Artigo Acesso aberto Revisado por pares

MDM2 recruitment of lysine methyltransferases regulates p53 transcriptional output

2010; Springer Nature; Volume: 29; Issue: 15 Linguagem: Inglês

10.1038/emboj.2010.140

ISSN

1460-2075

Autores

Lihong Chen, Zhenyu Li, Aleksandra Zwolińska, Matthew A. Smith, Brittany Cross, John M. Koomen, Zhi-Min Yuan, Thomas Jenuwein, Jean‐Christophe Marine, Kenneth L. Wright, Jiandong Chen,

Tópico(s)

Cancer-related Molecular Pathways

Resumo

Article29 June 2010free access MDM2 recruitment of lysine methyltransferases regulates p53 transcriptional output Lihong Chen Lihong Chen Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Zhenyu Li Zhenyu Li Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Aleksandra K Zwolinska Aleksandra K Zwolinska VIB-KULeuven, Laboratory for Molecular Cancer Biology, Leuven, Belgium Search for more papers by this author Matthew A Smith Matthew A Smith Immunotherapy Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Brittany Cross Brittany Cross Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author John Koomen John Koomen Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Zhi-Min Yuan Zhi-Min Yuan Department of Radiation Oncology, University of Texas Health Science Center, San Antonio, TX, USA Search for more papers by this author Thomas Jenuwein Thomas Jenuwein Department of Epigenetics, Max-Planck Institute of Immunobiology, Freiburg, Germany Search for more papers by this author Jean-Christophe Marine Jean-Christophe Marine VIB-KULeuven, Laboratory for Molecular Cancer Biology, Leuven, Belgium Search for more papers by this author Kenneth L Wright Kenneth L Wright Immunotherapy Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Jiandong Chen Corresponding Author Jiandong Chen Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Lihong Chen Lihong Chen Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Zhenyu Li Zhenyu Li Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Aleksandra K Zwolinska Aleksandra K Zwolinska VIB-KULeuven, Laboratory for Molecular Cancer Biology, Leuven, Belgium Search for more papers by this author Matthew A Smith Matthew A Smith Immunotherapy Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Brittany Cross Brittany Cross Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author John Koomen John Koomen Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Zhi-Min Yuan Zhi-Min Yuan Department of Radiation Oncology, University of Texas Health Science Center, San Antonio, TX, USA Search for more papers by this author Thomas Jenuwein Thomas Jenuwein Department of Epigenetics, Max-Planck Institute of Immunobiology, Freiburg, Germany Search for more papers by this author Jean-Christophe Marine Jean-Christophe Marine VIB-KULeuven, Laboratory for Molecular Cancer Biology, Leuven, Belgium Search for more papers by this author Kenneth L Wright Kenneth L Wright Immunotherapy Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Jiandong Chen Corresponding Author Jiandong Chen Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA Search for more papers by this author Author Information Lihong Chen1, Zhenyu Li1, Aleksandra K Zwolinska2, Matthew A Smith3, Brittany Cross1, John Koomen1, Zhi-Min Yuan4, Thomas Jenuwein5, Jean-Christophe Marine2, Kenneth L Wright3 and Jiandong Chen 1 1Molecular Oncology Department, Moffitt Cancer Center, Tampa, FL, USA 2VIB-KULeuven, Laboratory for Molecular Cancer Biology, Leuven, Belgium 3Immunotherapy Department, Moffitt Cancer Center, Tampa, FL, USA 4Department of Radiation Oncology, University of Texas Health Science Center, San Antonio, TX, USA 5Department of Epigenetics, Max-Planck Institute of Immunobiology, Freiburg, Germany *Corresponding author. Molecular Oncology Department, Moffitt Cancer Center, MRC3057A, 12902 Magnolia Drive, Tampa, FL 33612, USA. Tel.: +1 813 745 6822; Fax: +1 813 745 6817; E-mail: [email protected] The EMBO Journal (2010)29:2538-2552https://doi.org/10.1038/emboj.2010.140 PDFDownload PDF of article text and main figures. 