Elf4/Mef directly and positively regulates Mdm2 expression. (A) Elf4/Mef loss impairs H-Ras^V12-induced Mdm2 expression. H-Ras^V12-transduced KO mef's express 2.0-fold less Mdm2 mRNA and markedly less Mdm2 protein than H-Ras^V12-transduced WT mef's (P = 0.008; n = 3). The level of p19^ARF protein was the same in the Elf4/Mef KO mef's as the WT mef's, in the presence or absence of H-Ras^V12. (B) ELF4/MEF expression induces Mdm2, but not p19^ARF, thereby reducing p53 expression, while H-Ras^V12 induces p19, Mdm2, and p53 expression (as determined by densitometry studies). (C) Elf4/Mef is bound to the Mdm2 promoter in vivo. ChIP assays were performed using WT mef's. Protein-DNA complexes were precipitated with an anti-RNA polymerase II antibody, anti-Mef antibody, or preimmune rabbit immunoglobulin G (for a negative control). PCR was performed using primers specific for regions of the Mdm2 promoter: either 200 bp (upper row) or 1.5 kb (lower row) upstream of the start site. Binding of Elf4/Mef (and RNA polymerase II) to the MDM2 promoter was visualized by gel electrophoresis. (D) ELF4/MEF and p53 transactivate the Mdm2 promoter. The 200-bp MDM2 promoter region includes two ETS binding sites (GGAAG) and two p53 binding sites, the 100-bp promoter fragment contains only p53 binding sites, and the bp −200 to −100 promoter fragment contains two ETS binding sites (and TATA box). Transient transfection of p53 activated the 100-bp promoter activity 41-fold, the 200-bp promoter 10.1-fold, and the bp −200 to −100 promoter 0.4-fold (n = 6). Transient transfection of MEF activated the 100-bp promoter 3.0-fold, the 200-bp promoter 7.2-fold, and the bp −200 to −100 promoter 2.3-fold compared to the control vector (n = 6). (E) ELF4/MEF transactivates the Mdm2 promoter more potently than Ets1 or Ets2. MEF transfection activated the 200-bp Mdm2 promoter 7.2-fold, whereas Ets1 and Ets2 activated the 200-bp Mdm2 promoter 3.4-fold and 2.5-fold, respectively (n = 6). (F) Transient expression of ELF4/MEF increases the endogenous level of Mdm2 protein in a dose-dependent manner. The levels of Mdm2 protein were examined by Western blot analysis 24 h after the transfection of pCMV5-MEF or an empty vector control.

Elf4/Mef is required for transformation in the absence of p53. (A) Growth curves of WT, Elf4/Mef KO, p53 KO, and p53/Elf4 DKO mef's transduced with H-Ras^V12. WT and Elf4/Mef KO mef's cease to grow after the introduction of H-Ras^V12, whereas the Ras-transduced DKO and p53 KO mef's grow continuously. The vertical axis represents relative cell numbers measured by light absorption. A representative experiment is shown. (B) Oncogenic H-Ras^V12 can transform p53 KO mef's but not p53/Elf4 DKO mef's. A soft agar assay examined WT, Elf4/Mef KO, p53 KO, and DKO mef's transduced either with H-Ras^V12 or control vector. H-Ras^V12 transforms p53 KO mef's but not DKO mef's (P < 0.005; n = 4). Representative figures show photomicrographs of H-Ras^V12-transduced p53 KO and DKO mef's. (C) Elf4/Mef loss promotes H-Ras^V12-dependent p16 induction but impairs Mdm2 induction in cells lacking p53. The levels of Mdm2, p19^ARF, MDM2, p16, and Ets1 protein were examined in p53 KO and p53/Elf4 DKO mef's transduced with either the control vector or H-Ras^V12. The p53/Elf4 DKO mef's showed a marked accumulation of p16 protein and also reduced Mdm2 and Ets1 levels compared to p53 KO mef's in the presence H-Ras^V12. The DKO mef's expressed more p19^ARF protein than the p53 KO mef's. (D) Bmi-1 expression can restore transformation of p53/Elf4 DKO mef's by H-Ras^V12. DKO mef's were transduced with control vector, Bmi-1 alone, H-Ras^V12 alone, or H-Ras^V12 plus Bmi-1. While the Bmi-1 alone-transduced mef's showed very few soft agar colonies, the H-Ras^V12 plus Bmi-1-transduced p53/Elf4 DKO mef's showed more soft agar colonies than the H-Ras^V12-alone-transduced mef's (P < 0.005; n = 4). (E) Bmi-1 suppresses p16 expression but not p19^ARF expression in p53/Elf4 DKO mef's. The levels of p16, p19^ARF, and Bmi-1 were examined in the DKO mef's. (F) shRNA directed against Elf4/Mef led to the absence of detectable Elf4/Mef protein in the p53 KO mef's. The acute knockdown of Elf4/Mef led to a moderate increase in the level of p16, but not p19^ARF, protein.

