YY1 inhibits the activation of the p53 tumor suppressor in response to genotoxic stress

Abstract

The tumor suppressor p53 regulates cell-cycle progression and apoptosis in response to genotoxic stress, and inactivation of p53 is a common feature of cancer cells. The levels and activity of p53 are tightly regulated by posttranslational modifications, including phosphorylation, ubiquitination, and acetylation. Here, we demonstrate that the transcription factor Yin Yang 1 (YY1) interacts with p53 and inhibits its transcriptional activity. We show that YY1 disrupts the interaction between p53 and the coactivator p300 and that expression of YY1 blocks p300-dependent acetylation and stabilization of p53. Furthermore, expression of YY1 inhibits the accumulation of p53 and the induction of p53 target genes in response to genotoxic stress. YY1 also interacts with Mdm2 and the expression of YY1 promotes the assembly of the p53-Mdm2 complex. Consequently, YY1 enhances Mdm2-mediated ubiquitination of p53. Inactivation of endogenous YY1 enhances the accumulation of p53 as well as the expression of p53 target genes in response to DNA damage, and it sensitizes cells to DNA damage-induced apoptosis. Hence, our results demonstrate that YY1 regulates the transcriptional activity, acetylation, ubiquitination, and stability of p53 by inhibiting its interaction with the coactivator p300 and by enhancing its interaction with the negative regulator Mdm2. YY1 may, therefore, be an important negative regulator of the p53 tumor suppressor in response to genotoxic stress.

Fig. 1.

YY1 inhibits the transcriptional activity of p53. (A) Activity of the Mdm2-luc reporter gene in U2OS cells transfected with p53 (25 ng) in the absence or presence of YY1 (pcDNA3-YY1, 20–100 ng) or empty vector (pcDNA3). FL, full length. (B) Activity of the Mdm2-luc reporter gene in U2OS cells transfected with p53 (25 ng) in the absence or presence of an antisense YY1 construct (pcDNA3-AS-YY1, 100–250 ng) or empty vector (pcDNA3). FL, full length. (C) Activity of the G5E1b-luc reporter gene in U2OS cells transfected with Gal4-p53 (25 ng) in the absence or presence of YY1 (pcDNA3-YY1, 20–100 ng) or empty vector (pcDNA3). FL, full length. (D) Activity of the RGC-luc reporter gene in U2OS cells transfected in the absence or presence of YY1 (pcDNA3-YY1, 100 ng) or empty vector (pcDNA3, 100 ng). Where indicated, cells were treated with etoposide (ETOP, 5 μM) 12 h before analysis. (E) Activity of Mdm2-luc reporter gene in U2OS cells transfected with p53 (25 ng) in the absence or presence of Gal4-YY1 (10 and 20 ng), either full-length (FL) or the indicated deletion mutants. (F) p53-TetOn cells were transfected with Gal4-YY1 (250 ng), either full-length (FL) or the indicated deletion mutants. Where indicated, the cells were treated with doxycycline (DOX, 200 μg/ml) for 12 h to induce p53 expression. The levels of p21, p53, Gal4-YY1 and α-tubulin were estimated by Western blot analysis.

Fig. 2.

YY1 interacts with p53. (A) YY1, either full-length (1-414) or the indicated deletion mutants was translated in vitro and used in GST pull-down assays with GST alone or GST-p53. The amount of YY1 was analyzed by phosphorimage analysis. (B) Full-length YY1 was expressed in 293T cells and the cell lysates used in GST pull-downs with GST alone or the indicated GST-p53 proteins. FL, full length. The levels of YY1 were determined by Western blot analysis. (C) Cell lysates from p53-TetOn cells treated in the absence or presence of doxycycline (DOX) were immunoprecipitated (IP) with p53 antibodies (DO-1). Coimmunoprecipitated YY1 and the levels of p53 and YY1 in wholecell lysates (WCL) were determined by Western blot analysis. (D) Whole-cell lysates from U2OS cells treated in the absence or presence of etoposide (ETOP, 5 μM) or MG132 (25 μM) were immunoprecipitated (IP) with p53 antibodies (DO-1) or an unrelated antibody (anti-Gal4). Immunoprecipitated YY1 and p53 and the levels of p53 and YY1 and the phosphorylation of p53 on Ser-15 in whole-cell lysates (WCL) were determined by Western blot analysis.

Fig. 3.

