Transcriptional regulation of the p53 tumour suppressor gene plays an important role in the control of the expression of various target genes involved in the DNA damage response. However, the molecular basis of this regulation remains obscure. In the present study we demonstrate that RREB-1 (Ras-responsive-element-binding protein-1) efficiently binds to the p53 promoter via the p53 core promoter element and transactivates p53 expression. Silencing of RREB-1 significantly reduces p53 expression at both the mRNA and the protein levels. Notably, disruption of RREB-1-mediated p53 transcription suppresses the expression of the p53 target genes. We also show that, upon exposure to genotoxic stress, RREB-1 controls apoptosis in a p53-dependent manner. These findings provide evidence that RREB-1 participates in modulating p53 transcription in response to DNA damage.
- DNA damage
- Ras-responsive element-binding protein-1 (RREB-1)
The p53 tumour suppressor gene, which is frequently mutated in a wide variety of tumours, plays an important role in maintaining genomic integrity [1–4]. Following genotoxic insults the protein level of p53 is increased and p53 functions as a sequence-specific transcription factor that regulates the expression of downstream target genes required for cell cycle arrest, DNA repair or apoptosis [5–8].
As p53 plays a critical role in genomic stability, its expression must be tightly controlled for normal cell division, as well as for its ability to function as a tumour suppressor. The p53 protein is maintained at a very low level during normal cell growth by regulation of its protein stability. The E3 ubiquitin–protein ligase MDM2 (murine double minute 2), itself a downstream target of p53, binds to and ubiquitinates p53, thus targeting it for degradation by the proteasome [9–12]. Besides ubiquitination, p53 is also regulated by a wide variety of other post-translational modifications such as phosphorylation, acetylation and methylation . Moreover, it has been shown that the ribosomal protein L26 and nucleolin bind to the 5′ untranslated region of p53 mRNA to control p53 translation and induction after DNA damage . These findings indicate that p53 expression is regulated at both the translational and the post-translational levels .
On the other hand, an increasing number of studies have revealed that the transcriptional regulation of p53 is also important for regulating the overall level of the p53 protein. The finding that overexpression of HOXA5 (homeobox A5) induces wild-type p53 expression in human breast tumours suggests that it is possible that loss of HOXA5-dependent regulation of p53 transcription could be a crucial step in tumorigenesis . Likewise, previous studies have shown that p53 gene induction by interferon-α/β, Pitx1 (paired-like homeodomain 1) or PKCδ (protein kinase Cδ) contributes to tumour suppression [16–19]. In addition, overexpression of BCL6 (B-cell CLL/lymphoma 6) protects cells from DNA damage-induced apoptosis by binding to two specific sites within the p53 promoter region and suppressing p53 expression . Other studies have shown that impaired p53-mediated autoregulation of p53 transcription results in aberrant cell cycle regulation and suppression of p53-mediated apoptosis . However, the molecular basis for this transcriptional regulation remains largely unclear.
The present study demonstrates that transcription factor RREB-1 (Ras-responsive element-binding protein-1) up-regulates p53 gene transcription through the CPE-p53 (p53 core promoter element) following genotoxic stress. RREB-1 interacts with CPE-p53 and inhibition of RREB-1 by RNAi (RNA interference) reduces the expression of p53 and p53 downstream target genes. Intriguingly, RREB-1 is also involved in p53-dependent apoptosis. These findings thus demonstrate a pivotal role for RREB-1 in the transcriptional regulation of p53 during the DNA damage response.
The osteosarcoma cell line U2OS was cultured in RPMI 1640 medium containing 10% (v/v) heat-inactivated FBS (fetal bovine serum) and antibiotics. MCF-7 breast carcinoma cells, SaOS-2 osteosarcoma cells and HCT116 colon carcinoma cells were grown in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (v/v) and antibiotics. Cells were treated with 2 μg/ml ADR (adriamycin; Sigma–Aldrich).
