Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Xue Yuan Road 38, Beijing 100191, PR China.
Exp Cell Res. 2013 Dec 10;319(20):3104-15. doi: 10.1016/j.yexcr.2013.09.012. Epub 2013 Sep 26.
E2F1 is implicated in transcriptional activation of polo-like kinase-1 (PLK1), but yet the mechanism is not fully understood. PLK1 suppression plays an important checkpoint role in response to DNA damage. Suppression of the PLK1 gene by binding of p53 to upstream p53RE2 element in the promoter has been recently revealed. Here we report another mechanism, in which p53 interacts with E2F1 to form p53-E2F1-DNA complex repressing E2F1-dependent PLK1 expression. PLK1 was downregulated in cisplatin exposed HCT116p53(+/+) but not HCT116p53(-/-) cells, indicating p53-suppressed PLK1 upon DNA damage. Co-transfection and reporter enzyme assays revealed that p53 suppressed but E2F1 promoted PLK1 gene activation. 5'-Deletion and substitution mutations showed multiple positive cis-elements residing in the PLK1 promoter, of which at least two E2F1 sites at positions -75/-68 and -40/-32 were required for the full activity of the promoter. Combination of 5'-deletion and substitution mutations with over-expression of p53 showed that suppression of the PLK1 gene by p53 was E2F1-dependent: mutation of the E2F1 site at position -75/-68 partially abrogated suppression activity of p53; mutation of E2F1 site at position -40/-32 released from p53 suppression of PLK1 gene completely. Co-immunoprecipitation and electrophoretic mobility shift assay showed that DNA damage promoted p53-E2F1 interaction, thereby creating a p53-E2F1 complex assembly on the PLK1 promoter in vitro. The in vivo formation of p53-E2F1-PLK1 promoter complex upon DNA damage was further evidenced by chromatin immunoprecipitation (ChIP) and re-ChIP. In addition, we showed that suppression of PLK1 by p53 promoted apoptosis. Our data suggest that p53 may interact with E2F1 to form p53-E2F1-DNA complex suppressing E2F1-dependent PLK1 expression. The model of p53 action on E2F1-activated PLK1 gene may explain at least partly how p53 as a suppressor regulates the downstream effects of E2F1 in cellular stresses including DNA damage stress.
E2F1 被牵连到 Polo 样激酶-1（PLK1）的转录激活中，但机制尚未完全阐明。PLK1 的抑制在应对 DNA 损伤时起着重要的检查点作用。最近发现，p53 通过结合启动子上游的 p53RE2 元件与 PLK1 基因结合，从而抑制 PLK1 基因的表达。在这里，我们报告了另一种机制，即 p53 与 E2F1 相互作用形成 p53-E2F1-DNA 复合物，从而抑制 E2F1 依赖的 PLK1 表达。在顺铂暴露的 HCT116p53(+/+)细胞中，PLK1 下调，但在 HCT116p53(-/-)细胞中没有下调，这表明 p53 在 DNA 损伤时抑制 PLK1。共转染和报告酶分析显示，p53 抑制但 E2F1 促进 PLK1 基因的激活。5'缺失和取代突变显示，PLK1 启动子中存在多个正向顺式元件，其中至少两个 E2F1 位点位于-75/-68 和-40/-32 位，对于启动子的完全活性是必需的。5'缺失和取代突变与 p53 的过表达相结合表明，p53 通过 E2F1 抑制 PLK1 基因：E2F1 位-75/-68 点的突变部分消除了 p53 的抑制活性；E2F1 位-40/-32 点的突变完全解除了 p53 对 PLK1 基因的抑制。免疫共沉淀和电泳迁移率变动分析显示，DNA 损伤促进了 p53-E2F1 相互作用，从而在体外形成了 p53-E2F1 复合物在 PLK1 启动子上的组装。DNA 损伤后，p53-E2F1-PLK1 启动子复合物的体内形成进一步通过染色质免疫沉淀（ChIP）和再 ChIP 得到证实。此外，我们还表明，p53 通过抑制 PLK1 促进细胞凋亡。我们的数据表明，p53 可能与 E2F1 相互作用形成 p53-E2F1-DNA 复合物，抑制 E2F1 依赖的 PLK1 表达。p53 对 E2F1 激活的 PLK1 基因的作用模型至少部分解释了 p53 作为抑制剂如何调节 E2F1 在包括 DNA 损伤应激在内的细胞应激中的下游效应。
