EZH 2 cooperates with gain‐of‐function p53 mutants to promote cancer growth and metastasis
2019; Springer Nature; Volume: 38; Issue: 5 Linguagem: Inglês
10.15252/embj.201899599
ISSN1460-2075
AutoresYu Zhao, Liya Ding, Dejie Wang, Zhenqing Ye, Yundong He, Linlin Ma, Runzhi Zhu, Yunqian Pan, Qiang Wu, Kun Pang, Xiaonan Hou, Saravut J. Weroha, Conghui Han, Roger Coleman, Ilsa M. Coleman, R. Jeffery Karnes, Jun Zhang, Peter S. Nelson, Liguo Wang, Haojie Huang,
Tópico(s)RNA modifications and cancer
ResumoArticle5 February 2019free access Source DataTransparent process EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis Yu Zhao Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Liya Ding Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Dejie Wang Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Zhenqing Ye Division of Medical Informatics and Statistics, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Yundong He Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Linlin Ma Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Runzhi Zhu orcid.org/0000-0002-9565-7261 Center for Cell Therapy, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China Search for more papers by this author Yunqian Pan Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Qiang Wu Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, Tongji Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Kun Pang Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, Xuzhou Central Hospital and Medical College affiliated to Xuzhou Medical University, Xuzhou, Jiangsu, China Search for more papers by this author Xiaonan Hou Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Saravut J Weroha Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Conghui Han Department of Urology, Xuzhou Central Hospital and Medical College affiliated to Xuzhou Medical University, Xuzhou, Jiangsu, China Search for more papers by this author Roger Coleman Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Ilsa Coleman Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author R Jeffery Karnes Department of Urology, Mayo Clinic College of Medicine, Rochester, MN, USA Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Jun Zhang Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Peter S Nelson Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Liguo Wang Division of Medical Informatics and Statistics, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Haojie Huang Corresponding Author [email protected] orcid.org/0000-0003-2751-6413 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, Mayo Clinic College of Medicine, Rochester, MN, USA Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Yu Zhao Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Liya Ding Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Dejie Wang Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Zhenqing Ye Division of Medical Informatics and Statistics, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Yundong He Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Linlin Ma Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Runzhi Zhu orcid.org/0000-0002-9565-7261 Center for Cell Therapy, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China Search for more papers by this author Yunqian Pan Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Qiang Wu Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, Tongji Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Kun Pang Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, Xuzhou Central Hospital and Medical College affiliated to Xuzhou Medical University, Xuzhou, Jiangsu, China Search for more papers by this author Xiaonan Hou Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Saravut J Weroha Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Conghui Han Department of Urology, Xuzhou Central Hospital and Medical College affiliated to Xuzhou Medical University, Xuzhou, Jiangsu, China Search for more papers by this author Roger Coleman Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Ilsa Coleman Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author R Jeffery Karnes Department of Urology, Mayo Clinic College of Medicine, Rochester, MN, USA Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Jun Zhang Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Peter S Nelson Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Liguo Wang Division of Medical Informatics and Statistics, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Haojie