Construction of plasmids
RREB-1 cDNA was amplified by PCR using the Platinum Pfx DNA polymerase (Invitrogen) and was cloned into the pcDNA3-Flag vector, constructed using the pcDNA3 vector (Invitrogen) [22–26], or pFLAG-CMV-5a (Sigma–Aldrich). The p53 deletion mutants were constructed by inserting a PCR-amplified genomic region of the human p53 promoter [−431 to +111 nt (called SP1-p53-Luc), −90 to +111 nt (called −90-p53-Luc), or −60 to +111 nt (called −60-p53-Luc), relative to the p53 transcription start site] upstream of the luciferase coding sequence in the pGL-3 basic vector (Promega). Deletion mutants of ΔκB-p53-Luc, ΔCPE-p53-Luc or ΔCPEΔκB-p53-Luc were constructed by amplifying the SP1-p53-Luc plasmid and deleting from −45 to −39 nt, from −73 to −54 nt or from −77 to −39 nt, relative to the p53 transcription start site respectively.
Plasmid DNA was transfected by using FuGENE 6 transfection reagent (Roche). RREB-1 gene-specific siRNAs (short-interfering RNAs) were purchased from Invitrogen (Stealth RNAi). Stealth RNAi sequences were: for RREB-1 siRNA, 5′-CAUUGCCCAGAUCAUCUCAUCUGUA-3′; RREB-1 siRNA(a), 5′-CGUACACCAACUGCCUTCAGAAGAU-3′; and for RREB-1 siRNA(b), 5′-CCUCAGAGAAGAGCGACGAUGACAA-3′. Transfection of 50 nM siRNAs was performed using Lipo-fectamine RNAi MAX (Invitrogen).
Semi-quantitative RT–PCR (reverse transcription–PCR) analysis
Isolation of total RNA from cells was performed using TRIsure (Nippon Genes) according to the manufacturer's protocol. Total RNA (800 ng) was amplified using SuperScript III One Step RT–PCR System with the Platinum Taq Kit (Invitrogen). The RT–PCR was as follows: cDNA synthesis at 55 °C for 30 min, denaturation at 94 °C for 2 min, followed by 20 cycles at 94 °C for 15 s, 55 °C for 30 s and 68 °C for 30 s, with a final extension at 68 °C for 5 min. For quantitative real-time RT–PCR analysis, total RNA was reverse-transcribed into cDNA, using a Superscript III First-Strand Synthesis System for RT–PCR (Invitrogen), following the manufacturer's protocol. The PCR was performed using Power SYBR Green Master Mix (15 μl of Power SYBR Green Master Mix, 0.3 μl of each of 5 μM primer, 5 μl of cDNA and 9.4 μl of water; Applied Biosystems). The PCR programme was as follows: incubation for 10 min at 95 °C, denaturation for 15 s at 95 °C, annealing for 60 s at 60 °C, and extension for 30 s at 72 °C. Cumulative fluorescence was measured at the end of the extension phase of each cycle. Quantification was based on standard curves from the serial dilution of total RNA. The results were normalized to the level of β-actin. GADPH (glyceraldehyde-3-phosphate dehydrogenase) mRNA amplification was used as a loading control. Primer sequences are listed in Table 1.
Immunoblotting and antibodies
Cell lysates were prepared as described in [27–29]. Briefly, cultured cells were washed once with chilled PBS and resuspended in lysis buffer [50 mM Tris/HCl, pH 7.6, containing 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1 mM DTT (dithiothreitol), 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A and 1% (v/v) Tergitol NP-40]. Cell lysates were centrifuged at 12500 g for 10 min at 4 °C. The supernatants were separated by SDS/PAGE (6.5, 7.5 and 12.5% gels) and transferred to nitrocellulose membranes. The membranes were incubated with anti-p53 antibody (DO-1; Santa Cruz Biotechnology), anti-MDM2 (Santa Cruz Biotechnology), anti-p21 antibody (Calbiochem), anti-tubulin antibody (Sigma–Aldrich) or anti-FLAG antibody (Sigma–Aldrich) for 1–4 h at room temperature (25 °C). After washing, the membranes were incubated with anti-rabbit or anti-mouse IgG-peroxidase conjugate (Santa Cruz Biotechnology). The antigen–antibody complexes were visualized by chemiluminescence (PerkinElmer).
