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¹ Human Gene Sciences Center, and
² Section of Molecular Embryology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
³ Departments of Obstetrics and Gynecology and Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, 12800 East 19th Avenue, Aurora, CO 80045, USA

	Abstract

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The transcription factor E2F, the main target of the RB tumor suppressor pathway, plays crucial roles not only in cell proliferation but also in tumor suppression. The cyclin-dependent kinase inhibitor p27^Kip1 gene, an upstream negative regulator of E2F, is induced by ectopically expressed E2F1 but not by normal growth stimulation that physiologically activates endogenous E2F. This suggests that the gene can discriminate between deregulated and physiological E2F activity. To address this issue, we examined regulation of the p27^Kip1 gene by E2F. Here we show that p27^Kip1 promoter specifically senses deregulated E2F activity through elements similar to typical E2F sites. This E2F-like elements were activated by deregulated E2F activity induced by forced inactivation of pRb but not by physiological E2F activity induced by serum stimulation, contrary to typical E2F sites activated by both E2F activity. The endogenous p27^Kip1 gene responded to deregulated and physiological E2F activity in the same manner to the E2F-like elements. Moreover, the E2F-like elements bound ectopically expressed E2F1 but not physiologically activated E2F1 or E2F4 in vivo. These results suggest that the p27^Kip1 gene specifically senses deregulated E2F activity through the E2F-like elements to suppress inappropriate cell cycle progression in response to loss of pRb function.

	Introduction

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In response to inappropriate cell cycle promotion induced by oncogenic changes, cells commit apoptosis or enter into irreversible cell cycle arrest to avoid tumorigenesis (Lowe et al. 2004). However, the mechanism of how cells discriminate between normal and abnormal growth signals is largely unknown.

Defects in the retinoblastoma tumor suppressor (RB) pathway and consequent activation of the transcription factor E2F are observed in almost all tumors (Sherr 1996). E2F plays crucial roles in cell cycle progression by regulating a variety of growth-related genes in response to growth stimulation. E2F also plays important roles in tumor suppression likely through its ability to induce apoptosis or cell cycle arrest (Wu & Levine 1994; Dimri et al. 2000; Lomazzi et al. 2002). Consistent with this, E2F can also regulate a group of genes involved in apoptosis or suppression of cell cycle progression (DeGregori 2002; Ginsberg 2002; Trimarchi & Lees 2002). E2F regulation of these proapoptotic and cell cycle suppressive genes is inconvenient for normal cell proliferation, raising an important issue regarding how expression of these genes is regulated by E2F in response to normal and abnormal growth stimulations.

We previously showed that E2F regulation of the tumor suppressor gene p14^ARF, an upstream regulator of the tumor suppressor p53, is distinct from that of the growth-related target genes (Komori et al. 2005). The p14^ARF gene is activated by deregulated E2F activity induced by forced inactivation of pRb but not by physiological E2F activity induced by serum stimulation of fibroblasts, one of normal growth stimulations. In contrast, growth-related E2F targets are activated by both of E2F activity. The distinct regulation of the ARF gene is mediated through the unique E2F-responsive element of the p14^ARF promoter (EREA), whose sequence is diverged from that of typical E2F binding sites in growth-related E2F target genes. These results suggest that there is a distinct E2F-mediated transcriptional program, which specifically senses defective pRb function to activate the p53 pathway.

p27^Kip1 (hereafter p27) is a member of Cip/Kip family of CDK inhibitors, which are upstream negative regulators of E2F. Intriguingly, the p27 gene has been identified as a target of ectopically expressed E2F1, a member of activator E2Fs, suggesting a negative feedback in cell cycle control (Wang et al. 2005; Iwanaga et al. 2006). Importantly, expression of the p27 gene is not induced by serum stimulation of fibroblasts (Iwanaga et al. 2006), one of the normal growth stimulations that physiologically activate endogenous E2F inducing the growth-related genes. These observations suggest that the negative feedback functions only upon deregulation of E2F activity. To address this issue, we analyzed regulation of the p27 gene by physiological and deregulated E2F activity. Our results indicate that the p27 gene specifically senses deregulated E2F activity originating from defective pRb function through E2F-like elements in the promoter. This suggests that the distinct E2F-mediated transcriptional program activates not only the p53 pathway but also the p27 gene to suppress CDK activity to restrain inappropriate cell cycle progression in response to defective pRb function.

