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Cooperation between ARID3A and p53 in the transcriptional activation of p21WAF1 in response to DNA damage
Biochemical and Biophysical Research Communications 417 (2012) 710–716
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Cooperation between ARID3A and p53 in the transcriptional activation of p21WAF1 in response to DNA damage Widya Lestari a, Solachuddin J.A. Ichwan b, Megumi Otsu a, Shumpei Yamada a, Sachiko Iseki a, Shihoko Shimizu a, Masa-Aki Ikeda a,⇑ a b
Section of Molecular Craniofacial Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan Faculty of Dentistry, International Islamic University Malaysia, Kuantan, Malaysia
a r t i c l e
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Article history: Received 23 November 2011 Available online 8 December 2011 Keywords: ARID3A DRIL1 Bright p53 p21WAF1 Gene expression
a b s t r a c t ARID3A/DRIL1/Bright is a family member of the AT rich interaction domain (ARID) DNA-binding proteins that are involved in diverse biological processes. We have reported that p53 activates ARID3A transcription, and ARID3A overexpression induces G1 arrest. However, the role of ARID3A in the p53 pathway remains unclear. Here, we show that ARID3A cooperates with p53 to transcriptionally activate p21WAF1, a p53-target gene important for cell-cycle arrest. ARID3A bound to its binding sites in the p21WAF1 promoter in vivo and in vitro, and induced p21WAF1 transcription in U2OS cells expressing wild-type p53 but not Saos-2 cells lacking p53. The co-expression of ARID3A with p53 cooperates to activate p21WAF1 transcription and the stably transfected p21WAF1 promoter. Mutation of the ARID3A binding sites reduced the p21WAF1 promoter activity, and siRNA-based ARID3A knockdown suppressed the transcription of p21WAF1, but not the proapoptotic NOXA and PUMA in response to DNA damage. Furthermore, p53 knockdown decreased ARID3A transcription, and, conversely, ARID3A overexpression and knockdown resulted in an increase or decrease in p53 stability, respectively. These results indicate both cooperative and interdependent roles for ARID3A and p53 in the transcriptional activation of p21WAF1 in response to DNA damage. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction The ARID family member proteins share a conserved DNA binding domain, and they are involved in a wide range of biological processes including cell differentiation, cell cycle control, chromatin remodeling and epigenetic regulation (reviewed in [1,2]). In addition, ARID3A/DRIL1/Bright have additional conserved regions that extend outside the ARID domain, and the extended ARID domain is shared with the other ARID3 subfamily members. The ARID3 proteins bind specific AT-rich DNA sequences, while the other ARID proteins exhibit non-specific DNA binding activity. It has been reported that ARID3A binds to specific AT-rich sequences that are important for the nuclear matrix association regions (MARs) located in the immunoglobulin heavy chain (IgH) gene locus [3]. ARID3A is highly expressed in mature B cells and regulates IgH transcription [3,4]. Furthermore, a number of studies have reported diverse biological functions of ARID3A. For instance, ARID3A binds to the E2F transcription factor and activates E2F-dependent transcription [5]. ARID3A rescues Ras-induced premature senescence ⇑ Corresponding author. Address: Section of Molecular Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Fax: +81 3 5803 0213. E-mail address: [email protected] (M.-A. Ikeda). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.12.003
in primary murine fibroblasts [6] and plays a critical role in TGFb signaling in Xenopus embryos [7]. Recently, the inhibition of ARID3A has been shown to induce the reprogramming of somatic cells into pluripotent stem-like cells [8]. The p53 tumor suppressor protein plays key roles in responding to cellular stress by controlling genes that exert different cellular outcomes such as cell-cycle arrest and apoptosis (reviewed in [9,10]). Several lines of evidence indicate that a p53-responsible element exists in the human ARID3A gene [11–13]. It has been shown that ARID3A is transcriptionally up-regulated by p53 and DNA damage, and that the ectopic expression of ARID3A induces growth arrest in U2OS cells expressing normal p53, but not Saos-2 cells lacking p53 [11]. However, the role of ARID3A in p53-mediated growth arrest remains unclear. Here, we show that ARID3A cooperates with p53 to transcriptionally activate p21WAF1 in response to DNA damage.
2. Materials and methods 2.1. Cell culture and drug treatment A459, H1299 (human lung adenocarcinoma) U2OS, and Saos2 (human osteosarcoma), cells were maintained in Dulbecco‘s
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modified Eagle‘s medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in the presence of 5% CO2. Cells were treated with 0.2 lg/ml doxorubicin (Sigma–Aldrich) for 24 h.
2.2. Plasmids and transfections The p21 reporter plasmid and expression vectors for ARID3A and p53 were described previously [11]. To generate p21-luc_Mut, all of the putative ARID3A binding sites were mutated by sitedirected mutagenesis. To generate expression vectors for ARID3A short hairpin RNA (shRNA) (pshARID3A-1 and pshARID3A-2), double stranded oligonucleotides targeting ARID3A were cloned into the pcPURU6b vector downstream of the human U6 promoter. The shRNA sequences are available upon request. All constructs were confirmed by DNA sequencing. A control GFP shRNA expression vector was obtained from Takara Bio Inc. Cells were transfected using the Gene Juice transfection reagent (Merck) according to the manufacturer‘s instructions.
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2.6. Electrophoretic mobility shift assays (EMSAs) EMSAs were performed as described previously [15], except that Poly (dI-dC) (1 lg) was used as a non-specific competitor. The following 32P-labled double-stranded oligonucleotides were used as probes: 5’-GGACATTAATACATAAAAATTCATAAATTATAAA AATCTAG-3’. For the antibody supershift assay, an anti-ARID3A antibody (N-20) was added to the indicated samples. 2.7. Western blotting and antibodies Preparation of whole-cell extracts (WCE) and Western blotting were performed as described previously [16]. The following primary antibodies were used in this study: rabbit polyclonal anti-p53 (FL-393), anti-p21 (C-19; Santa Cruz Biotechnology), anti-ARID3A (N-20 [11]); mouse monoclonal anti-p53 (DO-1), anti-SV40 T Ag (Pab 108), anti-E1A (M73), anti-Omni-probe (D-8; Santa Cruz Biotechnology), anti-ARID3A (ARIDE9C11; Biomatrix Research), anti-X-press (Invitrogen, CA, USA) and anti-b-actin (AC-15; Sigma–Aldrich) antibodies.
2.3. Small interfering RNA 2.8. Semiquantitative RT-PCR analysis Stealth small interfering RNA (siRNA) duplexes targeting ARID3A (50 -AACAGAACUCCUGUGUACAUGAUGC-30 ) and p53 (50 -UAU UGGGUCCGCAGGGUGAAGGCUG-30 ), along with negative control duplexes were purchased from Invitrogen. Cells were reverse transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions, and incubated for 48 h prior to drug treatment.