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 MDM2 is a key regulator of the p53 tumor suppressor acting primarily as an E3 ubiquitin ligase to promote its degradation. MDM2 also inhibits p53 transcriptional activity by recruiting histone deacetylase and corepressors to p53. Here, we show that immunopurified MDM2 complexes have significant histone H3-K9 methyltransferase activity. The histone methyltransferases SUV39H1 and EHMT1 bind specifically to MDM2 but not to its homolog MDMX. MDM2 mediates formation of p53–SUV39H1/EHMT1 complex capable of methylating H3-K9 in vitro and on p53 target promoters in vivo. Furthermore, MDM2 promotes EHMT1-mediated p53 methylation at K373. Knockdown of SUV39H1 and EHMT1 increases p53 activity during stress response without affecting p53 levels, whereas their overexpression inhibits p53 in an MDM2-dependent manner. The p53 activator ARF inhibits SUV39H1 and EHMT1 binding to MDM2 and reduces MDM2-associated methyltransferase activity. These results suggest that MDM2-dependent recruitment of methyltransferases is a novel mechanism of p53 regulation through methylation of both p53 itself and histone H3 at target promoters. Introduction The p53 tumor suppressor pathway is functionally altered in the majority of human cancers. It is critical for the maintenance of genomic stability and protection against malignant transformation. P53 is stabilized and activated in response to a variety of stress signals, a property that is essential for its function in development and as a tumor suppressor (Harris and Levine, 2005; Vousden and Lane, 2007). P53 turnover is regulated by MDM2, which binds p53 and functions as an E3 ubiquitin ligase to promote proteasomal-dependent degradation of p53 (Zhang and Xiong, 2001). Mammalian cells also express an MDM2 homolog called MDMX (Shvarts et al, 1996). MDMX shares with MDM2 a common primary structure and, just like MDM2, interacts with p53 and inhibits its transcriptional activity. Knockout of either MDM2 or MDMX in mice results in embryonic lethality because of ectopic activation of p53 (Montes de Oca Luna et al, 1995; Parant et al, 2001). These experiments have highlighted a critical and non-redundant function for MDM2 and MDMX in the regulation of p53. Moreover, tissue-specific somatic knockout experiments suggest that these proteins have distinct biological functions. Loss of MDMX in different tissues consistently leads to milder phenotypes compared with tissues inactivated for MDM2 (Grier et al, 2006; Maetens et al, 2007). Biochemically, unlike MDM2, MDMX does not have significant intrinsic E3 ligase activity (Stad et al, 2001). Moreover, several ribosomal proteins and ARF specifically bind MDM2 but not MDMX (Wang et al, 2001; Gilkes et al, 2006), whereas casein kinase 1 α binds specifically MDMX and not MDM2 (Chen et al, 2005). Recent studies suggest that the major mechanism of p53 regulation by MDMX is the formation of transcriptionally inactive p53–MDMX complexes, whereas MDM2 primary functions to regulate p53 turnover (Francoz et al, 2006; Toledo et al, 2006). MDM2 contains an N-terminal p53-binding domain, a central acidic domain with regulatory functions, and a C-terminal RING domain that confers E3 ubiquitin ligase activity. MDM2 expression is highly inducible by p53, thus forming a negative feedback loop that limits p53 protein levels. DNA damage and mitogenic signals use several mechanisms to induce p53 activation. DNA damage induces phosphorylation of p53 and MDM2 on multiple residues that weaken p53–MDM2 binding and suppresses MDM2 E3 ligase function (Prives and Hall, 1999; Maya et al, 2001). Mitogenic signals induce expression of ARF, which binds to MDM2 and prevents MDM2-dependent p53 degradation (Zhang and Xiong, 2001). Ribosomal stress activates p53 by releasing several ribosomal proteins from the nucleolus that bind and inactivate MDM2 (Lohrum et al, 2003; Bhat et al, 2004; Dai and Lu, 2004; Jin et al, 2004). Although ubiquitination and degradation of p53 is the best-characterized function of MDM2, the ability of MDM2 to interact with other proteins suggests a more sophisticated mode of action. MDM2 binding to the p53 transactivation domain is sufficient to inactivate p53 by displacing coactivators and histone acetyltransferases such as p300 (Teufel et al, 2007). Furthermore, MDM2 forms a