	Results

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The p27 gene is activated by deregulated E2F activity but not by physiological E2F activity induced by serum stimulation

To explore the regulatory mechanism of the p27 gene by deregulated and physiological E2F activity, we first examined E2F regulation of p27 promoter by reporter assay in human foreskin fibroblasts (HFFs). As the source of deregulated E2F activity, we used ectopic expression of E2F1 and forced inactivation of pRb by the 2RG mutant form of adenovirus E1a, which binds and inhibits all members of the pRb family but does not interfere with p300/CBP co-activators (Alevizopoulos et al. 1998), and by short hairpin RNA (shRNA) against RB1 (RB shRNA), which was successfully used in previous study (Komori et al. 2005).

Ectopically expressed E2F1 increased p27 promoter (–1408) activity by 2.1-fold (Fig. 1A). A 5' deletion mutant (–635) was not activated at all, suggesting that the activation is specific to the p27 promoter (–1408). Although the increase in the p27 promoter activity is relatively small, it parallels the increase in the mRNA level and has a huge impact on protein expression (Iwanaga et al. 2006), possibly as a result of accumulation of the protein. This observation suggests that p27 gene expression should be finely regulated at the level of transcription.

View larger version (24K): [in this window] [in a new window]   Figure 1  The p27 gene is activated by deregulated E2F activity but not by physiological E2F activity induced by serum stimulation. (A, B) p27 promoter (–1408) was transfected into human foreskin fibroblasts (HFFs) together with expression vector for E2F1 (A) or 2RG form of E1a mutant (B), along with pCMV-β-gal by lipofection. The cells were then cultured under the serum-starved conditions for 1 day and were harvested for measurement of luciferase activity. Luciferase activity was normalized by β-galactosidase activity as an internal control. p27 promoter (–635), cdc6 promoter, ARF promoter (–736) and ARF core promoter (–66) were used as controls. All experiments were repeated at least three times in duplicate. Fold activation was presented relative to ARF core promoter as mean ±  SD. (C) HFFs were infected with RB shRNA virus or control virus at multiplicity of infection (MOI) of 500 and cultured under the serum-starved conditions for 2 days. Cells were then re-stimulated with serum for 1 day and were harvested. Expression of RB1 mRNA and pRb was examined by Northern and Western blot analyses, respectively. ARPP and -tubulin are internal controls, respectively. (D, E) HFFs were transfected with indicated reporter plasmids with (D) or without (E) expression vector for RB shRNA along with pCMV-β-gal. The cells were then cultured under the serum-starved conditions for 2 days, re-stimulated with serum for 1 day and harvested for measurement of luciferase activity normalized by β-galactosidase activity. *P < 0.01. (F) Responsiveness of the indicated reporter plasmids to E2F1 through E2F5 was similarly examined as in the legend to Fig. 1A. (G) The amount of E2F1 and E2F3 was examined by Western blot analysis. For serum stimulation and RB shRNA experiments, HFFs were not infected or infected with the RB shRNA virus at MOI of 500, cultured under the serum starved conditions for 2 days, re-stimulated with serum for 1 day and harvested. For E2F1 and E1a experiments, HFFs were infected with the E2F1 or E1a virus at MOI of 200, cultured under the serum-starved conditions for 1 day and harvested. 293A cells transfected with the expression vector for E2F1 or E2F3 were used as positive controls. (H) The level of p27 mRNA was examined by Northern blot analysis. HFFs were infected with the recombinant adenoviruses in the same manner as in legend to Fig. 1G. Fold induction of p27 gene expression, normalized by ARPP gene expression as an internal control, is indicated. (I) The expression of p27 protein was examined by Western blot analysis. HFFs were infected with the recombinant adenoviruses in the same manner as in legend to Fig. 1G.

