Identification of a nuclear localization signal in OCT4 and generation of a dominant negative mutant by its ablation
Identification of a nuclear localization signal in OCT4 and generation of a dominant negative mutant by its ablation
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DOI：
10.1074/jbc.m405117200
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发表时间：
2004-08-27
影响因子：
4.8
通讯作者：
Pei, DQ
中科院分区：
文献类型：
--
作者：
Pan, GJ;Qin, BM;Pei, DQ
OCT 4通过激活和抑制多个下游基因以剂量依赖性方式在维持干细胞多能性中起关键作用。OCT 4实现这些不同生物功能的确切机制仍然未知。在这份报告中，我们确定和表征（RKRKR）-R-195作为一个核定位信号，负责其在细胞核中的定位和所需的反式激活其靶基因。该基序内的点突变产生了一种突变体，该突变体随机定位于整个细胞中，并且在反式激活靶基因方面有缺陷。然而，用异源核定位信号恢复核定位未能挽救其反式激活功能，表明该（RKRKR）-R-195基序在介导反式激活功能中具有额外的功能。我们进一步证明，这种突变体是主管二聚体不仅本身，而且野生型OCT 4，并可以干扰野生型OCT 4的活性，从而作为一个显性负突变体。事实上，该突变体可以诱导P19细胞分化为滋养层样巨细胞。这些数据表明，这种显性负性形式的OCT 4可能是用于调节多能细胞（如胚胎干细胞）中OCT 4的活性以获得用于治疗应用的所需细胞类型的有用工具。
OCT4 plays a critical role in maintaining stem cell pluripotency in a dose-dependent manner by activating and repressing multiple downstream genes. The precise mechanism by which OCT4 achieves these diverse biological functions remains unknown. In this report, we identify and characterize (RKRKR)-R-195 as a nuclear localization signal responsible for its localization in the nuclei and required for the transactivation of its target genes. Point mutations within this motif yielded a mutant that localizes randomly throughout the cells and is defective in transactivating target genes. However, restoration of nuclear localization with a heterologous nuclear localization signal failed to rescue its transactivation function, suggesting that this (RKRKR)-R-195 motif has additional function in mediating transactivation function. We further demonstrate that this mutant is competent in dimerization with not only itself but also wild type OCT4 and can interfere with the activity of wild type OCT4, thus acting as a dominant negative mutant. Indeed, this mutant can induce the differentiation of P19 cells into trophoblast-like giant cells. These data suggest that this dominant negative form of OCT4 may be a useful tool for modulating the activity of OCT4 in pluripotent cells such as embryonic stem cells to achieve the desired cell types for therapeutic applications.