Rad9 modulates the P21 WAF1 pathway by direct association with p53
© Ishikawa et al; licensee BioMed Central Ltd. 2007
Received: 02 February 2007
Accepted: 21 May 2007
Published: 21 May 2007
Previous studies suggest that human RAD9 (hRad9), encoding a DNA damage checkpoint molecule, which is frequently amplified in epithelial tumor cells of breast, lung, head and neck cancer, participates in regulation of the tumor suppressor p53-dependent transactivation of pro-survival P21 WAF1 . This study examined the exact mechanism of the hRad9 function, especially through the phosphorylation of the C-terminus, in the transcription regulation of P21 WAF1 .
The transfection of phosphorylation-defective hRAD9 mutants of C-terminus resulted in reduction of the p53-dependent P21 WAF1 transactivation; the knockdown of total hRad9 elicited an increased P21 WAF1 mRNA expression. Immunoprecipitation and a ChIP assay showed that hRad9 and p53 formed a complex and both were associated with two p53-consensus DNA-binding sequences in the 5' region of P21 WAF1 gene. The association was reduced in the experiment of phosphorylation-defective hRAD9 mutants.
The present study indicates the direct involvement of hRad9 in the p53-dependent P21 WAF1 transcriptional mechanism, presumably via the phosphorylation sites, and alterations of the hRad9 pathway might therefore contribute to the perturbation of checkpoint activation in cancer cells.
DNA damage checkpoints are signal transduction pathways that maintain the proper order of cell cycle events. When DNA is damaged or perturbed during replication, the cells respond by the activation of evolutionarily conserved signal transduction pathways that delay the progression of the cell cycle and induce repair of the damaged DNA. These signal transduction pathways include protein sensors that recognize aberrant DNA structures and activate kinases, thereby inducing phosphorylation cascades that ultimately lead to cell cycle arrest and DNA repair [1, 2]. Failure of this cell cycle surveillance mechanism can cause genomic instability which eventually leads to the formation of cancer in mammals .
hRad9 protein is the human homologue of Schizosaccharomyces pombe Rad9, a member of the checkpoint rad genes (rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+) which are required for the S phase (DNA replication) and G2 (DNA mitosis) check points [4, 5]. Like its yeast counterpart, hRad9 forms a ring-shaped, heterotrimeric complex with the hRad1 and hHus1 proteins [6, 7]. Each member of hRad9-hRad1-hHus1 complex (also known as the 9-1-1 complex), shares sequence homology with proliferating cell nuclear antigen (PCNA), a homotrimer that encircles the DNA and tethers DNA polymerase δ during DNA synthesis [7–10]. PCNA is loaded onto DNA by the pentameric protein complex replication factor C (RFC) , which is composed of one large subunit and four smaller subunits. In a manner analogous to PCNA and RFC, 9-1-1 complex is loaded onto DNA by a complex between hRad17 and the four small subunits of RFC . Since DNA damage induces hRad17-dependent association of 9-1-1 complex with chromatin, the 9-1-1 complex is believed to be involved in the direct recognition of DNA lesions during the initial stages of the checkpoint response; the 9-1-1 complex may thus be associated with chromatin following DNA damage to transduce signals for DNA damage-activated checkpoint signaling pathways . In mammalian cells, the signal initiated by the sensors, two phosphatidylinositol 3-kinase-related kinases (PIKK), ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia mutated and Rad3-related (ATR), plays a central role in the checkpoint signaling pathways . ATM and ATR are activated by downstream signaling proteins, genotoxins and phosphorylation including Chk1 and Chk2, two protein kinases that regulate checkpoint responses [15–17]. hRad9 is highly modified by phosphorylation in at specific points of the cell cycle after DNA damage, and it also plays a critical role in checkpoint signaling . ATM-mediated phosphorylation at Ser-272 of hRad9 is required for IR-induced G1/S checkpoint activation [19, 20]; Other phosphorylation sites in the C-terminal region are also essential for Chk1 activation following hydroxyurea (HU), IR, and UV treatment , although the exact function of hRad9 in cell cycle control has not yet been completely characterized.
