Neural Stem Cell Laboratory, Institute of Medical Biology, Singapore 138678.
Biochem J. 2012 Nov 15;448(1):21-33. doi: 10.1042/BJ20120725.
Oct4 and Sox2 are two essential transcription factors that co-regulate target genes for the maintenance of pluripotency. However, it is unclear whether they interact prior to DNA binding or how the target sites are accessed in the nucleus. By generating fluorescent protein fusions of Oct4 and Sox2 that are functionally capable of producing iPSCs (induced pluripotent stem cells), we show that their interaction is dependent on the presence of cognate DNA-binding elements, based on diffusion time, complex formation and lifetime measurements. Through fluorescence correlation spectroscopy, the levels of Oct4 and Sox2 in the iPSCs were quantified in live cells and two diffusion coefficients, corresponding to free and loosely bound forms of the protein, were distinguished. Notably, the fraction of slow-diffusing molecules in the iPSCs was found to be elevated, similar to the profile in embryonic stem cells, probably due to a change in the nuclear milieu during reprogramming. Taken together, these findings have defined quantitatively the amount of proteins pertinent to the pluripotent state and revealed increased accessibility to the underlying DNA as a mechanism for Oct4 and Sox2 to find their target binding sites and interact, without prior formation of heterodimer complexes.
Oct4 和 Sox2 是两种重要的转录因子，它们共同调节维持多能性的靶基因。然而，目前尚不清楚它们在 DNA 结合之前是否相互作用，或者在核内如何访问靶位点。通过生成功能上能够产生 iPS 细胞（诱导多能干细胞）的 Oct4 和 Sox2 的荧光蛋白融合体，我们表明它们的相互作用取决于同源 DNA 结合元件的存在，这是基于扩散时间、复合物形成和寿命测量得出的。通过荧光相关光谱法，在活细胞中定量测定了 iPS 细胞中 Oct4 和 Sox2 的水平，并区分了对应于蛋白质游离和松散结合形式的两个扩散系数。值得注意的是，在 iPS 细胞中，慢扩散分子的分数被发现升高，类似于胚胎干细胞中的情况，可能是由于重编程过程中核环境的变化。总之，这些发现从定量上定义了与多能状态相关的蛋白质的数量，并揭示了 Oct4 和 Sox2 找到其靶结合位点并相互作用的基础 DNA 的可及性增加，而无需先形成异二聚体复合物。
