The Nucleolar Protein Myb-binding Protein 1A (MYBBP1A) Enhances p53 Tetramerization and Acetylation in Response to Nucleolar Disruption*

Background: Nucleolar disruption is involved in cellular stress response and is sufficient for p53 activation. p53 tetramerization is crucial to exert its activity. Results: The nucleolar protein MYBBP1A enhanced p53 tetramerization by directly interacting with p53. Conclusion: p53 tetramerization by MYBBP1A is indispensable for activating p53 under nucleolar stress. Significance: This is the first report to describe the possible mechanism underlying p53 tetramerization in cells under nucleolar stress. Tetramerization of p53 is crucial to exert its biological activity, and nucleolar disruption is sufficient to activate p53. We previously demonstrated that nucleolar stress induces translocation of the nucleolar protein MYBBP1A from the nucleolus to the nucleoplasm and enhances p53 activity. However, whether and how MYBBP1A regulates p53 tetramerization in response to nucleolar stress remain unclear. In this study, we demonstrated that MYBBP1A enhances p53 tetramerization, followed by acetylation under nucleolar stress. We found that MYBBP1A has two regions that directly bind to lysine residues of the p53 C-terminal regulatory domain. MYBBP1A formed a self-assembled complex that provided a molecular platform for p53 tetramerization and enhanced p300-mediated acetylation of the p53 tetramer. Moreover, our results show that MYBBP1A functions to enhance p53 tetramerization that is necessary for p53 activation, followed by cell death with actinomycin D treatment. Thus, we suggest that MYBBP1A plays a pivotal role in the cellular stress response.

The tumor suppressor p53 is a critical mediator of the cellular stress response because it maintains genomic integrity and prevents oncogenic transformation (1). The p53 protein regulates many target genes that induce cell cycle arrest or apoptosis (2,3). The p53 protein level is tightly regulated and maintained at low levels in unstressed cells by its negative regulator HDM2.
HDM2 is a ubiquitin ligase that ubiquitinates p53, thereby targeting p53 for proteasome-mediated degradation (4 -6). HDM2 is inactivated in response to various stressors such as DNA damage; thus, p53 is rapidly stabilized and activated in cells that sustain various types of stressors (3).
In addition, tetramerization regulates p53 post-translational modifications (40). p300 interacts and promotes acetylation of p53-WT but does not interact and promote p53 mutant proteins, which are unable to form tetramers. Activation of p53 in response to DNA damage begins with tetramerization of p53, which provides appropriate binding sites for p300 and leads to binding of p300 and subsequent acetylation of p53 C-terminal lysine residues. The acetylation, in turn, further tightens the p300-p53 complex, stabilizes the p53 protein, and binds to the promoter sequence to facilitate recruitment of coactivators and transactivation of target genes (28). Therefore, tetramerization of p53 is vital to its function and plays a pivotal role in regulating its activity.
We reported previously that nucleolar disruption induces acetylation and accumulation of p53 without phosphorylation. The nucleolar protein Myb-binding protein 1A (MYBBP1A) binds to p53-CRD lysine residues (57) and facilitates p53 acetylation to enhance p53-mediated transcription by enhancing p53-p300 interactions in a manner that differs from that of other nucleolar proteins (58). However, the mechanism by which MYBBP1A enhances p53 acetylation and induces p53 activity remains unclear.
In this study, we demonstrated that MYBBP1A has two p53binding regions and that it enhances p53 tetramerization by dimerizing itself, thereby promoting the interaction between p53 and p300 and acetylation of p53. Thus, we suggest that MYBBP1A plays a pivotal role in the cellular stress response.