2.4. Recombinant adenoviruses Ad-ARID3A, Ad-p53 and Ad-1w1 (Ad-con) viruses were purified and the viral titers were determined as described [11]. To generate the Ad-shARID3A-1 and Ad-shARID3A-2 viruses, the corresponding shRNA-expression cassettes were isolated from the shRNA expression plasmids and cloned into pShuttle, and then the recombinant adenoviruses were generated according to the manufacturer’s protocols of the AdEasy system (Quantum Biotechnologies). Cells were infected at the indicated multiplicity of infection (MOI) in serumfree DMEM containing 20 mM HEPES (pH 7.4) for 1 h at 37 °C with brief agitation every 15 min, after which cells were cultured in DMEM supplemented with 10% FCS.
2.5. Chromatin immunoprecipitation (ChIP) assay ChIP assay was performed according to a protocol described previously [14]. Briefly, U2OS cells were crosslinked with formaldehyde, lysed in lysis buffers and sonicated with a Bioruptor sonicator (Cosmo Bio) to shear the crosslinked DNA. After centrifugation, the resulting chromatin solution (2 106 cells per sample) was pre-cleared with anti-mouse IgG-conjugated magnetic beads (Invitrogen) for 2 h, and then incubated overnight at 4 °C with the magnetic beads that had been pre-incubated with either anti-p53 (DO-1) or control anti-E1A (M73) monoclonal antibodies. After washing, beads were incubated at 65 °C for 4 h for reverse crosslinking. Immunoprecipitated DNA was then treated with RNaseA and proteinase K. The resultant DNA was purified by phenol–chloroform extraction and ethanol precipitation, and dissolved in 50 ll H2O and subjected to semiquantitative PCR. For the input control, a 1/200 volume of chromatin was amplified. PCR products were resolved on 2% agarose gels, stained with ethidium bromide and visualized by UV transillumination. The primer sequences used for the ChIP assays are described in Supplementary Table 1.
Total RNA was prepared using the SV Total RNA Isolation Kit (Promega) according to the manufacturer‘s instructions. Complementary DNAs were synthesized from 0.5 lg of the total RNAs with the High Capacity RNA-to-cDNA kit (Applied Biosystem). RT-PCR was performed with GoTaq Hot Start Green Master Mix (Promega) under the following conditions: mixtures were denatured at 95 °C for 1 min, followed by 24 cycles for p21, 33 cycles for ARID3A, 29 cycles for NOXA and PUMA and 30 cycles for p53 and GAPDH at 95 °C for 40 s, 55 °C for 40 s, 73 °C for 10 s, with a final extension at 72 °C for 3 min. PCR products were resolved on 2% agarose gels, stained with ethidium bromide and visualized by UV transillumination. The primer sequences used for RT-PCR are described in Supplementary Table 1. 2.9. Quantitative real-time reverse transcription (RT)-PCR analysis Quantitative real-time RT-PCR analysis was performed on the LightCycler 480 instrument (Roche Applied Science) in triplicate using TaqMan Gene Expression Assays for PUMA, Noxa, p21Waf1, and 18s rRNA (Hs00248075_m1, Hs00560402_m1, Hs003557 82_m1, and Hs03003631_g1, respectively) (Applied Biosystems) and the LightCycler 480 Probes Master kit (Roche Applied Science), according to the manufacturer’s instructions. The relative expression of mRNA, normalized to 18s rRNA, was calculated using the 2(DDCP) method. 2.10. Northern blotting Northern blotting was performed as described [11]. The intensity of each band was quantified using a Bio-Imaging Analyzer BAS 2000 System (Fuji Film). The p21 band intensities were normalized to those of the corresponding GAPDH. 2.11. Luciferase assay Transient reporter assays were performed as described [17]. Luciferase and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega). Luciferase activity was normalized to Renilla-luciferase activity. For stable reporter assays, U2OS cells were transfected with p21-luc along with pcDNA3-neo at a ratio of 3:1, and then selected with 500 mg/ml of G418 over a period of 2 weeks. The resulting G418-resistant cells
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were re-plated at 50–70% confluency in 12 well plates 20–24 h prior to adenovirus infection or drug treatment. 3. Results and discussion 3.1. ARID3A binds the p21WAF1 promoter in vivo and in vitro We identified a p53-resposible element in the ARID3A gene [11]. To confirm that ARID3A is a downstream target of p53, we performed chromatin immunoprecipitation (ChIP) assays. U2OS cells were either treated or left untreated with doxorubicin and then cross-linked with formaldehyde. Immunoprecipitation with the anti-p53 antibody revealed that p53 was recruited to the p53-resposible element in the ARID3A gene following DNA damage in vivo (Fig. 1A). These results, along with the fact that ARID3A overexpression can induce G1 arrest in U2OS cells [11], prompted us to examine whether ARID3A is involved in the transcriptional regulation of p21WAF1, which plays an important role in p53-mediated cell-cycle arrest. It has been shown that the ARID3A/Bright consensus binding sequences contain a core hexamer within a region of more than 12 bp of AT/ATC-rich sequences in one strand of the duplex, together with a second AT dimer located close to the hexamer [3]. Three putative ARID3A binding sites were found in the p21WAF1 promoter region (Fig 1B and Suppl. Fig. 1). The binding sites (BS)A and BS-B match the consensus perfectly, and BS-C contains a degenerate hexamer (AATTAT) that is reportedly a native binding site for ARID3A/Bright [3]. ChIP assays with U2OS cells using an anti-ARID3A antibody revealed that ARID3A was recruited to these putative ARID3A binding sites following infection with an adenovirus expressing ARID3A (Ad-ARID3A) (Fig. 1C). To confirm these results, we performed EMSAs using a probe containing the BS-C
sequence. A shifted band was detected in the extracts from U2OS cells infected with Ad-ARID3A (Fig. 1D, left panel, lane 2). The band was competed by unlabeled competitor oligonucleotides containing the wild-type (WT) but not mutated (MUT-1, Suppl. Fig. 1) core hexamer sequence (lanes 3 and 4), and also super-shifted by an anti-ARID3A antibody (lane 6). Furthermore, a competitor mutated in the AT dimer close to the hexamer (MUT-2, Suppl. Fig. 1) lost the ability to compete with ARID3A for binding (Fig. 1D, right panel). However, competitors mutated in the AT dimers sites distant from the hexamer (MUT-3 and MUT-4, Suppl. Fig. 1) also failed to compete the binding, while the sequences of the MUT-3 and MUT-4 competitors match the ARID3A/Bright-binding consensus motif, suggesting that the reported consensus motif is not sufficient to bind ARID3A in this setting. Taken together, we conclude that ARID3A can bind the p21WAF1 promoter both in vivo and in vitro. 3.2. ARID3A transcriptionally activates p21WAF1 To determine if ARID3A induces the transcription of p21WAF1, A459 cells carrying wild-type p53 were infected with increasing amounts of adenovirus expressing ARID3A. Western blotting and RT-PCR analysis revealed that ARID3A overexpression up-regulated both the protein and mRNA expression levels of p21WAF1 in a dose-dependent manner (Fig 2A and B). Similar results were obtained with Northern blotting (Fig 2C). Furthermore, consistent with previous observations that ARID3A induces growth arrest in U2OS cells but not Saos-2 cells lacking p53 [11], infection of AdARID3A up-regulated the p21WAF1 protein and mRNA expression in U2OS cells, whereas p21WAF1 induction was undetectable in Saos-2 cells (Fig. 2D and E). Similar results were obtained with HCT116 cells expressing wild-type p53 (p53+/+) and p53-negative derivative HCT116 cells (p53/) (data not shown). These results