The tumor suppressor gene TP53 controls cell cycle checkpoints, apoptosis, and genomic stability . A defect in the pathway, involving p53, is essential for the malignant progression of cancer . When cells with wild-type TP53 are exposed to DNA-damaging agents, p53 is functionally activated, p53 protein level rises, and p53 binds to and transcriptionally activates the promoters of target genes [21, 23, 24]. These target genes include; P21, MDM2, GADD45, BAX, IGF-BP3 and CYCLIN G. The P21 gene product was originally identified, as a potential target for the p53 tumor suppressive activity (WAF1) . It is either an inhibitor of the G1 cyclin-dependent kinases (Cip1) , or an inhibitor of DNA synthesis that is expressed during cellular senescence (SDI1) . It is known that p21 is a major effector of the G1 cell cycle checkpoint. Therefore, p53 is a negative regulator of the cell cycle progression and it controls the transition from G1 to S phase of the cell cycle . This report demonstrates that hRad9 plays a role in the modulation of P21 transcription by direct interaction with p53. Furthermore, the substitution of the phosphorylation sites on hRad9 to Ala resulted in an alteration of the regulation of P21. The present study supports the hypothesis that hRad9 plays a role in the modulation of P21 transcription, presumably via competition with p53, that involves its C-terminus, which would be essential for the cellular response to DNA damage.
Results and discussion
P21 is activated immediately after UV treatment
The role of hRad9 in p53-dependent P21 activation through its C-terminus
Knock down of endogenous hRad9 increases the transcription of P21
hRad9 associates with p53
Previous studies have showed that p53 protein can be phosphorylated at Ser-15 within 1 hr after DNA damage . A Western blot analysis indicated that Ser-15 of p53 was phosphorylated 5 min after UV treatment in the present experiments (Fig. 4C). The time course of this reaction was examined to determine whether the association of hRad9 and p53 might be altered after UV irradiation. Figure 4D demonstrates that the phosphorylation of p53 at Ser-15 in the immunoprecipitated components increased at 5 min and reached a maximal level at 6 hr after UV treatment, and declined, consistent with the Western blot findings in Figure 4C, whereas the total amount of Rad9-p53 interaction did not increase. In addition, other phosphorylation sites of p53, including Ser-6, Ser-9, Ser-20, Ser-37, Ser-46, and Ser-392 were also examined and they were phosphorylated temporally, regardless of the positive association of hRad9 with p53 over the time course, as observed Ser-15 (Fig. 4D, and data not shown). These results indicate that the binding of hRad9 and p53 is not significantly affected by phosphorylation, though it is possible that the modifications of the phosphorylation in these amino acid residues are not involved in the binding of the components.
The present immunoprecipitation study revealed hRad9 to be associated with p53, and the association was detected regardless of degree of phosphorylation of p53 and presumably of hRad9. A previous report also demonstrated that constitutive phosphorylation of hRad9 does not influence the stability of the 9-1-1 complex . It is suggested that hRad9, as a complex with p53, may be involved in the transactivation of P21 and that the phosphorylations of hRad9 and p53 might modulate the transactivation activity of the complex. Whereas none of the phosphorylation sites of hRad9 targeted in the present study have been previously reported to be required for genotoxin-induced chromatin binding , the present data suggest that hRad9 phosphorylation might be involved in binding affinity for p53-consensus binding sites.