complex with histone deacetylase HDAC1 and inhibits p53 acetylation (Kobet et al, 2000; Ito et al, 2002; Jin et al, 2002). MDM2 also interacts with the nuclear corepressor KAP1, which promotes HDAC1 binding and regulates p53 acetylation (Wang et al, 2005). MDM2 contains a putative transcriptional repression domain and is associated with chromatin in a complex with p53 (Thut et al, 1997; Jin et al, 2002; Arva et al, 2005). These observations suggest that MDM2 actively suppress p53 transcriptional activity through the recruitment of various regulatory factors to p53 target promoters. Chromatin binding by transcription regulators and RNA polymerases is regulated by multiple modifications on core histones (Zhang and Reinberg, 2001). Although acetylation of lysine residues on histone tails are often associated with actively transcribed genes, histone methylation has both positive and negative effects on promoter activity. Histone H3-K4, -K36, -K79 methylation are associated with active transcription, whereas H3-K9 tri-methylation is a hallmark of silenced chromatin. H3-K9 tri-methylation is characteristic of condensed chromatin and transcriptional repression, and is also important for the subsequent establishment of heterochromatin domains by recruitment of HP1 and the associated DNA-methylating enzymes DNMTs (Martin and Zhang, 2005). Abnormal chromatin methylation on histones and DNA are suggested to have critical functions in cancer development by epigenetic silencing of tumor suppressor genes (Baylin and Ohm, 2006). Several enzymes are involved in the methylation of H3-K9, including SUV39H1, EHMT1 and G9a. SUV39H1 is the human ortholog of the Drosophila Su(var)3–9 histone methyltransferase that specifically mediates the tri-methylation of H3-K9 (Aagaard et al, 1999; Rea et al, 2000). Mice and men also express a second isoform SUV39H2 (O'Carroll et al, 2000). Mouse knockout experiments showed that Suv39h1/2 genes are required for viability and proper H3-K9 tri-methylation in pericentric heterochromatin regions (Peters et al, 2001). SUV39H1-null mice are viable but tumor prone (Peters et al, 2001). SUV39H1 forms a complex with pRb to inhibit E2F1 transcriptional activity through methylation of E2F1 target promoters (Nielsen et al, 2001). Rb-mediated recruitment of SUV39H1 to the E2F targets may be required for the formation of senescence-associated heterochromatin foci (Narita et al, 2003). However, H3-K9 methylation and HP1 recruitment also occurs in transcribed regions and may have a function in promoting transcription elongation by suppressing cryptic initiation (Vakoc et al, 2005; Berger, 2007). Therefore, compelling evidence indicate that SUV39H1 is involved in the regulation of H3-K9 methylation at heterochromatin but also, to a lesser extend, at euchromatin domains. EHMT1 (also called GLP) is a close relative to G9a (Ogawa et al, 2002). EHMT1 and G9a function as heterodimers to mediate H3-K9 methylation at euchromatin (Tachibana et al, 2005). Knockout mice have been generated for G9a and EHMT1. The phenotypes for EHMT1 and G9a deficiency are nearly identical, including very early embryonic lethality, drastic reduction of H3-K9 mono- and di-methylation, and HP1 relocalization (Tachibana et al, 2002, 2005). EHMT1 and G9a are thus critical for H3-K9 mono- and di-methylation at euchromatin. This function is distinct from the function of SUV39H1, which is mainly involved in H3-K9 tri-methylation at heterochromatin sites. Recent studies showed that p53 is positively regulated by mono-methylation of K372 by the SET9 methyltransferase (Chuikov et al, 2004), negatively regulated by mono-methylation of K370 by Smyd2 (Huang et al, 2006), positively regulated by di-methylation of K370 by an unknown enzyme (Huang et al, 2007), and negatively inhibited by mono-methylation of K382 by SET8 (Shi et al, 2007). Furthermore, di-methylation of R333, R335, and R337 by PRMT5 alters the promoter selectivity of p53 and favours cell cycle arrest over apoptosis (Jansson et al, 2008). In this report, we show that MDM2 specifically interacts with histone methyltransferase SUV39H1 and EHMT1. MDM2 mediates the formation of p53–MDM2–SUV39H1/EHMT1 complex