Fig. 1. Binding of ARID3A to the p21WAF1 promoter in vivo and in vitro. (A) U2OS cells were untreated or treated with 0.2 lg/ml doxorubicin (Doxo), fixed with 1% formaldehyde at 16 h post-treatment, and processed for ChIP analysis using anti-E1A (control) or anti-p53 antibodies. p53 occupancy on its binding sequences in the ARID3A gene was detected by PCR. (B) Schematic representation of ARID3A-binding sites in the p21WAF1 promoter reporter construct used in this study. The black and open boxes represent the p53 and ARID3A binding sites (BS-A, BS-B, and BS-C), respectively. Luc: luciferase gene. (C) U2OS cells were infected with either Ad-con or Ad-ARID3A (MOI = 200), fixed at 24 h post-infection, and processed for ChIP analysis using monoclonal anti-E1A (M73) and anti-ADRID3A (ARIDE9C11) antibodies. ADRID3A occupancy was detected as in A. (D) EMSAs were performed using cell lysates prepared from U2OS cells infected with Ad-con or Ad-ADRID3A and a 32P-labeled probe containing potential ARID3A-binding site (BS-C). (left panel) Competition assays were performed by adding 100-M excess of the unlabeled oligonucleotides to the reaction mixtures as indicated. Antibody supershift assays were performed by adding the following antibodies: normal rabbit IgG (lane 5) and a polyclonal anti-ARID3A N-20 antibody (lane 6). The positions of the ARID3A-DNA complexes and antibody-specific super-shifted complex are indicated by arrows. Asterisks indicate non-specific bands. (right panel) Competition assays were performed by adding a 50 or 200-M excess of the indicated unlabeled wild-type or mutant oligonucleotides.
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Fig. 2. ARID3A transcriptionally activates p21WAF1. (A, B, and C) A549 cells were treated with doxorubicin as in Fig. 1A or infected with increasing MOIs (20 and 80) of Ad-con or Ad-ARID3A. The total MOI of each sample was adjusted with Ad-con. (A) Western blotting analysis at 48 h post-infection using anti-p21 and anti-Xpress antibodies, the latter of which detects the Xpress-tagged ARID3A protein encoded by Ad-ARID3A. b-actin was used as the loading control. (B and C) Semiquantitative RT-PCR (B) and Northern blotting analysis (C) at 24 h post-infection. (C, right panel) Relative fold induction was determined as described in Section 2. Data represent means ± SD from three independent experiments. GAPDH was used as an internal control (B and C). (D and E) U2OS and Saos2 cells were treated with doxorubicin as in Fig. 1A or infected with Adcon or Ad-ARID3A at 80 MOI. (D) Western blotting analysis at 48 h post-infection using the indicated antibodies. b-actin was used as the loading control. (E) Semiquantitative RT-PCR analysis at 24 h post-infection.
indicate that ARID3A by itself cannot substitute for p53-mediated functions in the absence of wild-type p53. In addition, we detected an up-regulation of p53 protein but not mRNA following AdARID3A infection, indicating that the ectopic expression of ARID3A increases the stability of p53 protein (Fig. 2D and E). These results suggest that ARID3A needs to cooperate with p53 to induce p21WAF1 transcription. 3.3. ARID3A cooperates with p53 to activate p21WAF1 transcription We thus examined whether the coexpression of ARID3A with p53 activates p21WAF1 in a cooperative fashion. A549 cells were co-infected with Ad-p53 and Ad-ARID3A and the levels of p21WAF1 mRNA were examined by Northern blotting. Fig. 3A shows that the coinfection of Ad-ARID3A with a lower dose of Ad-p53 (MOI = 20) synergistically activates p21WAF1 transcription. We next asked whether the coexpression of these genes cooperate to activate the p21WAF1 promoter. To this end, we performed reporter assays using a construct encoding a luciferase reporter gene driven by the p21WAF1 promoter containing three ARID3A-binding sites (Fig. 1B). The p21WAF1 reporter construct was transiently transfected into U2OS cells along with either or both of the ARID3A and p53 expression plasmids. However, transfection of ARID3A failed to activate the p21WAF1 promoter, whereas p53 activated the reporter activity in a dose-dependent manner (Fig. 3B). Furthermore, co-transfection of ARID3A and p53 did not exhibit any cooperative activity, but rather, ARID3A reduced the transactivation capacity of p53. It has been shown that Bright can only transactivate the IgH S107 promoter, when it is integrated within chromatin [18]. Therefore, we asked whether ARID3A activates the chromatin-integrated p21WAF1 promoter. To this end, U2OS cells were transfected with the p21WAF1 reporter along with a plasmid expressing the neomycin-resistance gene, selected with G418, and then G418-resisant cells were subjected to reporter assay. Fig. 3C shows that infection of Ad-ARID3A enhanced the reporter activity in a dose dependent manner, which activity was comparable to that obtained by Adp53 infection and doxorubicin treatment. Furthermore, the coexpression of ARID3A with p53 cooperated to activate the p21WAF1
promoter (Fig. 3D), indicating that ARID3A can cooperate with p53 to activate the chromatin-integrated p21WAF1 promoter. To determine the role of the ARID3A binding sites in the transactivation of the p21WAF1 promoter, we mutated all three of the binding sites (BS-A, BS-B, and BS-C) in the p21WAF1 reporter construct (p21-luc_Mut). U2OS cells were stably transfected with either the wild-type or mutant reporter construct followed by the reporter assays. DNA damage caused a 3.5-fold activation of the wild-type reporter activity (Fig. 3E). Mutations in the ARID3A binding sites led to a partial reduction (2.5-fold) of the p21WAF1 promoter activity following DNA damage. However, the ARID3A binding site mutations had no significant effect on the transactivation of the p21WAF1 promoter by the ectopic expression of either ARID3A or p53 in both transient and stable reporter assays (data not shown), suggesting that the presence of the ARID3A binding sites affects the activity of the chromatin-integrated p21WAF1 promoter under conditions in which physiological levels of ARID3A and p53 are expressed in the cell. 3.4. ARID3A knockdown blocked transcriptional activation of p21WAF1 We then examined the role of ARID3A in p53-dependent transactivation of the p21WAF1 by using small interfering RNAs (siRNAs) that target ARID3A and p53 (siARID3A and sip53, respectively). A549 cells were reversely transfected with siRNAs followed by doxorubicin treatment. Consistent with previous observations, the ARID3A mRNA levels were increased following DNA damage, and suppressed not only by ARID3A siRNA, but also by p53 siRNA (Fig. 4A). ARID3A knockdown blocked the transcriptional activation of p21WAF1 but not the proapoptotic NOXA and PUMA following DNA damage, whereas p53 knockdown suppressed the induction of all three of the p53-target genes examined (Fig. 4A and B). Western blotting confirmed that the induction of p21WAF1 protein was inhibited by both ARID3A and p53 siRNAs (Fig. 4C). Although ARID3A knockdown inhibited p21WAF1 expression, it suppressed cell proliferation and the transcription of the E2F-target genes required for cell cycle progression in the absence of DNA damage (unpublished data), which is supported by the fact that ARID3A also plays a critical role in E2F-mediated cell cycle progression [5,6].