EXPERIMENTAL PROCEDURES
Cell Culture and Treatments-MCF-7 human breast cancer cells and H1299 p53-deficient human lung cancer cells were maintained in DMEM (Sigma). LNCaP human prostate cancer cells were maintained in RPMI 1640 (Nacalai Tesque, Kyoto, Japan). All media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution (Nacalai Tesque). Cells were maintained at 37°C in an atmosphere containing 5% CO 2 and 100% humidity. To induce nucleolar stress, cells were exposed to ActD (5 nM), UV irradiation (25 J/m 2 ), and adriamycin (0.5 mg/ml). Cell viability after ActD treatment was determined by the trypan blue dye exclusion test.
siRNA and Plasmid DNA Transfection-Cells at 30 -50% confluency were siRNA-transfected using Lipofectamine RNAi MAX (Invitrogen), according to the manufacturer's protocol. All siRNAs were purchased from Invitrogen. The siRNA duplex was siMYBBP1A, 5Ј-UCUUUCAGUCAGGUCGGCUGGU-GAA-3Ј. Stealth RNAi negative control medium or high GC was used as the negative control. Protein and RNA were extracted 48 h after siRNA transfection. Cells at 70 -80% confluency were transfected with plasmid DNA using Lipofectamine LTX (Invitrogen) according to the manufacturer's protocol. Protein was extracted 24 h after transfection of plasmid DNA.
Expression Vectors and Antibodies-cDNAs encoding fulllength and the indicated mutants of p53 and MYBBP1A were amplified by PCR and subcloned into the pcDNA3 plasmid (Invitrogen) containing sequences encoding FLAG, FLAG-HA, HA, or Myc sequences. ␤-Actin (Sigma) and anti-human-p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA) monoclonal antibodies and rabbit anti-p53-K382Ac (Cell Signaling Technology, Danvers, MA) polyclonal antibodies were used according to the manufacturer's instructions. Rabbit antihuman MYBBP1A antibody was raised against a synthetic peptide corresponding to amino acids 1265-1328 of human MYBBP1A.
Electrophoretic Mobility Shift Assay-EMSA was performed using the LightShift TM EMSA optimization and control kit and chemoluminescent nucleic acid detection modules (Pierce). Binding reactions containing 1 l of p53 and/or MYBBP1Aoverexpressing cell lysates, 10 mM Tris, 50 mM KCl, 1 mM DTT, 2.5% glycerol, 5 mM MgCl 2 , 0.05% Nonidet P-40, and the double-stranded oligonucleotide probe were incubated in a final volume of 10 l for 20 min at room temperature. After the incubation, the samples were separated on 5% SDS-PAGE gel, transferred to nylon membranes by electroblotting, and UV cross-linked. DNA-protein complexes were visualized with streptavidin-horseradish peroxidase, followed by chemiluminescence detection. According to a published procedure (59), EMSA was performed using p21 dsDNA (5Ј-CGCGAACAT-GTTCGAACATGTTCGCG-3Ј), which is similar to the p21-5Ј p53-binding site, as described under "Experimental Procedures" for the ChIP assay.

MYBBP1A Enhances p53 Tetramerization
Coimmunoprecipitation (Co-IP) and Immunoblotting-Cells were lysed in TNE buffer supplemented with 1 M phenylmethylsulfonyl fluoride and 1 g/ml aprotinin. Extracted proteins were immunoprecipitated with antibody-coated protein G-Sepharose (Amersham Biosciences) beads. Bound proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Milford, MA), and detected with appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualized using an enhanced chemiluminescence immunoblot detection system (Amersham Biosciences).
Protein Cross-linking Assay-Cells were transfected with the indicated siRNA or plasmids and lysed in TNE buffer after 6 h of ActD treatment. Glutaraldehyde (GA) was added to the lysates at the indicated concentrations. After incubating the lysates on ice for 20 min, the GA reactions were stopped by adding 2ϫ loading buffer, and the samples were heated at 100°C for 5 min and resolved by SDS-PAGE. Western blot analysis was performed with anti-p53 antibody (DO-1).