capable of methylating histone H3-K9, which may account for a paradoxical rise of H3-K9 methylation level during p53 activation. Furthermore, MDM2-dependent recruitment of EHMT1 promotes mono-methylation of p53 at K373. Our results suggest that MDM2–SUV39H1/EHMT1 interactions have a significant function in p53 regulation by methylating histone H3 at p53 target promoters, and to a lesser extent methylating p53 itself. Results MDM2-containing complexes possess histone methyltransferase activity MDM2 may directly inhibit p53 transcriptional activity through its ability to interact not only with transcriptional repressors such as YY1 (Sui et al, 2004) and Kap1 but also with histone methyltransferases. To test whether MDM2-containing complexes possess histone methyltransferase activity, MDM2 was immunoprecipitated from stably transfected H1299 cells and incubated with 3H-SAM and GST fusion containing N-terminal 56 amino acids of histone H3 (GST-H3). Significant methylation of GST-H3 was detected in the MDM2-immunopurified complexes on MG132 exposure (Figure 1A). This treatment was used to block proteasome-dependent degradation of MDM2. Control H1299 cells express very low levels of endogenous MDM2 and showed low background activity. Immunocomplexes of endogenous MDM2 from SJSA cells, a cell line with MDM2 gene amplification, also showed significant activity on MG132 exposure or induction of MDM2 expression using the p53 activator Nutlin (Vassilev et al, 2004) (Figure 1A). Bacteria expressed GST-MDM2 had no activity (data not shown), indicating that the methylation activity was likely because of the presence of cellular methyltransferases in the coprecipitates. Figure 1.MDM2 complex has histone methyltransferase activity. (A) MDM2 was immunoprecipitated from H1299 cells stably transfected with MDM2 or from SJSA cells with amplified MDM2 using antibody 5B10. Cells were treated with 30 μM MG132 for 4 h or with 8 μM Nutlin for 16 h to increase MDM2 level. The MDM2 complex was incubated with 3H-SAM and GST-histone H3-1-56 fusion protein. Methylation of GST-H3 was detected by autoradiography. (B) H1299 cells transiently transfected with FLAG-tagged MDM2, MDMX, and p53 were immunoprecipitated using indicated antibodies and analysed for methylation of GST-H3. Relative protein expression levels were determined by anti-FLAG western blot. Download figure Download PowerPoint MDMX is also an important regulator of p53 transcriptional activity. In transient transfection assays, MDMX IP showed much lower methyltransferase activity compared with MDM2 despite high level of expression (Figure 1B). P53 IP showed weak but detectable activity, which is likely because of interaction with endogenous MDM2 (see below). Furthermore, in contrast to MDM2, MDMX does not seem to interact with methylation enzymes in direct coimmunoprecipitation assays (see Figure 3B). These results indicate that MDM2 but not MDMX is specifically found in complexes together with methyltransferases. MDM2-containing complexes specifically methylate histone H3 lysine 9 To further test the substrate specificity of MDM2-associated methyltransferase activity, MDM2 immunoprecipitated from different cell lines were used in an in vitro methylation assay using a mixture of histones (H1, H2A, H2B, H3, H4) as substrates. The results showed that MDM2-containing complexes predominantly methylate histone H3 (Figure 2A), although weak methylation of H1 was also detected at high MDM2 levels. To identify the histone H3 residue modified by the MDM2 complex, GST-H3 mutants on various lysine residues were used as substrate in the in vitro methylation reaction. The results indicate that K9 is the target of MDM2-dependent methylation (Figure 2B). To further confirm the methylation site, histone H3 and H4 peptides were used as substrates for the in vitro reaction using non-radioactive SAM. The reaction products were analysed by mass spectrometry. The MDM2-containing complexes specifically methylated the H3 peptide but not the H4 peptide. The in vitro reaction generated mostly mono- and di-methylated H3 peptides (Figure 2C). Fragmentation analysis of the methylated H3 peptide produced Y ions that were