Single cell, genome wide dissection of dynamic transcription factor regulation

项目摘要 尽管有有限的信号成分和基因数量，细胞必须能够清楚地表达 响应大量的输入信号，如环境压力，以及执行不同的基因， 表达程序。至关重要的是，细胞接收，传输，过滤，并根据这些信号准确地触发 适当下游基因应答程序，如失调和异常信号传导， 在诸如免疫紊乱和癌症的疾病的开始或进展中的暗示。 具体地，单个转录因子（TF）可以通过改变其转录因子的表达来响应各种各样的输入信号。 核定位动力学以激活特异性启动子。然而，转录动力学的研究是 由于技术障碍，如细胞间变异性、多效性、 实验扰动的影响，掩盖异质性的表达平均值，以及基于荧光的 限制可测量的靶基因数量的技术。这项工作的中心假设是， TF动力学及其下游基因表达调控之间的功能联系 程序可以通过综合使用精心表征和控制的 单细胞RNA测序（scRNAseq）的光遗传学系统，增加了我们对 人类细胞中的基因调控，用于工程和生物医学应用。NF-κB（p65/RELA）， 调节数百个基因，并与免疫反应和癌症密切相关，将成为 我们的TF系统。 为了应对这一挑战和信息差距，拟议的工作将建立在一套复杂的 我们实验室开发的联合收割机将基于光遗传学的TF易位与scRNAseq相结合的工具。我们将建立一个 强大的，严格控制的系统，并检查直接扰动NF-κB的基因组范围的影响， 成千上万的细胞。将有三个主要目标：目标1将导致制定一个全面的 可控的光遗传学系统，通过着陆垫整合精确调节HeLa细胞中的p65易位， 优化定位动力学，并通过逆转录定量聚合酶链进行验证 已知靶基因的反应。目标2将侧重于确定全基因组基因转录组图谱 和细胞与细胞的异质性，响应于传统使用的化学刺激的p65动力学， scRNAseq.通过结合这两个组件，Aim 3将直接控制p65动力学， 光遗传学输入并使用scRNAseq评估所得的单细胞转录组学变化。总之，我们的目标是 提供一个有效的功能系统，以提供TF动态之间的直接因果关系， 人类细胞中的基因表达以及通过利用完全控制进行细胞工程的精确控制 通过光遗传学，哺乳动物合成生物学中的精确工具，以及转录组范围内的TF易位， scRNAseq提供的单细胞基因表达谱。

PROJECT SUMMARY Despite having limited sets of signaling components and number of genes, cells must be able to distinctly respond to a large number of input signals, such as environmental stresses, as well as execute diverse gene expression programs. It is vital that cells receive, transmit, filter, and act upon these signals accurately to trigger the appropriate downstream gene response program, as dysregulation and aberrant signaling have important implications in the initiation or progression of diseases such as immune disorders and cancer. Specifically, a single transcription factor (TF) can respond to a wide variety of input signals by changing its nuclear localization dynamics to activate specific promoters. However, the study of transcriptional dynamics is limited to largely correlational relationships due to technical barriers such as cell-to-cell variability, pleotropic effects of experimental perturbations, expression averages that mask heterogeneity, and fluorescence based techniques that limit the number of measurable target genes. The central hypothesis of this work is that the functional connections between TF dynamics and their downstream regulation of gene expression programs can be identified and tuned by the integrated use of carefully characterized and controlled optogenetic systems with single cell RNA sequencing (scRNAseq), increasing our understanding of gene regulation in human cells for engineering and biomedical applications. NF-κB (p65/RELA), which regulates hundreds of genes and is heavily implicated in immunological responses and cancer, will serve as the model TF for our system. To address this challenge and information gap, the proposed work will build upon a suite of sophisticated tools developed in our lab to combine optogenetic based TF translocation with scRNAseq. We will establish a robust, tightly controlled system and examine the genome wide effect of direct perturbations of NF-κB across thousands of cells. Three main objectives will be targeted: Aim 1 will result in the development of a fully controllable, optogenetic system to precisely regulate p65 translocation in HeLa cells by landing pad integration, optimization of localization dynamics, and validation by reverse transcription quantitative polymerase chain reaction of known target genes. Aim 2 will focus on determination of whole-genome gene transcriptomic profiles and cell-to-cell heterogeneity in response to traditionally used, chemically stimulated p65 dynamics using scRNAseq. By combining these two components, Aim 3 will directly control p65 dynamics with 15 distinct optogenetic inputs and assess resultant single cell transcriptomic changes using scRNAseq. In sum, we aim to provide an efficient, functional system to provide direct, causal relationships between TF dynamics and gene expression in human cells and exact control in cellular engineering by leveraging complete control of TF translocation through optogenetics, precise tools in mammalian synthetic biology, and transcriptome wide, single cell gene expression profiles provided by scRNAseq.