In vitro luciferase assay
Luciferase activities were measured at 48 h post-transfection using the Bright-Glo Luciferase assay system (Promega) according to the manufacturer's protocol. The relative increase in activity compared with cells transfected with pGL-3 basic vector was determined as described in [30,31]. The results represent means±S.D. from three or four independent transfection experiments, each performed in triplicate.
ChIP (chromatin immunoprecipitation) assay
U2OS cells [(1–5)×107] were harvested, washed once with chilled PBS, centrifuged at 500 g for 5 min at 4 °C and then incubated in 1% formaldehyde for 15 min at room temperature for chromatin cross-linking. Then the cells were collected by centrifugation at 500 g for 5 min at 4 °C and again washed with chilled PBS. After a further centrifugation at 500 g for 5 min at 4 °C, the cell pellets were resuspended in SDS lysis buffer [50 mM Tris/HCl, pH 8.0, containing 1 mM DTT, 1 mM PMSF, 1 mM Na3VO3, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1% (w/v) SDS and 10 mM EDTA] and the lysates were sonicated to obtain DNA fragments of 200–500 bp in length. After centrifugation, 50 μl of the supernatant was used as an input, and the remainder was diluted 2- to 2.5-fold in washing buffer (50 mM Tris/HCl, pH 7.6, containing 150 mM NaCl, 0.1% Tergitol NP-40 and protease inhibitors as described above). This diluted fraction was subjected to immunoprecipitation with anti-FLAG antibody or control IgG for at least 2 h, or overnight, at 4 °C with rotation. The immunocomplexes were collected by incubation with 30 μl of Protein G–Sepharose beads (Invitrogen) for 1–2 h at 4 °C with rotation. The beads were then pelleted by centrifugation at 1500 g for 5 s and washed sequentially with 300 μl of wash buffer I (20 mM Tris/HCl, pH 8.0, containing 500 mM NaCl, 0.1% SDS and 2 mM EDTA), 300 μl of wash buffer II (10 mM Tris/HCl, pH 8.0, containing 250 mM LiCl, 1 mM EDTA and 1% deoxycholate) and then with 300 μl of Tris/EDTA buffer twice. Precipitated chromatin complexes were removed from the beads by shaking twice with 100 μl of elution buffer (1% SDS and 0.1 M NaHCO4) for 15 min. All of the eluate was collected, and then the cross-linking was reversed by adding NaCl, to a final concentration of 200 mM, and incubating the mixture overnight at 65 °C. The remaining proteins were digested with the extraction buffer (50 mM Tris/HCl, pH 6.8, containing 10 mM EDTA and 40 μg/ml proteinase K) for 1 h at 45 °C. DNA was recovered by phenol/chloroform/isoamyl alcohol (25:24:1) extraction and precipitated with 0.1 volume of 3 M sodium acetate and 2.5 volumes of ethanol. PCR amplification was then performed using the chromatin immunoprecipitated-fragments. Quantitative real-time PCR was performed by using SYBR Green PCR Master Mix (Applied Biosystems) according to the instruction manual. The data was normalized for the level of input control. Primer sequences are listed in Table 1.
BrdU (5-bromo-2-deoxyuridine) incorporation assays
Cells cultured in poly-D-lysine-coated four-well chamber slides, at 5000 cells per well, were transfected with siRNAs and irradiated with UV at 20 J/m2 for 6 h. BrdU was then added into cells for 20 min. BrdU incorporation into cellular DNA was detected by the BrdU labelling and detection kit (Roche Applied Science).
TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assays
Cells cultured in poly-D-lysine-coated four-well chamber slides, at 5000 cells per well, were transfected with siRNAs and then treated with 2 μ ADR for 16 h. Apoptotic cells were detected by TUNEL assays using a DeadEnd Fluorometric TUNEL System (Promega).
RESULTS AND DISCUSSION
Identification of RREB-1 as a candidate transcription factor that participates in regulation of p53 gene transcription via CPE-p53
Previous studies  have demonstrated that the p53 promoter region extending from −70 to −46 nt relative to the p53 transcription start site [and overlapping the NF-κB (nuclear factor κB) motif], designated as the CPE-p53, is required for p53-basal and DNA damage-induced promoter activity. In order to identify transcription factors that are capable of binding to the CPE region of the p53 promoter and regulating its expression, we utilized the Matlnspector Professional Database (http://www.genomatix.de/site_map/index.html) to search for a putative transcription factor-binding site . The search yielded one candidate, the RREB-1 protein. The consensus binding site for RREB-1 and the sequence for CPE-p53 are shown in Figure 1.