Duolink in Situ Proximity Ligation Assay (PLA)-The Duolink in situ PLA was performed according to the manufacturer's protocol (Olink Bioscience, San Francisco, CA). In brief, H1299 cells transfected with the indicated plasmids and grown on chamber slides were rinsed three times with PBS (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , and 8.1 mM Na 2 HPO 4 ) and fixed in 4% formaldehyde in PBS for 10 min. After rinsing three times with PBS, the cells were permeabilized in 0.5% Triton X-100 in 20 mM HEPES (pH 7.5) with 150 mM KCl and blocked with TBS-T buffer containing 3% BSA for 1 h at 37°C. After blocking, the cells were incubated for 2 h at 37°C with mouse anti-p53 and rabbit anti-FLAG antibodies in PBS containing 1% BSA. Cells were washed three times with PBS and incubated with secondary rabbit PLUS and mouse MINUS antibodies for 1 h at 37°C in the dark. Cells were washed three times in PBS before detecting the probe using the in situ PLA detection kit (Olink Bioscience). Duolink and DAPI signals were detected using an LSM-700 microscope (Carl Zeiss, Oberkochen, Germany).

RESULTS
MYBBP1A Contains Two p53-binding Sites-To determine the region responsible for p53 binding to MYBBP1A, we performed the GST pulldown assay using full-length wild-type MYBBP1A (MYBBP1A-WT) or the indicated MYBBP1A deletion mutants (Fig. 1A). The GST pulldown assays indicated that both regions from amino acids (a.a.) 643 to 1150 and from a.a. 1151 to 1328 of MYBBP1A interacted with p53. To limit the MYBBP1A-responsive region for p53 binding, we generated four additional MYBBP1A deletion mutants (a.a. 1-1150, 643-1150, 775-1150, and 1151-1271). The GST pulldown assay revealed that MYBBP1A had bound to p53 via the middle region (a.a. 643-1150) and the basic repeat-rich region (a.a. 1151-1271) within the C-terminal region of MYBBP1A ( We subsequently tested the interaction of these domains with p53 in cells. We introduced p53 expression plasmids into H1299 cells with or without expression plasmids for the fulllength or deletion mutants of MYBBP1A. Co-IP assays showed the association between p53 and MYBBP1A-WT in H1299 cells under ActD treatment (Fig. 1B, lane 2). Interactions of p53 with MYBBP1A deletion mutants were also detectable ( Fig. 1B, lanes 3 and 4). To directly show the interaction between MYBBP1A and p53 in intact cells, we performed immunofluorescent staining using the Duolink in situ PLA, which enables visualization of the MYBBP1A-p53 interaction. Similar to MYBBP1A-WT, the N-terminal (a.a. 1-1150) and C-terminal (a.a. 1151-1328) regions of MYBBP1A, each of which contains a single p53-bindig site, bound to p53-mTET (Fig. 1C).
Taken together, these data indicate that MYBBP1A has two p53-binding sites. However, it remains to be determined whether these two regions of MYBBP1A simultaneously and cooperatively interact with the p53 dimer.
