E2F4 at the Promoters of Target Genes
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Oncogene (2012) 31, 4207–4220 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE p27Kip1 represses transcription by direct interaction with p130/E2F4 at the promoters of target genes R Pippa1, L Espinosa2, G Gundem3, R Garcı´a-Escudero4, A Dominguez1, S Orlando1, E Gallastegui1, C Saiz4, A Besson5, MJ Pujol1,NLo´pez-Bigas3, JM Paramio4, A Bigas2 and O Bachs1 1Department of Cell Biology, Immunology and Neurosciences, University of Barcelona, IDIBAPS, Barcelona, Spain; 2Institut Municipal d’Investigacions Me`diques-Hospital del Mar, Barcelona, Spain; 3Department of Experimental and Health Science, Research Unit on Biomedical Informatics, Universitat Pompeu Fabra, Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain; 4Molecular Oncology Unit, Division of Biomedicine, CIEMAT, Madrid, Spain and 5INSERM–UMR1037, Cancer Research Center of Toulouse, Toulouse, France The cyclin-cdk (cyclin-dependent kinase) inhibitor p27Kip1 Introduction (p27) has a crucial negative role on cell cycle progression. In addition to its classical role as a cyclin-cdk inhibitor, it The protein p27Kip1 (p27) is a cell cycle regulator whose also performs cyclin-cdk-independent functions as the main known function is the regulation of cyclin-depen- regulation of cytoskeleton rearrangements and cell dent kinase (cdk) activities (Sherr and Roberts, 1999). The motility. p27 deficiency has been associated with tumor importance of p27 as a cell cycle regulator in vivo was aggressiveness and poor clinical outcome, although the revealed by the generation of p27À/À mice that display an mechanisms underlying this participation still remain increase in body size and multiple organ hyperplasia (Fero elusive. We report here a new cellular function of p27 as et al., 1996; Nakayama et al., 1996) (Kiyokawa et al., a transcriptional regulator in association with p130/E2F4 1996). This protein was originally considered as a complexes that could be relevant for tumorigenesis. We ‘universal’ inhibitor of cyclin-cdk complexes whose observed that p27 associates with specific promoters of activity was exerted by the N-terminal half of the molecule genes involved in important cellular functions as proces- (Russo et al., 1996). However, recent reports revealed a sing and splicing of RNA, mitochondrial organization and dual role of p27, behaving as an inhibitor or and activator respiration, translation and cell cycle. On these promoters of cyclin D-cdk4/6 and cyclin E-cdk2 on depending p27 co-localizes with p130, E2F4 and co-repressors as of specific tyrosine phosphorylation (Chu et al., 2007; histone deacetylases (HDACs) and mSIN3A. p27 co- Grimmler et al., 2007; Blain, 2008; James et al., 2008). immunoprecipitates with these proteins and by affinity p27 deficiency is associated with tumorigenesis. Re- chromatography, we demonstrated a direct interaction of duced p27 levels are frequently observed in human cancers p27 with p130 and E2F4 through its carboxyl-half. We in association with tumor aggressiveness and poor clinical have also shown that p130 recruits p27 on the promoters, outcome (Slingerland and Pagano, 2000). In most of the and there p27 is needed for the subsequent recruitment of cases, low levels of p27 are the consequence of an HDACs and mSIN3A. Expression microarrays and increased protein degradation (Frescas and Pagano, luciferase assays revealed that p27 behaves as transcrip- 2008). In some tumors, cytoplasmic localization of p27 tional repressor of these p27-target genes (p27-TGs). is also associated with increased motility, aggressiveness Finally, in human tumors, we established a correlation and poor prognosis (Viglietto et al., 2002). with overexpression of p27-TGs and poor survival. Thus, Quiescent cells contain high levels of p27 mostly this new function of p27 as a transcriptional repressor located in the nucleus. After mitogenic stimulation, it could have a role in the major aggressiveness of tumors translocates to the cytoplasm where it is degraded via with low levels of p27. ubiquitin–proteasome (Shirane et al., 1999). In the Oncogene (2012) 31, 4207–4220; doi:10.1038/onc.2011.582; cytoplasm, p27 performs several cdk-independent activ- published online 19 December 2011 ities. It participates in both actin cytoskeleton rearran- gement and cell motility through the modulation of Keywords: p27; p130; E2F4; transcription RhoA activity, a role that relies in the C-terminal half of p27 (McAllister et al., 2003; Besson et al., 2004). p27 also interacts with stathmin, a microtubule-associated protein, thus regulating cell morphology and motility Correspondence: Professor O Bachs, Department of Cell Biology, (Baldassarre et al., 2005). These regulatory roles of p27 Immunology