YY1 inhibits the activation of p53 in response to genotoxic stress. (A) ChIP analysis of the p21 promoter after DNA damage. U2OS cells were infected with control (GFP) or YY1 adenovirus and either left untreated or treated with etoposide (ETOP, 5 μM) for 8 h. Cells were processed for ChIP analysis by using the indicated antibodies for immunoprecipitation (IP). (B) p53-TetOn cells were infected with control (GFP) or YY1 adenovirus and treated with doxycycline (DOX) for the indicated times to induce p53 expression. The expression of the p21, Mdm2, p53, and YY1 proteins and the phosphorylation of p53 on Ser-15 were determined by Western blot analysis. (C) U2OS cells were infected with control (GFP) or YY1 adenovirus and treated with etoposide (ETOP, 5 μM) for the indicated times. The expression of p21, p53, YY1, and α-tubulin and the phosphorylation of p53 on Ser-15 were determined by Western blot analysis.

Fig. 4.

YY1 interferes with the interaction between p53 and p300 and promotes Mdm2-mediated ubiquitination of p53. (A) p53-positive HCT116 cells were infected with control (GFP) or YY1 adenovirus. Endogenous p300 was immunoprecipitated (IP) from total cell lysates with anti-p300 antibodies. The amount of immunoprecipitated p53 and the levels of p53, p300, and YY1 in whole-cell lysates (WCL) were determined by Western blot analysis. (B) p53-deficient HCT116 cells were transfected with Flag-p53 (1 μg) in the absence or presence of p300-HA (2.5 μg) and YY1 (1 μg) or empty expression vector (pcDNA3). Flag-p53 was immunoprecipitated (IP) with Flag antibodies from whole-cell lysates. The acetylation of Flag-p53 was detected with anti-acetyl-lysine antibodies (α-AcK). The amount of immunoprecipitated Flag-p53 was determined by Western blot analysis. (C) The activity of the G5E1b-luc reporter gene was determined in H1299 cells transfected with Gal4-p53-AD (10 ng), a fusion protein containing the DNA-binding domain of Gal4 and the transactivation domain of p53 (amino acids 1–73), in the absence or presence of YY1 (20 and 40 ng) or empty expression plasmid (pcDNA3). (D) p53-negative HCT116 cells were transfected with Flag-p53 (0.1 μg) in the absence or presence of p300-HA (0.5 μg) and YY1 (0.25 μg) or empty expression vector (pcDNA3). The levels of Flag-p53, p300-HA, and YY1 in whole-cell lysates (WCL) were determined by Western blot analysis. (E) U2OS cells were infected with control (GFP) or YY1 adenovirus and treated with MG132 (25 μM) for 4 h before lysis. Whole-cell lysates were immunoprecipitated (IP) with anti-p53 antibodies, and the ubiquitination of p53 and the amount of p53 in the immunoprecipitates, as well as the levels of Mdm2 and YY1 in whole-cell lysates (WCL), were determined by Western blot analysis. (F) HEK293 cells were transfected with Mdm2 (0.5 μg) in the absence or presence of Flag-p53 (0.25 μg) and increasing amounts of YY1 (0.1 and 0.2 μg) or empty expression vector (pcDNA3). Whole-cell lysates were immunoprecipitated (IP) with anti-Flag antibodies, and coimmunoprecipitated Mdm2 was detected by Western blot analysis. The levels of Mdm2, Flag-p53, and YY1 in the whole-cell lysates (WCL) were determined by Western blot analysis. (G) HEK293 cells were transfected with Flag-p53 (0.25 μg) and Hisubiquitin (1 μg) in the absence or presence of Mdm2 (0.25 and 0.5) and YY1 (0.1 and 0.2 μg) or empty expression vector (pcDNA3). Whole-cell lysates were immunoprecipitated (IP) with anti-Flag antibodies and separated by SDS/PAGE. Ubiquitination of p53 and the amount of p53 in the immunoprecipitates, and the levels of Mdm2 and YY1 in whole-cell lysates (WCL) were determined by Western blot analysis.

Fig. 5.

Inactivation of YY1 promotes apoptosis in response to DNA damage. (A) siRNA-mediated inactivation of YY1. U2OS cells were transfected with control (GL2) or YY1 siRNA (20 nM) and treated with etoposide (5 μM) for the indicated times. The expression of p21, p53, YY1, and α-tubulin and the phosphorylation of p53 on Ser-15 were determined by Western blot analysis. (B) U2OS cells were transfected with control (GL2) or YY1 siRNA (20 nM) and treated with etoposide (5 μM) for 12 h. Apoptosis was determined after staining of fixed cells with an antibody directed against cleaved cytokeratin 18 (M30). (C) U2OS cells were transfected with siRNA and treated as described in B, and apoptosis was monitored by determination of cytoplasmic histone-associated DNA fragments by ELISA.