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Fig. 3. ARID3A cooperates with p53 to activate p21WAF1 transcription. (A) A549 cells were infected with Ad-con or the indicated combination of Ad-ARID3A and Ad-p53, and subjected to Northern blot analysis for p21WAF1 mRNA as in Fig. 2C (upper panel). (lower panel) Relative fold induction was determined as in Fig. 2C. (B) U2OS cells were transiently transfected with the p21WAF1 reporter along with increasing amounts of p53 (0.5, 2.5 and 10 ng) and/or ARID3A (50, 100 and 200 ng) expression vectors. Luciferase was assayed 24 h after transfection. (C) U2OS cells were transfected with the p21WAF1 reporter construct along with an expression vector for the neomycin resistance gene and selected with G418 for 2 weeks. The G418-resisant cells were re-plated and then infected with Ad-con (control) or increasing doses of Ad-ARID3A (MOI = 100, 200 and 300) and p53 (MOI = 15 and 30) as in Fig. 2, or treated with doxorubicin as in Fig. 1A. Luciferase was assayed 24 h after treatment. (D) U2OS cells stably transfected as in D with the p21WAF1 reporter were infected with the indicated combination of Ad-ARID3A (MOI = 100) and p53 (MOI = 15 and 30) as in Fig. 2. (E) U2OS cells stably transfected with the p21WAF1 reporter constructs containing the wild-type or mutated ARID3A-binding sites (p21-luc_WT and p21-luc_Mut, respectively) were treated with doxorubicin as in Fig. 1A. Luciferase was assayed 24 h after treatment. The data present the average of three independent experiments, each performed in duplicate (B, C, D, and E).
Collectively, these results demonstrate that ARID3A play an important role in the p53-mediated transcriptional transactivation of p21WAF1. 3.5. ARID3A knockdown reduces p53 stability Finally, as shown in Fig. 4A and B, ARID3A knockdown reduced the protein but not mRNA levels of p53. These results, along with the fact that ARID3A overexpression increases the stability of p53 protein (Fig. 2D and E), implies that ARID3A plays an important role in the regulation of p53 stability. These results raise the possibility that the reduction of p53 stability by ARID3A knockdown may affect the p53-mediated transactivation of the p21WAF1 promoter. To test this possibility, we performed transient reporter assays using expression vectors for two shRNAs that target different ARID3A sequences (shARID3A-1 and -2). RT-PCR analysis confirmed that each of the ARID3A shRNAs blocked the expression of p21WAF1 and ARID3A mRNA (Fig. 4D, left panel). Reporter assays revealed that both ARID3A shRNAs blocked the p53-mediated transactivation of the p21WAF1 promoter (Fig. 4D, right panel). Since
ARID3A overexpression and its binding site mutations had no effect of on the p53-mediated activation of the p21WAF1 promoter in transient reporter assays (Fig. 3B, data not shown), these results indicate that the inhibition of ARID3A blocks p53-mediated p21WAF1 promoter activation through a reduction of p53 stability. The mechanism underlying this phenomenon remains unknown. It has been shown that p53 co-localizes with the PML Nuclear Bodies (NBs) and PML potentiates p53 activity by regulating post-translational modifications, such as acetylation and phosphorylation (Reviewed in [19,20]). ARID3A also co-localizes with PML NBs and binds the Sp100 family protein, a component of the PML NBs [21]. We thus speculate that ARID3A might play a role in p53 post-translational modifications mediated by PML NBs. The results presented in his study, together with our previous study, show that p53 regulates ARID3A transcription and then cooperates with ARID3A to transcriptionally activate the common target gene, p21WAF1. Furthermore, we show that ARID3A plays critical roles in p53-mediated p21WAF1 transactivation and that modulation of the ARID3A level affects p53 protein stability, indicating cooperative and interdependent roles for these proteins in transcriptional
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Fig. 4. ARID3A knockdown blocked transcriptional activation of p21WAF1. (A, B and C) A549 cells were reversely transfected with scramble (control), ARID3A or p53 siRNA. At 24 h post-transfection, cells were either left untreated or treated with doxorubicin for 24 h. (A) Semiquantitative RT-PCR analysis using primers specific for the indicated transcripts. (B) Relative fold induction was determined by quantitative real-time RT-PCR analysis. The data represent means ± SD from three independent experiments. (C) Western blotting analysis using the indicated antibodies. (D) (left panel) 293 cells were infected with the indicated shRNA-expressing adenoviruses. Semiquantitative RT-PCR of the indicated transcripts at 48 h post-infection. (right panel) H1299 cells were transiently transfected with p53 along with the indicated shRNA-expression plasmids. Luciferase was assayed 24 h after transfection. The data present the average of three independent experiments, each performed in duplicate.
activation of p21WAF1. These data provide new insights into the mechanisms underlying the function of p53 and ARID3A in the transcriptional regulation of p53-tagert genes. Further experiments are required to elucidate specific roles of ARID3A in p53 stabilization and p53-mediated cell fate decision of cell cycle arrest. Acknowledgments This study was supported in part by Grants-in–Aid for Scientific Research (16209054, 21390502, and 21659454 to MAI) from the Japan Society for the Promotion of Science. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.12.003. References [1] D. Wilsker, A. Patsialou, P.B. Dallas, E. Moran, ARID proteins: a diverse family of DNA binding proteins implicated in the control of cell growth, differentiation, and development, Cell Growth Differ. 13 (2002) 95–106. [2] D. Wilsker, L. Probst, H.M. Wain, L. Maltais, P.W. Tucker, E. Moran, Nomenclature of the ARID family of DNA-binding proteins, Genomics 86 (2005) 242–251.
