Phage display screening reveals an association between germline-specific transcription factor Oct-4 and multiple cellular proteins
Phage display screening reveals an association between germline-specific transcription factor Oct-4 and multiple cellular proteins
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DOI：
10.1006/jmbi.2000.4238
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发表时间：
2000-12-08
影响因子：
5.6
通讯作者：
Pesce, M
中科院分区：
文献类型：
--
作者：
Butteroni, C;De Felici, M;Pesce, M
Oct-4是在小鼠胚胎干细胞及其衍生的细胞系中特异性表达的转录因子。在这些细胞中，Oct-4由于未知的共激活因子而激活来自远程结合位点的转录。Oct-4在分化细胞中的表达不足以从远处激活转录，而是需要共激活因子如腺病毒癌蛋白E1 A的共表达。在本文中，我们使用噬菌体展示来鉴定Oct-4相互作用蛋白。我们首先分析了Oct-4和E1 A之间的相互作用，以优化使Oct-4特异性与其他相互作用蛋白相互作用的生化条件。淘选方法用于从过量的非特异性底物的池中富集Oct-4相互作用底物。在我们的相互作用试验中建立的生化条件，然后被用来筛选在M13丝状噬菌体中的P19 EC细胞cDNA表达文库。获得了许多展示未知和已知转录因子部分的噬菌体克隆，从中鉴定了HMG-1转录因子。HMG-1和密切相关的因子HMG-2在哺乳动物细胞中共表达时与Oct-4相互作用。此外，HMG-1与Oct-4在P19 EC细胞中具有协同作用。这些结果提供了第一个证据的非病毒因子，增强Oct-4的距离依赖性的反式激活干细胞。(C)北京大学出版社.
Oct-4 is a transcription factor that is specifically expressed in mouse embryonic stem cells and in cell lines derived thereof. Ln these cells, Oct-4 activates transcription from remote binding sites due to as of yet unknown co-activators. Expression of Oct-4 in differentiated cells is not sufficient to activate transcription from a distance, rather it requires the co-expression of co-activators such as the adenoviral oncoprotein E1A. In this paper, we used phage display to identify Oct-4-interacting proteins. We first analyzed the interaction between Oct-4 and E1A in order to optimize the biochemical conditions that enable Oct-4-specific interactions with other interacting proteins. A panning approach was used to enrich Oct-4 interacting phages from a pool of excess unspecific phages. The biochemical conditions established in our interaction assays were then used to screen a P19 EC cell cDNA expression library in M13 filamentous phage. A number of phage clones displaying portions of unknown and known transcription factors were obtained, from which the HMG-1 transcription factor was identified. HMG-1, and the closely related factor HMG-2, interact with Oct-4 when co-expressed in mammalian cells. In addition, HMG-1 was found to cooperate with Oct-4 in P19 EC cells. These results provide the first evidence of a non-viral factor that enhances Oct-4 distance-dependent transactivation in stem cells. (C) 2000 Academic Press.