indicative of K9 methylation (data not shown). We conclude that MDM2 coprecipitates with methyltransferase specific for histone H3-K9. Figure 2.MDM2 associates with H3-K9-specific methyltransferase. (A) Endogenous and transfected MDM2 was immunoprecipitated from indicated cell lines and incubated with 3H-SAM and a mixture of core histones. The reaction products were fractionated by SDS–PAGE and transferred to membrane. The membrane was exposed against film (right panel), stained with Coomassie blue (left panel), and probed for MDM2 level (bottom panel). (B) MDM2 immunoprecipitated from SJSA cells were incubated with GST-H3-1-56 with K-to-R mutations at indicated sites and 3H-SAM. The results suggest that K9 is the target of MDM2-associated methylases. (C) H1299 (low MDM2 level) and SJSA (high MDM2 level) were immunoprecipitated using MDM2 antibody and incubated with histone H3 peptide (aa 1–20) and SAM. The reaction products were analysed by mass spectrometry and the peptides mono-methylated (Δm/z=14) and di-methylated by MDM2 (Δm/z=28) were indicated. Fragmentation analysis of the mono-methylated peptide indicated that K9 was modified (not shown). Download figure Download PowerPoint MDM2 forms a complex with SUV39H1 and EHMT1 To identify the histone methyltransferase responsible for the MDM2-associated enzymatic activity, the ability of MDM2 to bind to several enzymes known to target H3-K9 was tested by coimmunoprecipitation. FLAG-tagged histone methyltransferases and controls (Chk2 and KAP1) were cotransfected with MDM2 and immunoprecipitated using a FLAG antibody. The immunoprecipitates were resolved in denaturing gels and analysed by western blot using MDM2-specific antibody. Use of FLAG-tagged enzymes enabled us to compare the relative binding efficiency of each enzyme to MDM2. The results showed that SUV39H1 and EHMT1 bind significantly to MDM2 (Figure 3A). In contrast, MDMX interaction with the methyltransferases is negligible (Figure 3B), whereas its interaction with Chk2 (which phosphorylates MDMX) and KAP1 used here as positive controls could be detected in the same experimental conditions. These results are consistent with the lack of methyltransferase activity in MDMX IP (Figure 1B). The p53-modifying enzyme SET9 did not interact with MDM2 (Figure 3A). These results indicate that MDM2, but not MDMX, interacts with histone methyltransferase. Among the enzymes analysed, SUV39H1 and EHMT1 showed the highest affinity for MDM2. In separate experiments, the EHMT1 heterodimeric partner G9a also showed significant binding to MDM2 (Figure 3C). Figure 3.MDM2 specifically interacts with EHMT1 and SUV39H1. (A) MDM2 and (B) MDMX were transfected with FLAG-tagged enzymes into H1299 cells. Cells were immunoprecipitated using FLAG antibody and the coprecipitated MDM2 and MDMX were detected by western blot. The filters were probed with rabbit anti-FLAG antibody to determine the levels of enzyme expression. MDM2 and MDMX expression levels were confirmed by western blot of whole-cell extract (WCE). SUV39H1-324 K is a catalytically inactive mutant. (C) H1299 cells transiently transfected with MDM2 and indicated HA-tagged proteins were analysed by HA IP followed by MDM2 western blot to detect the coprecipitation of MDM2. HA-p73 serves as positive control for MDM2 binding. (D) Binding of endogenous MDM2, SUV39H1 and EHMT1 in SJSA cells were detected by IP using SUV39H1 and EHMT1 antibodies followed by MDM2 western blot. (E) MDM2 was immunoprecipitated from MEFs derived from SUV39H1/H2 double-null mice and analysed for methyltransferase activity using GST-H3 as substrate. MDM2-null MEF infected with MDM2 adenovirus served as control. Download figure Download PowerPoint When cell lysates were immunoprecipitated using SUV39H1 or EHMT1 antibodies and probed using MDM2 antibody, coprecipitation of MDM2 with endogenous SUV39H1 and EHMT1 were detectable in cells with high-level MDM2 (SJSA) (Figure 3D). This result indicates that the interactions between MDM2 and SUV39H1 or EHMT1 occur at endogenous levels of expression. Furthermore, using a separate approach, we found that MDM2 immunoprecipitates from Suv39h1/2 double-null MEFs contained