Our previous report also revealed that the p53-6KQ mutant (K370Q/K372Q/K373Q/K381Q/K382Q/K386Q), known as an acetylation mimicking mutation, does not bind to MYBBP1A (57). In contrast, the p53-6KR mutant (K370R/K372R/K373R/ K381R/K382R/K386R), which is generally used as an acetylation-deficient missense mutant, binds to MYBBP1A more strongly than p53-WT (57). Taken together, we conclude that MYBBP1A dissociates from p53 in an acetylation-dependent manner with the lysine residues within p53-CRD. was immunoprecipitated from the cell lysates using anti-FLAG antibodies, and the p53 association was detected by immunoblotting using anti-p53 antibody. C, MYBBP1A interacted with p53 via two independent regions in intact cells. H1299 cells transfected with the indicated plasmids were treated with ActD for 6 h. The cells were fixed and subjected to the Duolink in situ PLA using anti-FLAG and anti-p53 antibodies. The Duolink signal is shown in red. The nuclei were stained using DAPI and are shown in blue. FEBRUARY 21, 2014 • VOLUME 289 • NUMBER 8

JOURNAL OF BIOLOGICAL CHEMISTRY 4931
MYBBP1A Promotes Tetramerization of p5-p53 exerts its activity by oligomerization (33)(34)(35)(36)(37). MYBBP1A enhances p53 activity (58), and we found that MYBBP1A has two p53-binding domains. These findings suggest that MYBBP1A may regulate p53 oligomerization. We next performed gel filtration assay to examine p53 oligomerization. The gel filtration assay revealed that p53 was eluted in higher molecular weight fractions (fractions 1-9, 200 -669 kDa) in the presence of ectopically expressed MYBBP1A compared with that eluted in the absence of MYBBP1A (Fig. 4, (OE-Control) versus (OE-MYBBP1A-WT)). In contrast, we observed that the amount of p53 eluted in these fractions decreased in the presence of MYBBP1A-DN (a.a. 1-1150) compared with that eluted in the presence of MYBBP1A-WT (Fig. 4, (OE-MYBBP1A-WT) versus (OE-MYBBP1A-DN)). These results suggest that MYBBP1A affects p53 oligomerization. However, we could not exclude the effects of p53 interactants other than MYBBP1A because these assays were performed using cell lysates in which a large number of p53 interactants existed.
Next, we performed a protein cross-linking assay. Although siRNA knockdown of MYBBP1A hardly affected p53 dimerization under ActD treatment and other types of DNA damage, the p53 tetramer substantially decreased by knocking down MYBBP1A in MCF-7 (Fig. 5A) and LNCaP cells (data not shown). Similar results were also obtained by using the distinct siMYBBP1A (5Ј-GGUCCGAGAUGAAAUAUGCCCUGAA-3Ј) (data not shown).
On the other hand, we transfected expression plasmids encoding p53 and/or MYBBP1A into H1299 cells (Fig. 5B, panel a).
To perform a more convincing protein cross-linking assay, we titrated MYBBP1A to investigate the dose effect of MYBBP1A on p53 tetramerization. MYBBP1A was titrated by mixing two types of lysates: one prepared from cells in which MYBBP1A was overexpressed and the other prepared from cells in which MYBBP1A was knocked down. As expected, we found that p53 tetramerization was enhanced in a MYBBP1Adependent manner (Fig. 5C). Collectively, these findings indicate that p53 tetramerization is enhanced by MYBBP1A under nucleolar stress. These findings also indicate that p53 forms a dimer independent of MYBBP1A, in good agreement with a previous study suggesting that major p53 itself can autonomously form a dimer (32).   Fig. 1A. B, p53 interacted with MYBBP1A via its lysine residues in the CRD region in cells. H1299 cells were transfected with a combination of expression plasmids encoding F/H-MYBBP1A-WT, its various deletion mutants, and p53-WT, as indicated. The cells were treated with ActD for 6 h at 24 h post-transfection. MYBBP1A was immunoprecipitated from the cell lysates using anti-FLAG antibody and analyzed as in Fig. 1B. To further confirm the effects of MYBBP1A on p53 tetramerization because p53 exerts its DNA binding affinity under conditions in which it forms a tetramer (32,38), we next performed EMSA using the lysates extracted from p53-WToverexpressing H1299 cells. p53-WT interacted with the p21 promoter DNA probe (Fig. 7, lane 3), which was further poten-tiated by MYBBP1A co-overexpression (Fig. 7, lane 4). Moreover, the mobility of these bands was further retarded by anti-p53 antibody (Fig. 7, lanes 7 and 8). In contrast, p53-mTET did not associate with the DNA probe (Fig. 7, lanes 5 and 6). Therefore, these data indicate that MYBBP1A promotes not only p53 tetramerization but also binding to promoter DNA.  