Re-stimulation of serum-starved HFFs by serum induced about 70% of the cells to enter into S phase as examined by bromodeoxyuridine uptake (data not shown). Serum stimulation activated the cdc6 promoter, one of the growth-related E2F targets (Ohtani et al. 1998), 3.3-fold (Fig. 1E), indicating that endogenous E2F is physiologically activated. In sharp contrast, serum stimulation did not activate the p27 promoter at all (Fig. 1E). These results suggest that the p27 promoter specifically senses deregulated E2F activity like the p14^ARF promoter.

To investigate whether E2F activation of the p27 promoter is specific to E2F1 or common for other E2F family members, we examined whether E2F2 through E2F5 could activate the p27 promoter. E2F2 and E2F3, albeit slightly, activated the p27 promoter (Fig. 1F). However, E2F4 and E2F5 did not. This result suggests that activator type E2Fs, but not repressor type E2Fs, activate the p27 promoter upon deregulation. It is proposed that total amount of activator type E2Fs determines whether proapoptotic E2F targets are activated or not (threshold model) (Trimarchi & Lees 2002). We thus examined the amount of activator type E2Fs by Western blot analysis upon overexpression of E2F1 or forced inactivation of pRb by E1a or RB shRNA in HFFs. As transfection efficiency of HFFs is too low to perform biochemical analyses, we utilized recombinant adenovirus to introduce E2F1, E1a or RB shRNA that is though to infect the entire population of the HFFs. First, we titrated multiplicity of infection (MOI) of recombinant adenoviruses expressing E2F1, E1a or RB shRNA to determine the conditions, which give similar fold activation of the promoters by reporter assay. Using the conditions, we examined the amount of activator type E2Fs by Western blot analysis. As expected, the amount of E2F1 protein ectopically introduced was dramatically higher than that induced by serum stimulation (Fig. 1G upper panel). However, the amount of E2F1 protein induced by E1a was similar to that induced by serum stimulation. Moreover, the amount of E2F1 protein induced by RB shRNA was lower than that induced by serum stimulation. Similarly, although the amount of E2F3 protein induced by E1a was clearly higher than that induced by serum stimulation, the amount of E2F3 protein induced by RB shRNA was lower than that induced by serum stimulation (Fig. 1G lower panel). The amount of E2F2 protein was too low to detect under our conditions (data not shown). These results suggest that, inconsistent with the threshold model, the activation of the p27 promoter by deregulated E2F is not necessarily because of higher amounts of E2F protein compared to that induced by serum stimulation. They also suggest that there may be functional difference between the deregulated E2F activity and physiologically induced E2F activity.

To confirm that the endogenous p27 gene responds to deregulated and physiological E2F activity in a similar manner to the isolated promoter, we examined the expression of the p27 gene in HFFs infected with the recombinant adenoviruses. The expression of E2F1, adenovirus E1a and RB shRNA induced p27 gene expression 1.5-fold, 1.3-fold and 1.2-fold, respectively (Fig. 1H). However, serum stimulation slightly reduced p27 gene expression. We also examined whether p27 gene expression is reflected in protein expression. As expected, the expression of E2F1, adenovirus E1a and RB shRNA increased the p27 protein level whereas serum stimulation reduced the protein level (Fig. 1I). These results indicate that the endogenous p27 gene does indeed respond to deregulated E2F activity but not to physiological E2F activity induced by serum stimulation in a similar manner to the isolated promoter.

The regulation of the p27 gene by E2F is different from growth-related targets in that the p27 gene is not induced by physiological E2F activity, suggesting that the p27 gene discriminates deregulated from physiological E2F activity. However, there is an alternative possibility that, although physiological E2F activity induced by serum stimulation does activate the p27 promoter, the activation is negated by repression through another pathway caused by serum stimulation independent of E2F. To test these possibilities, we identified the E2F responsive element in the p27 promoter to examine whether the distinct regulation is directly mediated by E2F.

