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.

SummaryOncogenic transcription factors such as the leukemic fusion protein RUNX1/ETO, which drives t(8;21) acute myeloid leukemia (AML), constitute cancer-specific but highly challenging therapeutic targets. We used epigenomic profiling data for an RNAi screen to interrogate the transcriptional network maintaining t(8;21) AML. This strategy identified Cyclin D2 (CCND2) as a crucial transmitter of RUNX1/ETO-driven leukemic propagation. RUNX1/ETO cooperates with AP-1 to drive CCND2 expression. Knockdown or pharmacological inhibition of CCND2 by an approved drug significantly impairs leukemic expansion of patient-derived AML cells and engraftment in immunodeficient murine hosts. Our data demonstrate that RUNX1/ETO maintains leukemia by promoting cell cycle progression and identifies G1 CCND-CDK complexes as promising therapeutic targets for treatment of RUNX1/ETO-driven AML.

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Induction and activation of the p53 tumour suppressor protein occurs in response to a number of cellular stresses, including disruption of RNA polymerase II-mediated transcription. Both p53 itself and its principle negative regulator, the E3 ubiquitin ligase Mdm2, are substrates for phosphorylation by the protein kinase CK2 in vitro. CK2 phosphorylates Mdm2 within its central acidic domain, a region that is critical for making a second point of contact with p53 and mediating p53 ubiquitylation and turnover. Additionally, there is evidence that CK2 interacts with, and regulates, both p53 and Mdm2 within the cell but the molecular mechanisms through which CK2-mediated regulation of the p53 response can occur are only poorly understood. Previously, we showed that the basal transcription factor TAFII250, a critical component of TFIID, can interact with Mdm2 and promote the association of the Mdm2 acidic domain with p53. In the present study, we show that immunoprecipitates of TAFII250, either from mammalian cell extracts or from baculovirus-infected cells expressing elevated levels of HA-tagged TAFII250, can phosphorylate Mdm2 in vitro within its acidic domain. We show that CK2 is tightly associated with TAFII250 and is the principal activity responsible for TAFII250-mediated phosphorylation of Mdm2. Our data fit with recent observations that phosphorylation of the acidic domain of Mdm2 stimulates its association with p53 and are consistent with a model in which recruitment of CK2 by TAFII250 may play a contributory role in the maintenance of Mdm2 phosphorylation and function.