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[14] L.A. Boyer, T.I. Lee, M.F. Cole, et al., Core transcriptional regulatory circuitry in human embryonic stem cells, Cell 122 (2005) 947–956. [15] M.A. Ikeda, L. Jakoi, J.R. Nevins, A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation, Proc. Natl. Acad. Sci. USA 93 (1996) 3215–3220. [16] P. Sumrejkanchanakij, M. Tamamori-Adachi, Y. Matsunaga, K. Eto, M.A. Ikeda, Role of cyclin D1 cytoplasmic sequestration in the survival of postmitotic neurons, Oncogene 22 (2003) 8723–8730. [17] S.J. Ichwan, S. Yamada, P. Sumrejkanchanakij, E. Ibrahim-Auerkari, K. Eto, M.A. Ikeda, Defect in serine 46 phosphorylation of p53 contributes to acquisition of p53 resistance in oral squamous cell carcinoma cells, Oncogene 25 (2006) 1216–1224.
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Cooperation between ARID3A and p53 in the transcriptional activation of p21WAF1 in response to DNA damage Widya Lestari a, Solachuddin J.A. Ichwan b, Megumi Otsu a, Shumpei Yamada a, Sachiko Iseki a, Shihoko Shimizu a, Masa-Aki Ikeda a,⇑ a b
Section of Molecular Craniofacial Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan Faculty of Dentistry, International Islamic University Malaysia, Kuantan, Malaysia
a r t i c l e
i n f o
Article history: Received 23 November 2011 Available online 8 December 2011 Keywords: ARID3A DRIL1 Bright p53 p21WAF1 Gene expression
a b s t r a c t ARID3A/DRIL1/Bright is a family member of the AT rich interaction domain (ARID) DNA-binding proteins that are involved in diverse biological processes. We have reported that p53 activates ARID3A transcription, and ARID3A overexpression induces G1 arrest. However, the role of ARID3A in the p53 pathway remains unclear. Here, we show that ARID3A cooperates with p53 to transcriptionally activate p21WAF1, a p53-target gene important for cell-cycle arrest. ARID3A bound to its binding sites in the p21WAF1 promoter in vivo and in vitro, and induced p21WAF1 transcription in U2OS cells expressing wild-type p53 but not Saos-2 cells lacking p53. The co-expression of ARID3A with p53 cooperates to activate p21WAF1 transcription and the stably transfected p21WAF1 promoter. Mutation of the ARID3A binding sites reduced the p21WAF1 promoter activity, and siRNA-based ARID3A knockdown suppressed the transcription of p21WAF1, but not the proapoptotic NOXA and PUMA in response to DNA damage. Furthermore, p53 knockdown decreased ARID3A transcription, and, conversely, ARID3A overexpression and knockdown resulted in an increase or decrease in p53 stability, respectively. These results indicate both cooperative and interdependent roles for ARID3A and p53 in the transcriptional activation of p21WAF1 in response to DNA damage. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction The ARID family member proteins share a conserved DNA binding domain, and they are involved in a wide range of biological processes including cell differentiation, cell cycle control, chromatin remodeling and epigenetic regulation (reviewed in [1,2]). In addition, ARID3A/DRIL1/Bright have additional conserved regions that extend outside the ARID domain, and the extended ARID domain is shared with the other ARID3 subfamily members. The ARID3 proteins bind specific AT-rich DNA sequences, while the other ARID proteins exhibit non-specific DNA binding activity. It has been reported that ARID3A binds to specific AT-rich sequences that are important for the nuclear matrix association regions (MARs) located in the immunoglobulin heavy chain (IgH) gene locus [3]. ARID3A is highly expressed in mature B cells and regulates IgH transcription [3,4]. Furthermore, a number of studies have reported diverse biological functions of ARID3A. For instance, ARID3A binds to the E2F transcription factor and activates E2F-dependent transcription [5]. ARID3A rescues Ras-induced premature senescence ⇑ Corresponding author. Address: Section of Molecular Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Fax: +81 3 5803 0213. E-mail address: [email protected] (M.-A. Ikeda). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.12.003
in primary murine fibroblasts [6] and plays a critical role in TGFb signaling in Xenopus embryos [7]. Recently, the inhibition of ARID3A has been shown to induce the reprogramming of somatic cells into pluripotent stem-like cells [8]. The p53 tumor suppressor protein plays key roles in responding to cellular stress by controlling genes that exert different cellular outcomes such as cell-cycle arrest and apoptosis (reviewed in [9,10]). Several lines of evidence indicate that a p53-responsible element exists in the human ARID3A gene [11–13]. It has been shown that ARID3A is transcriptionally up-regulated by p53 and DNA damage, and that the ectopic expression of ARID3A induces growth arrest in U2OS cells expressing normal p53, but not Saos-2 cells lacking p53 [11]. However, the role of ARID3A in p53-mediated growth arrest remains unclear. Here, we show that ARID3A cooperates with p53 to transcriptionally activate p21WAF1 in response to DNA damage.
2. Materials and methods 2.1. Cell culture and drug treatment A459, H1299 (human lung adenocarcinoma) U2OS, and Saos2 (human osteosarcoma), cells were maintained in Dulbecco‘s
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modified Eagle‘s medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in the presence of 5% CO2. Cells were treated with 0.2 lg/ml doxorubicin (Sigma–Aldrich) for 24 h.
2.2. Plasmids and transfections The p21 reporter plasmid and expression vectors for ARID3A and p53 were described previously [11]. To generate p21-luc_Mut, all of the putative ARID3A binding sites were mutated by sitedirected mutagenesis. To generate expression vectors for ARID3A short hairpin RNA (shRNA) (pshARID3A-1 and pshARID3A-2), double stranded oligonucleotides targeting ARID3A were cloned into the pcPURU6b vector downstream of the human U6 promoter. The shRNA sequences are available upon request. All constructs were confirmed by DNA sequencing. A control GFP shRNA expression vector was obtained from Takara Bio Inc. Cells were transfected using the Gene Juice transfection reagent (Merck) according to the manufacturer‘s instructions.
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2.6. Electrophoretic mobility shift assays (EMSAs) EMSAs were performed as described previously [15], except that Poly (dI-dC) (1 lg) was used as a non-specific competitor. The following 32P-labled double-stranded oligonucleotides were used as probes: 5’-GGACATTAATACATAAAAATTCATAAATTATAAA AATCTAG-3’. For the antibody supershift assay, an anti-ARID3A antibody (N-20) was added to the indicated samples. 2.7. Western blotting and antibodies Preparation of whole-cell extracts (WCE) and Western blotting were performed as described previously [16]. The following primary antibodies were used in this study: rabbit polyclonal anti-p53 (FL-393), anti-p21 (C-19; Santa Cruz Biotechnology), anti-ARID3A (N-20 [11]); mouse monoclonal anti-p53 (DO-1), anti-SV40 T Ag (Pab 108), anti-E1A (M73), anti-Omni-probe (D-8; Santa Cruz Biotechnology), anti-ARID3A (ARIDE9C11; Biomatrix Research), anti-X-press (Invitrogen, CA, USA) and anti-b-actin (AC-15; Sigma–Aldrich) antibodies.