RREB-1 up-regulates p53 expression at a transcriptional level
RREB-1 is a transcription factor that is expressed ubiquitously. It contains four Cys2His2 zinc fingers and plays a role in Ras and Raf signal transduction in medullary thyroid cancer and other cells [34,35]. However, the precise function of RREB-1 remains obscure. To test whether RREB-1 can functionally regulate p53 gene transcription, U2OS cells were transfected with RREB-1 siRNA, followed by treatment with ADR. The results demonstrated that the level of mRNA expression of p53 after genotoxic stress, and hence induced after DNA damage, was significantly attenuated when the level of RREB-1 was knocked down compared with the control cells (Figure 2A). To exclude the possibility that this finding is due to the off-target effect of siRNA transfection, we prepared additional two different siRNAs targeting RREB-1, then examined the effect of these siRNAs on the expression of RREB-1, as well as p53. The results demonstrated that transfection of these siRNAs contributes to robust knockdown of RREB-1 (Figure 2B). More importantly, p53 expression was significantly attenuated in cells transfected with these siRNAs (Figure 2B). Notably, immunoblotting with anti-p53 antibody revealed that the induced p53 protein expression, following ADR treatment, was also suppressed by depletion of RREB-1. To directly assess the effect of RREB-1 on p53 transcription, MCF-7 cells were transfected with a FLAG vector or FLAG–RREB-1, then treated with ADR. Analysis by RT–PCR revealed that, in contrast with the FLAG vector-transfected cells, p53 mRNA expression was substantially increased, at each time point, by the expression of RREB-1 (Figure 2C). To examine the involvement of RREB-1 on p53 expression in response to UV irradiation damage, U2OS cells were transfected with RREB-1 siRNA, followed by treatment with UV. The results demonstrated that p53 expression was induced after UV exposure, whereas RREB-1 silencing abrogated the p53 augment after UV irradiation (Figure 2D). Taken together, these observations indicate that RREB-1 regulates p53 expression at a transcriptional level.
To further define the role of RREB-1 in the regulation of p53 gene transcription, U2OS cells were co-transfected with the luciferase reporter construct (SP1-p53-Luc) driven by the 543-bp genomic DNA regions containing the human p53 promoter (Figure 3A) together with scramble siRNA or RREB-1 siRNA. Cells were then left untreated, or were treated with ADR for 8 h, and the luciferase activity of the promoters was measured. RREB-1 silencing was significantly associated with decreases in both the basal and DNA damage-induced promoter activity compared with the control of scramble siRNA (Figure 3B). To confirm whether RREB-1 enhances p53 promoter activity through CPE-p53, we generated two reporter mutants that have deletions of the 5′ region of the p53 promoter and co-transfected them with scramble siRNA or RREB-1 siRNA. Inhibition of RREB-1 was still associated with attenuation of the promoter activity for the mutant construct including the CPE-p53 (−90-p53-Luc), whereas down-regulation of RREB1 had little, if any, repressive effect on the mutant excluding the CPE-p53 (−60-p53-Luc) (Figure 3B). It should be noted that the reason why the activity of −90-p53-Luc was so much lower than that of SP1-p53-Luc remains obscure. However, it is conceivable that SP1-p53-Luc includes a number of transcription-responsive regions besides the CPE, which could be associated with high luciferase activity. To further assess whether RREB-1 directly targets the CPE-p53 for activation of the p53 promoter, U2OS cells were transfected with three further promoter contrusts, in which the sequence for κB binding had been deleted (ΔκB-p53-Luc), the CPE-p53 was deleted (ΔCPE-p53-Luc) or both were deleted (ΔCPEΔκB-p53-Luc) (Figure 3C). Transfection with ΔκB-p53-Luc retained a positive response to the DNA-damaging agent, and inhibition of RREB-1 decreased the basal and DNA damage-induced activity (Figure 3D). In contrast, deletion of CPE-p53 completely abolished DNA damage-induced activation of the promoter, and inhibition of RREB-1 had little if any repressive effect on the promoter activity (Figure 3D). Taken together, these results collectively indicate that RREB-1 functionally regulates p53 gene transcription via CPE-p53.
