Cdc25b Is Negatively Regulated by P53 Through Sp1 and NF-Y Transcription Factors
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Oncogene (2011) 30, 2282–2288 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc SHORT COMMUNICATION Cdc25B is negatively regulated by p53 through Sp1 and NF-Y transcription factors M Dalvai1,2,5, O Mondesert1,2, J-C Bourdon3, B Ducommun1,2,4 and C Dozier1,2 1Universite´ de Toulouse, LBCMCP, Toulouse, France; 2CNRS, LBCMCP—UMR 5088 CNRS, University of Toulouse, Toulouse, France; 3Inserm-European Associated Laboratory U858, Department of Surgery and Molecular Oncology, College of Medicine, University of Dundee, Dundee, UK and 4CHU de Toulouse, Toulouse, France Cdc25B phosphatases function as key players in G2/M CDK–cyclin complexes (Boutros et al., 2006). Cdc25B is cell cycle progression by activating the CDK1–cyclinB1 thought to act as the starter of mitosis, being responsible complexes. They also have an essential role in recovery for the initial dephosphorylation and activation of the from the G2/M checkpoint activated in response to DNA CDK1–cyclinB1 complex (Lindqvist et al., 2005). damage. Overexpression of Cdc25B results in bypass of Cdc25B is also essential for cell cycle recovery after the G2/M checkpoint and illegitimate entry into mitosis, DNA-damage induced checkpoint and its overexpres- and also causes replicative stress, leading to genomic sion results in bypass of the G2/M checkpoint and instability. Thus, fine-tuning of Cdc25B expression level illegitimate entry into mitosis, and also causes replica- is critical for correct cell cycle progression and G2 tive stress, leading to genomic instability (Miyata et al., checkpoint recovery. However, the transcriptional regula- 2001; Bugler et al., 2006). Thus, fine-tuning of Cdc25B tion of Cdc25B remains largely unknown. Earlier studies expression level is critical for correct cell cycle progres- have shown that the tumor suppressor p53 overexpression sion and for scheduled recovery from a G2 checkpoint transcriptionally represses Cdc25B; however, the mole- activated in response to DNA damage. cular mechanism of this repression has not yet been Upregulation of Cdc25B has been documented in a elucidated, although it was suggested to occur through the wide variety of human cancers, often associated with induction of p21. Here we show that Cdc25B is down- high-grade tumors and poor prognosis (Boutros et al., regulated by the basal level of p53 in multiple cell types. 2007). The mechanism leading to this upregulation is This downregulation also occurs in p21À/À cell lines, as yet unknown, but excludes gene amplification or indicating that p21 is not required for p53-mediated rearrangement. The components of the signaling path- regulation of Cdc25B. Deletion and mutation analyses of ways regulating Cdc25B levels are therefore likely to the Cdc25B promoter revealed that downregulation by be altered in cancer cells. This raises the major issue of p53 is dependent on the presence of functional Sp1/Sp3 understanding how and when Cdc25B levels are and NF-Y binding sites. Furthermore, chromatin immuno- regulated during cell cycle to allow entry into mitosis precipitation analyses show that p53 binds to the Cdc25B and recovery from a checkpoint arrest. promoter and mediates transcriptional attenuation The transcription factor p53 is activated in response through the Sp1 and NF-Y transcription factors. Our to a variety of cellular stresses, such as DNA damage results suggest that the inability to downregulate Cdc25B or oncogenic signal, and regulates expression of genes after loss of p53 might contribute to tumorigenesis. impinging on cell cycle arrest, senescence and apoptosis Oncogene (2011) 30, 2282–2288; doi:10.1038/onc.2010.588; (Riley et al., 2008). In particular, p53 represses impor- published online 17 January 2011 tant G2/M regulators such as cyclin B, Plk1, cdc2, Cdc25C, survivin and FoxM1 (Wang et al., 2010), Keywords: Cdc25B; p53; Sp1; NF-Y establishing a role for p53 at the G2/M transition. Recent studies show that p53 can regulate expression of genes in the absence of any cellular stresses, uncovering Introduction an additional role of the p53 protein that is expressed under basal physiological and cell-culture stress condi- The Cdc25 phosphatases (Cdc25A, B and C) regulate tions (Vousden and Prives, 2009). Using microarray eukaryotic cell-cycle progression through the depho- analysis, Cdc25B has been recently identified as a gene sphorylation and activation of their substrates, the transcriptionally repressed upon p53 overexpression (Scian et al., 2008). However, no molecular mechanism Correspondence: Dr C Dozier, LBCMCP—UMR 5088 CNRS, has been described concerning this repression. In part of University of Toulouse, Bat 4R3B1, 118 Route de Narbonne, our effort to decipher the regulation of the Cdc25B Toulouse 31062, France. phosphatase, we have analyzed the repression of E-mail: [email protected] Cdc25B by p53. Here we show that Cdc25B is down- 5Current address: CNRS, LBME-UMR5099, F-31062 Toulouse, France regulated by the basal level of the tumor suppressor p53 Received 