2.3. Small interfering RNA 2.8. Semiquantitative RT-PCR analysis Stealth small interfering RNA (siRNA) duplexes targeting ARID3A (50 -AACAGAACUCCUGUGUACAUGAUGC-30 ) and p53 (50 -UAU UGGGUCCGCAGGGUGAAGGCUG-30 ), along with negative control duplexes were purchased from Invitrogen. Cells were reverse transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions, and incubated for 48 h prior to drug treatment.
2.4. Recombinant adenoviruses Ad-ARID3A, Ad-p53 and Ad-1w1 (Ad-con) viruses were purified and the viral titers were determined as described [11]. To generate the Ad-shARID3A-1 and Ad-shARID3A-2 viruses, the corresponding shRNA-expression cassettes were isolated from the shRNA expression plasmids and cloned into pShuttle, and then the recombinant adenoviruses were generated according to the manufacturer’s protocols of the AdEasy system (Quantum Biotechnologies). Cells were infected at the indicated multiplicity of infection (MOI) in serumfree DMEM containing 20 mM HEPES (pH 7.4) for 1 h at 37 °C with brief agitation every 15 min, after which cells were cultured in DMEM supplemented with 10% FCS.
2.5. Chromatin immunoprecipitation (ChIP) assay ChIP assay was performed according to a protocol described previously [14]. Briefly, U2OS cells were crosslinked with formaldehyde, lysed in lysis buffers and sonicated with a Bioruptor sonicator (Cosmo Bio) to shear the crosslinked DNA. After centrifugation, the resulting chromatin solution (2 106 cells per sample) was pre-cleared with anti-mouse IgG-conjugated magnetic beads (Invitrogen) for 2 h, and then incubated overnight at 4 °C with the magnetic beads that had been pre-incubated with either anti-p53 (DO-1) or control anti-E1A (M73) monoclonal antibodies. After washing, beads were incubated at 65 °C for 4 h for reverse crosslinking. Immunoprecipitated DNA was then treated with RNaseA and proteinase K. The resultant DNA was purified by phenol–chloroform extraction and ethanol precipitation, and dissolved in 50 ll H2O and subjected to semiquantitative PCR. For the input control, a 1/200 volume of chromatin was amplified. PCR products were resolved on 2% agarose gels, stained with ethidium bromide and visualized by UV transillumination. The primer sequences used for the ChIP assays are described in Supplementary Table 1.
Total RNA was prepared using the SV Total RNA Isolation Kit (Promega) according to the manufacturer‘s instructions. Complementary DNAs were synthesized from 0.5 lg of the total RNAs with the High Capacity RNA-to-cDNA kit (Applied Biosystem). RT-PCR was performed with GoTaq Hot Start Green Master Mix (Promega) under the following conditions: mixtures were denatured at 95 °C for 1 min, followed by 24 cycles for p21, 33 cycles for ARID3A, 29 cycles for NOXA and PUMA and 30 cycles for p53 and GAPDH at 95 °C for 40 s, 55 °C for 40 s, 73 °C for 10 s, with a final extension at 72 °C for 3 min. PCR products were resolved on 2% agarose gels, stained with ethidium bromide and visualized by UV transillumination. The primer sequences used for RT-PCR are described in Supplementary Table 1. 2.9. Quantitative real-time reverse transcription (RT)-PCR analysis Quantitative real-time RT-PCR analysis was performed on the LightCycler 480 instrument (Roche Applied Science) in triplicate using TaqMan Gene Expression Assays for PUMA, Noxa, p21Waf1, and 18s rRNA (Hs00248075_m1, Hs00560402_m1, Hs003557 82_m1, and Hs03003631_g1, respectively) (Applied Biosystems) and the LightCycler 480 Probes Master kit (Roche Applied Science), according to the manufacturer’s instructions. The relative expression of mRNA, normalized to 18s rRNA, was calculated using the 2(DDCP) method. 2.10. Northern blotting Northern blotting was performed as described [11]. The intensity of each band was quantified using a Bio-Imaging Analyzer BAS 2000 System (Fuji Film). The p21 band intensities were normalized to those of the corresponding GAPDH. 2.11. Luciferase assay Transient reporter assays were performed as described [17]. Luciferase and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega). Luciferase activity was normalized to Renilla-luciferase activity. For stable reporter assays, U2OS cells were transfected with p21-luc along with pcDNA3-neo at a ratio of 3:1, and then selected with 500 mg/ml of G418 over a period of 2 weeks. The resulting G418-resistant cells
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were re-plated at 50–70% confluency in 12 well plates 20–24 h prior to adenovirus infection or drug treatment. 3. Results and discussion 3.1. ARID3A binds the p21WAF1 promoter in vivo and in vitro We identified a p53-resposible element in the ARID3A gene [11]. To confirm that ARID3A is a downstream target of p53, we performed chromatin immunoprecipitation (ChIP) assays. U2OS cells were either treated or left untreated with doxorubicin and then cross-linked with formaldehyde. Immunoprecipitation with the anti-p53 antibody revealed that p53 was recruited to the p53-resposible element in the ARID3A gene following DNA damage in vivo (Fig. 1A). These results, along with the fact that ARID3A overexpression can induce G1 arrest in U2OS cells [11], prompted us to examine whether ARID3A is involved in the transcriptional regulation of p21WAF1, which plays an important role in p53-mediated cell-cycle arrest. It has been shown that the ARID3A/Bright consensus binding sequences contain a core hexamer within a region of more than 12 bp of AT/ATC-rich sequences in one strand of the duplex, together with a second AT dimer located close to the hexamer [3]. Three putative ARID3A binding sites were found in the p21WAF1 promoter region (Fig 1B and Suppl. Fig. 1). The binding sites (BS)A and BS-B match the consensus perfectly, and BS-C contains a degenerate hexamer (AATTAT) that is reportedly a native binding site for ARID3A/Bright [3]. ChIP assays with U2OS cells using an anti-ARID3A antibody revealed that ARID3A was recruited to these putative ARID3A binding sites following infection with an adenovirus expressing ARID3A (Ad-ARID3A) (Fig. 1C). To confirm these results, we performed EMSAs using a probe containing the BS-C
sequence. A shifted band was detected in the extracts from U2OS cells infected with Ad-ARID3A (Fig. 1D, left panel, lane 2). The band was competed by unlabeled competitor oligonucleotides containing the wild-type (WT) but not mutated (MUT-1, Suppl. Fig. 1) core hexamer sequence (lanes 3 and 4), and also super-shifted by an anti-ARID3A antibody (lane 6). Furthermore, a competitor mutated in the AT dimer close to the hexamer (MUT-2, Suppl. Fig. 1) lost the ability to compete with ARID3A for binding (Fig. 1D, right panel). However, competitors mutated in the AT dimers sites distant from the hexamer (MUT-3 and MUT-4, Suppl. Fig. 1) also failed to compete the binding, while the sequences of the MUT-3 and MUT-4 competitors match the ARID3A/Bright-binding consensus motif, suggesting that the reported consensus motif is not sufficient to bind ARID3A in this setting. Taken together, we conclude that ARID3A can bind the p21WAF1 promoter both in vivo and in vitro. 3.2. ARID3A transcriptionally activates p21WAF1 To determine if ARID3A induces the transcription of p21WAF1, A459 cells carrying wild-type p53 were infected with increasing amounts of adenovirus expressing ARID3A. Western blotting and RT-PCR analysis revealed that ARID3A overexpression up-regulated both the protein and mRNA expression levels of p21WAF1 in a dose-dependent manner (Fig 2A and B). Similar results were obtained with Northern blotting (Fig 2C). Furthermore, consistent with previous observations that ARID3A induces growth arrest in U2OS cells but not Saos-2 cells lacking p53 [11], infection of AdARID3A up-regulated the p21WAF1 protein and mRNA expression in U2OS cells, whereas p21WAF1 induction was undetectable in Saos-2 cells (Fig. 2D and E). Similar results were obtained with HCT116 cells expressing wild-type p53 (p53+/+) and p53-negative derivative HCT116 cells (p53/) (data not shown). These results