Phosphorylation of hRad9 affects the preference of p53 for binding sites
Recent studies have demonstrated the multifunctional roles of hRad9 a DNA damage sensor in the 9-1-1 complex, a G2/M checkpoint via the phosphorylation of Chk1, in DNA repair via DNA polymerase β  or flap endonuclease 1 , and in apoptosis via potential binding to Bcl-2 or Bcl-xL . The present study, demonstrated the direct association of Rad9-p53 and the regulatory role of phosphorylation in the activation of P21 transcription, thus indicating that hRad9 is an important modulator, but not a unique player with a single function.
hRad9 has a complex role in response to DNA damage, acting not only as an activator but also as a modulator in P21 transcription and contributing to the regulation of genomic integrity. If hRad9 was regulated inaccurately, the P21 could not regulate the appropriate G1/S transition and replication, thus resulting in the occurrence of unscheduled replications after DNA damage. Such events can contribute to the accumulation of pathological conditions and genomic instability in carcinogenesis and tumor progression.
Primers and Probes for Each Experiments
Names of Primers and Probes
Target sequence of pBAsi-mU6 vector (TAKARA; Code: 3225)
5'-CCA CAC TCT TAG AGC AAG A-3'
PCR primers for wild-type and RAD9 mutants
5'-AAA AGC GGC CGC GCA TGA AGT GCC TGG TCA CGG G-3' and 5'-TTT TCT AGA TCA GCC TTC ACC CTC ACT GTC-3'
RT-PCR primers for P21 corresponding to an amplified product of 335 bp
5'-ACC CTC TCA TGC TCC AGG T-3' and 5'-CCT TGT TCC GCT GCT AAT CA-3'
RT-PCR primers for glyceraldehydes-3-phosphate dehydrogenase (G3PDH), to an amplified product of 451 bp product
5'-ACC ACA GTC CAT GCC ATC AC-3' and 5'-TCC ACC ACC CTG TTG CTG TA-3'
ChIP primers for human P21 downstream promoter
5'-GAG GTC AGC TGC GTT AGA GG-3' and 5'-TGC AGA GGA TGG ATT GTT CA-3'
ChIP primers for human P21 upstream promoter
5'-CCT ATG CTG CCT GCT TCC CAG GAA-3' and 5'-TAG CCA CCA GCC TCT TCT ATG CCA G-3'
Antibodies and plasmids
The following antibodies were used: anti-Rad9 (Alexis), anti-p53 (BD Transduction Laboratories), anti-acetylated histone H4 (Upstate Biotechnology), anti-FLAG (Sigma-Aldrich), and anti-phosphorylated p53 (Ser-15) antibody (Cell Signaling Technology).
We used the wild-type RAD9 plasmid and the phosphorylation-defective RAD9 mutants, kindly given from Dr. L. M. Karnitz . To construct FLAG-tagged RAD9 expression plasmids, the DNAs were amplified by the PCR method with the advantage Clontaq system with high fidelity (Clontech) according to the manufacture's instruction, by using wild-type and RAD9 mutant vectors as templates and a set of primers (Table 1). The amplified products were separated by the electrophoresis, cut and purified with gel purification kit (Qiagen). After digestion with NotI and XbaI, the samples were ligated to the cloning site of pcDNA3.1-FLAG vector. WWP-Luc-P21 promoter vector, kindly given from Dr. B. Vogelstein , pcDNA-TP53 expression plasmid, kindly given from Dr. J. Yokota .
Plasmids were transfected with LipofectAMINE2000 according to manufacturer's instructions (Invitrogen). Double-stranded siRNAs for hRAD9 and TP53 (Santa Cruz Biotechnology) were transfected twice 24 h apart using TransIT-TKO transfection reagent (Mirus).
