TCF3: A Wnt Pathway Effector in Pluripotent Stem Cell Self-Renewal

描述(由申请人提供)：摘要尽管人类胚胎干细胞(HESCs)在推进人类疾病的新疗法方面具有巨大的潜力，但在理解干细胞状态方面仍存在重要的未知因素。解决这些未知因素可能会提高建立多能干细胞的能力：如何维持它们，以及如何将它们转变为特定的细胞。拟议的研究策略将通过研究Wnt途径效应器、T细胞因子和淋巴增强因子(TCF/LEF)来解决多能性问题。其中一个成员TCF3是一种转录因子，在小鼠胚胎干细胞(MESCs)中抑制主要多能因子Oct4、Sox2和Nanog(OSN)的表达。重要的是，小鼠Tcf3在mESC分化过程中限制了这些基因的前馈表达。虽然对mESC的研究是有用的，并表明Tcf3应该如何被认为是第四个核心干细胞调节因子，但推断hESCs的作用是内在的。 由于mESCs和hESCs之间的关键信号差异而造成的限制。但TCF3在人胚胎干细胞中的功能完全未知。我们的初步数据表明，TCF3在hESCs中动态表达。与其他核心因素相比，TCF3蛋白水平在分化后迅速下降，然后才能看到其他变化，因此可能是一个关键的第一事件。TCF3在hESC克隆中的表达差异很大，提示hESCs可能具有分化的潜能。分化伴随着其他TCF/LEF的快速上调，这表明Wnt靶基因启动子的占位发生了转换。最重要的假设是hESCs表达TCF3，TCF3抑制Wnt信号，维持“干细胞”染色质结构，并维持Activin信号(调节hESC多能性的关键途径)，以保持hESCs处于稳定状态；多能，但“准备就绪”。在有分化信号的情况下，TCF3被激活的TCF/LEF取代，TCF/LEF改变染色质结构，驱动基因表达和细胞分化。具体目标1将测试TCF3在调节激活素信号中的作用。我们会 研究控制激活素抑制剂，卵泡抑素和抑制素E，在初步研究中被确定为潜在的TCF3靶点。特定目的2将测试TCF3在调节染色质重塑因子SMARCA2中的作用。这些研究可以确定TCF3下调与hESC多能性和分化的相关性，并将其置于更大的hESC调控网络中。TCF3和主调节器(OSN)之间的关系也将使用微阵列和芯片序列来定义。特异靶蛋白3将检测TCF3相关基因在hESC分化中的作用。我们发现hESC分化伴随着TCF3相关基因(Lef-1/TCF-1/TCF-4)的快速上调。这些因子的过度表达将被用来测试它们在推动hESC分化中的作用。利用ChIPseq，我们将测试TCF/LEF如何占据基因启动子，以特定细胞类型的方式控制基因。这项研究可以更好地从分子上表征多能性，并确定信号通路如何通过迅速改变转录因子的活性来影响命运决定。这些研究可以为调节早期人类发育的机制提供新的见解。

DESCRIPTION (provided by applicant): Summary Despite the great potential of human embryonic stem cells (hESCs) for advancing new treatments for human disease, there are important unknowns in understanding the stem cell state. Addressing these unknowns could improve the ability to establish pluripotent stem cells: how to maintain them and how to turn them into specific cells. The proposed research strategy will address questions about pluripotency through study of the Wnt pathway effectors, T cell factor and Lymphoid enhancer factors (TCF/LEFs). One member, TCF3, is a transcription factor that in mouse ESCs (mESCs) represses expression of master pluripotency factors Oct4, Sox2, and Nanog (OSN). Importantly, mouse Tcf3 limits pro-pluripotency feedforward expression of these genes during mESC differentiation. While studies with mESC have been useful and show how Tcf3 should be considered the fourth core stem cell regulatory factor, extrapolating the role to hESCs has inbuilt limitations due to key signaling differences between mESCs and hESCs. But TCF3 function in human ESCs is entirely unknown. Our preliminary data show that TCF3 is dynamically expressed in hESCs. By comparison with other core factors, TCF3 protein levels decline rapidly upon differentiation before other changes are seen and may therefore be a critical, first event. TCF3 expression is highly varied in hESC colonies indicating that hESCs may be poised to differentiate. Differentiation is accompanied by rapid upregulation of other TCF/LEFs suggesting switching of Wnt target gene promoter occupancy. The overarching hypothesis is that hESCs express TCF3 which inhibits Wnt signaling, maintains a "stem cell" chromatin structure and maintains Activin signaling (a key pathway regulating hESC pluripotency), to hold hESCs in a poised state; pluripotent but "ready to go". With differentiation signals TCF3 is replaced by activating TCF/LEFs that alter chromatin structure, driving gene expression and cellular differentiation. Specific Aim 1 will test the role of TCF3 in regulating Activin signaling. We will study control of Activin inhibitors, Follistatin and Inhibin E, identified as potential TCF3 target in preliminary studies. Specific Aim 2 will test the role of TCF3 in regulating the chromatin remodeling factor SMARCA2. These studies could define the relevance of TCF3 downregulation for hESC pluripotency and differentiation and place it in the larger hESC regulatory network. Relationships between TCF3 and master regulators (OSN) will also be defined using microarray and ChIP-Seq. Specific Aim 3 will test the role of TCF3 relatives in hESC differentiation. We find hESC differentiation is accompanied by rapid up-regulation of TCF3 relatives, (LEF-1/TCF-1/TCF-4). Overexpression of these factors will be used to test their role in driving hESC differentiation. Using ChIPseq we will test how TCF/LEFs occupy gene promoters to control genes in cell type- specific ways. This research could better characterize pluripotency molecularly and establish how signaling pathways can instigate fate decisions by rapidly altering transcription factor activity. These studies could provide new insights into mechanisms regulating early human development.