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

项目摘要 WNT信号通路调节许多发育过程，并在发育过程中起关键作用。 维持成年人的健康组织和细胞。此外，WNT信号传导的功能障碍也与 在许多发育障碍、神经变性和肿瘤发生中。典型的WNT信号是 通常被描述为“二元”系统，即所谓的“双态”模型。在“关闭”状态下， 复合物指导β-连环蛋白的持续蛋白水解降解。在“开”状态下，在存在WNT的情况下， 配体时，这种蛋白质复合物被分解，使β-连环蛋白积聚并易位到细胞核中， 从而改变基因转录。然而，这个模型并不能完全解释WNT信号的梯度是如何 在许多组织的发育和形成过程中存在的活动导致了 转录反应和细胞身份。此外，该模型并不能充分解释WNT 信号传导阈值导致癌症和其它病理状况的表现。更好地了解 WNT信号在人类发育和疾病中的复杂，多方面的作用，我们已经设计了一个在 基于体外人多能干细胞（hPSC）的模型，模拟相同的早期体内发育效应 WNT信号梯度对神经管前后（A/P）模式的影响。使用该系统，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的机制：（i）直接通过 与A/P神经管模式相关的基因的转录调节，以及（ii）间接通过以下作用 转录抑制因子SP 5。在本研究的第一个目的中，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）， 定义β-连环蛋白调节hPSC衍生的神经细胞A/P身份的转录机制， 细胞在第二个目标中，我们将利用一系列新的敲低和过表达的hPSC系， 用ChIP-seq分析来研究单个TCF/LEF蛋白在调节TCF/LEF区域特性中的作用。 hPSC衍生的神经细胞。最后，在第三个目标中，我们将使用工程化的敲除和敲入hPSC系， 沿着ChIP-seq以建立SP 5作为WNT信号传导的介体，以指定A/P区域身份。 hPSC衍生的神经细胞。总的来说，从这项研究中获得的新见解不仅会带来更多的 深入了解WNT信号传导如何调节早期神经发育， 对我们理解WNT信号在疾病发生和发展中的作用产生了影响。

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.