Title: Arginine methylation controls growth regulation by E2F-1
Abstract: Article10 February 2012free access Source Data Arginine methylation controls growth regulation by E2F-1 Er-Chieh Cho Er-Chieh Cho Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UKJoint first authors Search for more papers by this author Shunsheng Zheng Shunsheng Zheng Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UKJoint first authors Search for more papers by this author Shonagh Munro Shonagh Munro Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Geng Liu Geng Liu Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Simon M Carr Simon M Carr Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Jutta Moehlenbrink Jutta Moehlenbrink Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Yi-Chien Lu Yi-Chien Lu Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Lindsay Stimson Lindsay Stimson Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Omar Khan Omar Khan Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Rebecca Konietzny Rebecca Konietzny Nuffield Department of Clinical Medicine, University of Oxford, Henry Wellcome Building for Molecular Physiology, Headington, Oxford, UK Search for more papers by this author Joanna McGouran Joanna McGouran Nuffield Department of Clinical Medicine, University of Oxford, Henry Wellcome Building for Molecular Physiology, Headington, Oxford, UK Search for more papers by this author Amanda S Coutts Amanda S Coutts Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Benedikt Kessler Benedikt Kessler Nuffield Department of Clinical Medicine, University of Oxford, Henry Wellcome Building for Molecular Physiology, Headington, Oxford, UK Search for more papers by this author David J Kerr David J Kerr Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Nicholas B La Thangue Corresponding Author Nicholas B La Thangue Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Er-Chieh Cho Er-Chieh Cho Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UKJoint first authors Search for more papers by this author Shunsheng Zheng Shunsheng Zheng Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UKJoint first authors Search for more papers by this author Shonagh Munro Shonagh Munro Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Geng Liu Geng Liu Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Simon M Carr Simon M Carr Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Jutta Moehlenbrink Jutta Moehlenbrink Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Yi-Chien Lu Yi-Chien Lu Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Lindsay Stimson Lindsay Stimson Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Omar Khan Omar Khan Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Rebecca Konietzny Rebecca Konietzny Nuffield Department of Clinical Medicine, University of Oxford, Henry Wellcome Building for Molecular Physiology, Headington, Oxford, UK Search for more papers by this author Joanna McGouran Joanna McGouran Nuffield Department of Clinical Medicine, University of Oxford, Henry Wellcome Building for Molecular Physiology, Headington, Oxford, UK Search for more papers by this author Amanda S Coutts Amanda S Coutts Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Benedikt Kessler Benedikt Kessler Nuffield Department of Clinical Medicine, University of Oxford, Henry Wellcome Building for Molecular Physiology, Headington, Oxford, UK Search for more papers by this author David J Kerr David J Kerr Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Nicholas B La Thangue Corresponding Author Nicholas B La Thangue Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK Search for more papers by this author Author Information Er-Chieh Cho1, Shunsheng Zheng1, Shonagh Munro1, Geng Liu1, Simon M Carr1, Jutta Moehlenbrink1, Yi-Chien Lu1, Lindsay Stimson1, Omar Khan1, Rebecca Konietzny2, Joanna McGouran2, Amanda S Coutts1, Benedikt Kessler2, David J Kerr1 and Nicholas B La Thangue 1 1Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK 2Nuffield Department of Clinical Medicine, University of Oxford, Henry Wellcome Building for Molecular Physiology, Headington, Oxford, UK *Corresponding author. Department of Oncology, Laboratory of Cancer Biology, University of Oxford, Old Road Campus Research Building, Old Road Campus, Off Roosevelt Drive, Oxford OX3 7DQ, UK. Tel.: +44 1865 617090; Fax: +44 1865 617092; E-mail: [email protected] The EMBO Journal (2012)31:1785-1797https://doi.org/10.1038/emboj.2012.17 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 E2F transcription factors are implicated in diverse cellular functions. The founding member, E2F-1, is endowed with contradictory activities, being able to promote cell-cycle progression and induce apoptosis. However, the mechanisms that underlie the opposing outcomes of E2F-1 activation remain largely unknown. We show here that E2F-1 is directly methylated by PRMT5 (protein arginine methyltransferase 5), and that arginine methylation is responsible for regulating its biochemical and functional properties, which impacts on E2F-1-dependent growth control. Thus, depleting PRMT5 causes increased E2F-1 protein levels, which coincides with decreased growth rate and associated apoptosis. Arginine methylation influences E2F-1 protein stability, and the enhanced transcription of a variety of downstream target genes reflects increased E2F-1 DNA-binding activity. Importantly, E2F-1 is methylated in tumour cells, and a reduced level of methylation is evident under DNA damage conditions that allow E2F-1 stabilization and give rise to apoptosis. Significantly, in a subgroup of colorectal cancer, high levels of PRMT5 frequently coincide with low levels of E2F-1 and reflect a poor clinical outcome. Our results establish that arginine methylation regulates the biological activity of E2F-1 activity, and raise the possibility that arginine methylation contributes to tumourigenesis by influencing the E2F pathway. Introduction E2F is a complex family of transcription factors implicated in different cell fates, including proliferation and apoptosis (Stevens and La Thangue, 2003; Frolov and Dyson, 2004; Polager and Ginsberg, 2008; van den Heuvel and Dyson, 2008). The first family member identified, E2F-1, physically interacts with the retinoblastoma tumour suppressor protein pRb, which negatively regulates E2F-1 activity (Bandara and La Thangue, 1991; Zamanian and La Thangue, 1992; Weinberg, 1995; Stevens and La Thangue, 2003; Frolov and Dyson, 2004). While it has been established that E2F-1 promotes proliferation and therefore is potentially oncogenic, it has also become clear that E2F-1 is endowed with apoptotic activity (Polager and Ginsberg, 2008; van den Heuvel and Dyson, 2008). Thus, many E2F target genes are connected with apoptosis (Ren et al, 2002), and in Rb−/− mice the enhanced level of tissue-specific apoptosis reflects deregulated E2F-1 activity (Tsai et al, 1998; Iaquinta and Lees, 2007). Further, E2F-1−/− mice suffer from an increased incidence of tumours, which is consistent with a role for E2F-1 as a tumour suppressor, possibility reflecting its role in apoptosis (Field et al, 1996; Yamasaki et al, 1996). In contrast, the frequency of tumours seen in Rb+/− mice is reduced upon inactivating E2F-1, arguing that E2F-1 also contributes to tumour progression (Yamasaki et al, 1998). The molecular mechanisms that dictate the opposing outcomes of E2F-1 activity, and basis of the context dependency of these events, remain largely unknown. Not only is E2F-1 regulated during cell-cycle progression through its cyclical interactions with pRb and phosphorylation by Cdk kinases (Stevens and La Thangue, 2003; van den Heuvel and Dyson, 2008), but also under conditions of DNA damage (Pediconi et al, 2003; Stevens and La Thangue, 2003; Stevens et al, 2003). In DNA-damaged cells, E2F-1 is induced in a fashion that follows similar kinetics to p53 (Blattner et al, 1999; Hofferer et al, 1999; Pediconi et al, 