p53/Elf4 DKO mice survive longer than p53 KO mice. (A) Kaplan-Meier survival curves show longer median survival for the p53/Elf4 DKO mice than the p53 KO mice (142 days versus 102 days; P = 0.001). (B) Time to death from lymphoma is shown by Kaplan-Meier curves for the p53 KO and the p53/Elf4 DKO mice. Results show the median survival of the p53 KO mice, who develop lymphoma in 105 days, versus 149 days for the p53/Elf4 DKO mice who develop lymphoma (P = 0.036). (C) Similar Ki67 indexes in lymphomas isolated from p53/Elf4 DKO and p53 KO mice (44% ± 19% versus 39% ± 18%; P = 0.25). (D) H&E staining of splenic lymphoma tissue in a p53 KO mouse and a p53/Elf4 DKO mouse, together with representative staining of Mdm2 and p16 protein by IHC. (E) Correlation between Mdm2 and p16 expression in lymphoma tissue obtained from p53 KO and p53/Elf4 DKO mice. Each score shows the number of lymphoma samples with low, moderate (mod), and high Mdm2 staining or with negative and positive p16 staining by IHC.

ELF4/MEF can prevent cellular senescence by suppressing the p53 pathway. (A) ELF4/MEF overexpression blocks oncogenic Ras-induced senescence. SA β-galactosidase staining of control vector, ELF4/MEF alone, H-Ras^V12 alone, and ELF4/MEF plus H-Ras^V12-transduced WT mef's was assessed. ELF4/MEF plus H-Ras^V12-transduced mef's showed less SA β-Gal-positive staining than the H-Ras^V12-alone-transduced mef's (P = 0.003, n = 5). (B) The enhanced senescence seen in cells lacking Elf4/Mef is dependent on p53. SA β-galactosidase staining of WT, Elf4/Mef KO, p53 KO, and p53/Elf4 DKO mef's was assessed at passages 5 and 6, and Elf4/Mef KO mef's showed significantly more positive staining cells at passage 6 (54% versus 35% for WT mef's; P = 0.007; n = 3). DKO mef's showed very few positive cells (0.5% ± 1.0%; n = 3). (C) The rate of DNA synthesis was examined using BrdU incorporation into WT and Elf4/Mef KO mef's at passage 6. BrdU incorporation was 1.8-fold less in Elf4/Mef KO mef's than in WT mef's (P = 0.020; n = 3). (D) Elf4/Mef loss promotes p53 accumulation. The levels of cell cycle regulators were examined at passages 4 and 6 in WT and Elf4/Mef KO mef's. The level of p16 accumulation over time was the same in the WT and Elf4/Mef KO mef's. Elf4/Mef KO mef's showed greater accumulation of both p53 and p21 at passage 6 than WT mef's. The level of Mdm2 protein showed a slight increase in passage 6 Elf4/Mef KO mef's compared to WT mef's. (E) Knockdown of Elf4/Mef by shRNA enhances senescence at passages 5 and 6. Growth curve assays showed that the Elf4/Mef-directed shRNA transduced WT mef's cease to grow at passage 5. (F) SA β-galactosidase staining of Elf4/Mef-directed shRNA-transduced and control empty vector-transduced WT mef's were assessed at passages 5 and 6, and Elf4/Mef-directed shRNA-transduced mef's showed significantly more positive staining cells at passage 5 (33.9% versus 3.3% for the control; P = 0.0098, n = 3). (G) Acute knockdown of Elf4/Mef activates the p53 pathway. The levels of p19^ARF, Mdm2, p53, p21, and p16 were examined at passage 5 in Elf4/Mef-directed shRNA-transduced and control (empty vector)-transduced WT mef's. The Elf4/Mef-directed shRNA-transduced cells showed increases in p53, p21, and p16 and a slight increase in p19^ARF and Mdm2 protein levels. (H) Elf4/Mef expression is not induced by serum in WT mef's that undergo senescence (at passage 5). Passage 3 WT mef's, but not passage 5 WT mef's, upregulate Elf4/Mef mRNA expression 4 hours after serum restimulation, leading to increased Mdm2 protein expression.

Mdm2 is required for the transformation of p53 KO mefs by ELF4/MEF. (A) Elf4/Mef, Mdm2, and p21 protein levels were assessed in p53 KO mef's after transduction with control vectors, Mdm2-directed shRNA alone, ELF4/MEF alone, or ELF4/MEF plus Mdm2-directed shRNA. The ELF4/MEF plus Mdm2-directed shRNA-transduced mef's showed increased p21 protein expression compared to the ELF4/MEF-alone or the shRNA-alone-transduced mef's. (B) Growth curves of the p53 KO mef's transduced with ELF4/MEF- and/or Mdm2-directed shRNA. The ELF4/MEF-alone-transduced p53 KO mef's grew continuously, as did the control mef's. The Mdm2-directed shRNA severely impaired the cell growth in the presence or absence of ELF4/MEF expression. The vertical axis represents relative cell numbers as measured by light absorption. A representative experiment is shown. (C) Mdm2 knockdown partially blocks transformation of p53 KO mef's by ELF4/MEF. The ELF4/MEF alone-transduced p53 KO mef's formed many more colonies in soft agar than the ELF4/MEF plus Mdm2-directed shRNA-transduced mef's (P < 0.005; n = 4). (D) Analysis of ELF4/MEF and MDM2 mRNA levels in 38 human lymphoma patient tissue samples, stratified by p53 IHC staining and level of MDM2 expression. The highest level of ELF4/MEF expression was seen in the samples with high MDM2 expression and wild-type p53.

