YY1/BCCIP Coordinately Regulates P53-Responsive Element (p53RE)-Mediated Transactivation of p21Waf1/Cip1

Abstract

Transactivation of p21 (cyclin-dependent kinase inhibitor 1A, CDKN1A) is closely related to the recruitment of transcription cofactors at the p53 responsive elements (p53REs) in its promoter region. Human chromatin remodeling enzyme INO80 can be recruited to the p53REs of p21 promoter and negatively regulates p21. As one of the key subunits of the INO80 complex, YY1 has also been confirmed to bind to the p53RE sites of p21 promoter. Importantly, YY1 was recently reported to be bound and stabilized by BCCIP (BRCA2 and CDKN1A-interacting protein). Therefore, we hypothesized that the YY1/BCCIP complex plays an important role in regulating the transactivation of p21. Here we present evidence that the YY1/BCCIP complex coordinatively regulates p53RE-mediated p21 transactivation. We first confirmed the cross-interaction between YY1, BCCIP, and p53, suggesting an intrinsic link between three proteins in the regulation of p21 transcription. In dual luciferase assays, YY1 inhibited p53RE-mediated luciferase activity, whereas BCCIP revealed the opposite effect. More interestingly, the region 146-270 amino acids of YY1, which bound to BCCIP, increased p53-mediated luciferase activity, indicating the complexity of the YY1/BCCIP complex in co-regulating p21 transcription. Further in-depth research confirmed the co-occupancy of YY1/BCCIP with p53 at the p53RE-proximal region of p21. Lentiviral-mediated knockdown of BCCIP inhibited the recruitment of p53 and YY1 at the p53RE proximal region of p21; however, this phenomenon was reversed by expressing exogenous YY1, suggesting the collaborative regulation of YY1/BCCIP complex in p53RE-mediated p21 transcription. These data provide new insights into the transcriptional regulation of p21 by the YY1/BCCIP complex.

Keywords: BCCIP; YY1; gene transcription; p21; p53; transactivation.

Figure 1

BCCIP stabilized p53 in HCT116 (p53+/+) cells. (A,B) Increase in endogenous p53 protein by transfection of BCCIP plasmids. Cells were transfected with 1.5 and 4.5 μg Myc tagged BCCIPα or BCCIPβ plasmids. Endogenous p53 protein in whole cell lysate and p53 mRNA levels were measured 72 h later with western blot methods (A, GAPDH is an internal control) and RT-qPCR (B, relative mRNA levels were normalized by GAPDH). (C,D) Decrease of endogenous p53 proteins in shBCCIP-treated cells. BCCIP shRNA (2 μg) and an shNT control was transfected into HCT116 cells. Indicated proteins were detected 72 h after transfection by western blot and cell staining. DAPI staining shows total nuclei. Scale bar indicates 200 µm. (E) Stabilization of exogenous p53 by transfection of BCCIPα plasmids. Cells were co-transfected with Flag-p53 (2.8 μg), Myc-BCCIPα (1.2 and 3.6 μg), and HA-ubiquitin (2 μg) in the presence of MG132 (10 μM). Exogenous p53 proteins were measured 48 h after transfection by western blot using anti-Flag antibody. The degradation of Flag-p53 was measured with anti-HA antibody.

Figure 2

Stabilization of p53 by BCCIP was impacted by YY1 in HCT116 (p53+/+) cells. (A) Negative regulation of p53 on YY1. Cells were transiently transfected with shYY1 (12 μg/10 cm culture plate) and a shNT control for 72 h. Then, endogenous YY1, p53, and p21 proteins were immune-stained by indicated antibodies. Scale bar indicates 200 µm. (B) Impact of Flag-YY1 on p53-stabilization by BCCIP. Cells were co-transfected with Myc-BCCIPα (1.5 and 4.5 μg) and Flag-YY1 (2 μg) or deletion mutant Flag-YY1/∆146-270 (0.4 μg) for 48 h. Indicated proteins were analyzed by western blot with specific antibodies. (C) Impact of shYY1 on p53 stabilization by BCCIP. The experiments were designed as shown. Whole cell lysate was prepared and endogenous p53 protein levels were detected 72 h after shYY1 transfection using anti-p53 antibody. (D) Endogenous p53 protein levels in stably expressing shYY1 cells. Endogenous p53 protein was measured at 72 h after transfection with BCCIPα in stably expressing shYY1 cells.

Figure 3

Cross-interaction between BCCIP, YY1, and p53 was verified in HCT116 (p53+/+) cells. (A) Interaction between BCCIP and YY1. In vitro co-transfection/coimmunoprecipitation (CoIP) experiments with anti-Flag M2 and anti-c-Myc affinity agarose were carried out in HCT116 (p53+/+) and HCT116 (p53−/−) whole-cell lysates. Bound proteins were visualized by western blot. (B) Interaction between p53 and BCCIP in shNT- and shYY1-treated HCT116 cells. CoIP experiments with anti-Flag M2 and anti-Myc antibodies were performed in HCT116 cells, and bound proteins were detected by western blot. (C) Interaction between p53 and YY1 in shNT- and shBCCIP-treated HCT116 cells. CoIP experiments with anti-Flag M2 and anti-Myc antibodies were done as designed.