2003; Stevens and La Thangue, 2003). DNA damage activates phosphokinases, such as ATM/ATR and Chk1/Chk2, which in turn phosphorylate effector proteins that mediate the outcome of the DNA damage response (Jackson and Bartek, 2009). Both families of DNA damage responsive kinases phosphorylate E2F-1 and augment apoptosis (Stevens and La Thangue, 2003; Stevens et al, 2003). Because tumour cells remain sensitive to the apoptotic effects of E2F-1 (Rodicker et al, 2001; Polager and Ginsberg, 2008), it is possible that mechanisms exist that counter balance and suppress any apoptosis that could result from the inadvertent E2F-1 activity. Arginine methylation is becoming increasingly recognized as an important type of modification in protein control (Bedford and Richard, 2005; Bedford and Clarke, 2009; Kowenz-Leutz et al, 2010), and a variety of processes are know to be influenced by arginine methylation, including RNA processing, chromatin regulation and transcriptional control (Meister et al, 2001; Pal et al, 2004; Kowenz-Leutz et al, 2010). The methylation of arginine residues is catalysed by two groups of protein arginine methyltransferases (PRMT), the type I enzymes that catalyse formation of asymmetric modifications, and type II enzymes that catalyse symmetric modifications (Bedford and Clarke, 2009). In previous studies, p53 was shown to undergo arginine methylation, and a role established for PRMT5 in the methylation process; PRMT5 directed methylation of p53 occurred in cells upon DNA damage, and coincided with activation of the p53 response (Jansson et al, 2008). Further, PRMT5 activity is enhanced by cyclin D/Cdk4 kinase, which thereby contributes to oncogenesis (Aggarwal et al, 2010). In exploring the wider role of arginine methylation in proliferation control, we reasoned that there might be an influence on other key growth regulating pathways, and considered the E2F pathway as one such possibility. Here, we demonstrate that E2F-1 is a direct target for arginine methylation mediated by PRMT5, which occurs on a central motif in the protein, which shares similarity to p53. Arginine methylation regulates E2F-1 DNA-binding and transcriptional activity. Depleting PRMT5 causes an E2F-1-dependent decrease in cell growth, and renders cells sensitive to E2F-1-dependent apoptosis, which coincides with increased levels of E2F-1 protein and expression of downstream E2F target genes. Significantly, E2F-1 is methylated in tumour cells, and demethylated under DNA damage conditions, which cause E2F-1 stabilization and associated apoptosis. Human biopsies taken from malignant disease frequently exhibit high expression of PRMT5, and in colorectal cancer (CRC) a subgroup of tumours exist where high levels of PRMT5 and low levels of E2F-1 correlate with poor prognosis. Our results establish a new mechanism that influences the biological properties and physiological outcome of E2F-1 activity, and highlight the interplay between arginine methylation and E2F-1 that might promote tumourigenesis. Results E2F-1 is methylated by PRMT5 A short sequence motif exists in E2F-1, RGRGR, which is similar to the site of arginine methylation in p53, namely RGRER (Jansson et al, 2008; Figure 1A), and wild-type (WT) E2F-1 could be methylated in vitro by the arginine methyltransferase PRMT5 (Figure 1B). To assess whether the RGRGR motif was a direct target for PRMT5 methylation, a series of mutations were made in which each arginine (R) residue was substituted with a lysine (K) residue (Figure 1A), and the ability of each mutant derivative to be methylated by PRMT5 in vitro then measured. Both R111K and R113K exhibited a greater level of reduction compared with the minimal effect of mutating R109 (Figure 1B). The double R111/113K (referred to as KK) and triple R109/111/113K (KKK) mutants could not be methylated (Figure 1B), and when a short E2F-1 peptide containing the RGRGR sequence was methylated, the same residues (R111 and R113) were required (Figure 