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.

项目摘要摘要 Wnt信号通路调节了许多发展过程，并在 维持健康组织和成人细胞。此外，已经实现了WNT信号的功能障碍 规范的Wnt信号传导有许多发育障碍，神经退行性和肿瘤发生。 通常被描述为“二进制”系统，即所谓的“两态”模型。在“ OFF”状态下，蛋白质破坏 复合物指导β-catenin的连续蛋白水解降解。在“ on”状态下，在有wnt的情况下 配体，该蛋白质复合物被拆卸，使β-catenin可以积聚并转移到细胞核中， 从而改变基因转录。但是，该模型无法完全解释Wnt信号的梯度如何 在许多组织的开发和模式期间存在的活动导致精确变化 转录响应和细胞身份。此外，该模型无法充分说明不同的Wnt 信号传导阈值导致癌症和其他病理状况的表现。更好地理解 Wnt信号在人类发展和疾病中的复杂，多方面的作用，我们设计了 体外人类多能干细胞（HPSC）基于体内发育效果的同一早期模型 神经管的前后（a/p）上的Wnt信号传导梯度的梯度。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活性被转化为精度 通过两种完整机制的转录反应和细胞表型：（i）直接通过 转录调节与A/P神经管形成型和（ii）的基因的转录调节，并通过（ii）间接地通过 转录复制品SP5。在拟议的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）与 定义β-catenin调节HPSC衍生神经元的A/P同一性的转录机制 细胞。在第二个目标中，我们将使用一系列新颖的敲低和过表达HPSC线结合使用 通过CHIP-seq分析来研究单个TCF/LEF蛋白在确定区域身份的作用 HPSC衍生的神经细胞。最后，在第三个目标中，我们将使用工程的淘汰赛和敲除HPSC线条 与chip-seq一起建立SP5作为Wnt信号的中介，以指定A/P区域身份 HPSC衍生的神经细胞。总体而言，这项研究获得的新见解不仅会导致更多 透彻了解Wnt信号如何调节早期神经发育，但也将具有重要的 影响我们对Wnt信号在疾病倡议和进展中的作用的理解。