p53-dependent repression of polo-like kinase-1 (PLK1).

PLK1 is a critical mediator of G2/M cell cycle transition that is inactivated and depleted as part of the DNA damage-induced G2/M checkpoint. Here we show that downregulation of PLK1 expression occurs through a transcriptional repression mechanism and that p53 is both necessary and sufficient to mediate this effect. Repression of PLK1 by p53 occurs independently of p21 and of arrest at G1/S where PLK1 levels are normally repressed in a cell cycle-dependent manner through a CDE/CHR element. Chromatin immunoprecipitation analysis indicates that p53 is present on the PLK1 promoter at two distinct sites termed p53RE1 and p53RE2. Recruitment of p53 to p53RE2, but not to p53RE1, is stimulated in response to DNA damage and/or p53 activation and is coincident with repression-associated changes in the chromatin. Downregulation of PLK1 expression by p53 is relieved by the histone deacetylase inhibitor, trichostatin A, and involves recruitment of histone deacetylase to the vicinity of p53RE2, further supporting a transcriptional repression mechanism. Additionally, wild type, but not mutant, p53 represses expression of the PLK1 promoter when fused upstream of a reporter gene. Silencing of PLK1 expression by RNAi interferes with cell cycle progression consistent with a role in the p53-mediated checkpoint. These data establish PLK1 as a direct transcriptional target of p53, independently of p21, that is required for efficient G2/M arrest.

Regulation of p53 tumour suppressor target gene expression by the p52 NF-kappaB subunit.

The p52/p100 nuclear factor kappa B (NF-kappaB) subunit (NF-kappaB2) is aberrantly expressed in many tumour types and has been implicated as a regulator of cell proliferation. Here, we demonstrate that endogenous p52 is a direct regulator of Cyclin D1 expression. However, stimulation of Cyclin D1 expression alone cannot account for all the cell cycle effects of p52/p100 and we also find that p52 represses expression of the Cyclin-dependent kinase inhibitor p21(WAF/CIP1). Significantly, this latter effect is dependent upon basal levels of the tumour suppressor p53. By contrast, p52 cooperates with p53 to regulate other known p53 target genes such as PUMA, DR5, Gadd45alpha and Chk1. p52 associates directly with these p53-regulated promoters where it regulates coactivator and corepressor binding. Moreover, recruitment of p52 is p53 dependent and does not require p52-DNA-binding activity. These results reveal a complex role for p52 as regulator of cell proliferation and p53 transcriptional activity. Furthermore, they imply that in some cell types, p52 can regulate p53 function and influence p53-regulated decision-making following DNA damage and oncogene activation.

Regulation of NF-kappaB and p53 through activation of ATR and Chk1 by the ARF tumour suppressor.

The ARF tumour suppressor is a central component of the cellular defence against oncogene activation. In addition to activating p53 through binding Mdm2, ARF possesses other functions, including an ability to repress the transcriptional activity of the antiapoptotic RelA(p65) NF-kappaB subunit. Here we demonstrate that ARF induces the ATR- and Chk1-dependent phosphorylation of the RelA transactivation domain at threonine 505, a site required for ARF-dependent repression of RelA transcriptional activity. Consistent with this effect, ATR and Chk1 are required for ARF-induced sensitivity to tumour necrosis factor alpha-induced cell death. Significantly, ATR activity is also required for ARF-induced p53 activity and inhibition of proliferation. ARF achieves these effects by activating ATR and Chk1. Furthermore, ATR and its scaffold protein BRCA1, but not Chk1, relocalise to specific nucleolar sites. These results reveal novel functions for ARF, ATR and Chk1 together with a new pathway regulating RelA NF-kappaB function. Moreover, this pathway provides a mechanism through which ARF can remodel the cellular response to an oncogenic challenge and execute its function as a tumour suppressor.