Huang Corresponding Author [email protected] orcid.org/0000-0003-2751-6413 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA Department of Urology, Mayo Clinic College of Medicine, Rochester, MN, USA Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Author Information Yu Zhao1,‡, Liya Ding1,†,‡, Dejie Wang1, Zhenqing Ye2, Yundong He1, Linlin Ma1, Runzhi Zhu3, Yunqian Pan1, Qiang Wu1,4, Kun Pang1,5, Xiaonan Hou6, Saravut J Weroha6, Conghui Han5, Roger Coleman7, Ilsa Coleman7, R Jeffery Karnes8,9, Jun Zhang10, Peter S Nelson7, Liguo Wang2 and Haojie Huang *,1,8,9 1Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA 2Division of Medical Informatics and Statistics, Mayo Clinic College of Medicine, Rochester, MN, USA 3Center for Cell Therapy, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China 4Department of Urology, Tongji Hospital, Tongji University School of Medicine, Shanghai, China 5Department of Urology, Xuzhou Central Hospital and Medical College affiliated to Xuzhou Medical University, Xuzhou, Jiangsu, China 6Department of Oncology, Mayo Clinic College of Medicine, Rochester, MN, USA 7Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 8Department of Urology, Mayo Clinic College of Medicine, Rochester, MN, USA 9Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN, USA 10Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA †Present address: Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 507 2931311; E-mail: [email protected] EMBO J (2019)38:e99599https://doi.org/10.15252/embj.201899599 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In light of the increasing number of identified cancer-driven gain-of-function (GOF) mutants of p53, it is important to define a common mechanism to systematically target several mutants, rather than developing strategies tailored to inhibit each mutant individually. Here, using RNA immunoprecipitation-sequencing (RIP-seq), we identified the Polycomb-group histone methyltransferase EZH2 as a p53 mRNA-binding protein. EZH2 bound to an internal ribosome entry site (IRES) in the 5′UTR of p53 mRNA and enhanced p53 protein translation in a methyltransferase-independent manner. EZH2 augmented p53 GOF mutant-mediated cancer growth and metastasis by increasing protein levels of mutant p53. EZH2 overexpression was associated with worsened outcome selectively in patients with p53-mutated cancer. Depletion of EZH2 by antisense oligonucleotides inhibited p53 GOF mutant-mediated cancer growth. Our findings reveal a non-methyltransferase function of EZH2 that controls protein translation of p53 GOF mutants, inhibition of which causes synthetic lethality in cancer cells expressing p53 GOF mutants. Synopsis In addition to its well-established oncogenic roles as SET-domain containing polycomb-group family member, EZH2 is here shown to promote cap-independent translation of p53 GOF mutant mRNA to drive prostate cancer progression, possibly explaining the paradoxical EZH2 deletion observed in various malignancies. EZH2 binds to the 5′UTR IRES site of p53 mRNA in human prostate cancer cells independent of its methyltransferase activity. EZH2 interacts with eIF factors and increases p53 mRNA binding with polysomes. Depletion of EZH2 decreases p53 mRNA expression and protein stability in vitro and in a prostate cancer in vivo model. Expression of EZH2 and p53 positively correlate in human cancers. EZH2 depletion induces synthetic vulnerability and inhibits growth in p53 GOF mutant-expressing cancer cells and tumors in vivo. Introduction EZH2 is a SET domain-containing protein that belongs to the Polycomb-group (PcG) family (Margueron & Reinberg, 2011). Working coordinately with other PcG proteins in the Polycomb repressive complex 2 (PRC2), EZH2 primarily functions as an methyltransferase by catalyzing histone H3 lysine 27 trimethylation (H3K27me3; Cao et al, 2002; Czermin et al, 2002; Kuzmichev et al, 2002; Muller et al, 2002). The Polycomb-dependent (PcD) function of EZH2 is not only important for developmental patterning, X chromosome inactivation, stem cell maintenance, and cell-fate decision (Plath et al, 2003; Boyer et al, 2005; Ezhkova et al, 2009; Margueron & Reinberg, 2011), but also implicated in cancer (Varambally et al, 2002; Cha et al, 2005; Chen et al, 2010). Overexpression of EZH2 often correlates with aggressive, metastatic solid tumors such as prostate and breast cancers (Varambally et al, 2002; Kleer