1B). Thus, R111 and R113 are the predominant sites of methylation by PRMT5 in vitro. Figure 1.E2F-1 undergoes arginine methylation by PRMT5. (A) Location of the RGRGR sequence motif in E2F-1, highlighting its similarity with the region in p53 targeted by PRMT5 (Jansson et al, 2008). The arginine (R) residues mutated to lysines (K) in the E2F-1 mutants are indicated (in red), together with the designation of the different mutant derivatives. (B) Each of the indicated GST–E2F-1 proteins (about 1 μg; ii) was used in the methylation reaction (i). In vitro methylated samples were analysed by SDS–PAGE as described. Quantitation of either GST–E2F-1 (iv) and associated mutant derivatives (A) or a 20-residue peptide (from residue 100 to 120) together with the equivalent KK and KKK peptides (v) after in vitro methylation by PRMT5. Incorporation of 3H-methyl groups was measured as DPM. Coomassie stain of the GST–E2F-1 proteins is shown in (ii), and the Flag–PRMT5 immunoprecipitated from transfected cells in (iii), which was used in (i, iv, v). (C) Endogenous E2F-1 or PRMT5 was immunoprecipitated with either anti-E2F-1 or anti-PRMT5 from untransfected U2OS cells and subsequently immunoblotted with anti-PRMT5 or anti-E2F-1 as indicated. The input (In) and control (C) immunoprecipitations (IPs) are indicated. (D) Binding of E2F-1 to PRMT5. The indicated GST–E2F-1 proteins were incubated with U2OS cell lysate, and bound proteins immunoblotted using anti-DP-1 or PRMT5 antibodies. The level of GST–E2F-1 input protein (top anti-GST immunoblot) is indicated. (E) Either WT E2F-1 or the indicated mutant derivative expression vectors were transfected into U2OS cells and HA11 antibody was used for IP followed by immunoblotting with HA11 (E2F-1), PRMT5 or DP-1 as indicated. The level of ectopic E2F-1 input (In) protein is shown, and (−) indicates empty vector control transfected cells. Figure source data can be found with the Supplementary data Download figure Download PowerPoint We assessed whether E2F-1 could interact with PRMT5 and thereafter the role of the arginine residues in mediating the interaction. E2F-1 and PRMT5 exist in a complex in cells (e.g., U2OS cells; Figure 1C), and GST–E2F-1 bound to PRMT5 in cell extracts (Figure 1D). When expressed ectopically in cells, each of R109K, R111K and R113K could interact with PRMT5, although the KK and KKK mutants failed to bind to PRMT5 (Figure 1E). Binding to DP-1, the major heterodimeric partner for E2F-1 (Girling et al, 1993; Stevens and La Thangue, 2003), was not affected since it bound equally well to each mutant E2F-1 protein (Figure 1E). These results show that E2F-1 and PRMT5 interact in cells, and that R111 and R113 are the principal sites modified by PRMT5 and required for E2F-1 to interact with PRMT5. E2F-1 is methylated in cells We investigated whether E2F-1 is methylated in cells using a modification-specific peptide antibody that we prepared against a methylated RGRGR peptide derived from E2F-1, in which R111 and R113 were symmetrically methylated. The anti-MeR–E2F-1 antibody recognized ectopic WT E2F-1 immunoprecipitated from cells, but failed to react with either the KK or KKK mutants (Figure 2A), indicating that the antibody recognized the relevant methylated region in E2F-1. Each ectopic protein was immunoprecipitated at an equivalent level and bound equally well to the DP-1 subunit control (Figure 2A). Figure 2.E2F-1 is methylated on arginine residues under physiological conditions. (A) Anti-MeR–E2F-1 recognizes WT E2F-1 but not the KK or KKK mutants. Either WT E2F-1, KK or KKK expression vectors were transfected into HeLa cells and immunoprecipitated (IP) with HA11, followed by immunoblotting (IB) with anti-MeR–E2F-1, HA11 (E2F-1) or DP-1 antibodies. The level of input (In) protein is indicated. (B) Anti-MeR–E2F-1 recognizes arginine methylated E2F-1. Either WT E2F-1, KK or KKK expression vectors were transfected to HeLa cells and IP with HA11, followed by immunoblotting with