Distinct Contributions of Tryptophan Residues within the Dimerization Domain to Nanog Function.
Distinct Contributions of Tryptophan Residues within the Dimerization Domain to Nanog Function.
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DOI：
10.1016/j.jmb.2016.12.001
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发表时间：
2017-05-19
影响因子：
5.6
通讯作者：
Chambers I
中科院分区：
文献类型：
--
作者：
Mullin NP;Gagliardi A;Khoa LTP;Colby D;Hall-Ponsele E;Rowe AJ;Chambers I
转录因子Nanog的水平直接决定小鼠胚胎干细胞自我更新的效率。Nanog蛋白作为二聚体存在，二聚化结构域由简单重复区组成，其中每五个残基是色氨酸，即色氨酸重复（WR）。虽然WR是必要的，使Nanog赋予LIF-独立的自我更新，二聚化的机制和调节二聚化强度的效果一直不清楚。在这里，我们夫妇诱变功能和二聚化试验表明，WR内的的多聚体的数量与同源二聚化，Sox 2异源二聚化和自我更新活性的强度。色氨酸残基数目的减少最初导致活性的逐渐降低，然后在色氨酸数目减少到低于8时发生活性的急剧降低。进一步的功能损耗随后色氨酸数减少，取代所有色氨酸残基，完全消除二聚化和自我更新功能。一个强有力的位置影响存在，在WR末端的残基，特别是在N-末端的Nanog功能贡献更多。有限的蛋白水解表明，Nanog的结构核心，包括同源结构域和色氨酸重复可以支持LIF独立的集落形成。这些结果增加了对多能性基因调控网络核心转录因子亚基之间分子相互作用的理解，并将增强我们控制多能性细胞自我更新和分化的能力。Nanog色氨酸重复序列（WR）是LIF非依赖性小鼠胚胎干细胞自我更新所必需的。WR内的色氨酸在自我更新和二聚化中产生梯度增加。WR末端的色氨酸对Nanog功能有贡献。含有WR的Nanog的结构核心可以驱动独立于LIF的自我更新。结果阐明了WR内色氨酸残基对Nanog功能的贡献。
The level of the transcription factor Nanog directly determines the efficiency of mouse embryonic stem cell self-renewal. Nanog protein exists as a dimer with the dimerization domain composed of a simple repeat region in which every fifth residue is a tryptophan, the tryptophan repeat (WR). Although WR is necessary to enable Nanog to confer LIF-independent self-renewal, the mechanism of dimerization and the effect of modulating dimerization strength have been unclear. Here we couple mutagenesis with functional and dimerization assays to show that the number of tryptophans within the WR is linked to the strength of homodimerization, Sox2 heterodimerization and self-renewal activity. A reduction in the number of tryptophan residues leads initially to a gradual reduction in activity before a precipitous reduction in activity occurs upon reduction in tryptophan number below eight. Further functional attrition follows subsequent tryptophan number reduction with substitution of all tryptophan residues ablating dimerization and self-renewal function completely. A strong positional influence of tryptophans exists, with residues at the WR termini contributing more to Nanog function, particularly at the N-terminal end. Limited proteolysis demonstrates that a structural core of Nanog encompassing the homeodomain and the tryptophan repeat can support LIF-independent colony formation. These results increase understanding of the molecular interactions occurring between transcription factor subunits at the core of the pluripotency gene regulatory network and will enhance our ability to control pluripotent cell self-renewal and differentiation. The Nanog tryptophan repeat (WR) is required for LIF-independent mouse embryonic stem cell self-renewal. Tryptophans within WR give a graded increase in self-renewal and dimerization. Tryptophans at the WR extremities contribute disproportionally to Nanog function. A structural core of Nanog containing WR can drive LIF-independent self-renewal. Results clarify the contribution of tryptophan residues within WR to Nanog function.