et al, 2003; Karanikolas et al, 2009). Active mutations in the SET domain of EZH2 frequently occur in human lymphomas (Morin et al, 2010; Sneeringer et al, 2010; McCabe et al, 2012a), resulting in aberrant activation of PcD and elevation of H3K27me3. In a Polycomb-independent (PcI) manner, EZH2 acts in “solo” to regulate actin polymerization and the oncogenic activities of transcription factors such as the androgen receptor (AR), but the PcI function remains methyltransferase-dependent (Su et al, 2005; Xu et al, 2012). Because of the importance of the methyltransferase-dependent PcD and PcI functions in cancer, targeting the enzymatic activity of EZH2 is the current focus to develop small molecule inhibitors of EZH2 for cancer treatment (McCabe et al, 2012b). A few such inhibitors have been developed and exhibit apparent anti-cancer activities by decreasing cell proliferation and tumor growth in various cancer models (McCabe et al, 2012b; Wu et al, 2016). While the methyltransferase activities of EZH2 are well studied, it remains unexplored whether or not EZH2 also possesses non-methyltransferase function(s) that might also be important for oncogenesis. TP53 is a well-studied tumor suppressor gene (Levine, 1997; Li et al, 2012). It is commonly mutated in advanced tumors. While losing the tumor suppressor activity, some mutants of p53 acquire a dominant negative function to inhibit the activity of the remaining wild-type p53 or gain completely new functions (GOF) to drive cancer progression, which include the functions to promote cell proliferation, migration, metastasis, and metabolism in various types of cancer (Dittmer et al, 1993; Olive et al, 2004; Freed-Pastor et al, 2012; Weissmueller et al, 2014). Due to the presence of many different types of p53 GOF mutations with distinctive roles in driving cancer progression, various strategies have been explored to target mutant p53 for cancer therapy, including the degradation of p53 mutant proteins, conversion back to the wild-type p53 or targeting downstream signaling pathways of p53 mutants (Adorno et al, 2009; Muller & Vousden, 2014; Weissmueller et al, 2014; Zhu et al, 2015). However, such approaches largely depend on the types of p53 mutations a tumor carries, which potentially limits the broad use of each strategy in clinic. Thus, it becomes very critical to identify common regulators of different p53 GOF mutants for effective treatment of cancers. Protein translation can be carried out by both cap-dependent and cap-independent pathways. When cap-dependent translation is globally inhibited under conditions such as cellular stresses, cells can continuously synthesize full-length p53 protein and ΔNp53 isoform by utilizing two IRES elements residing in the 5′UTR (hereafter termed IRES1) and the coding region (hereafter termed IRES2) of p53 mRNA, respectively (Candeias et al, 2006; Ray et al, 2006; Yang et al, 2006). The importance of IRES-dependent p53 protein production is further manifested in unstressed cells (Weingarten-Gabbay et al, 2014). A few proteins, including polypyrimidine tract binding protein (PTB), translation initiation factor DAP5, HDMX, and HDM2, have been identified as putative IRES trans-acting factors (ITAFs) that preferentially bind to IRES2 of p53 mRNA (Grover et al, 2008; Malbert-Colas et al, 2014; Weingarten-Gabbay et al, 2014). To date, however, proteins that preferentially bind to p53 IRES1 remain unidentified. In the present study, we identified EZH2 as a p53 mRNA-binding protein. We demonstrated that EZH2 bound to IRES1 of p53 mRNA and enhanced p53 protein translation in a methyltransferase-independent manner. We further showed that inhibition of such function of EZH2 induced synthetic lethality in p53 GOF-driven cancer cells. Results RIP-seq analysis identifies EZH2 as a p53 mRNA-binding protein Previous studies suggest that EZH2 regulates gene expression through interaction with long non-coding RNAs (lncRNAs; Wang et al, 2015; Chen et al, 2018). To define previously unrecognized oncogenic functions of EZH2, we sought to identify new EZH2-interacting RNAs in cancer cells. A previous study determined that EZH2 nonselectively binds to RNAs, at least under in vitro conditions while findings from other studies suggest that the PRC2 complex as a whole may not do the same in live cells (Davidovich et al, 2013; Cifuentes-Rojas et al, 2014). Since the crosslink-based RIP may be susceptible to contamination with non-specific