Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
Wallenberg Centre for Molecular Medicine (WCMM) and Department of Biomedical and Clinical Sciences, Faculty of Health Science, Linköping University, Linköping, Sweden.
Stem Cells. 2021 Aug;39(8):1107-1119. doi: 10.1002/stem.3373. Epub 2021 Mar 29.
The Sox2 transcription factor is necessary for the long-term self-renewal of neural stem cells (NSCs). Its mechanism of action is still poorly defined. To identify molecules regulated by Sox2, and acting in mouse NSC maintenance, we transduced, into Sox2-deleted NSC, genes whose expression is strongly downregulated following Sox2 loss (Fos, Jun, Egr2), individually or in combination. Fos alone rescued long-term proliferation, as shown by in vitro cell growth and clonal analysis. Furthermore, pharmacological inhibition by T-5224 of FOS/JUN AP1 complex binding to its targets decreased cell proliferation and expression of the putative target Suppressor of cytokine signaling 3 (Socs3). Additionally, Fos requirement for efficient long-term proliferation was demonstrated by the reduction of NSC clones capable of long-term expansion following CRISPR/Cas9-mediated Fos inactivation. Previous work showed that the Socs3 gene is strongly downregulated following Sox2 deletion, and its re-expression by lentiviral transduction rescues long-term NSC proliferation. Fos appears to be an upstream regulator of Socs3, possibly together with Jun and Egr2; indeed, Sox2 re-expression in Sox2-deleted NSC progressively activates both Fos and Socs3 expression; in turn, Fos transduction activates Socs3 expression. Based on available SOX2 ChIPseq and ChIA-PET data, we propose a model whereby Sox2 is a direct activator of both Socs3 and Fos, as well as possibly Jun and Egr2; furthermore, we provide direct evidence for FOS and JUN binding on Socs3 promoter, suggesting direct transcriptional regulation. These results provide the basis for developing a model of a network of interactions, regulating critical effectors of NSC proliferation and long-term maintenance.
Sox2 转录因子对于神经干细胞（NSC）的长期自我更新是必需的。其作用机制仍不清楚。为了鉴定 Sox2 调节的分子，并作用于维持小鼠 NSC，我们将表达强烈下调的基因（Fos、Jun、Egr2）分别或组合转导到 Sox2 缺失的 NSC 中。单独的 Fos 即可挽救长期增殖，如体外细胞生长和克隆分析所示。此外，通过 T-5224 抑制 FOS/JUN AP1 复合物与其靶标的结合，降低细胞增殖和推定靶标细胞因子信号转导抑制因子 3（Socs3）的表达。此外，通过 CRISPR/Cas9 介导的 Fos 失活后能够长期扩增的 NSC 克隆减少，证明了 Fos 对有效长期增殖的需求。先前的工作表明，Socs3 基因在 Sox2 缺失后强烈下调，其通过慢病毒转导的重新表达可挽救长期 NSC 增殖。Fos 似乎是 Socs3 的上游调节剂，可能与 Jun 和 Egr2 一起；事实上，Sox2 在 Sox2 缺失的 NSC 中的重新表达逐渐激活 Fos 和 Socs3 的表达；反过来，Fos 转导激活 Socs3 的表达。基于现有的 SOX2 ChIPseq 和 ChIA-PET 数据，我们提出了一个模型，其中 Sox2 是 Socs3 和 Fos 的直接激活剂，也可能是 Jun 和 Egr2 的直接激活剂；此外，我们提供了 FOS 和 JUN 结合 Socs3 启动子的直接证据，表明直接转录调控。这些结果为开发调节 NSC 增殖和长期维持的关键效应子的相互作用网络模型提供了基础。
