Investigating the mechanisms of a multi-state model of Wnt signaling

项目总结摘要 WNT信号通路调节多种发育过程，并在 维护成人的健康组织和细胞。此外，WNT信号转导功能障碍也牵涉其中。 在许多发育障碍、神经退行性变和肿瘤形成中。规范的WNT信令是 通常被描述为“二进制”系统，即所谓的“两状态”模型。在关闭状态下，蛋白质被破坏 复合体指导β-连环蛋白的持续蛋白降解。在WNT存在的情况下，处于‘开’状态 配体，这种蛋白质复合体被分解，使β-连环蛋白积累并转移到细胞核中， 从而改变基因转录。然而，该模型并不能完全解释WNT信号的梯度 在许多组织的发育和图案形成过程中存在的活动会导致 转录反应和细胞特性。此外，该模型不能充分解释不同的WNT 信号阈值会导致癌症和其他病理情况的出现。为了更好地理解 WNT信号在人类发育和疾病中的复杂、多方面的作用，我们设计了一种 基于人类多能干细胞(HPSC)的体外模型，模拟体内早期发育效应 在神经管的前后(A/P)模式上的WNT信号梯度。使用这个系统，我们 将测试我们提出的模型和假设，即特定水平的WNT活动被转化为精确的 转录反应和细胞表型通过两种互补机制：(I)直接通过 A/P神经管构型相关基因的转录调控和(Ii)通过 转录抑制因子SP5。在拟议研究的第一个目标中，我们将使用单细胞基因 表达分析、全基因组表达分析(RNA-SEQ)和DNA结合分析(CHIP-SEQ) 确定β-连环蛋白调节hPSC来源神经的A/P特性的转录机制 细胞。在第二个目标中，我们将利用一系列新的基因敲除和过表达hPSC株系相结合 用CHIP-SEQ分析研究单个TCF/Lef蛋白在调节人类免疫缺陷病毒区域同一性中的作用 HPSC来源的神经细胞。最后，在第三个目标中，我们将使用工程敲除和敲击hPSC生产线 与CHIP-SEQ一起建立SP5作为WNT信令的中介者，以指定A/P区域标识 HPSC来源的神经细胞。总体而言，从这项研究中获得的新见解不仅将导致更多 彻底了解WNT信号如何调节早期神经发育，也将具有重要意义 影响我们对WNT信号在疾病发生和发展中的作用的理解。

PROJECT SUMMARY ABSTRACT The WNT signaling pathway regulates numerous developmental processes and plays a critical role in the maintenance of healthy tissue and cells in adults. Moreover, dysfunction in WNT signaling has been implicated in numerous developmental disorders, neurodegeneration, and tumorigenesis. Canonical WNT signaling is typically described as a ‘binary’ system, the so-called ‘two-state’ model. In the ‘off’ state, a protein destruction complex directs the continual proteolytic degradation of β-catenin. In the ‘on’ state, in the presence of a WNT ligand, this protein complex is disassembled, allowing β-catenin to accumulate and translocate into the nucleus, thereby altering gene transcription. However, this model does not fully explain how gradients of WNT signaling activity that are present during the development and patterning of many tissues lead to precise changes in transcriptional response and cell identity. In addition, this model does not adequately explain how different WNT signaling thresholds lead to the manifestation of cancer and other pathological conditions. To better understand the complex, multifaceted role of WNT signaling in human development and disease, we have engineered an in vitro human pluripotent stem cell (hPSC)-based model that mimics the same early in vivo developmental effects of the WNT signaling gradient on the anterior-posterior (A/P) patterning of the neural tube. Using this system we will test our proposed model and hypothesis that specific levels of WNT activity are translated into precise transcriptional responses and cell phenotypes through two complementary mechanisms: (i) directly through the transcriptional regulation of genes related to A/P neural tube patterning and (ii) indirectly through the actions of the transcriptional repressor SP5. In the first aim of the proposed research, we will use single cell gene expression analysis, genome-wide expression analysis (RNA-seq), and DNA binding analysis (ChIP-seq) to define the transcriptional mechanisms by which β-catenin regulates the A/P identity of hPSC-derived neural cells. In the second aim, we will utilize a series of novel knockdown and overexpression hPSC lines in conjunction with ChIP-seq analysis to investigate the role of individual TCF/LEF proteins in regulating the regional identity of hPSC-derived neural cells. Finally, in the third aim, we will use engineered knockout and knockin hPSC lines along with ChIP-seq to establish SP5 as a mediator of WNT signaling in specifying the A/P regional identity of hPSC-derived neural cells. Overall, the new insights gained from this research will not only lead to a more thorough understanding of how WNT signaling regulates early neurodevelopment but also will have significant impact on our understanding of the role of WNT signaling in disease initiation and progression.