Figure 4

P53RE-mediated luciferase activity was regulated by YY1 and BCCIP in HCT116 (p53+/+) cells. (A) Schematic diagram of p53RE-Luc plasmid. Multiple response elements of p53 were inserted into a pp53-TA-Luc vector. (B,C) Effects of YY1 on p53RE-Luc luciferase activity. P53RE-Luc dual luciferase activity was measured after co-transfection with p53RE-Luc and Flag-YY1 or siYY1 (48 h) (n = 3). ** p < 0.01, t-test, comparison to p53RE-Luc-transfected group, while ## p < 0.01, t-test, comparison to Flag-YY1-transfected group. (D,E) Effects of BCCIPα/β on p53RE-Luc luciferase activity. P53RE-Luc dual luciferase activity was measured after co-transfection of p53RE-Luc and BCCIPα/β or shBCCIP (48 h) (n = 3). ** p < 0.01, t-test, comparison to p53RE-Luc-transfected group. shBCCIP-311 and shBCCIP-730 target different sequences.

Figure 5

YY1 and BCCIP co-regulated the p53RE-mediated luciferase activity in HCT116 (p53+/+) cells. (A) Effects of BCCIP on p53RE-Luc activity in the Flag-YY1 transfected cells. Experiments were performed with the indicated design. ** p< 0.01, t-test, comparison to empty vector-transfected group, while ## p < 0.01, t-test, comparison to Flag-YY1-transfected group. (B) Effects of BCCIP on p53RE-Luc activity in the YY1 knockdown cells. Experiments were performed with the indicated design. ** p < 0.01, t-test, comparison to empty vector-transfected group. (C) Effect of YY1 on p53RE-Luc activity in the BCCIP knockdown cell. Experiments were performed with the indicated design. (D) Impact of deletion mutants of YY1 on p53RE-Luc activity. The upper panel shows a schematic diagram of deletion mutants of YY1. p53RE-Luc luciferase activities were measured after co-transfection of p53RE-Luc and deletion mutants of YY1 (48 h) (n = 3). ** p < 0.01, t-test, comparison to p53RE-Luc-transfected group. (E) Coordinative roles of BCCIP and YY1 on p53RE-Luc activity. Experiments were performed with the indicated design.

Figure 6

P53 downstream target gene p21 was coordinately regulated by YY1 and BCCIP in HCT116 (p53+/+) cells. (A) Induction of p53 by 250 µM 5FU. Indicated proteins were analyzed by western blot with specific antibodies. (B) Impact of shBCCIP on 5FU-induced p53. shNT- and shBCCIP-transfected cells were treated with 0 and 250 µM 5FU, and indicated proteins were measured by western blot with antibodies. (C) Coordinative effects of YY1 and BCCIP on p53. The experiment was designed as shown. Proteins in prepared lysate were analyzed by western blot. (D) Immune staining. Cells were transfected with indicated plasmids in the presence or absence of 5FU. Then, cells were stained with anti-p21 antibody. DAPI staining shows total nuclei. Scale bar indicates 200 µm.

Figure 7

Co-occupancy of YY1, BCCIP, and p53 at p53RE sites in p21 gene in HCT116 cells. (A) Six primer sets at the p21 locus designed for amplifying chromatin immunoprecipitation (ChIP) DNA. Chromatin lysates were prepared from cells with or without 5FU treatment. 5FU-induced proteins were analyzed by western blot (B). ChIP experiments were performed using anti-p53 (C), anti-YY1 (D), and anti-BCCIP (E) antibodies. ChIP DNA amplified with qPCR. The y-axis shows the ratio of ChIP DNA signals to IgG, and all signals were normalized to input (n = 3). * p < 0.05, ** p < 0.01, t-test, comparison to 5FU untreated group.

Figure 8

YY1 and BCCIP coregulation of p53 target gene p21 in shBCCIP-treated HCT116 cells. BCCIP knockdown cells with or without 5FU exposure were used in the following ChIP experiments. (A,B) p53 and YY1 ChIP experiments were carried out using chromatin lysate in the presence of shNT or shBCCIP expressing cells. Chip DNA was analyzed with qPCR (n = 3). (C,D) Rescue experiments. Stably expressing shNT or shBCCIP cells were transiently transfected with Flag-YY1 for 48 h. Then, prepared chromatin lysate was applied to ChIP assays. The y-axis shows the ratio of ChIP DNA signals to IgG, and all signals were normalized to input (n = 3). * p < 0.05, ** p < 0.01, t-test, comparison to shNT group. (E) Indicated protein levels in A, B, C and D.

Figure 9

Coordinated regulation of p53RE-dependent gene transcription by p53/BCCIP/YY1. BCCIP promotes p53RE-mediated transactivation by binding to and stabilizing p53 and further activates p21 transcription. This pathway is regulated by intracellular YY1 protein levels. Increased YY1 inhibits p53RE-mediated transactivation, and the opposite effect is seen in transient knockdown of YY1. However, stable knockdown of YY1 attenuates BCCIP transcription, further affecting p53 stability, which suggests that there may be feedback inhibition. Black solid arrows, published data [17]; black T dotted arrow, indirect inhibition; blue solid arrows, possible pathway of p53RE-dependent gene transcription; blue T dotted arrow, stably expressing shYY1 affect the INO80/YY1-BCCIP pathway.