Reverse transcription (RT)-PCR and real time PCR
RNA was extracted using ISOGEN protocol (Nippon Gene). First-strand cDNA was prepared from total RNA (5 μg) and oligo (dT) using the Superscript First-Strand Synthesis System (Invitrogen). The synthesized cDNA was amplified by PCR. The oligonucleotides used were shown in Table 1[42, 43]. The PCR conditions were: for P21, an initial denaturation at 94°C for 1 min, followed by 37 cycles of 94°C for 8 sec, 53°C for 30 sec, and 72°C for 1 min; for G3PDH, the denaturation at 94°C for 1 min, followed by 28 cycles of 94°C for 10 sec, 60°C for 15 sec, and 72°C for 1 min. PCR products were separated by electrophoresis and visualized by ethidium bromide staining. The intensity of each band corresponding to PCR was quantified by densitometry analysis (Quantity One, BIO-RAD). The negative control without RTase showed no amplifications. Each experiment was repeated at least thrice. PCR-Southern blot analysis was performed as described , with minor modifications. Briefly, after four different cycles (24, 28, 32 and 36) of PCR, reactions (20 ul) in separate tubes were subjected to electrophoresis, transferred to nylon filter and were hybridized with [32P] dCTP-labeled P21 or G3PDH probe, which was amplified by RT-PCR. After washes, filter was exposed on x-ray film. For Real time RT-PCR assessment of P21 expression, total RNA was extracted and cDNA was synthesized. Primers and TaqMan probe were used for amplification and assessment according to the manufacture's instruction (Mm00432448_m1, Applied Biosystems).
For Western blotting, cells were extracted with lysis buffer [20 mM Tris-Hcl (PH 7.4), 1% Triton × 100, 10% glycerol, and 0.1 mM PMSF]. The cleared extracts were resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Immunoblotting was performed by standards methods and signal was detected enhanced chemiluminescence system (ECL, Amarsham Biosciences). The intensity of each band was quantified by densitometry analysis (Quantity One, BIO-RAD). For immunoprecipitation (IP), cells were harvested with IP-lysis buffer [25 mM Tris-HCl (PH 7.5), 0.2% NP40, 250 mM NaCl, and 1 mM EDTA] and 500 μg of cell lysates, after being precleared with protein G-Sepharose beads, were incubated with 3 μg of specific antibody overnight. Antigen-antibody complex was immobilized on protein G-Sepharose beads, and washed five times in lysis buffer. Bound proteins were eluted by boiling and subjected to SDS-PAGE and immunoblotting.
For luciferase reporter assay, transfected cells were cultured in a complete growth medium for 24 h and harvested, performed according to the manufacture's instruction (Promega). Each plasmids including empty expression vector were transfected with the same amounts (60 ng). Luciferase activity was measured on a Fluoroskan Ascent FL Luminometer (Thermo Labsystems Oy). As the internal control, each sample was co-transfected with pRL-TK, and the relative luciferase activity was figured out as the ratio of Firefly to Renille to adjust the tansfection rates. Each experiment was repeated at least thrice. Chromatin immunoprecipitation (ChIP) assay was performed using the Upstate Biotechnology kit. Briefly, ~1 × 106 cells were cross-linked with 1% formaldehyde, resuspended in 200 μL of SDS-lysis buffer, and sonicated to disrupt chromatin at an average length of 200 to 1,000 bp. After centrifugation, 20 μL of each supernatant were heated at 65°C for 4 h after the addition of 1 μL of 5 M NaCl, which was used as an input. The rest of the supernatant was added to 300 μL of ChIP dilution buffer containing 1 μg of either anti-Rad9 or anti-p53 antibody. After incubation at 4°C for 16 h, the immunoprecipitates were washed, eluted, heat treated, and digested with proteinase K. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. We used one tenth (2 μL) of the final suspension for PCR using primers corresponding to different regions of the human P21 promoter. Primers used were shown in Table 1.
We thank Bert Vogelstein (The Johns Hopkins Oncology Center, Baltimore, USA) for giving WWP-Luc-P21 promoter plasmid kindly, Jun Yokota (National cancer Center Research Institute, Tokyo, Japan) for giving TP53 plasmid kindly, and Larry M. Karnitz (Developmental Oncology Research and Radiation Oncology, Mayo Clinic and Foundation, Rochester, Minnesota, USA) for giving RAD9 plasmids (wild-type RAD9, RAD9-S272A, RAD9-8A, RAD9-9A) kindly. We thank Larry M. Karnitz and Jun Yokota for critical reading the manuscript and helpful suggestions.
This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and The Research Award to JMS Graduate Student.
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