significantly lower methyltransferase activity compared with MDM2 complexes from wild-type MEFs (Figure 3E). Endogenous Suv39h1 or Suv39h2 are therefore significant mediators of the MDM2-associated methyltransferase activity in MEFs. As EHMT1 levels are variable in different cell lines (e.g. very low in MEFs, Supplementary Figure S3B), the relative contributions of SUV39H1 and EHMT1 to MDM2-associated methylase activity are likely to be cell type dependent. Given the ability of MDM2 to interact with SUV39H1, it was somewhat surprising that MDM2 IP did not produce tri-methylated H3 peptide in vitro (Figure 2C). Our control experiments using purified FLAG-SUV39H1 showed that high levels of SUV39H1 were needed to produce tri-methylated H3 peptide in the in vitro reaction (data not shown), which was not attainable by MDM2 co-IP of endogenous SUV39H1. In the presence of low levels of SUV39H1, the primary products were mono- and di-methylated H3 peptide because of the sequential nature of the reaction. Mapping of MDM2 and SUV39H1 interaction domains To determine the domain of SUV39H1 that interacts with MDM2, FLAG-tagged deletion mutants of SUV39H1 were constructed and cotransfected with MDM2 in H1299 cells. IP-western blot assay show that a region of SUV39H1 that includes the nuclear localization signal and pre-SET domain is critical for MDM2 binding (Supplementary Figure S1). This binding evidently has a recruiting function and does not affect SUV39H1 enzymatic activity, as shown below. Using a panel of MDM2 deletion mutants, we found that SUV39H1 and EHMT1 interact with the MDM2 acidic domain (Supplementary Figure S2). On the basis of the fact that MDMX does not bind SUV39H1 and EHMT1 (Figure 2B), a panel of MDM2–MDMX hybrid constructs were also tested, which resulted in the same conclusions (data not shown) (Kawai et al, 2003). These results further expand the repertoire of MDM2 acidic domain as a multi-functional protein-binding region. MDM2 mediates the formation of active p53–SUV39H1/EHMT1 complexes We next tested whether MDM2 promotes the binding of SUV39H1 and EHMT1 to p53, which is needed for their recruitment to p53 target promoters. To this end, p53 was cotransfected with MDM2, SUV39H1, and EHMT1. P53 was immunoprecipitated with Pab1801 and tested for its ability to methylate GST-H3. A longer exposure showed that p53 IP contain weak but readily detectable methyltransferase activity for GST-H3 (Figure 4A). SUV39H1 and EHMT1 were not efficiently coimmunoprecipitated with p53, but cotransfection of MDM2 resulted in efficient p53–SUV39H1 and p53–EHMT1 coprecipitation. This observation indicates that MDM2 may act as abridging molecule to allow the formation of trimeric complexes. The p53-associated methyltransferase activity was also significantly stimulated after coexpression of SUV39H1/EHMT1 and MDM2 (Figure 4A). These results indicate that MDM2 mediates complex formation between p53, SUV39H1, and EHMT1. Furthermore, the p53-containing complexes have the ability to methylate histone H3. Quantitatively, SUV39H1 and EHMT1 enzymatic activity were fully retained after interaction with MDM2 and p53 (Figure 4A, compare p53 IP and MDM2 IP). The critical function of MDM2 acidic domain in mediating trimeric complex formation was further confirmed using the 1–200 fragment of MDM2 that does not have the acidic domain (Supplementary Figure S3A). Figure 4.MDM2 mediates formation of active p53–SUV39H1/EHMT1 complex. (A) H1299 cells were transfected with p53, MDM2, FLAG-SUV39H1, and FLAG-EHMT1. P53 and MDM2 IPs were analysed for GST-H3 methylation activity. Coprecipitation of SUV39H1 and EHMT1 was confirmed by probing the p53 and MDM2 IP using FLAG antibody. P53 alone did not coprecipitate SUV39H1 or EHMT1. Coexpression of MDM2 stimulated p53-SUV39H1/EHMT1 binding and increased p53-associated methyltransferase activity. (B) MDM2 is recruited to DNA by p53. H1299 cells were transfected with p53 and MDM2 for 24 h and analysed by ChIP using anti-MDM2 antibody and PCR detection of the p21 promoter DNA. (C) Promoter recruitment of SUV39H1 and EHMT1 by MDM2 and p53. H1299 cells were transfected with p53, MDM2, Myc-SUV39H1, and Myc-EHMT1 for 