Identification of the E2F responsive element of p27 promoter

We first assessed the E2F1 responsive region using 5' deletion mutants of the p27 promoter (Fig. 2A). A remarkable decrease in E2F1-responsiveness was observed between (–814) and (–635) mutants, suggesting that the E2F1-responsive element resides in the –814 to –635 region. To narrow down the E2F1-responsive element in this region, we isolated parts of the region and examined E2F1-responsiveness in the context of the ARF core promoter (Komori et al. 2005). The analysis identified relatively short regions, –813 to –802 (12 bp) and –801 to –785 (21 bp), being responsive to ectopically expressed E2F1 (Fig. 2B). These regions contain sequences similar to the E2F binding consensus sequence (TTT^C/_G^G/_CCGC), TTAGGCGC and GTTGGCGG, which are located in opposing directions as often observed in typical E2F-target promoters (Fig. 2C). Two point mutations in the GC stretches in these sequences completely abolished E2F1-responsiveness (Fig. 2B). Introduction of the same mutations into the p27 promoter (–1408) almost abolished E2F1-responsiveness (Fig. 2D), confirming that the two regions are the main E2F1-responsive regions. Here, we refer the –813 to –802 region as EREK1 (E2F-Responsive Element of Kip1 part 1) and the –801 to –785 region as EREK2.

View larger version (15K): [in this window] [in a new window]   Figure 2  Identification of E2F-responsive element of p27Kip1 promoter (EREK). (A) A set of 5' deletion mutants of p27(–1408) was examined for E2F1-responsiveness as in legend to Fig. 1A. (B) Isolated p27 promoter fragments were similarly examined for E2F1-responsiveness in the context of ARF core promoter (–66). EREK1m and EREK2m indicate mutants of EREK1 and EREK2, respectively. EREK(1+2) indicates EREK1 + EREK2. (C) Sequences were compared between typical E2F site, EREK1, EREK2 and EREA (E2F-responsive element of p14ARF promoter). (D) p27(–1408) mutant p27(–1408)pm was similarly examined for E2F1-responsiveness. (E) p27(–1408) and p27(–1408)pm were examined for serum-responsiveness as in legend to Fig. 1E.

EREKs are specifically activated by deregulated E2F activity

We discovered that EREKs play a pivotal role in regulation of the p27 promoter in response to ectopically expressed E2F1. Thus, we examined whether EREKs are activated by deregulated endogenous E2F induced by E1a or RB shRNA, and by serum stimulation, representing normal growth stimulation. E1a activated EREK2 1.7-fold, but did not remarkably affect EREK1 (Fig. 3A). As EREK1 and EREK2 are located next to each other, and the responsiveness of EREKs to E1a is relatively small and may hinder further analyses, we used –813 to –785 fragment containing EREK1 and EREK2 thereafter, and referred to this element as EREK(1+2). As expected, EREK(1+2) showed bigger activation (2.2-fold) by E1a than each EREK alone. Two point mutations in EREK1 and EREK2 in EREK(1+2) completely abolished responsiveness to E1a and to ectopically expressed E2F1 (Fig. 3A,B). Basal promoter activity of the EREK(1+2) mutant was not elevated under the serum-starved conditions (data not shown), confirming that EREKs are not under repression in the resting state unlike E2F sites of typical E2F target promoters (Dyson 1998; Ohtani 1999).

View larger version (17K): [in this window] [in a new window]   Figure 3  EREKs are activated by deregulated E2F activity but not by physiological E2F activity induced by serum stimulation. (A) EREK1 and EREK2, along with their mutants, were examined for E1a-responsiveness in combination with ARF core promoter (-66) as in the legend to Fig. 1B. EREK(1+2) m indicates mutant form of EREK(1+2), in which both EREK1 and EREK2 were mutated. Three-tandem repeat of EREA was used as a positive control. (B) EREK(1+2) and EREK(1+2)m were examined for E2F1-responsiveness as in the legend to Fig. 1A. (C) EREK(1+2) and EREK(1 + 2)m were examined for responsiveness to knockdown of pRb expression as in the legend to Fig. 1D. *P < 0.01. (D) EREK(1+2) and EREK(1+2)m were examined for serum-responsiveness as in the legend to Fig. 1D. (E) Responsiveness of the indicated reporter plasmids to E2F1 through E2F5 was similarly examined as in the legend to Fig. 1A.

p27 promoter binds ectopically expressed E2F1 but not physiologically activated E2F1 or E2F4

