection of plasmids and is expressed as fold activation over the pcDNA3.1 vector (con) in each promoter construct. Values are mean ± SE of triplicate platings. **P< 0.01; ***P< 0.001 versus control, assessed by Student’s t-test. (C) The –849 bp 5 -flanking region of MEF. Nucleotide sequence of 5 -flanking region of human MEF gene is shown. The site indicated (+1) denotes the start site of the first exon. The predicted binding sites for E2F1 are marked on the sequence. Nucleic Acids Research, 2011, Vol. 39, No. 1 83 Figure 4. E2F1 upregulates MEF expression in human cell lines. (A–E) Cells were transiently transfected with pcDNA3.1 vector (con) or the indicated amount of E2F1 (A) or 1.0 mg E2F1 (B–E). Forty-eight hours after transfection, total RNA was extracted and analyzed for MEF mRNA expression. (F–H) si-GL2 or si-E2F1 (50 nM) was transfected into the indicated cell lines and MEF mRNA expression was assessed 48 h post-transfection. For (A–H) MEF mRNA level, determined by quantitative RT–PCR, was normalized to GAPDH or 18S rRNA (internal control) and expressed as relative amount of mRNA. Values are mean ± SD of triplicate measurements. *P< 0.05; **P< 0.01; ***P< 0.001 versus control, assessed by ANOVA with Tukey–Kramer (A) or Student’s t-test (B–H). (I–J) MEF and E2F1 protein expressions were examined by western blotting in nuclear extracts of HCT116 p53 (I) or 16HBE14o- cells (J) transiently transfected with E2F1 or pcDNA3.1 empty vector. g-tubulin was used as internal control. +/+ doxorubicin (dox). Treatment with moderate dose p53 cells was also downregulated upon senescence (100 nM) of dox for 24 h and additional incubation for induction (Figure 7C). Next, we assessed the associ- 5 days was reported to induce cellular senescence in ation between E2F1 and p53 during cellular senescence HCT116 wild-type cells (21). Intense SA-b-gal staining by performing IP analysis. IP using antibody specific +/+ was observed in HCT116 p53 cells while faint to E2F1 and probing with p53 antibody revealed that staining was seen in p53 cells (Figure 7A), consistent cellular senescence augmented the physical interaction with the results obtained by Chang et al. (21). between E2F1 and p53 (Figure 7D). Notably, we Interestingly, under senescent condition, concomitant detected by ChIP assay that steady-state binding of with an enhanced level of p53, MEF protein expression E2F1 to MEF promoter was abolished in HCT116 +/+ +/+ was reduced compared with control in HCT116 p53 p53 senescent cells (Figure 7E). Taken together, these cells (Figure 7B). On the other hand, dox treatment did results suggested that under cellular senescence condition, not suppress MEF in HCT116 p53 cells (Figure 7B), activated p53 downregulates MEF expression by arguing for p53-dependency of MEF downregulation associating with E2F1 and inhibiting E2F1 binding to during senescence. MEF mRNA expression in HCT116 MEF promoter. 84 Nucleic Acids Research, 2011, Vol. 39, No. 1 Figure 5. E2F1 increases MEF promoter activity by binding to E2F site. (A) HCT116 p53 cells were transiently transfected with MEF (–849/ +181 bp) promoter construct or pGL3b vector (0.2 mg) and the indicated amounts of E2F1 plasmid or pcDNA3.1 empty vector. Luciferase activity was determined 48 h after transfection and is expressed as fold activation over the pGL3b vector. Values are the mean ± SE of triplicate platings. yyy / P< 0.001 versus pGL3b; ***P< 0.001 versus pcDNA3.1, determined by ANOVA with Tukey–Kramer. (B) HCT116 p53 cells were transiently transfected with the indicated MEF promoter constructs (0.2 mg) and E2F1 plasmid or pcDNA3.1 vector (0.1 mg). Luciferase activity was determined 48 h after transfection and is expressed as fold activation over the pcDNA3.1 vector in each promoter construct. Values are mean ± SE of triplicate platings. ***P< 0.001 versus control, assessed by Student’s t-test. (C) HCT116 p53 cells were transfected with 0.2 mg pGL3b vector or MEF (– 200/+181 bp) promoter wild-type (WT), MT1, MT2 or MT1&2. (Left) MT1, MT2 represent MEF (–200/+181 bp) promoter containing mutated E2F site. Luciferase activity was determined 48 h after transfection and is expressed as fold activation over pGL3b vector. Values are mean ± SE of triplicate platings. ***P< 0.001 versus WT, assessed by ANOVA with Tukey–Kramer test. (D) E2F1 binding on MEF promoter was determined by ChIP assay using nuclear extracts of HCT116 p53 cells transiently transfected with E2F1 or pcDNA3.1 vector (1.0 mg). CDC6 promoter was used as positive control and GAPDH promoter was used as negative control for E2F1 binding. Upper panel illustrates the MEF promoter region (– 204 bp/+181 bp) in which binding was assessed. (E) Nuclear extract from HEK293 cells was used to assess the endogenous binding of E2F1 on MEF promoter. CDC6 and GAPDH promoters were used as positive and negative controls, respectively. DISCUSSION in MEF promoter while p53 antagonizes this positive interaction by associating with E2F1 and reducing its We have identified a dual, opposing transcriptional regu- lation mechanism of MEF by E2F1 and p53. E2F1 acti- DNA-binding activity. The extensive crosstalk between vates MEF transcription by binding to E2F consensus site p53 and E2F pathways is well documented (27). It was Nucleic Acids Research, 2011, Vol. 39, No. 1 85 Figure 6. p53 inhibits E2F1 binding to MEF promoter and reduces E2F1-induced MEF promoter activation. (A and B) HCT116 p53 and HEK293 cells were transiently transfected with MEF (–204/+181 bp) promoter construct or pGL3b vector (0.2 mg), pcDNA3.1 or E2F1 (0.1 mg) and the indicated amounts of p53 plasmid. Luciferase activity was determined 48 h after transfection and is expressed as fold activation over the pGL3b yyy ### vector. Values are the mean ± SE of triplicate platings. P< 0.001 versus pGL3b; P< 0.001 versus pcDNA3.1; *P< 0.05; ***P< 0.001 versus E2F1, determined by ANOVA with Tukey–Kramer. (C) HCT116 p53 cells were transiently transfected with p53 and E2F1 plasmids. Nuclear extracts were analyzed for p53-E2F1 association by IP using antibody specific to p53, E2F1 or control mouse IgG. Immunoprecipitates were loaded onto SDS–PAGE gel, blotted and probed with E2F1 or p53 antibodies. (D) HCT116 p53 cells were transiently transfected with E2F1 and p53 plasmid or pcDNA3.1 empty vector. Nuclear extracts were isolated and used for ChIP assay to determine E2F1 binding on MEF promoter and CDC6 promoter in the presence or absence of p53. (E) Endogenous protein level of E2F1 was examined in nuclear extracts of HCT116 +/+ / p53 and p53 cells. g-Tubulin was used as internal control. described earlier by O’Connor et al. (24) that E2F1 and gene that regulates the early steps of DNA replication p53 reciprocally inhibit each other’s transcriptional (28–30), raising the possibility that p53 interferes with activity through their physical association, probably the transcription of genes other than MEF that are forming a protein–protein complex and thereby prevent- targeted by E2F1. Being two pivotal regulators of cell ing DNA binding. E2F1 and p53 are also known to proliferation, the functional interaction between E2F1 control genes that influence cell cycle progression and p53 most likely affects cell fate. (24,27). Indeed, we found here that their interaction Sashida et al. (16) has demonstrated that MEF de- (Figures 6C and 7D) affects the regulation of MEF, a creases p53 protein stability by inducing the transcription molecule that is involved in the transition of cells from of MDM2, and MEF also downregulates Rb, an endogen- G1 to S phase (7) and in driving quiescent hematopoietic ous inhibitor of E2F1. Because the loss of MEF substan- stem cells to G1 phase (14). We also noted that exogenous tially enhanced the senescent phenotype of murine addition of p53 slightly lessened the recruitment of E2F1 embryonic fibroblasts and activated the p53 function, it to the promoter of CDC6 (Figure 6D), an E2F1 target was evident that MEF inhibits cellular senescence by 86 Nucleic Acids Research, 2011, Vol. 39, No. 1 +/+ / Figure 7. p53 suppresses MEF expression during cellular senescence. (A and B) HCT116 p53 and p53 cells were treated with doxorubicin (Dox) for 24 h and incubated for 5 days to induce cellular senescence. Cells were fixed and stained with X-gal to detect SA-b-gal activity. Stained cells were photographed at phase contrast with 20-fold magnification (Scale bar, 100 mm) (A). The nuclear