Fig. 1. Binding of ARID3A to the p21WAF1 promoter in vivo and in vitro. (A) U2OS cells were untreated or treated with 0.2 lg/ml doxorubicin (Doxo), fixed with 1% formaldehyde at 16 h post-treatment, and processed for ChIP analysis using anti-E1A (control) or anti-p53 antibodies. p53 occupancy on its binding sequences in the ARID3A gene was detected by PCR. (B) Schematic representation of ARID3A-binding sites in the p21WAF1 promoter reporter construct used in this study. The black and open boxes represent the p53 and ARID3A binding sites (BS-A, BS-B, and BS-C), respectively. Luc: luciferase gene. (C) U2OS cells were infected with either Ad-con or Ad-ARID3A (MOI = 200), fixed at 24 h post-infection, and processed for ChIP analysis using monoclonal anti-E1A (M73) and anti-ADRID3A (ARIDE9C11) antibodies. ADRID3A occupancy was detected as in A. (D) EMSAs were performed using cell lysates prepared from U2OS cells infected with Ad-con or Ad-ADRID3A and a 32P-labeled probe containing potential ARID3A-binding site (BS-C). (left panel) Competition assays were performed by adding 100-M excess of the unlabeled oligonucleotides to the reaction mixtures as indicated. Antibody supershift assays were performed by adding the following antibodies: normal rabbit IgG (lane 5) and a polyclonal anti-ARID3A N-20 antibody (lane 6). The positions of the ARID3A-DNA complexes and antibody-specific super-shifted complex are indicated by arrows. Asterisks indicate non-specific bands. (right panel) Competition assays were performed by adding a 50 or 200-M excess of the indicated unlabeled wild-type or mutant oligonucleotides.
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Fig. 2. ARID3A transcriptionally activates p21WAF1. (A, B, and C) A549 cells were treated with doxorubicin as in Fig. 1A or infected with increasing MOIs (20 and 80) of Ad-con or Ad-ARID3A. The total MOI of each sample was adjusted with Ad-con. (A) Western blotting analysis at 48 h post-infection using anti-p21 and anti-Xpress antibodies, the latter of which detects the Xpress-tagged ARID3A protein encoded by Ad-ARID3A. b-actin was used as the loading control. (B and C) Semiquantitative RT-PCR (B) and Northern blotting analysis (C) at 24 h post-infection. (C, right panel) Relative fold induction was determined as described in Section 2. Data represent means ± SD from three independent experiments. GAPDH was used as an internal control (B and C). (D and E) U2OS and Saos2 cells were treated with doxorubicin as in Fig. 1A or infected with Adcon or Ad-ARID3A at 80 MOI. (D) Western blotting analysis at 48 h post-infection using the indicated antibodies. b-actin was used as the loading control. (E) Semiquantitative RT-PCR analysis at 24 h post-infection.
indicate that ARID3A by itself cannot substitute for p53-mediated functions in the absence of wild-type p53. In addition, we detected an up-regulation of p53 protein but not mRNA following AdARID3A infection, indicating that the ectopic expression of ARID3A increases the stability of p53 protein (Fig. 2D and E). These results suggest that ARID3A needs to cooperate with p53 to induce p21WAF1 transcription. 3.3. ARID3A cooperates with p53 to activate p21WAF1 transcription We thus examined whether the coexpression of ARID3A with p53 activates p21WAF1 in a cooperative fashion. A549 cells were co-infected with Ad-p53 and Ad-ARID3A and the levels of p21WAF1 mRNA were examined by Northern blotting. Fig. 3A shows that the coinfection of Ad-ARID3A with a lower dose of Ad-p53 (MOI = 20) synergistically activates p21WAF1 transcription. We next asked whether the coexpression of these genes cooperate to activate the p21WAF1 promoter. To this end, we performed reporter assays using a construct encoding a luciferase reporter gene driven by the p21WAF1 promoter containing three ARID3A-binding sites (Fig. 1B). The p21WAF1 reporter construct was transiently transfected into U2OS cells along with either or both of the ARID3A and p53 expression plasmids. However, transfection of ARID3A failed to activate the p21WAF1 promoter, whereas p53 activated the reporter activity in a dose-dependent manner (Fig. 3B). Furthermore, co-transfection of ARID3A and p53 did not exhibit any cooperative activity, but rather, ARID3A reduced the transactivation capacity of p53. It has been shown that Bright can only transactivate the IgH S107 promoter, when it is integrated within chromatin [18]. Therefore, we asked whether ARID3A activates the chromatin-integrated p21WAF1 promoter. To this end, U2OS cells were transfected with the p21WAF1 reporter along with a plasmid expressing the neomycin-resistance gene, selected with G418, and then G418-resisant cells were subjected to reporter assay. Fig. 3C shows that infection of Ad-ARID3A enhanced the reporter activity in a dose dependent manner, which activity was comparable to that obtained by Adp53 infection and doxorubicin treatment. Furthermore, the coexpression of ARID3A with p53 cooperated to activate the p21WAF1
promoter (Fig. 3D), indicating that ARID3A can cooperate with p53 to activate the chromatin-integrated p21WAF1 promoter. To determine the role of the ARID3A binding sites in the transactivation of the p21WAF1 promoter, we mutated all three of the binding sites (BS-A, BS-B, and BS-C) in the p21WAF1 reporter construct (p21-luc_Mut). U2OS cells were stably transfected with either the wild-type or mutant reporter construct followed by the reporter assays. DNA damage caused a 3.5-fold activation of the wild-type reporter activity (Fig. 3E). Mutations in the ARID3A binding sites led to a partial reduction (2.5-fold) of the p21WAF1 promoter activity following DNA damage. However, the ARID3A binding site mutations had no significant effect on the transactivation of the p21WAF1 promoter by the ectopic expression of either ARID3A or p53 in both transient and stable reporter assays (data not shown), suggesting that the presence of the ARID3A binding sites affects the activity of the chromatin-integrated p21WAF1 promoter under conditions in which physiological levels of ARID3A and p53 are expressed in the cell. 3.4. ARID3A knockdown blocked transcriptional activation of p21WAF1 We then examined the role of ARID3A in p53-dependent transactivation of the p21WAF1 by using small interfering RNAs (siRNAs) that target ARID3A and p53 (siARID3A and sip53, respectively). A549 cells were reversely transfected with siRNAs followed by doxorubicin treatment. Consistent with previous observations, the ARID3A mRNA levels were increased following DNA damage, and suppressed not only by ARID3A siRNA, but also by p53 siRNA (Fig. 4A). ARID3A knockdown blocked the transcriptional activation of p21WAF1 but not the proapoptotic NOXA and PUMA following DNA damage, whereas p53 knockdown suppressed the induction of all three of the p53-target genes examined (Fig. 4A and B). Western blotting confirmed that the induction of p21WAF1 protein was inhibited by both ARID3A and p53 siRNAs (Fig. 4C). Although ARID3A knockdown inhibited p21WAF1 expression, it suppressed cell proliferation and the transcription of the E2F-target genes required for cell cycle progression in the absence of DNA damage (unpublished data), which is supported by the fact that ARID3A also plays a critical role in E2F-mediated cell cycle progression [5,6].
