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

研究 Wnt 信号传导多状态模型的机制

• 批准号: 9900020
• 负责人: DAVID A BRAFMAN
• 金额: $ 29.63万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900020
• 关键词: Adult; Alleles; Anterior; Binding; Binding Proteins; Biological Models; Cell Line; Cell Nucleus; Cells; ChIP-seq; Chimeric Proteins; Complex; DNA; DNA Binding; Data; Development; Developmental Process; Disease; Engineering; Family; Functional disorder; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic Transcription; Homeostasis; Human Development; In Vitro; Individual; Knock-in; Knock-out; Lead; Ligands; Lymphoid; Maintenance; Malignant Neoplasms; Mediating; Mediator of activation protein; Modeling; Nerve Degeneration; Neural tube; Neurons; Nucleic Acid Regulatory Sequences; Pathologic; Pathway interactions; Pattern; Phenotype; Play; Protein Family; Proteins; Regulation; Research; Role; Series; Signal Transduction; Signaling Molecule; Specific qualifier value; System; TCF Transcription Factor; TCF7L2 gene; Testing; Tissues; Transcription Repressor; Transcriptional Regulation; Translating; WNT Signaling Pathway; base; beta catenin; developmental disease; genome-wide; glycogen synthase kinase 3 beta; human disease; human pluripotent stem cell; in vitro Model; in vivo; insight; knock-down; loss of function; neural patterning; neurodevelopment; novel; overexpression; prevent; protein complex; receptor; recruit; relating to nervous system; response; therapeutic development; transcription factor; transcriptome sequencing; tumorigenesis

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.

项目概要摘要 WNT信号通路调节许多发育过程，并在发育过程中发挥着关键作用。 此外，WNT 信号传导功能障碍也与维持成人健康组织和细胞有关。 典型的 WNT 信号传导在许多发育障碍、神经退行性变和肿瘤发生中发挥作用。 通常被描述为“二元”系统，即所谓的“双态”模型。在“关闭”状态下，蛋白质被破坏。 在 WNT 存在的情况下，复合物在“开启”状态下指导 β-catenin 的持续蛋白水解降解。 配体，该蛋白质复合物被分解，使 β-连环蛋白积累并转移到细胞核中， 然而，该模型并没有完全解释 WNT 信号传导的梯度是如何改变的。 许多组织的发育和模式化过程中存在的活动会导致精确的变化 此外，该模型没有充分解释WNT有何不同。 信号阈值导致癌症和其他病理状况的表现。 鉴于 WNT 信号在人类发育和疾病中复杂、多方面的作用，我们设计了一种 基于人多能干细胞 (hPSC) 的体外模型，模拟相同的早期体内发育效应 我们使用该系统研究了神经管前后 (A/P) 模式的 WNT 信号梯度。 将测试我们提出的模型和假设，即 WNT 活性的特定水平被转化为精确的 转录反应和细胞表型通过两种互补机制：（i）直接通过 与 A/P 神经管模式相关的基因的转录调控，以及 (ii) 间接通过 转录抑制子 SP5 在本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) 定义 β-连环蛋白调节 hPSC 衍生神经元 A/P 身份的转录机制 在第二个目标中，我们将结合使用一系列新型敲除和过表达 hPSC 系。 通过 ChIP-seq 分析研究单个 TCF/LEF 蛋白在调节区域特征中的作用 最后，在第三个目标中，我们将使用工程敲除和敲入 hPSC 系。 与 ChIP-seq 一起建立 SP5 作为 WNT 信号传导的中介者，指定 A/P 区域身份 总的来说，从这项研究中获得的新见解不仅会带来更多的结果。 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 信号在疾病发生和进展中的作用的理解。