To further explore the molecular mechanism of E2F regulation of the p27 gene, we examined the ability of EREK(1+2) to bind ectopically expressed E2F1 and endogenous E2F1 physiologically induced by serum stimulation in vitro and in vivo. First, we carried out gel mobility shift assays using a lysate from serum-starved normal human fibroblast WI-38 cells infected with recombinant adenovirus expressing E2F1 (Ad-E2F1) or control virus (Ad-Con). When a fragment from the DHFR promoter containing typical E2F sites (DHFR E2F sites) was used as a probe, E2F4 and the E2F4/p130 complex were observed with the lysate from WI-38 cells infected with the control virus (Fig. 4A left). With the lysate from WI-38 cells infected with the E2F1 virus, ectopically expressed E2F1 and E2F1/pRb complex were evident. Although EREK(1+2) had the ability to compete out these E2F complexes, competition was less efficient compared to typical E2F sites from adenovirus E2 enhancer (E2WT). When EREK(1+2) was used as a probe, it showed weaker but specific E2F binding activity only with lysate from Ad-E2F1 infected cells (Fig. 4A right). Binding of ectopically expressed E2F1 was confirmed by antibody supershift assay (Fig. 4B). These results indicate that EREK(1+2) specifically binds ectopically expressed E2F1 in vitro with lower affinity than typical E2F sites.

View larger version (27K): [in this window] [in a new window]   Figure 4  EREKs bind deregulated E2F but not physiologically activated E2F. (A) Gel mobility shift assay was carried out with typical E2F binding sites from DHFR promoter (left panel) or EREK(1+2) (right panel) as a probe using cell lysate from serum-starved normal human fibroblast WI-38 cells infected with E2F1-expressing (Ad-E2F1) or control adenovirus (Ad-Con). Asterisk indicates E2F-specific binding activity. Competition experiments were carried out with 100 or 500 molar excess of cold competitors. E2WT and E2Mut indicate adenovirus E2 enhancer fragment containing typical E2F sites and its mutant form, respectively. (B) Antibody supershift experiments. Mnt is a negative control. Asterisks indicate supershifts. (C) Chromatin immunoprecipitation (ChIP) experiment was carried out using serum starved WI-38 cells infected with control virus or E2F1 virus, or stimulated with serum.

RB-deficient tumor cell lines retain the deregulated E2F activity to activate EREKs

Our results indicate that forced inactivation of pRb in normal human fibroblasts generate deregulated E2F activity to activate EREKs. We further examined whether RB-deficient tumor cells retain the deregulated E2F activity to activate EREKs. For this purpose, we examined whether EREKs have activity in RB-deficient human tumor cell lines, 5637, Saos-2 and C-33A by reporter assay. Combination of EREK(1+2) to ARF core promoter (–66) enhanced the promoter activity in all of the RB-deficient tumor cell lines, whereas EREK(1+2) mutant did not (Fig. 5A). In contrast, combination of EREK(1+2) to ARF core promoter (–66) did not enhance the promoter activity in normal human fibroblast HFFs. These results suggest that the RB-deficient tumor cell lines, but not HFFs, harbor the deregulated E2F activity to activate EREKs. To confirm the activity of EREK(1+2) in the RB-deficient tumor cells is because of deregulated E2F activity, we examined whether re-introduction of pRb into these cells suppressed activity of EREK(1+2). Co-expression of constitutive active form of pRb (PSM.7 LP) reduced the activity of EREK(1+2) but not its mutant in all of the RB-deficient tumor cell lines. In contrast, co-expression of pRb did not reduce the activity of EREK(1+2) in HFFs, whereas it clearly suppressed that of cdc6 promoter. These results indicate that the RB-deficient tumor cell lines retain the deregulated activity to activate EREKs.

View larger version (18K): [in this window] [in a new window]   Figure 5  RB-deficient tumor cell lines retain the deregulated E2F activity to activate EREKs. (A) RB-deficient human tumor cell lines 5 637, Saos-2 and C-33A were transfected with EREK reporter plasmids along with an expression vector for constitutive active form of pRb (PSM.7-LP). The cells were cultured for 1 day and harvested for measurement of luciferase activity. HFFs were used as a negative control and EREAx3 and cdc6 reporter plasmids were used as positive controls. (B) A model for the role of E2F regulation of the p27 gene upon loss of pRb function.

