Catchpole Steven, Tavner Fiona, Le Cam Laurent, Sardet Claude, Watson Roger J
Ludwig Institute for Cancer Research and the Section of Virology and Cell Biology, Imperial College of Science, Technology and Medicine, Faculty of Medicine, Norfolk Place, London W2 1PG, United Kingdom.
J Biol Chem. 2002 Oct 11;277(41):39015-24. doi: 10.1074/jbc.M202960200. Epub 2002 Jul 29.
Transcription from the B-myb (MybL2 gene) promoter is strictly cell cycle-regulated by repression mediated through an E2F site during G(0)/early G(1). We report here the characterization of a corepressor site (downstream repression site (DRS)) required for this activity that is closely linked to the E2F site. Systematic mutagenesis of the DRS enabled a consensus to be derived, and it is notable that this sequence is compatible with cell cycle gene homology region sequences associated with cell cycle-dependent elements in the cyclin A, cdc2, and CDC25C promoters. The B-myb promoter is inappropriately active during G(0) in mouse embryo fibroblasts lacking the p107 and p130 pocket proteins, and we show that the ability of transfected p107 and p130 to re-impose repression on the promoter is dependent on the DRS. In contrast, transfected Rb was unable to repress the B-myb promoter. Consistent with the notion that Rb.E2F complexes are unable to bind the B-myb promoter E2F site in vivo, footprinting showed that this site is unoccupied in cells lacking p107 and p130. Chromatin immunoprecipitation assays showed a requirement for the DRS in recruiting p107 and p130 complexes to the B-myb promoter, indicating that in vivo the DRS governs the occupancy of the adjacent E2F site by transcriptional repressors.
B-myb（MybL2基因）启动子的转录在G(0)/早期G(1)期间通过E2F位点介导的抑制作用受到严格的细胞周期调控。我们在此报告了该活性所需的一个共抑制因子位点（下游抑制位点（DRS））的特征，该位点与E2F位点紧密相连。对DRS进行系统诱变得出了一个共有序列，值得注意的是，该序列与细胞周期蛋白A、cdc2和CDC25C启动子中与细胞周期依赖性元件相关的细胞周期基因同源区域序列相符。在缺乏p107和p130口袋蛋白的小鼠胚胎成纤维细胞中，B-myb启动子在G(0)期间异常活跃，并且我们表明，转染的p107和p130对启动子重新施加抑制的能力取决于DRS。相比之下，转染的Rb无法抑制B-myb启动子。与Rb.E2F复合物在体内无法结合B-myb启动子E2F位点的观点一致，足迹分析表明，在缺乏p107和p130的细胞中该位点未被占据。染色质免疫沉淀分析表明，将p107和p130复合物募集到B-myb启动子需要DRS，这表明在体内DRS通过转录抑制因子控制相邻E2F位点的占据情况。