RNAs (Kaneko et al, 2014), we performed native EZH2 RIP-seq in two prostate cancer cell lines without crosslink. In addition to binding with lncRNAs, EZH2 selectively bound to a subset of messenger RNA (mRNA) encoding proteins highly relevant to cancer such as p53 (Fig 1A and B, and Table EV1). Using ultraviolet crosslinked RNA immunoprecipitation (UV-RIP) and quantitative polymerase chain reaction (qPCR), we confirmed EZH2 selective association with p53 mRNA (Fig 1C). EZH2 specific binding with mRNA of other genes such as AKT1 and KDM1A, but not the unbound target SKP2 was also confirmed by RIP-qPCR (Figs 1A and EV1A–D). These data indicate that EZH2 protein selectively binds to mRNA of a subset of cancer-relevant genes including TP53 in cells. Figure 1. EZH2 binds to 5′UTR of p53 mRNA A. Heat map showing a subset of mRNAs of genes immunoprecipitated by anti-EZH2 antibody in LNCaP and C4-2 prostate cancer cell lines, which was generated based on the distinct RIP-seq reads on specific gene exons. The units of the heatmap values were reads per kilobase million (RPKM). B. Screen shots from the UCSC genome browser showing signal profiles of p53 mRNA immunoprecipitated by anti-EZH2 antibody in LNCaP and C4-2 cells. C. C4-2 cells were subjected to UV-RIP assay. RT–qPCR measurement of p53 mRNA immunoprecipitated by IgG or anti-EZH2 antibody. Data shown as means ± SD (n = 3). Statistical significance was determined by two-tailed Student's t-test. **P < 0.01. D. Schematic diagram of four GST-EZH2 recombinant proteins (EZ1–EZ4). EBD, EED binding domain; SANT1(/2)L, “Swi3, Ada2, N-Cor, TFIIIB”1(/2) like; MCSS, motif connecting SANT1L and SANT2L; CXC, cysteine-rich domain; SET, catalytic domain. E. Top, RT–qPCR analysis of p53 mRNA in C4-2 cell lysate pulled down by GST or GST-EZH2 recombinant proteins EZ1-EZ4. Bottom, Western blotting analysis of GST or GST-EZH2 proteins used for GST pull-down assay. Asterisks indicate the protein bands at expected molecular weight. Data shown as means ± SD (n = 3). Statistical significance was determined by two-tailed Student's t-test.**P < 0.01. F. C4-2 cells were transfected with Myc-tagged EZH2-WT or EZH2-∆336–554 for 24 h, and cells were harvested for RIP with IgG or anti-Myc-tag antibody. Transfected proteins and pull-down p53 mRNAs were analyzed by Western blot and RT–qPCR, respectively. Data shown are means ± SD (n = 3). *P < 0.01. ERK2, a loading control. G. GST pull-down assay using in vitro transcribed different fragments of p53 mRNA and indicated GST proteins followed by RT–qPCR analysis of pull-down p53 mRNA. FL, full length; ORF, open reading frame; UTR, untranslated region. H, I. RNA EMSA evaluation of EZh2 binding of p53 mRNA. GST-EZH2 recombinant proteins (EZ1–EZ4) were incubated with biotin-labeled in vitro transcribed p53 5′UTR (biotin-labeled probe) in the presence or absence of 100-fold unlabeled p53 5′UTR (unlabeled probe), followed by PAGE and immune blotting with HRP-conjugated streptavidin. Source data are available online for this figure. Source Data for Figure 1 [embj201899599-sup-0006-SDataFig1.tif] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Assessment of binding and regulation of mRNAs by EZH2. Related to Fig 1 RT–qPCR analysis of mRNAs of AKT1 and KDM1A immunoprecipitated by IgG or EZH2 antibody in C4-2 cells. These genes are on the top of the list of mRNAs highly enriched by anti-EZH2 antibody. Data shown are mean values ± SD from three replicates. *P < 0.01. Screen shots from UCSC genome browser showing signal profiles of SKP2 mRNA immunoprecipitated by anti-EZH2 antibody in LNCaP and C4-2 cells. Total RNA was used as the input control of RIP-seq. RIP-qPCR analysis of SKP2 mRNA immunoprecipitated by IgG and anti-EZH2 antibody in LNCaP and C4-2 cells. Data shown are mean values ± SD from three replicates. Effect of EZH2 on SKP2 mRNA expression. C4-2 prostate cancer and U2OS osteosarcoma cell lines were transfected with non-specific control (siC) or two independent EZH2-specific siRNAs. 