	Discussion

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In this study, we showed that the p27 promoter specifically senses deregulated E2F activity through EREKs. EREK(1+2) responded to deregulated E2F activity induced by ectopically expressed E2F1 through E2F3, inactivation of pRb by adenovirus E1a or RB shRNA, but not to physiological E2F activity induced by serum stimulation (Fig. 3A–E). Moreover, our gel shift and ChIP assays showed that the p27 promoter preferentially bound ectopically expressed E2F1 and not endogenous E2F4 or physiologically activated E2F1 in vitro and in vivo (Fig. 4A–C). These results indicate that the p27 gene is activated by deregulated E2F activity but not by physiological E2F activity in a similar manner to the tumor suppressor ARF gene (Komori et al. 2005). Indeed, the ARF gene was also induced under the same condition that the p27 gene was induced (Komori et al. 2005 and data not shown). In addition, all of the RB-deficient tumor cell lines that harbor the deregulated E2F activity to activate EREA (Komori et al. 2005) also retained the activity to activate EREKs (Fig. 5A). Thus the distinct E2F-mediated transcriptional program may not only activate the p53 pathway but also activate the p27 gene to suppress CDK activity to restrain inappropriate cell cycle progression in response to defective pRb function. This notion is supported by our previous observation that mouse embryonic fibroblasts obtained from p27^–/– mice showed accelerated cell cycle progression upon expression of E1a but not upon serum stimulation (Iwanaga et al. 2006). Moreover, p27^–/–RB^+/– mice showed accelerated tumor formation compared to RB^+/– mice (Park et al. 1999). These observations indicate that the p27 gene is crucial for suppression of cell cycle progression in response to loss of pRb function and suggest the importance of the distinct regulation of the p27 gene by deregulated E2F in tumor suppression in vivo.

It has been proposed that the total amount of activating E2Fs (E2F1 through E2F3) is important for activation of proapoptotic E2F targets (the threshold model) (Trimarchi & Lees 2002). Although the amount of E2F proteins induced by E1a was similar or slightly higher than that induced by serum stimulation, the amount induced by RB shRNA was not. This result suggests that a higher amount of E2F protein is not necessarily required for activation of atypical E2F targets such as p27 and ARF, and that there is qualitative difference between deregulated and physiological E2F activity. Further studies are required for elucidation of the qualitative difference.

Recent reports indicate that pRb stabilizes p27 at the protein level by interacting with Skp2 and APC/C, and that this activity of pRb is important for suppression of cell cycle progression by pRb (Ji et al. 2004; Binne et al. 2007). Accordingly, loss of pRb function reduces the stability of p27, leading to reduced p27 protein levels. In this context, our finding that the p27 promoter specifically senses deregulated E2F activity induced by defective pRb function could serve as a fail-safe mechanism to maintain p27 protein levels (Fig. 5B).

The mode of E2F regulation of the p27 gene is similar to that of the ARF gene (Komori et al. 2005). Interestingly, however, the E2F-responsive element of the ARF gene is GC rich and lacking a T stretch but that of the p27 gene is similar to typical E2F sites (Fig. 2C). There may be yet unidentified consensus sequence between these two elements to specifically sense deregulated E2F activity. Alternatively, E2F binding sequence alone may not be sufficient to discriminate between deregulated and physiological E2F activity. Further studies are required to elucidate the mechanism of how E2F-mediated transcriptional program distinguishes between deregulated and physiological E2F activity to suppress tumorigenesis against loss of pRb function.

	Experimental procedures

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Cell culture

Human foreskin fibroblasts (HFFs) (ATCC), normal human fibroblasts WI-38 (RIKEN Bioresource Center Cell Bank), 293A cells (Invitrogen), RB-deficient human tumor cell lines Saos-2 and C-33A were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS). RB-deficient human tumor cell line 5637 was cultured in RPMI 1640 medium containing 10% FCS. HFFs were synchronized in resting state by starvation of serum for 48 h and then stimulated to re-enter the cell cycle by addition of FCS to a final concentration of 20%.

