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

导致体细胞身份丧失的机制的功能剖析

• 批准号: 9148350
• 负责人: Bernadett Papp
• 金额: $ 7.63万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-06 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9148350
• 关键词: Adult; Alpha Cell; Behavior; Binding; Binding Sites; Biological Models; Cell Differentiation process; Cells; Data; Development; Developmental Process; Disease; Dissection; Embryonic Development; Enhancers; Epigenetic Process; Foundations; Generations; Genes; Genome; Genomics; Goals; In Vitro; Lead; Link; Location; Maintenance; Mediating; Medical; Methods; Nucleic Acid Regulatory Sequences; Pathway interactions; Patients; Play; Population; Process; Regulator Genes; Replacement Therapy; Repression; Role; Signal Pathway; Signal Transduction; Site; Somatic Cell; Stem cells; T cell factor 3; Testing; Therapeutic; Transcription Repressor/Corepressor; WNT Signaling Pathway; Work; base; c-myc Genes; cell type; design; embryonic stem cell; epigenome; genome-wide; in vitro Model; induced pluripotent stem cell; inhibitor/antagonist; overexpression; small molecule; small molecule libraries; transcription factor; transcriptome

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.

抽象的 成体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) 状态。对 iPSC 的重编程可以通过过表达 四种转录因子 (TF) Oct4、Sox2、Klf4 和 cMyc (OSKM)。重新编程需要两周时间 iPSC 通常源自不到 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或 抑制剂。在这方面，我们最近证明了 Wnt 信号的逐步调节 不同阶段的信号通路及其下游转录因子（Tcf1、Tcf3、Tcf4 和 Lef1） 重编程促进 iPSC 的建立。我们的研究还强调，重新编程 体细胞为研究细胞底层的信号传导机制提供了强大的体外模型系统 命运改变。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发挥作用。 细胞并限制重编程的各个阶段。目前对细胞命运的理解强调，不 不仅需要表达正确的基因组，而且还必须表达正确的基因调控区域 由细胞类型特异性 TF 和表观遗传标记参与。信号通路 TF 通常表达于 细胞类型特定的方式必须针对其正确的位置，以维持细胞身份或 在信号刺激下创造新的细胞命运，但它们的靶向机制很大程度上未知。我们的 初步数据表明，Wnt 信号传导 TF Tcf3 在 重新编程。通过研究 Tcf3 的这一阶段特定行为，我们揭示了一个以前未知的早期 重编程屏障，控制体细胞 Tcf3 位点的擦除及其新位点的生成 重新编程站点。在本提案中，我们将研究新发现的重编程抑制蛋白的作用 TF 作为 Tcf3 结合的调节剂。此外，我们建议确定信号通路，以维持 以特定阶段的方式识别体细胞并控制重编程。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，我们的工作最终将导致（i）更好地理解特定细胞如何 命运由信号通路维持，并且（ii）还有助于识别发挥作用的关键信号通路 在特定的细胞命运变化期间。信号通路控制发育和发育过程中的许多细胞命运选择 他们的放松管制可能导致疾病。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至关重要，这样我们就可以调节这些信号传导 创造或维持所需细胞命运的途径。