TCF7 is a key regulator of the switch of self-renewal and differentiation in a multipotential hematopoietic cell line. Mus musculus

A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34- cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a key regulator in this self-renewal/differentiation switch, and it operates in the absence of canonical Wnt signaling. We found that TCF7 is the most downregulated transcription factor when CD34+ cells switch into CD34- cells using RNA-Seq. We subsequently identified the target genes bound by TCF7 using ChIP-Seq. We show that TCF7 binds to Runx1 (Aml1) promoter region, and RUNX1 and TCF7 co-regulate. Gene Set Enrichment Analysis suggests that TCF7 primarily acts as a positive regulator of genes preferentially expressed in CD34+ cells. Consistent with this possibility, knocking-down TCF7 represses many up-regulated genes in Lin-CD34+ cells. Finally a network of up-regulated transcription factors of CD34+ cells which defines the self-renewing state was constructed. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems. Overall design: Examining the transcription factor binding targets of TCF7 and RUNX1.生物学领域的一项核心难题，在于解析细胞如何在自我更新与分化之间做出抉择。为全面阐明调控早期造血前体细胞自我更新与分化的分子机制，我们采用了基于系统生物学的研究方法，并以小鼠EML多能造血前体细胞作为核心实验模型。EML细胞可通过细胞自主方式，产生由自我更新型Lin-SCA+CD34+细胞与部分分化且丧失自我更新能力的Lin-SCA-CD34-细胞组成的混合群体。我们鉴定并验证了HMG盒蛋白TCF7是这一自我更新/分化转换过程的关键调控因子，且其发挥功能不依赖经典Wnt信号通路。通过RNA测序（RNA-Seq）分析，我们发现当CD34+细胞向CD34-细胞转换时，TCF7是表达下调幅度最大的转录因子。后续我们借助染色质免疫沉淀测序（ChIP-Seq）鉴定了TCF7所结合的靶基因。我们证实TCF7可结合至Runx1（Aml1）的启动子区域，且RUNX1与TCF7存在共调控关系。基因集富集分析（Gene Set Enrichment Analysis）结果显示，TCF7主要作为偏好性表达于CD34+细胞中的基因的正向调控因子发挥作用。与此结论一致的是，敲低TCF7会抑制Lin-CD34+细胞中诸多上调基因的表达。最终我们构建了一套定义自我更新状态的CD34+细胞上调转录因子调控网络。上述基于EML细胞的研究，阐明了细胞自我更新与分化转换过程中固有的核心细胞内在特性，并为调控造血干细胞（Hematopoietic Stem Cells, HSCs）及其他分化相关系统提供了极具价值的理论参考。实验整体设计：探究TCF7与RUNX1的转录因子结合靶标。