RREB-1 interacts with the p53 promoter via CPE-p53
To determine if RREB-1 binds to CPE-p53 in vivo, we performed ChIP assays on the p53 promoter. Chromatin was isolated from U2OS cells expressing FLAG, from a control vector, or FLAG–RREB-1 and immunoprecipitated with anti-FLAG or control IgG antibodies. Immunoprecipitated DNA was analysed by PCR, with primers amplifying the p53 promoter region encompassing CPE-p53 or another control region. DNA fragments containing CPE-p53, but not the control region, were specifically immunoprecipitated using anti-FLAG antibodies in FLAG–RREB-1-transfected cells, but not in the FLAG vector-transfected cells (Figure 4A). Importantly, occupancy of RREB-1 to CPE-p53 was substantially increased following DNA damage. We quantified the ChIP results by real-time PCR analysis, clearly indicating increased occupancy of RREB-1 on CPE-p53 after DNA damage (Figure 4B). These results imply that RREB-1 is recruited to the CPE-p53 site on the p53 promoter and is thus a strong candidate for the transcription factor that induces p53 transcription.
RREB-1 silencing diminishes p53-mediated transcription
The impact of RREB-1 levels on the transcriptional induction of p53 prompted us to investigate the cellular function that promotes p53-mediated transcription in response to DNA damage. To characterize the role for endogenous RREB-1 in regulating the target genes of p53, RT–PCR was performed with U2OS cells that were transfected with scramble siRNA or RREB-1 siRNA, followed by treatment with ADR. Silencing of RREB-1 suppressed the mRNA expression of not only p53, but also target genes of p53, such as MDM2, p21 and Bax (BCL2-associated X protein) (Figures 5A and 5B). To quantify expression levels, we performed real-time RT–PCR and obtained comparable results (Figure 5C). To further determine if abrogation of RREB-1 affects the protein expression of p53 target genes, cell lysates were subjected to immunoblot analysis. As shown with the RT–PCR, protein levels of MDM2 and p21 were substantially attenuated in RREB-1-depleted cells (Figure 5D). These findings indicate that activation of p53 gene transcription by RREB-1 probably has important functional consequences resulting from alterations in p53-mediated transcription. Taken together, we demonstrate that RREB-1 is a positive transcriptional regulator of p53 via CPE-p53.
RREB-1 silencing diminishes p53-dependent cell cycle arrest and apoptosis
To define the biological significance of RREB-1-dependent regulation of p53 transcriptional activity, we examined UV-induced cell cycle arrest. U2OS cells were transfected with scramble siRNA or RREB-1 siRNA, followed by UV exposure. Analysis of BrdU incorporation revealed that, upon exposure to UV, the population of S-phase cells is significantly diminished in control cells (Figure 6). By contrast, the UV-induced decrease in the distribution of S-phase cells was much less in cells with attenuated RREB-1 expression, indicating that induction of the G1-phase arrest was markedly impaired in the absence of RREB-1 expression (Figure 6). These results suggest the involvement of RREB-1 in DNA damage-induced cell cycle arrest, which is primarily governed by p53 and its downstream target p21. In this context, depletion of RREB-1 was associated with the attenuation of p21 expression in response to DNA damage (Figure 5).
To further pursue the functional significance of RREB-1 on p53 transcription, we investigated DNA damage-induced apoptotic cell death. U2OS (p53 wild-type) or SaOS-2 (p53 null) cells were transfected with scramble siRNA or RREB-1 siRNA, followed by treatment with ADR. Depletion of RREB-1 markedly suppressed induction of apoptosis after ADR stimulation in U2OS cells (Figure 7A). In contrast, the induction of apoptosis was not significantly reduced in SaOS-2 cells (Figure 7B), mainly owing to the absence of p53 . Importantly, the finding that knockdown of RREB-1 significantly attenuated DNA damage-induced apoptosis in U2OS cells, but not in SaOS-2 cells, indicates that the contribution of endogenous p53 to apoptosis is triggered by RREB-1 expression. Similar results were obtained from HCT116/p53+/+ and HCT116/p53−/− cells (Figures 7C and 7D). These findings provide evidence that the activation of p53 transcription by RREB-1 has important functional consequences with respect to DNA damage-induced apoptosis.