8 July 2010; revised 1 November 2010; accepted 30 November in multiple cell types and elucidate the mechanism 2010; published online 17 January 2011 of this downregulation. Cdc25B is negatively regulated by p53 M Dalvai et al 2283 Results and discussion accumulation of p53 by nutlin-3 led to the down- regulation of Cdc25B at both the mRNA and protein We observed that Cdc25B protein level was lower levels in p21À/À HCT116, indicating that p53 can in HCT116 human colon carcinoma cells expressing downregulate Cdc25B independently of p21. However, wild-type (WT) p53 than in their isogenic counterparts the downregulation was not as pronounced in p21À/À lacking p53 (HCT116 p53À/À) (Figure 1a). Cdc25B HCT116 as that observed with WT cells. This results is an unstable protein degraded in a proteasome- probably from the upregulation of the p53 target gene dependent manner (Baldin et al., 1997a). Treatment of p21 in nutlin-treated WT HCT116 cells, as in earlier HCT116 WT or p53 null cells with the translational studies Cdc25B was also repressed by overexpression inhibitor, cycloheximide, or the proteasomal inhibitor, of p21 (Scian et al., 2008). Therefore, it is likely that MG132, at various times showed that the higher there exist p21-dependent and -independent pathways of Cdc25B protein level observed in HCT116 p53 null repression. cells was not the result of deregulation of protein It has been previously shown that a 941-bp fragment degradation or synthesis (Supplementary Figure S1). of the human Cdc25B promoter was able to mediate Analysis of the Cdc25B expression at the mRNA level the repression by overexpressed p53 (Scian et al., 2008). showed that HCT116 WT cells also express a lower In silico analyses of this sequence revealed no consensus Cdc25B mRNA level than p53À/À cells (Figure 1b). p53-binding sites. However, p53 can repress indirectly To examine further whether the correlation observed by interfering with transcriptional factors that usually between absence of p53 and increased Cdc25B levels in transactivate the genes (Riley et al., 2008; Wang et al., HCT116 p53À/À cells stems from p53-dependent effect, 2010). To identify promoter elements involved in p53- we invalidated p53 expression by RNA interference in mediated regulation of Cdc25B, progressively deleted HCT116 WT cells. Although p53 expression was fragments of the Cdc25B promoter were linked to the significantly downregulated in p53 siRNA-transfected luciferase reporter gene and the resulting constructs cells compared with mock- or scr siRNA-transfected were transfected in HCT116 cells previously transfected cells, increased levels of Cdc25B protein and mRNA with p53 siRNA or scr siRNA as control. Luciferase were observed (Figure 1c). No change in Cdc25B assays showed that invalidation of p53 increased the expression was observed in HCT116 p53À/À cells activities of the –911, –575, –227 and –122 luciferase transfected with p53 siRNA (Figure 1c). To determine constructs, but not that of the –51 luciferase construct, whether this observation could be extended to other suggesting the presence of p53 response elements within cell types, we invalidated p53 in IMR-90 and MCF-7 the –122 to –51 region of the Cdc25B promoter cells and found that Cdc25B was upregulated in p53 (Figure 3a). This region contains two GC-rich motifs siRNA-transfected cells (Figure 1c and Supplementary for binding Sp1/Sp3 and one CAAT box for binding Figure S2). As the expression of Cdc25B is cell cycle NF-Y, both conserved between human and mouse regulated, being initiated during S-phase, peaking in G2 genomes (data not shown), and important for transcrip- and declining in mitosis (Baldin et al., 1997b; Korner tional activity of the Cdc25B promoter (Korner et al., et al., 2001), we analysed the cell cycle distribution of the 2001), as well as two juxtaposed cell cycle-dependent siRNA-transfected cells and found that both scr and p53 element motifs (Figure 3b). There have been several siRNA-transfected cells showed similar cell-cycle dis- reports of p53 repressing genes via Sp1/Sp3, NF-Y or tribution profile (Figure 1d). Thus, increased Cdc25B cell cycle-dependent element sequences (Muller and expression in p53-depleted cells is a direct effect of p53 Engeland, 2009; Wang et al., 2010). To clarify the role and not due to alteration of cell-cycle progression. These of these elements in the p53-mediated regulation of data indicate that Cdc25B is downregulated by the basal Cdc25B, we mutated the binding sites for Sp1/Sp3 and level of p53. NF-Y as well as the cell cycle-dependent element motifs, It has been suggested that overexpression of p53 may singly or in combinations, within the –122 luciferase repress Cdc25B expression through the induction of p21 construct. The promoter mutants were then transfected (Scian et al., 2008). To investigate the role of p21 in the into HCT116 cells invalidated for p53 expression. p53-dependent regulation of Cdc25B, we invalidated Luciferase assays (Figure 3c and Supplementary Figure p53 in the p21 null derivative of HCT116 cells (HCT116 S3) showed that invalidation of p53 into HCT116 cells p21À/À).