Key Laboratory of Biodiversity and Conservation of Aquatic Organisms, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, PR China.
Development. 2013 Mar;140(5):1067-78. doi: 10.1242/dev.086157. Epub 2013 Jan 30.
Eaf factors play a crucial role in tumor suppression and embryogenesis. To investigate the potential mechanism of Eaf activity, we performed loss- and gain-of-function assays in zebrafish using morpholino and mRNA injections, respectively. We found that eaf1 and eaf2 inhibit Wnt/β-catenin signaling, thereby modulating mesodermal and neural patterning in the embryo. Moreover, ectopic expression of eaf1 and eaf2 in embryos and cultured cells blocked β-catenin reporter activity. By immunoprecipitation, we also observed that Eaf1 and Eaf2 bound to the Armadillo repeat region and C-terminus of β-catenin, as well as to other β-catenin transcription complex proteins, such as c-Jun, Tcf and Axin, suggesting the formation of a novel complex. In addition, the N-terminus of Eaf1 and Eaf2 bound to β-catenin and exhibited dominant-negative activity, whereas the C-terminus appeared to either harbor a suppression domain or to recruit a repressor. Both the N- and C-terminus must be intact for Eaf1 and Eaf2 suppressive activity. Lastly, we demonstrate a conservation of biological activities for Eaf family proteins across species. In summary, our evidence points to a novel role for Eaf1 and Eaf2 in inhibiting canonical Wnt/β-catenin signaling, which might form the mechanistic basis for Eaf1 and Eaf2 tumor suppressor activity.
Eaf 因子在肿瘤抑制和胚胎发生中发挥着关键作用。为了研究 Eaf 活性的潜在机制，我们分别使用 morpholino 和 mRNA 注射在斑马鱼中进行了失活和功能获得实验。我们发现 eaf1 和 eaf2 抑制 Wnt/β-catenin 信号通路，从而调节胚胎中的中胚层和神经模式。此外，eaf1 和 eaf2 在胚胎和培养细胞中的异位表达阻断了 β-catenin 报告基因的活性。通过免疫沉淀，我们还观察到 Eaf1 和 Eaf2 与 Armadillo 重复区和 β-catenin 的 C 末端以及其他 β-catenin 转录复合物蛋白（如 c-Jun、Tcf 和 Axin）结合，表明形成了一个新的复合物。此外，Eaf1 和 Eaf2 的 N 末端与 β-catenin 结合并表现出显性负活性，而 C 末端似乎含有抑制结构域或招募抑制物。Eaf1 和 Eaf2 的抑制活性都需要 N 端和 C 端完整。最后，我们证明了 Eaf 家族蛋白在物种间具有保守的生物学活性。总之，我们的证据表明 Eaf1 和 Eaf2 在抑制经典 Wnt/β-catenin 信号通路中具有新的作用，这可能是 Eaf1 和 Eaf2 肿瘤抑制活性的机制基础。