To validate the efficiency of overexpression of p53-WT or p53-6KA with or without MYBBP1A, the protein abundance of p53 and MYBBP1A was determined by immunoblotting with anti-p53 or anti-MYBBP1A antibodies in panels a. The lysates were subjected to a cross-linking assay in GA at final concentrations of 0, 0.005, 0.01, or 0.02% in panels b-d. C, dose effect of MYBBP1A on p53 tetramerization. The H1299 cell lysate of OE-MYBBP1A was mixed with that of siMYBBP1A#1 at the indicated ratio and was incubated for 1 h on ice. Following this, the mixed lysates were cross-linked by GA at a final concentration of 0.04%. FEBRUARY 21, 2014 • VOLUME 289 • NUMBER 8

MYBBP1A Forms a Dimer via the C-terminal Region, Which
Is Necessary for p53 Tetramerization-It has been previously reported that MYBBP1A forms a dimer (63). Therefore, we investigated whether MYBBP1A could bind to two p53 molecules and whether MYBBP1A dimerization enhances p53 tetramerization. First, we examined the MYBBP1A-MYBBP1A interaction by Co-IP assays and observed that Myc-MYBBP1A coimmunoprecipitated with F/H-MYBBP1A (Fig. 8A). This result indicates that MYBBP1A interacts with MYBBP1A. To further investigate the mode of the MYBBP1A-MYBBP1A interaction, we generated MYBBP1A deletion mutants and tested the interaction in the GST pulldown assay (Fig. 8B, panel  a). The GST pulldown assay determined that the region from a.a. 1151 to 1328 of MYBBP1A interacted with MYBBP1A-WT. However, the regions from a.a. 1 to 642 and from a.a. 643 to 1150 of MYBBP1A did not interact with MYBBP1A-WT (Fig.  8B, panel b, upper panel). Moreover, the region from a.a. 1151 to 1328 of MYBBP1A associated with the region itself (Fig. 8B,  panel b, lower panel). Taken together, these results show that MYBBP1A interacts with itself via the region from a.a. 1151 to 1328.
To test whether MYBBP1A forms a self-assembled complex via a.a. 1151-1328 in intact cells, we transfected expression plasmids for MYBBP1A-WT or MYBBP1A-DN (a.a. 1-1150) into H1299 cells. Co-IP assays demonstrated that MYBBP1A-DN did not interact with MYBBP1A-WT (Fig. 8C). These data indicate that the region from a.a. 1151 to 1328 is indispensable for MYBBP1A dimerization in cells.
Although MYBBP1A-DN (a.a. 1-1150), which contains a single p53-binding site (Fig. 1), can only interact with the p53 monomer (Fig. 2), the mutant did not form a dimer in MYBBP1A-WT cells (Fig. 8C). Thus, we expected that MYBBP1A-DN would act as a dominant-negative MYBBP1A mutant by inhibiting p53 tetramerization. To examine this possibility, we performed a protein cross-linking assay using MYBBP1A-DN-overexpressing cells (Fig. 8D, panel a). Overexpression of MYBBP1A-DN significantly reduced tetramerized p53 in MCF-7 (Fig. 8D, panel b) and LNCaP cells (data not shown). We also found that MYBBP1A-DN inhibited the effect of exogenous MYBBP1A-WT on p53 tetramerization (Fig. 8D, panel c). These data indicate that MYBBP1A dimerization and/or interactions with two p53 molecules are necessary for p53 tetramerization.
MYBBP1A Enhances the p53-p300 Interaction and p53 Acetylation by Regulating p53 Tetramerization-It has been previously demonstrated that tetramerization of p53 is necessary for p300-mediated p53 acetylation (28). Consistent with these findings, our results indicate that acetylation of p53-mTET was not enhanced by overexpressing p300 (Fig. 9A). Therefore, we next examined whether overexpression of MYBBP1A-DN, which inhibited p53 tetramerization (Fig. 8), affected p53 acetylation. Overexpression of MYBBP1A-DN reduced protein and Lys-382 acetylation (K382Ac) levels of p53 compared with those of the control (Fig. 9B, lane 3 versus lane  4). We further increased the amount of total extract (Fig. 9B,  lanes 4 -7) and found that MYBBP1A-DN significantly reduced K382Ac levels of p53 rather than the protein levels of p53 (Fig.  9B, lane 3 versus lane 7). These results indicate that p53 is not acetylated without tetramerization promoted by MYBBP1A. Next, a combination of p53 and p300 expression plasmids was introduced into H1299 cells in the presence or absence of the MYBBP1A-DN expression plasmid to test the effects of MYBBP1A on the interaction between p53 and p300. Co-IP assays showed that the association between p53 and p300 in the presence of ActD significantly decreased by overexpressing MYBBP1A-DN (Fig. 9C).