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Fig. 3. ARID3A cooperates with p53 to activate p21WAF1 transcription. (A) A549 cells were infected with Ad-con or the indicated combination of Ad-ARID3A and Ad-p53, and subjected to Northern blot analysis for p21WAF1 mRNA as in Fig. 2C (upper panel). (lower panel) Relative fold induction was determined as in Fig. 2C. (B) U2OS cells were transiently transfected with the p21WAF1 reporter along with increasing amounts of p53 (0.5, 2.5 and 10 ng) and/or ARID3A (50, 100 and 200 ng) expression vectors. Luciferase was assayed 24 h after transfection. (C) U2OS cells were transfected with the p21WAF1 reporter construct along with an expression vector for the neomycin resistance gene and selected with G418 for 2 weeks. The G418-resisant cells were re-plated and then infected with Ad-con (control) or increasing doses of Ad-ARID3A (MOI = 100, 200 and 300) and p53 (MOI = 15 and 30) as in Fig. 2, or treated with doxorubicin as in Fig. 1A. Luciferase was assayed 24 h after treatment. (D) U2OS cells stably transfected as in D with the p21WAF1 reporter were infected with the indicated combination of Ad-ARID3A (MOI = 100) and p53 (MOI = 15 and 30) as in Fig. 2. (E) U2OS cells stably transfected with the p21WAF1 reporter constructs containing the wild-type or mutated ARID3A-binding sites (p21-luc_WT and p21-luc_Mut, respectively) were treated with doxorubicin as in Fig. 1A. Luciferase was assayed 24 h after treatment. The data present the average of three independent experiments, each performed in duplicate (B, C, D, and E).
Collectively, these results demonstrate that ARID3A play an important role in the p53-mediated transcriptional transactivation of p21WAF1. 3.5. ARID3A knockdown reduces p53 stability Finally, as shown in Fig. 4A and B, ARID3A knockdown reduced the protein but not mRNA levels of p53. These results, along with the fact that ARID3A overexpression increases the stability of p53 protein (Fig. 2D and E), implies that ARID3A plays an important role in the regulation of p53 stability. These results raise the possibility that the reduction of p53 stability by ARID3A knockdown may affect the p53-mediated transactivation of the p21WAF1 promoter. To test this possibility, we performed transient reporter assays using expression vectors for two shRNAs that target different ARID3A sequences (shARID3A-1 and -2). RT-PCR analysis confirmed that each of the ARID3A shRNAs blocked the expression of p21WAF1 and ARID3A mRNA (Fig. 4D, left panel). Reporter assays revealed that both ARID3A shRNAs blocked the p53-mediated transactivation of the p21WAF1 promoter (Fig. 4D, right panel). Since
ARID3A overexpression and its binding site mutations had no effect of on the p53-mediated activation of the p21WAF1 promoter in transient reporter assays (Fig. 3B, data not shown), these results indicate that the inhibition of ARID3A blocks p53-mediated p21WAF1 promoter activation through a reduction of p53 stability. The mechanism underlying this phenomenon remains unknown. It has been shown that p53 co-localizes with the PML Nuclear Bodies (NBs) and PML potentiates p53 activity by regulating post-translational modifications, such as acetylation and phosphorylation (Reviewed in [19,20]). ARID3A also co-localizes with PML NBs and binds the Sp100 family protein, a component of the PML NBs [21]. We thus speculate that ARID3A might play a role in p53 post-translational modifications mediated by PML NBs. The results presented in his study, together with our previous study, show that p53 regulates ARID3A transcription and then cooperates with ARID3A to transcriptionally activate the common target gene, p21WAF1. Furthermore, we show that ARID3A plays critical roles in p53-mediated p21WAF1 transactivation and that modulation of the ARID3A level affects p53 protein stability, indicating cooperative and interdependent roles for these proteins in transcriptional
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Fig. 4. ARID3A knockdown blocked transcriptional activation of p21WAF1. (A, B and C) A549 cells were reversely transfected with scramble (control), ARID3A or p53 siRNA. At 24 h post-transfection, cells were either left untreated or treated with doxorubicin for 24 h. (A) Semiquantitative RT-PCR analysis using primers specific for the indicated transcripts. (B) Relative fold induction was determined by quantitative real-time RT-PCR analysis. The data represent means ± SD from three independent experiments. (C) Western blotting analysis using the indicated antibodies. (D) (left panel) 293 cells were infected with the indicated shRNA-expressing adenoviruses. Semiquantitative RT-PCR of the indicated transcripts at 48 h post-infection. (right panel) H1299 cells were transiently transfected with p53 along with the indicated shRNA-expression plasmids. Luciferase was assayed 24 h after transfection. The data present the average of three independent experiments, each performed in duplicate.
activation of p21WAF1. These data provide new insights into the mechanisms underlying the function of p53 and ARID3A in the transcriptional regulation of p53-tagert genes. Further experiments are required to elucidate specific roles of ARID3A in p53 stabilization and p53-mediated cell fate decision of cell cycle arrest. Acknowledgments This study was supported in part by Grants-in–Aid for Scientific Research (16209054, 21390502, and 21659454 to MAI) from the Japan Society for the Promotion of Science. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.12.003. References [1] D. Wilsker, A. Patsialou, P.B. Dallas, E. Moran, ARID proteins: a diverse family of DNA binding proteins implicated in the control of cell growth, differentiation, and development, Cell Growth Differ. 13 (2002) 95–106. [2] D. Wilsker, L. Probst, H.M. Wain, L. Maltais, P.W. Tucker, E. Moran, Nomenclature of the ARID family of DNA-binding proteins, Genomics 86 (2005) 242–251.
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