24 h. Cells were analysed by chromatin immunoprecipitation using Myc antibody and PCR detection of the p21 promoter DNA. Download figure Download PowerPoint As reported earlier (Jin et al, 2002; Minsky and Oren, 2004; White et al, 2006), MDM2 can be recruited to promoters by p53 as shown by chromatin immunoprecipitation (ChIP) assay (Figure 4B). To test whether MDM2 in turn recruits methyltransferases to p53 target promoters, H1299 cells were transfected with p53, MDM2, SUV39H1, and EHMT1. SUV39H1 and EHMT1 recruitment to the p21 promoter was analysed by ChIP. The results showed that in the absence of p53, SUV39H1, and EHMT1 did not bind to the p21 promoter. P53 expression led to weak binding of EHMT1 to p21 promoter. Importantly, MDM2 expression stimulated SUV39H1 and EHMT1 binding to the p21 promoter (Figure 4C). These results showed that MDM2 recruits SUV39H1 and EHMT1 to p53-responsive promoter and may repress p53-mediated transcription by methylating histone H3. MDM2 stimulates EHMT1 methylation of p53 K373 To test whether MDM2-dependent recruitment of SUV39H1 and EHMT1 promotes methylation of p53 itself, p53 was cotransfected with MDM2, SUV39H1, and EHMT1 and immunoprecipitated using specific p53 antibody. The p53 complexes were incubated with 3H-SAM. The data showed that p53 was methylated by EHMT1 in vitro (Figure 5A). Furthermore, MDM2 stimulated EHMT1–p53 interaction and increased p53 methylation (Figure 5A). The E3 ligase deficient MDM2-457S mutant retained its ability to promote p53 methylation. The EHMT1 dimeric partner G9a also showed significant p53-methylation activity, which was further stimulated by high levels of MDM2-457S (Figure 5A). Despite efficient recruitment of SUV39H1 by MDM2, p53 was not methylated by SUV39H1 (Figure 5A). We also did not observe MDM2 methylation by EHMT1 or SUV39H1 in these experiments despite high MDM2 levels in the IP complex (data not shown). We conclude that MDM2 specifically promotes EHMT1- and G9a-mediated p53 methylation. Figure 5.MDM2 stimulates p53 methylation by EHMT1. (A) H1299 cells were transfected with indicated plasmids. P53 was immunoprecipitated and incubated with 3H-SAM. P53 methylation was detected by autoradiography, and coprecipitated proteins were detected by western blot. (B) FLAG-EHMT1 was transfected alone or with MDM2 into H1299 cells. EHMT1 was purified by anti-FLAG IP; MDM2–EHMT1 complex was purified by anti-MDM2 IP and analysed for methylation of His6-p53. EHMT1 was used at different amounts to compare its methyltransferase activity to MDM2–EHMT1 complex. Download figure Download PowerPoint The ability of MDM2 to stimulate p53 methylation by EHMT1 was also tested using a titration assay. Purified EHMT1-methylated His6-p53 in vitro, suggesting that the reaction can occur in the absence of MDM2 (Figure 5B). However, purified EHMT1–MDM2 complex showed 4-fold higher activity in methylating p53 compared with EHMT1 alone. EHMT1 self-methylation was not affected by MDM2 binding, suggesting that MDM2 does not alter EHMT1 catalytic activity (Figure 5B). The data therefore favour a mechanism in which stimulation of p53 methylation by MDM2 depends on its ability to recruit EHMT1 to p53 rather than to stimulate its catalytic activity. EHMT1 did not methylate GST-p53 with C-terminal deletions (Figure 6A) or K-to-R mutation of nine C-terminal lysine residues (Figure 6B). These results indicate that EHMT1 methylates p53 on C-terminal lysines. To more precisely identify the methylation site, single K-to-R mutants of p53 were tested for methylation by the EHMT1–MDM2 complex. The results showed that K373 is critical for the modification (Figure 6B). Similar analysis indicated that G9a methylation of p53 also required K373 (data not shown). Therefore, MDM2 recruitment of EHMT1 stimulates p53 methylation specifically at K373. Figure 6.MDM2 stimulates p53 K373 methylation by EHMT1. (A) MDM2–EHMT1 complex was precipitated from transfected H1299 cells using MDM2 antibody and incubated with recombinant His6-p53 deletion mutants and 3H-SAM. P53 methylation was detected by autoradiography. (B) H1299 cells were transfected w

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