Plasmids

The p27 reporter plasmid p27 (–1408) (formerly pGL3-1408S) and its 5' deletion mutants (Ito et al. 1999), expression vectors for E2F1 through E2F3, pCMV-β-gal, the expression vector for 2RG form of E1a mutant that inactivates all members of the pRb family but does not interfere with CBP/p300 (Iwanaga et al. 2006), pCDC6-Luc(-570) (Ohtani et al. 1998), pCDNA3-E2F4, pCDNA3-E2F5 (Sardet et al. 1995), pARF-Luc(-736), pARF-Luc(-66), pEREAx3-(-66)-Luc, the shRNA expression vector against RB1 pshRB, the expression vector for constitutive active form of pRb PSM.7 LP (Komori et al. 2005), have been described. Isolated fragments of p27 promoter, EREK1, EREK2 and EREK (1 + 2) were ligated to ARF core promoter in pARF-Luc(-66). Two point mutations were introduced into EREK1 and EREK2 that changed TTAGGCGCcgct to TTAGatGCcgct (EREK1m) and tgcGTTGGCGGgttcgc to tgcGTTGatGGgttcgc (EREK2m), respectively. The mutant form of EREK(1+2) and p27 reporter pGL3-1408S, in which both EREK1 and EREK2 were mutated, were referred to EREK(1+2)m and p27(–1408)m, respectively.

Transfection and reporter assay

The reporter and effector plasmids were transfected by lipofection using FuGENE 6 (Roche). Luciferase assays proceeded as previously described (Komori et al. 2005). All assays were repeated three times in duplicate and values are shown as means ± SE. Statistically significance was determined with student's t-test.

Recombinant adenoviruses

Recombinant adenoviruses expressing human E2F1 (Ad-E2F1), d2-11 mutant form of adenovirus E1a, which binds and inhibits all members of the pRb family but does not interfere with p300/CBP co-activators (Ad-12SE1A(d2-11)), and the control virus (Ad-Con) have been described (Iwanaga et al. 2006). Recombinant adenovirus expressing the shRNA against RB1 (Ad-shRB) was produced using pshRB (Komori et al. 2005) and ViraPower Adenoviral Expression System (Invitrogen) according to the supplier's protocol. Infection with recombinant adenoviruses proceeded as previously described (Iwanaga et al. 2006).

Northern blot analysis

Total RNA was extracted using Isogen (Nippon Gene) and poly (A)+ RNA was purified using PolyA Tract (Promega) according to the manufacturer's protocol. Northern blot analysis was carried out as described (Ohtani et al. 1998). Human acidic ribosomal phosphoprotein P0 (ARPP) cDNA was the control probe. The radioactivity of the signals was measured with an image analyzer BAS 1500 (Fuji Film).

Immunoblot analysis

Immunoblot analysis was carried out as described (Iwanaga et al. 2001). The antibodies used were anti-E2F1 (sc-251, Santa Cruz, 1 : 60 with ChemiBlocker, Millipore Corporation), anti-E2F3 (sc-878, Santa Cruz, 1 : 500 with BlockAce, Dainippon Pharmaceutical), anti-p27 (sc-1641, Santa Cruz, 1 : 300 with skim milk), and anti--tubulin (DM1A; Oncogene Research Products, 1 : 500 with skim milk) as an internal control.

Gel mobility shift assay

Gel mobility shift assay proceeded as described (Ohtani et al. 1998). The antibodies used for supershift experiments were E2F1 (sc-251 X; Santa Cruz), E2F4 (sc-512 X; Santa Cruz), pRb (14 031A; Pharmingen) and Mnt (sc-769X; Santa Cruz) as a negative control.

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was carried out as described (Komori et al. 2005) using specific primer sets for p27 promoter (Iwanaga et al. 2006) and cdc6 promoter (Komori et al. 2005). Antibodies for immunoprecipitating protein-DNA complexes were anti-E2F1 (KH95), anti-E2F4 (C-108), and anti-p50 (NLS) as a negative control (all from Santa Cruz).

	Acknowledgements

 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. H.K. was supported by a fellowship from the Japan Society for the Promotion of Science.

	Footnotes

 
Communicated by: Yoshiaki Ito

^* Correspondence: kiyo.gene{at}cmn.tmd.ac.jp

	References

Top Abstract Introduction Results Discussion Experimental procedures References

 
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Received: 19 July 2008
Accepted: 14 October 2008

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