Functional Dissection of the Mechanism that Lead to Loss of Somatic Cell Identity

摘要 成年细胞可以被迫改变其细胞身份，并重新编程为胚胎干细胞（ESC）样细胞。 诱导多能干细胞（iPSC）。重编程为iPSC可以通过过表达 四种转录因子（TF）Oct4、Sox2、Klf4和cMyc（OSKM）。重新编程需要2周时间， iPSC通常来源于少于1%的起始细胞群。然而，重新编程 当我们用小分子激活剂调节信号通路的活性时， 抑制剂的在这方面，我们最近已经证明，Wnt信号的逐步调节， Tcf1、Tcf3、Tcf4和Lef1等转录因子在不同发育阶段的表达变化 重编程促进iPSC的建立。我们的研究还强调， 体细胞为研究细胞信号传导机制提供了强大的体外模型系统 命运会改变重要的是，我们仍然不知道Wnt通路和其他信号通路如何在体细胞中起作用， 细胞，并限制了重编程的各个阶段。目前对细胞命运的理解强调， 只有正确的基因组必须表达，而且正确的基因调控区域必须被表达。 由细胞类型特异性转录因子和表观遗传标记参与。信号通路转录因子通常表达于 细胞类型特定的方式必须以它们的适当位置为目标， 在信号刺激下产生新的细胞命运，然而它们的靶向机制在很大程度上是未知的。我们 初步数据显示，Wnt信号转导TF Tcf3在转录过程中动态改变其全基因组结合。 重新编程通过研究Tcf3的这种阶段特异性行为，我们揭示了一个以前未知的早期Tcf3的表达。 重编程的障碍，控制体细胞Tcf3位点的擦除和其新的Tcf3位点的产生。 重编程网站在这项提案中，我们将研究我们新发现的重编程抑制基因的作用， TF作为Tcf3结合的调节剂。此外，我们建议确定信号通路，维持 体细胞身份和控制以阶段特异性方式重编程。使用我们新的单细胞水平 免疫染色方法，我们将确定这些信号通路如何调节不同的细胞命运， 重新编程我们相信，我们的工作将最终导致（i）更好地了解特定细胞如何 命运是由信号通路维持的，（ii）也有助于识别起作用的关键信号通路， 在特定的细胞命运的变化。在发育过程中，信号通路控制着许多细胞命运的选择， 它们的失调会导致疾病。因此，了解信号通路在维持 体细胞状态和调节体细胞身份的丧失是至关重要的， 这些途径可以产生或维持所需的细胞命运。

Abstract Adult cells can be forced to change their cell identity and reprogrammed to an embryonic stem cell (ESC)-like induced pluripotent stem cells (iPSCs) state. Reprogramming to iPSCs can be achieved by overexpression of the four transcription factors (TFs) Oct4, Sox2, Klf4, and cMyc (OSKM). Reprogramming takes 2 weeks and the iPSCs are generally derived from less than 1% of the starting cell population. However, the reprogramming is more efficient when we modulate the activity of signaling pathways with small molecule activators or inhibitors. In this regard, we have recently demonstrated that the stepwise-modulation of the Wnt signaling pathway and its downstream transcriptional factors (Tcf1, Tcf3, Tcf4 and Lef1) during different stages of reprogramming promotes the establishment of iPSCs. Our studies also highlight that reprogramming of somatic cells provides a powerful in vitro model system for studying the signaling mechanisms underlying cell fate changes. Importantly, we still do not know how Wnt pathway and other signaling pathways act in somatic cells and limit the various stages of reprogramming. Current understanding of cell fates emphasize that not only the proper set of genes has to be expressed, but also the correct gene regulatory regions have to be engaged by cell type specific TFs and epigenetic markings. Signaling pathway TFs that are often expressed in a cell type specific manner must be targeted to their proper locations in order to maintain cell identity or to create new cell fates upon signal stimulation, however their targeting mechanisms are largely unknown. Our preliminary data show that the Wnt signaling TF Tcf3 dynamically changes its genome-wide binding during reprogramming. By studying this stage-specific behavior of Tcf3, we revealed a previously unknown early barrier of reprogramming, which controls the erasure of somatic Tcf3 sites and the generation of its new reprogramming sites. In this proposal, we will study the role of our newly identified reprogramming-inhibiting TFs as regulators of Tcf3 binding. Furthermore, we propose to identify signaling pathways, which maintain the somatic cell identity and control reprogramming in a stage specific manner. Using our new single cell level immunostaining method, we will determine how these signaling pathways regulate the distinct cell fates during reprogramming. We believe that our work will ultimately lead to (i) a better understanding of how specific cell fates are maintained by signaling pathways, and (ii) also help identifying critical signaling pathways that act during specific cell fate changes. Signaling pathways control many cell fate choices during development and their deregulation can cause diseases. Therefore, understanding the role of signaling pathways in maintaining somatic state and regulating the loss of somatic identity is crucial so that we can modulate these signaling pathways to create or maintain desired cell fates.