Association of p21 with NF-YA suppresses the expression of Polo-like kinase 1 and prevents mitotic death in response to DNA damage

Y-C Lin,M-Y Chen,F-F Wang,Y-N Chen,T-Y Chou,K-F Lin

doi:10.1038/cddis.2013.527

Abstract

Polo-like kinase 1 (PLK1) is an important mitotic kinase and its expression is tightly regulated in the cell cycle and in the DNA damage response. PLK1 expression is previously shown to be suppressed by p53 and/or p21. Here, we demonstrate that the CCAAT box in the PLK1 promoter is pivotal for p53/p21-mediated PLK1 repression. Chromatin immunoprecipitation showed that cyclin-dependent kinase 2 (CDK2) associated with the CCAAT box-containing region of PLK1 promoter in unstressed cells, whereas adriamycin (ADR) induced the recruitment of p21 with a concomitant reduction in the occupancy of CDK2 in this region. Expression of p21 inhibited the interaction between CDK2 and the nuclear factor YA (NF-YA) subunit of the CCAAT box-binding transcription factor NF-Y. A mutant p21 that is defective in CDK2 binding was unable to disrupt the CDK2–NF-YA interaction or suppress PLK1 transcription. Co-immunoprecipitation experiments demonstrated the interaction between NF-YA and p21, and in vitro assays showed that p21 could directly bind to NF-YA. Knockdown of NF-YA decreased the amount of PLK1 promoter-associated p21 and abolished p21-mediated PLK1 repression in cells treated with ADR. Depletion of NF-YA diminished the p53-regulated transcriptional activation and suppressed the p53-mediated protection from mitotic death after DNA damage, and these effects of NF-YA deletion were alleviated by PLK1 depletion. Our findings have uncovered a novel p21/NF-YA/PLK1 axis critical for maintaining the checkpoint function of p53 to prevent mitotic death in the DNA damage-induced response.

Highlights

G1 and S phases, start to increase in the G2 phase and peak during mitosis; the concordant changes in transcript and protein levels suggest that Polo-like kinase 1 (PLK1) expression is primarily controlled transcriptionally in these phases of cell cycle.[4,5]
We found that ADR treatment could increase p53 expression in HCT116 cells with or without p21 deletion, whereas levels of PLK1 protein and transcript were only decreased in the wild-type but not in the p21 À / À cells (Figure 1a)
We have uncovered that beyond its well-known role as a p53 downstream checkpoint component that binds Cyclin-dependent kinase 2 (CDK2) and halts cell cycle progression, p21 can directly bind to NF-YA on the PLK1 promoter and actively suppress PLK1 transcription to inhibit mitotic entry and prevent mitotic catastrophe

Summary

Introduction

G1 and S phases, start to increase in the G2 phase and peak during mitosis; the concordant changes in transcript and protein levels suggest that PLK1 expression is primarily controlled transcriptionally in these phases of cell cycle.[4,5]. The cell cycle-dependent element (CDE)/cell cycle gene homology region (CHR) in the PLK1 promoter represents a key transcriptional repression element; the responsible trans-acting factors remain elusive, mutations in this bipartite cis element hinder the cell cycle-specific regulation of PLK1.4,5. PLK1 is regulated at the protein level by anaphase-promoting complex/cyclosome-mediated ubiquitination, which destines PLK1 for proteosomal degradation.[6]. ATM and ATR, and is dependent on functional p53 and/or p21.10–12 The repressive effect of p53 on PLK1 expression appears to be CDE/CHR independent and involves the binding of p53 to a response element (p53RE2) B800-bp upstream of the CDE/CHR element and the recruitment of histone deacetylase to the vicinity of p53RE2.13 Deletion studies suggest that the CDE/CHR element is important in p21-mediated repression of the PLK1,14 but the underlying mechanism has not been well elucidated. The PLK1 promoter contains one single CCAAT box, which is essential for the promoter activity;[15] the role of this element in stressresponsive PLK1 regulation has not been explored yet

Methods

Results

Conclusion