Next, we tested the effects of MYBBP1A-DN overexpression on recruitment of p53 and p300 to the promoter region of p21 using the ChIP assay (Fig. 9D). Recruitment of p53 and p300 were abrogated in the presence of MYBBP1A-DN as compared with those in the presence of MYBBP1A-WT (lanes 2 versus 6 for p53 and lanes 3 versus 7 for p300). By comparison, MYBBP1A itself did not bind to the promoter region (lanes 4 and 8). Consistent with these results, we previously revealed that the recruitment of p53 was abrogated by knocking down MYBBP1A or by overexpressing MYBBP1A-DN. In contrast, overexpression of MYBBP1A-WT increased the recruitment of p53 to the p21 promoter, whereas MYBBP1A was not recruited to the promoter (57).  Taken together, these data indicate that MYBBP1A enhances the tetramerization, p300-mediated acetylation, and recruitment of p53 to the target gene promoters. We conclude that MYBBP1A itself is dissociated from p53 by p300-mediated acetylation of p53 and that MYBBP1A is absent from DNAbound p53 on the promoter.
MYBBP1A Regulates p53 Activation by Enhancing Tetramerization-Our results indicate that MYBBP1A promotes p53 tetramerization (Fig. 5). A previous report has suggested that p53 tetramerization is indispensable for the transactivating capabilities of p53 (64). Therefore, we examined whether promotion of tetramerization by MYBBP1A contributes to p53 function under cell stress.
The effects of MYBBP1A on the induction levels of p53 target gene products were examined. mRNA and protein levels of p21, HDM2, and PUMA in MCF-7 (Fig. 10A) and LNCaP (data not shown) cells substantially decreased in cells in which MYBBP1A was knocked down. Similarly, overexpression of MYBBP1A-DN reduced mRNA and protein levels.
We then investigated the effects of MYBBP1A-dependent p53 tetramerization on apoptosis induced under cell stress (Fig.  10B). In agreement with the previous results, ActD treatment increased the number of dead MCF-7 cells (Fig. 10B, panel a, OE-control or siControl). ActD-induced cell death was enhanced by overexpressing MYBBP1A-WT (Fig. 10B, panel a, OE-control versus OE-MYBBP1A-WT). However, cell death induced by ActD was significantly suppressed by knockdown of MYBBP1A or by overexpression of MYBBP1A-DN in MCF-7 (Fig. 10B, panel a, siMYBBP1A or OE-MYBBP1A-DN) and LNCaP cells (data not shown). Furthermore, we investigated this effect on p53-null H1299 cells by overexpressing MYBBP1A-WT in H1299 cells. In contrast to the p53-positive MCF-7 cells (Fig. 10B, panel a), ActD-induced cell death did not increase by overexpressing MYBBP1A-WT in p53-null H1299 cells (Fig. 10B, panel b, OE-control versus OE-MYBBP1A-WT). These results clearly indicate that promotion of p53 tetramerization by MYBBP1A is a fundamental mechanism for activating p53 under cell stress.