The role of RREB-1 on the p53 expression level in response to DNA damage
The normal cellular function of RREB-1 remains unclear, although transcripts for this gene have been identified in all tissues expect brain . RREB-1 was originally identified as a protein that binds to the upstream Ras-responsive element of the human calcitonin promoter to activate transcription in the presence of Ras . The c-erbB2 and secretin promoters were also attributed as targets for this protein . A previous study showed that RREB-1 lacks an intrinsic activation function, yet it potentiates the transcriptional activity of NeuroD (neurogenic differentiation) that binds in the promoter region . In contrast, other studies have shown that RREB-1 down-regulates several promoters, such as p16INK4a (cyclin-dependent kinase inhibitor 2A) or the human angiotensinogen gene [38,39]. In the present study, we have shown for the first time that the p53 tumour suppressor gene is a novel target of RREB-1. Given that the transcriptional function of RREB-1 depends on the cellular or promoter context, it is conceivable that other proteins that bind to RREB-1 may influence its activity. It is interesting to note that RREB-1 was shown to be a component of a multiprotein complex with the transcriptional corepressor CtBP (C-terminal binding protein) and other chromatin modifying enzymes . Many of these enzymes are able to repress transcription through modification of chromatin structure. In this context, the results in the present study suggest it is possible that RREB-1 could be a negative regulator of MDM2 transcription, independently of p53, in the unstressed condition (Figures 5A and 5C). Thorough identification of RREB-1 interacting modifiers could open a new avenue for precise elucidation of the modulation of p53 transcription, as well as that of other genes, in response to genotoxic stress. We also found that RREB-1 induces apoptotic cell death, at least in part, via a p53-dependent mechanism. However, although not statistically significant, the finding that RREB-1 could affect apoptosis induction even in SaOS-2 cells raises the possibility that RREB-1 is capable of inducing apoptosis independently of p53. Further studies are needed to clarify this mechanism. Nevertheless, to our best knowledge, this is the first study that identifies RREB-1 as a positive regulator of p53 transcription and shows that RREB-1 transactivates p53 to induce apoptosis in response to DNA damage.
In summary, we demonstrate that RREB-1 interacts with the p53 promoter and induces p53 transactivation. Further studies of the mechanism for RREB-1-mediated transcriptional regulation of p53, and the identification of upstream molecules of the RREB-1 signalling pathway, will provide additional insight into the potential role of RREB-1 as a critical signalling mediator in response to DNA damage.
Hanshao Liu and Kiyotsugu Yoshida designed the research; Hanshao Liu, Hoi Chin Hew, Zheng-Guang Lu and Tomoko Yamaguchi performed the research; Hanshao Liu, Hoi Chin Hew, Yoshio Miki and Kiyotsugu Yoshida analysed results; Hanshao Liu and Kiyotsugu Yoshida wrote the manuscript; Kiyotsugu Yoshida supervised and co-ordinated the project.
This work was supported by the Ministry of Education, Science and Culture of Japan [grant numbers 19657038, 20015017, 20390091, 17013027, 19390083]; the Yasuda Memorial Foundation; the Life Science Foundation of Japan; the Uehara Memorial Foundation; the Cell Science Research Foundation; and by the Senri Life Science Foundation.
Abbreviations: ADR, adriamycin; Bax, BCL2-associated X protein; BrdU, 5-bromo-2-deoxyuridine; ChIP, chromatin immunoprecipitation; CPE-p53, p53 core promoter element; DTT, dithiothreitol; FBS, fetal bovine serum; GADPH, glyceraldehyde-3-phosphate dehydrogenase; HOXA5, homeobox A5; MDM2, murine double minute 2; RNAi, RNA interference; RREB-1, Ras-responsive element-binding protein-1; RT–PCR, reverse transcription–PCR; siRNA, short-interfering RNA; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling
- © The Authors Journal compilation © 2009 Biochemical Society