Lavrrar Jennifer L, Farnham Peggy J
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
J Biol Chem. 2004 Oct 29;279(44):46343-9. doi: 10.1074/jbc.M402692200. Epub 2004 Aug 24.
The E2F family of transcription factors regulates the expression of genes involved in cell cycle progression, DNA synthesis, repair, and recombination, and a variety of other cellular processes. Although E2F proteins are often redundant in function, specificity of binding and activity can occur. For example, E2F1, but not other E2F family members, was shown previously to bind the murine carboxylesterase promoter in chromatin immunoprecipitation studies. This promoter region lacks a consensus E2F binding site, suggesting that E2F1 may be recruited to the DNA in a unique fashion. To further investigate this E2F1-specific binding, we have employed a "transient chromatin immunoprecipitation" approach. Using various deletions and mutations of the promoter region, we localized the E2F1-specific binding site and demonstrated that it was required for E2F1-mediated transcription of the carboxylesterase promoter. The identified site was similar to the 8-bp consensus E2F site but differed from the consensus at a crucial position. To address whether E2F1 directly bound to this non-consensus site, we demonstrated that the DNA binding domain of E2F1 is necessary for E2F1-mediated activation of the carboxylesterase promoter. Interestingly, a "UP" mutation of the site, making it more similar to the consensus element, did not improve the ability of E2F1 to bind the promoter. Rather, E2F1 could no longer bind to the carboxylesterase promoter that contained the consensus E2F site. We propose a model in which E2F1-specific regulation of the carboxylesterase promoter requires both E2F1/DNA interactions and protein-protein interaction between E2F1 and a factor that binds adjacent to the non-consensus site.
转录因子E2F家族调控参与细胞周期进程、DNA合成、修复和重组以及多种其他细胞过程的基因表达。尽管E2F蛋白在功能上常常冗余，但结合和活性的特异性仍可能出现。例如，在染色质免疫沉淀研究中，E2F1能结合小鼠羧酸酯酶启动子，而其他E2F家族成员则不能。该启动子区域缺乏一致的E2F结合位点，这表明E2F1可能以独特方式被招募到DNA上。为进一步研究这种E2F1特异性结合，我们采用了“瞬时染色质免疫沉淀”方法。通过对启动子区域进行各种缺失和突变，我们定位了E2F1特异性结合位点，并证明它是E2F1介导的羧酸酯酶启动子转录所必需的。所确定的位点与8碱基对的一致E2F位点相似，但在关键位置与一致序列不同。为确定E2F1是否直接结合到这个非一致位点，我们证明E2F1的DNA结合结构域是E2F1介导的羧酸酯酶启动子激活所必需的。有趣的是，该位点的“UP”突变使其更类似于一致元件，但并未提高E2F1结合启动子的能力。相反，E2F1不再能结合含有一致E2F位点的羧酸酯酶启动子。我们提出一个模型，其中E2F1对羧酸酯酶启动子的特异性调控既需要E2F1/DNA相互作用，也需要E2F1与结合在非一致位点相邻位置的因子之间的蛋白质-蛋白质相互作用。
