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

项目总结/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命运和功能。 目前的光学显微镜技术现在能够直接观察活细胞中的转录因子， 不能调节转录因子的活性，这是描述基因型的贡献所必需的。 调节和表型反应。新兴的非神经元光遗传学提供了一种新的策略， 调节基因转录，或者通过将转录激活结构域募集到特定启动子，或者通过 光解开一个被隔离的转录因子。与调节数百种转录因子的天然转录因子不同 和数千个靶基因，目前的光遗传学策略仅适用于单个或少数基因靶点， 在黑暗中可能会受到高基础活动的影响。用更大的基因文库控制多重基因转录 因此，转录因子，呼吁一种替代策略，使新的光学控制模式， 基因转录该项目的目标是通过制定一项基于受控的 拯救蛋白质降解。在这种策略中，基础水平的蛋白质活性被恒定蛋白质抑制 降解直到光照引发蛋白质的爆发。该策略不依赖于激活 研究目的蛋白质的作用机制，并将显著增强非神经元光遗传学的能力。 在这个项目中，我们提出了一个计划，在四年的预算期内开发和验证控制本地 转录因子我们将证明蓝光控制的T细胞因子（TCF）下游的孔- 建立了Wnt信号通路（Aim 1），并建立了一个调控Notch的正交光遗传学系统 细胞内结构域（NICD）介导的转录与红光（目的2）。利用我们最近开发的空间 光调制器，我们将在空间和时间上实现精确的多路复用转录控制， 实现在单细胞水平上控制天然转录因子（目标3）。我们最近在 为哺乳动物细胞和非洲爪蟾胚胎开发光遗传学工具， 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时空调控。

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.