extracts from Dox-treated or untreated cells +/+ were subjected to immunoblotting using the indicated antibodies. g-tubulin was used as internal control (B). (C) HCT116 p53 cells were untreated or treated with Dox for 24 h and incubated for 3 days. Total RNA was recovered and MEF mRNA expression was examined by Q-PCR. Amount of MEF mRNA was normalized to GAPDH and expressed as relative amount compared to control cells. Values are mean ± SD of triplicate platings. +/+ **P< 0.01, determined by Student’s t-test. (D) Cellular senescence was induced in HCT116 p53 cells by doxorubicin treatment similar to (A). p53 binding to E2F1 was examined by immunoprecipitation of nuclear extracts. (E) E2F1 binding on MEF promoter was examined by ChIP assay using +/+ nuclear extracts of control or Dox-induced senescent HCT116 p53 cells. (F) Negative regulatory mechanism between p53 and MEF. (Left) DNA damage stimulates p53 that enhances its binding to E2F1. This leads to reduced recruitment of E2F1 to the MEF promoter and the suppression of MEF transcription. (Right) MEF upregulates MDM2 (described in ref. 16), which inhibits p53. suppressing p53 pathway (16) (Figure 7F, right diagram). combined with our data here showing that MEF mRNA However, whether this occurs at steady state or induced and protein levels were suppressed by p53 implies that a by pathogenic/pathological condition is still unclear (16). negative regulatory mechanism exists between MEF and The previous study showing that MEF downregulates p53 p53 pathways. We propose that during DNA damage, Nucleic Acids Research, 2011, Vol. 39, No. 1 87 p53 is activated and interacts with E2F1, which minimizes now shown that p53 reciprocally opposes MEF transactivation. E2F1 recruitment to MEF promoter (Figure 7F, left diagram). The lessening of MEF levels may partly contrib- ute to the modulation of cell cycle progression. We SUPPLEMENTARY DATA showed here that p53 affects MEF transcription indirectly, but the possibility that p53 can also directly affect MEF at Supplementary Data are available at NAR online. the post-translational level cannot be fully ruled out. Until now, the regulation of MEF protein stability mediated by cyclin A is the only known molecular event ACKNOWLEDGEMENTS by which MEF is controlled especially during cell cycle +/+ / HCT116 p53 and HCT116 p53 cell lines were (7). Our data here indicated that MEF transcription is provided by Dr. Bert Vogelstein (Johns Hopkins also influenced by E2F1, an activator E2F family University). 16HBE14o- cells were donated by member that affects the cell cycle (31,32). Thus, mechan- Dr. Dieter C. Gruenert (California Pacific Medical isms of controlling MEF exist at both transcriptional and Center). Wild-type p53 plasmid was supplied by post-translational levels. Because E2F1 binds to MEF Dr. Hideyuki Saya (Keio University). p53 mice were promoter and mutation of E2F binding site inhibited made available by Dr. Shin Aizawa (Laboratory for basal MEF promoter activity, MEF may be considered Animal Resources and Genetic Engineering, Center for a target of E2F1 transcription factor. While it is possible Developmental Biology, RIKEN). that E2F2 and E2F3, which are also trans-activating E2Fs, can affect MEF, we found that specific silencing of E2F1 efficiently suppressed endogenous MEF mRNA FUNDING level, suggesting a considerable specificity of E2F1 where transactivation is concerned. However, although we The Ministry of Education, Science, Sports and Culture observed that MEF transcription was reduced by p53 at (MEXT) of Japan (Grant # 19390045); and the Global the organismal level (Figure 2), the effect of E2F1 on COE Program (Cell Fate Regulation Research and MEF in vivo still awaits verification. As far as we know, Education Unit), MEXT, Japan. Funding for open MEF is the first member of the Ets transcription factor access charge: The Ministry of Education, Science, family to be identified as a direct E2F target gene despite Sports and Culture of Japan. the fact that Ets transcription factors are notable for their Conflict of interest statement. 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Published: Jan 30, 2011

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