anti-MeR–E2F-1 or HA11 (E2F-1), either in the presence of methylated (MeR–E2F-1) or unmethylated E2F-1 peptide (1 μg), as indicated. Two different (short and long) exposures of the HA11 (E2F-1) blot are shown. (C) Endogenous E2F-1 is methylated in cells. HeLa or U2OS cell lysates were harvested and IP with anti-E2F-1 (KH95) or control (C) antibody, followed by immunoblotting with anti-MeR–E2F-1 or anti-E2F-1 antibodies. The level of input (In) E2F-1 protein is shown. (D) U2OS cells transfected with E2F-1 expression vector were immunostained with anti-E2F-1 or anti-MeR–E2F-1 in the presence or absence of competing methylated E2F-1 peptide as indicated. DAPI shows the location of nuclei. (E) U2OS cells were transfected with PRMT5 (P5), PRMT1 (P1) or control (C) non-targeting siRNA (50 nM) and harvested at 72 h. Extracts were immunoblotted as indicated. Actin served as the loading control. (F) The extracts described in (E) were IP with anti-E2F-1 (KH95) or control (C) antibody, followed by immunoblotting with anti-MeR–E2F-1 or anti-E2F-1 antibodies as indicated. The level of input (In) E2F-1 protein is shown. (G) U2OS cells were transfected with the indicated E2F-1 mutant derivatives (i, ii) and immunoprecipitated with anti-HA or control (C) antibody, followed by immunoblotting with anti-MeR–E2F-1 or anti-E2F-1 antibodies as indicated. The level of input (In) E2F-1 protein is shown; (iii) shows the vector transfected control. (H) E2F-1 expressed in MCF7 cells was immunopurified as described and subjected to analysis by tandem mass spectrometry. Analysis by LC-MS/MS revealed the presence of four methyl groups present in the peptide fragment GRGR (110–113) in E2F-1 (Sprot Acc Nr: Q01094/ IPI00005630). The MS/MS spectrum of the modified tryptic peptide RLDLETDHQYLAESSGPARGRGR + 4 x CH2-[M+5H]5+ 528.8783 Da (MW 2639.3551 Da) is shown. Fragment ions are indicated as b and y ions. ++ represents loss doubly charged ions. Figure source data can be found with the Supplementary data Download figure Download PowerPoint The specificity of the antibody was established by immunoprecipitating ectopic E2F-1, followed by immunoblotting with anti-MeR–E2F-1 in the presence of competing E2F-1 peptides that differed only in the methylation status of R111 and R113. Anti-MeR–E2F-1 recognized the WT protein, and its binding activity was competed by the methylated but not the unmodified E2F-1 peptide (Figure 2B, compare tracks 6 and 10). As expected, neither the KK nor KKK mutants were recognized by the anti-MeR–E2F-1 antibody (Figure 2B). These results establish that the anti-MeR–E2F-1 peptide antibody detects methylated E2F-1 and, further, that E2F-1 expressed ectopically in cells undergoes arginine methylation. We used the anti-MeR–E2F-1 antibody to study the methylation of endogenous E2F-1 by immunoprecipitation of E2F-1 followed by immunoblotting with anti-MeR–E2F-1. E2F-1 was methylated in different cancer cell lines, including HeLa and U2OS cells (Figure 2C), and further localized to nuclei (Figure 2D). These results indicate that endogenous E2F-1 undergoes arginine methylation at the RGRGR motif. It was important to establish that PRMT5 is responsible for methylating E2F-1 in cells. To pursue this question, we depleted PRMT5 with siRNA (Figure 2E) and thereafter assessed the level of E2F-1 methylation. Methylated E2F-1 was readily detected when E2F-1 was immunoprecipitated from cells, which was reduced when a parallel immunoprecipitation was performed from PRMT5 depleted cells (Figure 2F). The level of E2F-1 methylation was not affected upon depleting another member of the PRMT family, the asymmetric arginine methyltransferase PRMT1 (Figure 2E), as methylation remained at a similar level to the control-treated cells (Figure 2F). Further, methylation of R111K and R113K, in addition to KK and KKK, was reduced compared with WT E2F-1 (Figure 2A and G), thus establishing the methylation of R111 and R113 in cells. Additional analysis by tandem mass spectrometry (Taylor and Goodlett, 2005; Batycka et al, 2006; Fischer et al, 2011) provided further support for di-methylation at R111 and R113 upon in vitro methylation of E2F-1 (Supplementary Figure S1G), and an analysis of E2F-1 immunopurified from MCF7 cells further substantiated methylation at R111 and R113 (Figure 2H). Altogether, these results indicate that E2F-1 undergoes symmetrical arginine methylation under physiological conditions and identify PRMT5 as the enzyme involved. Arginine methylation regulates E2F-1 stability and target gene expression We considered that arginine methylation may play an important role in controlling E2F-1 in DNA-damaged cells, where E2F-1 levels increase (Stevens et al, 2003). When the level of ectopic protein was studied under conditions where each protein underwent equivalent nuclear accumulation (Supplementary Figure S1B), KK and KKK were expressed at increased levels compared with WT E2F-1 (Figure 3A). In part, the difference in protein level was caused by the shorter half-life of WT E2F-1 compared with the mutant derivatives (25–75 min respectively; Figure 3B), which corresponded to the increased steady-state level of KK and KKK compared with E2F-1 (Figure 3A). The R111K and R113K mutant also exhibited an extended half-life relative to WT E2F-1 (Supplementary Figure S2C). One type of modification that was affected by PRMT5, and which might account for different stabilities, was ubiquitination because the mutants exhibited a lower level of ubiquitination relative to WT E2F-1 (Figure 3C). In support of this idea, the level of ubiquitination that occurred on WT E2F-1 was reduced upon PRMT5 depletion, to a level similar to that seen with the KKK mutant (Figure 3D). Figure 3.Properties of arginine methylated E2F-1. (A) Levels of WT E2F-1, KK and KKK after transfection of the indicated expression vectors (1 μg) into U2OS cells and immunoblotting with HA11; – indicates untransfected cells. (B) Stability of WT E2F-1, and the KK and KKK mutants. Expression vectors (1 μg) encoding WT E2F-1, KK and KKK mutants were transfected into U2OS cells for 48 h. Cells were treated with 100 μg/ml of cyclohexamide and then harvested at 0, 2, 4, 6 h post-treatment time points as indicated for subsequent immunoblotting (i), and further quantitated (ii). The HA11 antibody was used for immunoblotting and actin served as protein loading control; n=2. Both the KKK (red) and KK (yellow) mutants had similarly increased half-life compared with WT (blue) E2F-1 (from 75 to 25 min, respectively). (C) Ubiquitination of WT E2F-1 and the KKK mutant. Cells were transiently transfected with expression vectors encoding WT E2F-1 or KKK (2 μg), together with His6-ubiquitin (4 μg) as indicated, and treated with MG132 (20 nM) for 4 h before harvesting. Cell lysates and Ni2+ pull-down eluates were analysed as described. (D) Effect of PRMT5 on E2F-1 ubiquitination: U2OS cells were transfected with PRMT5 (P) or control (C) siRNA. After 24 h, cells were transfected with expression vectors encoding WT E2F-1 or the KKK mutant (2 μg) and His6-ubiquitin (4 μg) as indicated. Cells were treated with MG132 (20 nM) for 4 h before being collected. Cell lysates and Ni2+ pull-down eluates were analysed as described. Graphical presentation of quantitation of ubiquitin signals was performed using ImageJ 1.43u, and the input protein levels are shown underneath. (E) PRMT5 siRNA increases E2F-1 protein levels. PRMT5 (P) or control (C) non-targeting siRNA was transfected into U2OS cells, and cells harvested 72 h post-transfection with or without etoposide (Et) treatment (10 μM) in the last 16 h. Extracts were immunoblotted with anti-PRMT5 and E2F-1, and GAPDH levels served as a loading control. (F) Protein levels of E2F-1 target genes in PRMT5 siRNA-treated cells. PRMT5 (P) or control (C) non-targeting siRNA was transfected into U2OS cells and cells were harvested 72 h post-transfection with or without etoposide (Et; 10 μM) or doxorubicin (Dox; 2 μM) treatment