MYBBP1A Enhances p53 Tetramerization and Acetylation in
Response to Nucleolar Disruption-Based on the findings of our studies and those of previous reports, we propose the following model in which MYBBP1A enhances tetramerization and activation of p53 (Fig. 11). Under nucleolar stress, rRNA transcription is inhibited and rRNA content in the nucleolus decreases. Loss of nucleolar rRNA, which serves as a scaffold for nucleolar proteins, results in nucleolar disruption and translocation of MYBBP1A to the nucleoplasm (58). At the same time, p53 is stabilized by stress-induced inactivation of HDM2 (3) and forms a dimer independent of MYBBP1A. The nucleoplasmlocated MYBBP1A may bind to the nonacetylated p53 dimer via the middle (a.a. 643-1150) and C-terminal (a.a. 1151-1328) regions of MYBBP1A. Following this, tetramerization of nonacetylated p53 is enhanced by dimerizing MYBBP1A to which the p53 dimer is bound. The transcriptional coactivator p300 binds to the nonacetylated p53 tetramer in a complex with the MYBBP1A dimer. After p300 acetylates p53 within the complex, MYBBP1A dissociates from the acetylated p53 tetramer in a complex with p300 (57). Finally, the acetylated p53 tetramer in a complex with p300 binds to the promoters and induces expression of target genes related to apoptosis or cell cycle arrest. However, whether p53 acetylation by p300 precedes DNA binding of the p300-p53 complex (dashed arrow) remains a significant future issue.
Nucleolar Stress and p53 Activation-A number of external and internal insults induce nucleolar stress by disrupting the nucleolar structure (41,42). Nucleolar stress inhibits rRNA transcription and impairs ribosomal biogenesis, thereby leading to nucleolar disruption. Grummt et al. demonstrated that loss of rRNA transcription resulting in nucleolar disruption is sufficient for inducing p53-dependent apoptosis (65).
Tetramerization of p53 and Its Regulatory Proteins-Tetramerization of p53 is regulated by the TET domain in the C terminus, spanning residues 325-356, which can by itself form tetramers in vitro (38). p53 forms a homodimer or a tetramer by FIGURE 9. MYBBP1A enhances the p53-p300 interaction and p53 acetylation by regulating p53 tetramerization. A, p53-mTET was not acetylated by overexpressing p300. The expression plasmids encoding p53-WT or p53-mTET were transfected into H1299 cells with or without p300, 24 h before treatment with ActD for 6 h. Cell lysates were analyzed by immunoblotting using anti-p53 or site-specifically acetylated p53 antibodies. B, overexpression of MYBBP1A-DN decreased Lys-382 acetylation (K382Ac) levels of p53. MCF-7 cells were transfected with the expression plasmid encoding EGFP (OE-Control) or MYBBP1A-DN (OE-MYBBP1A-DN), 24 h before treatment with ActD for 6 h. Cell lysates were analyzed by immunoblotting using anti-p53 or site-specifically acetylated p53 antibodies. ϫ1, ϫ2, ϫ3, and ϫ4 indicate the ratios of total extract volumes applied to SDS-PAGE. C, overexpression of MYBBP1A-DN decreased binding of p300 to p53. H1299 cells were transfected with a combination of expression plasmids encoding FLAG-p300, Myc-MYBBP1A-DN, and p53-WT, as indicated. The cells were treated with ActD for 6 h at 24 h after transfection. FLAG-p300 was immunoprecipitated using anti-FLAG antibody, and the immunoprecipitates were eluted using FLAG peptides and analyzed by immunoblotting using the indicated antibodies. D, overexpression of MYBBP1A-DN reduced the recruitment of p53 and p300 to the p21 promoter in ActD-treated cells. MCF-7 cells were transfected with the expression plasmid encoding EGFP (OE-Control) or FH-MYBBP1A-DN (OE-MYBBP1A-DN) for 24 h before treatment with ActD for 6 h. The CHIP assay was performed using normal rabbit IgG, anti-p53, and anti-p300 antibodies. The p53-binding region of the p21 promoter was amplified and analyzed by RT-qPCR. The values are given as the means Ϯ standard deviation for triplicate assays.
directly interacting with TET domains (32). TET domain deletion mutants can neither form oligomers nor be activated under DNA damage conditions because they cannot associate with DNA (66).