48-h post-transfection expression of SKP2 mRNA was measured by RT–qPCR. Error bar showing SD from three replicates. NS, no significance. Download figure Download PowerPoint EZH2 binds to IRES1 of p53 mRNA The p53 protein is a cellular gatekeeper that plays essential roles in maintaining genomic integrity and regulating cell growth, survival, and energy metabolism (Levine, 1997; Li et al, 2012). We chose to further characterize the molecular basis of the interaction between EZH2 protein and p53 mRNA and the biological consequences. We first examined which region of EZH2 protein is responsible for p53 mRNA binding. Glutathione S-transferase (GST)-EZH2 recombinant proteins were purified from bacteria as described (Chen et al, 2010; Fig 1D) and incubated with lysate of LNCaP cells which express endogenous wild-type p53. GST pull-down products were subjected to RNA purification and reverse transcription quantitative PCR (RT–qPCR) analysis. The p53 mRNA-binding domain is the central region (amino acids 336–554, aa336–554; Fig 1D and E). RIP assays demonstrated that deletion of aa336–554 abolished EZH2 binding of p53 mRNA (Fig 1F). In vitro RNA binding assay showed that the aa336–554 region in EZH2 bound primarily to the 5′UTR, but not the coding region and the 3′UTR of p53 mRNA (Fig 1G). These data suggest that EZH2 binds directly to p53 mRNA 5′UTR. To further validate this observation, we performed in vitro RNA electrophoretic mobility shift assay (EMSA) using purified human EZH2 and biotin-labeled p53 5′UTR as a probe. Consistent with GST pull-down results (Fig 1E and F), GST-EZ3 (aa336–554), but not GST alone or other GST-EZH2 recombinant proteins formed an RNA–protein complex (RPC) with p53 5′UTR (Fig 1H). The binding was dose-dependent and blocked by excessive amount of unlabeled p53 5′UTR (Figs 1I and EV2A), confirming that the interaction between EZH2 and p53 mRNA 5′UTR is specific. Together, these data suggest that EZH2 protein directly binds to the 5′UTR of p53 mRNA. Click here to expand this figure. Figure EV2. EZH2 regulation of expression of p53 downstream target genes. Related to Fig 1 A. EZH2 fragment binding to p53 5′UTR determined by RNA EMSA. Different doses of GST-EZH2 recombinant proteins (GST-EZ3) were incubated with 1 μg of biotin-labeled p53 mRNA 5′UTR probe for 1 h on ice. The RNA–protein complex (RPC) was detected by PAGE followed by immune blotting with HRP-conjugated streptavidin. B. pcDNA3.1-based expression vectors for Flag-p53 FL and/or Flag-p53/47 in combination with empty vector or Myc-EZH2 were transfected into PC3 cells. Forty-eight hours after transfection cells was lysed in RIPA buffer for Western blots with indicated antibodies. ERK2, a loading control. C. PC3 cells were transfected with indicated plasmids. Forty-eight hours after transfection cells was lysed for Western blot. D. Diagram of the map for pp53-5′UTR-F/Rluc vector. F means firefly luciferase gene. R means Renilla luciferase gene. E, F. Expression of mRNAs for p21CIP1, BAX, and MDM2 genes was measured by RT–qPCR in C4-2 (E) and U2OS (F) cells 48 h after transfection with non-specific control (siC) or two independent EZH2-specific siRNAs. GAPDH was used as internal control. Data shown are mean values ± SD (error bar) from three replicates. *P < 0.01 comparing EZH2 siRNA-transfected with siC-transfected cells. G. C4-2 cells were transfected with indicated plasmids for 24 h, and cells were harvested for co-IP and Western blot analysis. Asterisks indicate different EED isoforms. H–J. C4-2 cells treated with vehicle (DMSO) or different concentrations (5 and 20 μM) of GSK126 for 24 h were harvested for analysis of expression of p53 mRNA and protein using RT–qPCR and Western blot, respectively (H), mRNA expression of EZH2 repressed genes DAB2IP and BRACHYURY (I), and EZH2-activated genes TEME48, CKS2, and KIAA0101 (J). The GAPDH was used as internal control. Data shown are mean values ± SD (error bar) from three replicates. *P < 0.01 comparing GSK126-treated with mock-treated cells. Source data are available online for this figure. Download figure Download PowerPoint EZH2 enhances IRES1-mediated p53 protein translation Consistent with the results of in vitro RNA binding assay (Fig 1G), reciprocal biotin-labeled RNA pull-down assays showed that endogenous EZH2 protein from LNCaP cell lysate were bound strongly by p53 mRNA 5′UTR, but very weakly by the 3′UTR and ORF (Fig 2A). As a positive control, EZH2 was readily pulled down by the HOTAIR lncRNA (Fig 2A). We further demonstrated that a 122-nucleotide (nt) region (−122 to −1 nt) immediately adjacent to the translation start in the 5′UTR of p53 mRNA is critical for EZH2 binding (Fig 2B). Notably, this region almost completely overlaps with IRES1 (−130 to −1 nt) reported previously (Ray et al, 2006; Yang et al, 2006). To determine the IRES1 effect on p53 protein level, p53-null PC3 cells were