The resistance of p53/Elf4 DKO mef's to transformation depends on p16. (A) p16 knockdown allows transformation of p53/Elf4 DKO mef's by H-Ras^V12. The protein levels of p16 and p19^ARF were examined in p53/Elf4 DKO mef's after transduction with the control vector, shRNAp16 alone, H-Ras^V12 alone, or H-Ras^V12 plus shRNAp16. Soft agar transformation assays were performed using p53/Elf4 DKO mef's transduced with control vector, shRNAp16 alone, H-Ras^V12 alone, or H-Ras^V12 plus shRNAp16. Introduction of shRNAp16 plus H-Ras^V12 resulted in an 8.3-fold increase in soft agar colonies (compared to H-Ras^V12-alone-transduced mef's; P < 0.005; n = 4). (B) Knockdown of p16 promotes transformation of p53 KO mef's by ELF4/MEF. The protein levels of p16 and Elf4/Mef were examined in p53 KO mef's after transduction with the control vector, shRNAp16 alone, ELF4/MEF alone, or ELF4/MEF plus shRNAp16. Introduction of shRNAp16 plus ELF4/MEF resulted in a 1.5-fold increase in soft agar colony formation compared to ELF4/MEF alone-transduced p53 KO mef's (P = 0.008; n = 4). (C) Dose-dependent reduction of Ets1-induced p16 promoter activation by ELF4/MEF. Ets1, but not ELF4/MEF, activated the p16 promoter activity, and increasing amounts of ELF4/MEF inhibited Ets1-induced p16 promoter activity by 80% (n = 3). (D) ELF4/MEF expression can rescue the transformation of p53/Elf4 DKO mef's by H-Ras^V12. When p53/Elf4 DKO mef's were transduced with control vector, ELF4/MEF alone, H-Ras^V12 alone, or ELF4/MEF plus H-Ras^V12, the ELF4/MEF plus H-Ras^V12-transduced DKO mef's showed more soft agar colonies than the H-Ras^V12-alone-transduced DKO mef's (P < 0.005; n = 4). (E) ELF4/MEF expression suppresses p16 expression but enhances Mdm2 expression in p53/Elf4 DKO mef's. The protein levels of Ets1, p16, Rb, phospho-Rb (Ser 807/811), p19^ARF, and Mdm2 were examined in the DKO mef's. ELF4/MEF plus H-Ras^V12-transduced DKO mef's showed a 1.4-fold decrease in p16 protein, compared to H-Ras^V12-alone-transduced DKO mef's (as determined by densitometries of two independent studies), despite increased Ets1 expression. The ELF4/MEF plus H-Ras^V12-transduced DKO mef's showed a significant increase in phospho-Rb protein, compared to the H-Ras^V12 alone. The ELF4/MEF plus H-Ras^V12-transduced DKO mef's also showed a significant increase in Mdm2 protein expression compared to the H-Ras^V12 alone. (F) ELF4/MEF expression does not promote transformation of p16/p19^ARF DKO mef's by H-Ras^V12. p16/p19^ARF DKO mef's were transduced with control vector, ELF4/MEF alone, H-Ras^V12 alone, or ELF4/MEF plus H-Ras^V12. The ELF4/MEF plus H-Ras^V12-transduced cells had similar soft agar colony-forming abilities as the H-Ras^V12-alone-transduced p16/p19^ARF DKO mef's (18.7 ± 6.3 versus 15.5 ± 5.8; P = 0.19; n = 4). (G) ELF4/MEF induction suppresses p53 expression in p16/p19^ARF DKO mef's. The protein levels of Mef, Mdm2, p53, and p21 were examined in p16/p19^ARF DKO mef's. ELF4/MEF plus H-Ras^V12-transduced p16/p19^ARF DKO mef's showed reduced levels of p53 and p21 protein compared to H-Ras^V12-alone-transduced mef's.

Model for how ELF4/MEF functions to suppress senescence. The model shows how Elf4/Mef can regulate Mdm2 expression and also Ets1-induced p16 expression. Arrows represent the expression levels of the genes indicated. (Left) In Elf4/Mef null cells, Elf4/Mef loss reduces Mdm2 expression, which leads to an accumulation of p53. Together with p53, p16 promotes Ras-induced senescence when it is activated by Ras-induced Ets1 expression. (Right) In the Elf4/Mef-overexpressing cells, Elf4/Mef not only enhances Mdm2 expression (overriding the opposite effect of p19^ARF on p53) but also inhibits Ets1-induced p16 induction, which is provoked by oncogenic Ras. Lastly, Elf4/Mef overexpression inhibits oncogene-induced senescence and promotes tumorigenesis.