and Neurosciences, Faculty of Medicine, University of on cell migration have been associated with the cdk- Barcelona, Institut d’Investigacions Biome` diques August Pi i Sunyer independent oncogenic functions of cytoplasmic p27 (IDIBAPS), Casanova 143, 08036-Barcelona, Spain. E-mail: [email protected] (Besson et al., 2008). Received 15 August 2011; revised 29 October 2011; accepted 11 It is assumed that the main function of nuclear p27 November 2011; published online 19 December 2011 during early G1 is to prevent premature entry into S Transcriptional regulatory role of p27Kip1 R Pippa et al 4208 phase by maintaining cyclin E/cdk2 complexes inactive TRANSFAC database, we found that target sequences (Chu et al., 2008). However, whether nuclear p27 in of ETS1, E2F4 and GABP were significantly enriched in quiescent cells solely acts as a CDK inhibitor or may the promoters of these genes (Figure 2a). We subse- fulfill other nuclear functions remains unclear. It has quently analyzed whether p27-TGs were also targeted been postulated that p27 may participate in the by these three mentioned TFs (Hollenhorst et al., 2007; regulation of transcription independently of cyclin-cdk Lopez-Bigas et al., 2008). Results revealed that p27 regulation. It has been shown that the interaction of p27 shared a significant number of target genes with these with neurogenin-2 leads to the differentiation of neural TFs and additionally also with ELF1 and RBP2 progenitors in the cortex. Specifically, p27 stabilizes (Figure 2b and web Supplementary analysis S1). neurogenin-2 by a mechanism that depends on the To analyze the putative interaction of p27 with E2F4, integrity of its N-terminal half but does not require we performed immunoprecipitation (IP) experiments interactions with cyclins and cdks (Nguyen et al., 2006). with anti-p27 and observed that E2F4 co-immunopre- Moreover, the overexpression of p27 in C2C12 cells cipitated with p27 (Figure 2c). This interaction was induces myogenic differentiation, whereas elimination of confirmed by IPs with anti-E2F4 in mice embryo p27 prevents differentiation (Munoz-Alonso et al., fibroblasts (MEFs) p27WT or p27À/À (Supplementary 2005). In another example it has been shown that Figures S2a and b). Moreover, by ChIP, we demon- overexpression of p27 induces the expression of ery- strated that p27 and E2F4 simultaneously associated throid markers in the K562 cell line (Acosta et al., 2008). with several p27-TG promoters enriched with E2Fs- All these data suggested that the nuclear role of p27 in binding sequences (Supplementary Figures S3 and S4) quiescence might rely not only on the inhibition of but not with the control (HoxB8) (Figure 2d). We cyclin-cdk complexes but also on the transcriptional subsequently studied whether p27 interacted with repression of specific target genes. specific co-repressors known to be associated with Thus, we hypothesize that p27 could interact with E2F4. IP experiments revealed that p27 co-immunopre- transcriptional regulators at specific gene promoters. We cipitated with endogenous p130, mSIN3A and histone explored this possibility by chromatin immunoprecipita- deacetylases (HDACs) 1, 4 and 5, indicating that they tion (ChIP) analysis followed by promoter microarrays form complexes in vivo (Figure 2e). The interaction of (chip). Results revealed that p27 associates with a p27 with p130 was confirmed by IPs with anti-p130 number of promoters in quiescent cells. These p27 (Supplementary Figure S2c). Finally, we checked target genes (p27-TGs) are mainly involved in RNA whether all these repressors associated with p27 on processing, translation, cell cycle and respiration. We p27-TG promoters. As observed in Figure 2f, in found that p27 has a role as a transcriptional repressor quiescent cells p27 associated with p130, mSIN3A and in coordination with p130 and E2F4. We have shown HDACs 1, 4 and 5 on several p27-TG promoters but not that p27-TGs are overexpressed in different tumors and on the control gene promoter. We also observed that this is associated with poor clinical outcome. p27 co-immunoprecipitated with ETS1 (Figure 2g). Moreover, ChIP experiments revealed the simultaneous presence of p27 and ETS1 on some p27-TG promoters Results enriched with Ets-binding sites (Figure 2h and Supple- mentary Figure S5). p27 binds to specific gene promoters Quiescent cells contain high levels of p27 mostly in the p130 directly interacts with p27 and recruits it nucleus (Supplementary Figure S1) (Shirane et al., on the p27-TG promoters 1999). Whether p27 has a role, aside from cyclin-cdk To analyze the functional relationship between p27 and inhibition in the nucleus of quiescent cells still remains p130/E2F4 in transcriptional regulation, we performed unclear. We therefore tested the possibility