Precise regulation of native transcription factor at the single-cell level

在单细胞水平上精确调控天然转录因子

• 批准号: 10379570
• 负责人: Kai Zhang
• 金额: $ 9.97万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10379570
• 关键词: 15 year old; Animals; Automobile Driving; Biochemistry and Cellular Biology; Budgets; Calibration; Cells; Color; Devices; Embryo; Embryonic Development; Equipment; Face; Fluorescence; Funding; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genotype; Goals; Grant; Hemorrhage; Human Resources; Image; Letters; Libraries; Light; Luciferases; Mammalian Cell; Mediating; Microscopy; Modality; Molecular; Molecular and Cellular Biology; National Institute of General Medical Sciences; Optics; Phenotype; Positioning Attribute; Production; Proteins; Reader; Regulation; Request for Applications; Research; Research Personnel; Research Project Grants; Schools; Signal Transduction; Students; System; TCF Transcription Factor; Techniques; Technology; Time; Transcriptional Activation Domain; Transcriptional Regulation; Uncertainty; Visualization; WNT Signaling Pathway; Work; Xenopus; base; beta catenin; cell type; cellular imaging; cost; design; experimental study; imaging system; innovation; instrument; interest; notch protein; optogenetics; pandemic disease; parent grant; programs; promoter; protein degradation; recruit; response; spatiotemporal; success; tool; transcription factor

Project Summary/Abstract Transcription factors drive dynamic, cell-type specific, gene expression to define cell fate and functionality. Current optical microscopy technologies now enable direct visualization of transcription factors in live cells but cannot modulate transcription factor activity, which is required for delineating the contribution of genotypic modulation and phenotypic response. The emerging non-neuronal optogenetics provides a new strategy to regulate gene transcription, either by recruiting a transcription activation domain to a specific promoter or by photo-uncaging a sequestered transcription factor. Unlike native transcription factors, which regulates hundreds and thousands of target genes, the current optogenetic strategy only works for single- or a few gene targets and could suffer from high basal activity in the dark. Controlling multiplexed gene transcription with a larger library of transcription factors, thus, calls for an alternative strategy that empowers new modalities of optical control of gene transcription. The goal of this project is to fill this gap by developing a strategy based on the controlled rescue of protein degradation. In this strategy, base-level protein activities are suppressed by constant protein degradation until light triggers a burst of protein production. This strategy does not depend on the activation mechanism of the protein of interests and will significantly enhance the capacity of non-neuronal optogenetics. In this project, we present a plan within a four-year budget period to develop and validate the control native transcription factors. We will demonstrate blue-light-controlled T cell factor (TCF) downstream of the well- established Wnt signaling pathway (Aim 1) and develop an orthogonal optogenetic system to regulate the Notch intracellular domain (NICD)-mediate transcription with red light (Aim 2). Using our recently developed spatial light modulator, we will achieve precise multiplexing transcription control in space and time and ultimately achieve controlling the native transcription factors at the single-cell level (Aim 3). Our recent success in developing optogenetic tools for mammalian cells and Xenopus embryos well positions the applicant to carry out the proposed project. Results of this project will provide valuable assets to researchers who are interested in dissecting the spatial and temporal regulation of signal transduction during early embryonic development.

项目概要/摘要 转录因子驱动动态的、细胞类型特异性的基因表达，以定义细胞的命运和功能。 当前的光学显微镜技术现在可以直接可视化活细胞中的转录因子，但 不能调节转录因子活性，这是描述基因型贡献所必需的 调制和表型反应。新兴的非神经元光遗传学提供了一种新策略 通过将转录激活结构域招募到特定启动子或通过 光解封隔离的转录因子。与调节数百个的天然转录因子不同 和数千个目标基因，目前的光遗传学策略仅适用于单个或少数基因目标， 在黑暗中可能会遭受高基础活动的影响。用更大的文库控制多重基因转录 因此，转录因子需要一种替代策略，以实现光学控制的新模式 基因转录。该项目的目标是通过制定基于受控的策略来填补这一空白。 挽救蛋白质降解。在该策略中，基础水平的蛋白质活性被恒定的蛋白质抑制 降解直到光触发蛋白质的大量产生。该策略不依赖于激活 感兴趣的蛋白质的机制将显着增强非神经元光遗传学的能力。 在这个项目中，我们提出了一个在四年预算期内开发和验证本机控制的计划 转录因子。我们将展示蓝光控制的 T 细胞因子 (TCF) 下游 建立Wnt信号通路（目标1）并开发正交光遗传学系统来调节Notch 胞内结构域 (NICD) - 用红光介导转录（目标 2）。使用我们最近开发的空间 光调制器，我们将实现空间和时间上的精确多重转录控制，最终 实现在单细胞水平上控制天然转录因子（目标 3）。我们最近的成功 为哺乳动物细胞和非洲爪蟾胚胎开发光遗传学工具，使申请人能够很好地开展 拟议的项目。该项目的结果将为有兴趣的研究人员提供宝贵的资产 剖析早期胚胎发育过程中信号转导的空间和时间调节。