Nucleolar disruption reportedly induces p53 activation without phosphorylation (58). Therefore, nucleolar stress-mediated p53 activation may not be accounted for by 14-3-3. In this study, we observed enhanced p53 tetramerization through the p53 CRD region by interaction with MYBBP1A, which acts as a platform for tetramerization. This regulatory cascade begins with nucleolar disruption, followed by translocation of MYBBP1A. This is the first report to describe a possible mechanism underlying p53 tetramerization under nucleolar stress.
Relationship between Tetramerization and Acetylation of p53-Gu and co-workers (26) showed that acetylation, among many other p53 post-translational modifications, plays a crucial role in regulating p53 activity. It has also been shown that p53 tetramerization plays an essential role in regulating p53 activity (28). Recent evidence indicates that post-translational modifications, including p53 acetylation, are regulated by p53 tetramerization (40). Tetramerization provides appropriate binding sites for p300 and leads to p300 binding and subsequent acetylation of p53 C-terminal lysine residues (28). Our findings highlight the relationship between tetramerization and acetylation in the mechanisms of p53 activation. We demonstrated that knockdown of MYBBP1A-WT or overexpression of MYBBP1A-DN reduced p53 tetramerization and acetylation. In contrast, immunoprecipitation assays have revealed that MYBBP1A does not associate with acetylated p53 (57). These FIGURE 10. MYBBP1A regulates p53 activation by enhancing tetramerization. A, overexpression of MYBBP1A-DN and knockdown of MYBBP1A reduced mRNA and protein levels of p53 target genes, p21, HDM2, and PUMA. MCF-7 cells were transfected with indicated siRNAs or plasmids for 48 or 24 h, respectively, followed by ActD treatment. In panel a, total RNAs were prepared and expression of the indicated genes was analyzed by RT-qPCR. In panel b, total extracts were prepared, and expression of the indicated genes was analyzed by immunoblotting. B, overexpression of MYBBP1A-DN and knockdown of MYBBP1A decreased the percentage of apoptosis induced by ActD treatment. p53-positive MCF-7 (panel a) and p53-null H1299 (panel b) cells were treated with the indicated siRNAs or plasmids and cultured for 24 h. Following this, the cells were treated with or without ActD at a final concentration of 5 nM. The percentage of dead cells was measured by a trypan blue exclusion assay at the indicated times after ActD treatment. The values are means Ϯ standard deviation for triplicate assays. FEBRUARY 21, 2014 • VOLUME 289 • NUMBER 8 results suggest that MYBBP1A interacts with p53 and facilitates p53 tetramerization, subsequently enhancing acetylation of p53. Thus, consistent with previous reports, our findings also suggest that tetramerization and subsequent acetylation are essential for p53 activation.

MYBBP1A Enhances p53 Tetramerization
Physiological Significance of p53 Activation by MYBBP1A-p53 plays a critical role in cancer prevention, given that p53 suppresses tumorigenesis by inducing cell cycle arrest and apoptosis through its transcriptional activity (71). We recently demonstrated that MYBBP1A plays a role in tumor prevention in the context of p53 activation (72). We conducted extensive analysis and demonstrated that MYBBP1A expression is associated with breast cancer tumorigenesis in clinical samples. In vivo and in vitro assays demonstrated that MYBBP1A regulates p53-dependent tumorigenesis. These studies showed that promotion of p53 tetramerization by MYBBP1A is essential for p53 activation.
Approximately half of human tumors carry inactivating mutations in TP53 (73,74). Numerous reports have shown that many mutations occur in the p53 DNA-binding domain (75,76). Correspondingly, TET domain mutations occur in patients with Li-Fraumeni and Li-Fraumeni-like syndromes, suggesting the importance of p53 tetramerization in cancer progression (77)(78)(79). In contrast, a mutation in the p53-CRD region, which interacts with MYBBP1A, rarely occurs in cancer (74). In view of these observations and our results, inactivation of the p53-CRD region during tumorigenesis may be caused by down-regulation of a protein such as MYBBP1A rather than by the emergence of a mutation in the CRD region.