Molecular Characterization of the Human NANOG Protein
Molecular Characterization of the Human NANOG Protein
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DOI：
10.1634/stemcells.2008-0657
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发表时间：
2009-01-01
期刊：
影响因子：
5.2
通讯作者：
Lutzko, Carolyn
中科院分区：
文献类型：
--
作者：
Chang, David F.;Tsai, Steven C.;Lutzko, Carolyn
NANOG是多能干细胞（PSC）自我更新的关键转录调节因子。NANOG占据了在自我更新过程中有活性的启动子和其他被抑制的启动子;然而，NANOG调节转录抑制和激活的机制尚不清楚。我们假设NANOG的单个蛋白质结构域控制其与启动子及其辅助调节子的相互作用。我们使用一组缺失突变体和点突变体构建体对人（h）NANOG蛋白中的功能结构域进行了详细表征。我们确定同源结构域中的6个氨基酸（（136）YKQVKT（141））足以用于hNANOG的核定位。我们还确定hNANOG的富含色氨酸的区域（W）包含用于核输出的CRM 1独立信号，这表明可能的细胞穿梭行为尚未报道hNANOG。我们还表明，核出口至少需要4个钚。我们还确定，类似于鼠（m）NANOG，hNANOG的W区含有同源二聚化结构域。最后，在体外反式激活分析确定了不同的区域，增强或减弱在自我更新过程中活跃的基因启动子的活性。具体地，N-末端区域干扰转录并去除该区域，产生具有增强的转录活性的“超活性”hNANOG。我们还证实，在hNANOG中的转录激活因子包含在C-末端区域，类似于小鼠NANOG。总之，这项研究的特点是hNANOG蛋白的结构和功能，从而增加了对hNANOG在PSC自我更新过程中调节转录激活和抑制的机制的理解。干细胞2009;27：812-821
NANOG is a key transcriptional regulator of pluripotent stem cell (PSC) self-renewal. NANOG occupies promoters that are active and others that are repressed during self-renewal; however, the mechanisms by which NANOG regulates transcriptional repression and activation are unknown. We hypothesized that individual protein domains of NANOG control its interactions with both the promoters and its coregulators. We performed a detailed characterization of the functional domains in the human ( h) NANOG protein, using a panel of deletion-mutant and point-mutant constructs. We determined that six amino acids in the homeodomain ((136)YKQVKT(141)) are sufficient for the nuclear localization of hNANOG. We also determined that the tryptophan-rich region (W) of hNANOG contains a CRM1-independent signal for nuclear export, suggesting a possible cellular shuttling behavior that has not been reported for hNANOG. We also show that at least four tryptophans are required for nuclear export. We also determined that similar to murine (m) NANOG, the W region of hNANOG contains a homodimerization domain. Finally, in vitro transactivation analyses identified distinct regions that enhance or diminish activity at gene promoters that are active during self-renewal. Specifically, the N-terminal region interferes with transcription and removal of this region that produced a "super-active'' hNANOG with enhanced transcriptional activity. We also confirmed that the transcriptional activator in hNANOG is contained in the C-terminal region, similar to murine NANOG. In summary, this study has characterized the structure and function of hNANOG protein leading to an increased understanding of the mechanism by which hNANOG regulates both transcriptional activation and repression during PSC self-renewal. STEM CELLS 2009;27:812-821