Identification of a regulatory region which is involved in Sox-2 expression in embryonic and neural stem cells and its molecular basis

我们一直有兴趣了解ES细胞和神经干细胞之间的共同特性，如在分子水平上的自我更新能力。为了了解这一点，我们认为重要的是确定一个在这两种干细胞中都起作用的调控区域。由于已知Sox-2基因在ES细胞和神经干细胞中都有表达，因此我们探讨了Sox-2基因具有该调控区的可能性。我们之前已经根据它们在多能ES细胞中的活性确定了两个增强子，SRR1和SRR2。在此背景下，我们研究了这些SOX-2调节区是否也在神经干细胞/祖细胞中表现出它们的活性。事实上，所有病例的胚胎干细胞巢蛋白阳性细胞、神经球法和宫内电穿孔实验都表明，SRR1和SRR2在神经干细胞/祖细胞和ES细胞中都具有相当强的转录刺激活性。此外，与ES细胞的情况一样，神经干/祖细胞中的SRR1和SRR2的活性在其多潜能状态下是特异的，当神经干/祖细胞与血清诱导分化时，这些增强子失去了全部活性。突变分析表明，SRR2通过利用共同的核心序列在这两种不同类型的干细胞中展示了其活性。我们的分析还表明，BRN-1-SOX-2和BRN-2-SOX-2复合体参与了神经细胞中SRR2的活性，而Oct-3/4-SOX-2复合体在ES细胞中起主要作用。从这些分析中，我们发现SRR2一般不在神经干/祖细胞中发挥作用，而是在端脑中的神经干/祖细胞中特异性地发挥作用。

We have been interested in understanding common properties between ES cells and neural stem cells such as self-renewal ability in a molecular level. To gain insight about it, we considered that it is important to identify a regulatory region which works in both of these two kinds of stem cells. Since it is known that Sox-2 gene is expressed in both ES cells and neural stem cells, we pursued the possibility that the Sox-2 gene bears such regulatory region. We have previously identified two enhancers, SRR1 and SRR2 based on their activities in pluripotent ES cells. In this context, we examined whether these Sox-2 regulatory regions exhibit their activities also in neural stem/progenitor cells. Indeed, experiments with nestin-positive cells derived from ES cells, neurosphere methods, and in utero electroporation in all cases revealed that both of SRR1 and SRR2 exert rather strong transcription stimulating activities in neural stem/progenitor cells as well as in ES cells. Moreover, as in the case of ES cells, activities of SRR1 and SRR2 in neural stem/progenitor cells are specific in their multipotent state and these enhancers lose their entire activities when neural stem/progenitor cells are induced to differentiate with serum. Mutagenesis analyses demonstrated that SRR2 exhibits its activities in these two different types of stem cells by utilizing a common core sequence. Our analyses also revealed that Brn-1-Sox-2 and Brn-2-Sox-2 complexes are involved in SRR2 activities in neural cells, while Oct-3/4-Sox-2 complex plays major role in ES cells.We also performed the transgenic analyses about the SRR2 enhancer. From these analyses, we found that the SRR2 do not function in neural stem/progenitor cells in general, but exerts its activity rather specifically in neural stem/progenitor cells residing in telencephalon.