Analysis of pluripotency transcription factor interactions reveals preferential binding of NANOG to SOX2 rather than NANOG or OCT4

Analysis of pluripotency transcription factor interactions reveals preferential binding of NANOG to SOX2 rather than NANOG or OCT4

The pluripotency transcription factors (TFs) Nanog, Sox2, and Oct4 are at the centre of the gene regulatory network that controls cell identity in embryonic stem (ES) cells. However, the mechanisms by which these factors control cell fate, and their interactions with one another are not fully understood. Here we combine biophysical and novel biochemical assays to assess how these factors interact with each other quantitatively. A confocal microscopy method to detect binding of a target protein to a fluorescently labelled partner (coimmunoprecipitated bead imaging microscopy [CBIM]) is presented and used to demonstrate homotypic binding of Nanog and heterotypic binding between Nanog and Sox2 and between Nanog and Oct4. Using fluorescence correlation spectroscopy we show that in solution, Nanog but not Oct4 or Sox2 can form homomultimers. However, both Sox2 and Oct4 can form heterotypic multimers with Nanog in a manner that depends on the presence of tryptophan residues within the Nanog tryptophan repeat. Fluorescence Cross Correlation Spectroscopy shows the affinity of Nanog for dimer formation is in the order Sox2 > Nanog > Oct4. Importantly, live cell analysis demonstrates the existence of Nanog homomultimers in vivo. Together these findings extend understanding of the molecular interactions occurring between these central mediators of the pluripotency gene regulatory network at the single-molecule level.

多能性转录因子（TFs）Nanog、Sox2和Oct4处于基因调控网络的核心位置，该网络控制着胚胎干细胞（ES）的细胞特性。然而，这些因子控制细胞命运的机制以及它们之间的相互作用尚未完全被理解。在此，我们结合生物物理和新型生化分析来定量评估这些因子如何相互作用。我们提出了一种共聚焦显微镜方法，用于检测目标蛋白与荧光标记的伙伴的结合（共免疫沉淀磁珠成像显微镜[CBIM]），并用于证明Nanog的同型结合以及Nanog与Sox2和Nanog与Oct4之间的异型结合。利用荧光相关光谱技术，我们表明在溶液中，Nanog而非Oct4或Sox2能够形成同源多聚体。然而，Sox2和Oct4都能够以依赖于Nanog色氨酸重复序列内色氨酸残基存在的方式与Nanog形成异型多聚体。荧光互相关光谱显示Nanog形成二聚体的亲和力顺序为Sox2 > Nanog > Oct4。重要的是，活细胞分析证明了体内存在Nanog同源多聚体。总之，这些发现从单分子水平扩展了对多能性基因调控网络的这些核心介质之间发生的分子相互作用的理解。