co-transfected with EZH2 in combination with pcDNA-p53-FL (5′UTR + CDS + 3′UTR) and/or pcNDA-p53/47 (ΔNp53), an IRES1-deletion mutant encoding a protein translated from the start codon at +40. While EZH2 overexpression increased protein level of p53-FL, its expression had no overt effect on p53/47 in PC cells (Fig EV2B). Similar results were obtained for p53ΔIRES1, an IRES1 deletion (deletion of −130 to −1 nt) mutant in PC3 cells, although p53ΔIRES1 encodes a full-length p53 (Fig EV2C). These data suggest that EZH2 regulates p53 protein level through IRES1. Figure 2. EZH2 upregulates p53 expression via binding to the IRES motif in 5′UTR Biotin pull-down assay by incubating biotin-labeled different fragments of p53 mRNA and HOTAIR (positive control) with C4-2 cell lysate followed by Western blot with EZH2 antibody. Biotin pull-down assay as in (A) using unmutated and various internally deleted mutants of 5′UTR of p53 mRNA. Top, diagram of different p53 5′UTR deletion mutants. Upper, the linear map of the pRF bicistronic report plasmid. The SV40 promoter (purple box) was used to drive firefly luciferase (Fluc) and Renilla luciferase (Rluc) gene transcription. Different p53 5′UTR fragments were inserted between the Fluc and Rluc genes. Lower, at 24 h after transfection, cells were lysed and luciferase activities were measured using a dual-luciferase kit and the ratio of Fluc/Rluc was calculated. Data shown as means ± SD (n = 3). Statistical significance was determined by two-tailed Student's t-test. *P < 0.01. EZH2 knockdown C4-2 cells were transfected with the bicistronic reporter vector in combination with empty vector, Myc-tagged EZH2 WT, or deletion mutants followed by Western blot analysis with indicated antibodies (bottom) and luciferase assay (top). Data shown as means ± SD (n = 3). *P < 0.01. C4-2 and U2OS cell lines were transfected with non-specific control (siC) or two independent EZH2-specific siRNAs and harvested for Western blot analysis with indicated antibodies. ERK2 and β-TUBULIN, loading controls. U2OS cells were transfected with control (siC) or EZH2-specific siRNAs and treated with 200 nM of CPT followed by Western blot analysis for indicated proteins. C4-2 cells were transfected with control (siC) or EZH2-specific siRNAs and plasmids for empty vector, EZH2 WT, or deletion mutants followed by Western blot analysis for indicated proteins. Source data are available online for this figure. Source Data for Figure 2 [embj201899599-sup-0007-SDataFig2.tif] Download figure Download PowerPoint We further employed a dual reporter assay to determine whether EZH2 regulates p53 protein translation by binding to IRES1. We generated a series of luciferase reporter constructs by cloning different portions of p53 5′UTR into a bicistronic plasmid (Figs 2C and EV2D). Similar to the previous reports in MCF7 and HeLa cells (Ray et al, 2006; Yang et al, 2006), both the entire 5′UTR and the IRES1 region of p53 mRNA exhibited translation-promoting activity in comparison with the empty vector (Fig 2C). However, deletion of 60 nt in the 5′ end of IRES1 (IRES1∆60) largely diminished the activity of IRES1 (Fig 2C). Knockdown of endogenous EZH2 by small interference RNAs (siRNAs) significantly reduced the luciferase activity of the IRES-F/RLuc construct, and this effect was completely reversed by restored expression of siRNA-resistant wild-type EZH2 (EZH2-WTSR), SET domain deletion (methyltransferase-deficient) mutant (EZH2∆SETSR), but not the aa336–554-deletion mutant (EZH2∆336–554SR; Fig 2D). These data indicate that EZH2 enhances IRES1-dependent translation of p53 mRNA and this effect is mediated through the RNA-binding function, but not the methyltransferase activity of EZH2. We also determined the effect of EZH2 on the steady-state level of p53 protein under physiological conditions. We knocked down EZH2 using two independent siRNAs in C4-2 prostate cancer cell line and U2OS osteosarcoma cell line, both of which express wild-type p53. Knockdown of endogenous EZH2 decreased expression of endogenous p53 proteins in both cell lines and p53 downstream targets p21CIP1, MDM2, and BAX at both protein and mRNA levels (Figs 2E, and EV2E and F). Thus, EZH2 regulates expression of p53 protein and its downstream genes in unstressed cells. p53 remains at low activity under normal conditions and becomes highly activated in response to genotoxic stresses. We treat
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