Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells
Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells
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DOI：
10.1073/pnas.1502855112
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发表时间：
2015-04-14
影响因子：
11.1
通讯作者：
Fletterick, Robert J.
中科院分区：
文献类型：
--
作者：
Hayashi, Yohei;Caboni, Laura;Fletterick, Robert J.
NANOG（来自爱尔兰神话Tir na nOg）转录因子在维持多能性方面起着核心作用，与OCT 4（也称为POU 5 F1或OCT 3/4），SOX 2和其他多能性因子合作。虽然NANOG蛋白的生理作用已被广泛研究，但与其结构分析相关的生物化学和生物物理特性知之甚少。在这里，我们确定了与OCT 4启动子DNA结合的人NANOG同源结构域（hNANOG HD）的晶体结构，这揭示了参与DNA识别的氨基酸残基，这些氨基酸残基可能在功能上很重要。基于蛋白质-DNA相互作用和进化保守性，我们构建了一系列hNANOG HD丙氨酸取代突变体，并测定了它们的生物学活性。一些突变蛋白质不太稳定，导致DNA结合亲和力丧失或降低。在没有白血病抑制因子的情况下，正常小鼠NANOG（mNANOG）突变体的过表达未能维持小鼠胚胎干细胞的自我更新。这些结果表明，这些残基对于NANOG转录活性至关重要。有趣的是，一种突变体hNANOG L122 A相反地增强了蛋白质稳定性和DNA结合亲和力。当mNANOG L122 A在小鼠胚胎干细胞中过表达时，即使在添加视黄酸以强制驱动分化时，也保持其自我更新标记的表达。当在外胚层干细胞或人诱导多能干细胞中过表达时，L122 A突变体增强重编程为基态多能性。这些发现表明，关键转录因子的结构和生物物理信息提供了深入了解干细胞行为的操纵和合理的蛋白质工程框架。
NANOG (from Irish mythology Tir na nOg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein-DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.