in the last 16 h. Extracts were immunoblotted with anti-PRMT5, E2F-1, p73, Chk2 and Chk1 as indicated. Levels of GAPDH served as the loading control. Download figure Download PowerPoint To explore this possibility in greater detail and establish whether PRMT5 had an effect on endogenous E2F-1, we depleted PRMT5 in cells using siRNA and thereafter measured E2F-1 protein levels. Depleting PRMT5 under conditions that reduced arginine methylation of E2F-1 (Figure 2E and F) caused a coincident increase in E2F-1 protein (about three-fold; Figure 3E), a result that is compatible with the increased stability observed for each of the E2F-1 mutants (Figure 3B; Supplementary Figure S2C). Significantly, the increased E2F-1 levels that occurred upon PRMT5 depletion also coincided with an increase in the expression of a variety of E2F target genes, including p73, Chk2 and Chk1 (Figure 3F). Together, these results establish that PRMT5 regulates the level of E2F-1 protein through altered stability. Functional consequences of arginine methylation We reasoned that the transcriptional activity of E2F target genes might be affected by PRMT5, and tested this idea on a small group of genes in a reporter-based transfection assay. When the WT E2F-1 and KKK mutant were compared on E2F responsive promoters under conditions of equivalent protein expression (Figure 4Aii), the KKK mutant consistently exhibited increased transcriptional activity compared with WT E2F-1 (Figure 4Ai); the R111K, R113K and KK mutants behaved in a similar fashion (Supplementary Figure S2E). Furthermore, when endogenous PRMT5 was depleted, E2F responsive promoters were similarly more active compared with the control treatment (Supplementary Figure S1B). These results suggest, therefore, that arginine methylation also impacts on the ability of E2F-1 to activate transcription. Figure 4.Functional properties of arginine methylated E2F-1. (A) Transcription properties of WT E2F-1 and the KKK mutant. U2OS cells were transfected with expression vectors encoding WT E2F-1 or the KKK mutant, together with p73-luciferase, Cdc6-luciferase, E2F-1-luciferase, Apaf1-luciferase, DHFR-luciferase or cyclin E-luciferase for 48 h as indicated, and pCMV-βgal to monitor transfection efficiency. Relative luciferase activity (luciferase/βgal) is shown together with the expression level of the ectopic proteins underneath; n=3. (B) ChIP of U2OS cells transfected with expression vectors encoding HA-tagged WT E2F-1 or the KKK mutant as described, followed by immunoprecipitation with anti-HA on the E2F target genes, and quantitation by real-time PCR shown in (ii); n=4. (C) ChIP of U2OS cells transfected with PRMT5 (P) or control (C) siRNA followed by immunoprecipitation with anti-E2F-1 antibodies on the indicated E2F target genes, and quantitation by real-time PCR (i); the level of the input protein is shown below (ii); n=2. (D) Effect of PRMT5 siRNA on E2F-1 RNA: PRMT5 (P) or control (C) siRNA was transfected into U2OS cells in the presence or absence of etoposide (Et) and cells harvested at 72 h post-transfection. RNA levels for E2F-1, 18S and GAPDH were assayed as indicated. (E) Effect of PRMT5 (P) or control (C) siRNA on E2F-1 and p73 RNA at 72 h post-transfection and quantitation by real-time PCR. Download figure Download PowerPoint Significantly, by chromatin immunoprecipitation (ChIP), increased binding of the methylation defective mutants compared with WT E2F-1 was evident on the promoter region of E2F target genes when each protein was expressed at an equivalent level (Figure 4B). Upon PRMT5 depletion, increased E2F-1 DNA binding was also apparent on a variety of target genes (Figure 4C). The level of histone H3 K4 acetylation, reflecting an active chromatin environment, also occurred upon PRMT5 depletion (Supplementary Figure S1C). Importantly, the enhanced binding of E2F-1 to endogenous promoters reflected, as expected, increased levels of E2F target gene RNA (Figure 4D and E). These result