Single cell, genome wide dissection of dynamic transcription factor regulation

单细胞、全基因组动态转录因子调控的剖析

• 批准号: 10665592
• 负责人: Leandra Caywood
• 金额: $ 3.47万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-11 至 2025-07-10
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10665592
• 关键词: Address; Affect; Autoimmune Diseases; Biomedical Engineering; Bypass; Cell Line; Cells; Chemical Stimulation; Cues; Development; Disease; Disease Progression; Dissection; Ensure; Environment; Eukaryotic Cell; Exposure to; Fibrinogen; Fluorescence; Fluorescence Microscopy; Foundations; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Hela Cells; Heterogeneity; Human; Immune System Diseases; Immune response; Individual; Inflammation; Knock-out; Knowledge; Lead; Light; Malignant Neoplasms; Maps; Masks; Measurable; Methods; Modeling; Modernization; NF-Kappa B p65; NF-kappa B; Nature; Nuclear; Nuclear Translocation; Pathway interactions; Pattern; Perception; Phenotype; Polymerase Chain Reaction; Population Heterogeneity; Proliferating; RELA gene; Regulation; Research; Reverse Transcription; Signal Transduction; Stimulus; Stretching; System; TNF gene; Techniques; Technology; Therapeutic; Transcriptional Regulation; Tumor Suppression; Validation; Work; biological systems; cellular engineering; differential expression; environmental stressor; experience; extracellular; genome-wide; interest; optogenetics; p65; phenomenological models; programs; promoter; response; single-cell RNA sequencing; synthetic biology; tool; transcription factor; transcriptome; transcriptomics; transmission process; whole genome

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.

项目总结 尽管信号组件和基因数量有限，但细胞必须能够明显地 响应大量的输入信号，如环境胁迫，以及执行各种基因 表情程序。细胞准确地接收、传输、过滤和作用于这些信号以触发是至关重要的 适当的下游基因反应程序，如失调和异常信号具有重要的 在免疫紊乱和癌症等疾病的发生或发展过程中的影响。 具体地说，单个转录因子(Tf)可以通过改变其转录因子(Tf)来响应各种各样的输入信号 激活特定启动子的核定位动力学。然而，转录动力学的研究是 由于技术障碍，如细胞间的可变性、多效性等，限制在很大程度上相关关系 实验扰动、掩饰异质性的表达平均值和基于荧光的影响 限制可测量的目标基因数量的技术。这项工作的中心假设是 转铁蛋白动力学与其下游基因表达调控的功能联系 程序可以通过综合使用精心刻画和控制的 光遗传系统与单细胞RNA测序(ScRNAseq)，增加了我们对 用于工程和生物医学应用的人类细胞的基因调控。核因子-κB(p65/rela)，它 调节数百个基因，并与免疫反应和癌症密切相关，将作为 我们系统的TF型。 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 我们实验室开发的工具将基于光遗传的转铁蛋白易位与scRNAseq相结合。我们将建立一个 强健、严密控制的系统，并检查核因子-κB的直接扰动在全基因组范围内的影响 成千上万的细胞。将以三个主要目标为目标：目标1将导致制定一个 可控的光遗传系统通过着陆台整合精确调控HeLa细胞中的p65转位， 定位动力学的优化及逆转录定量聚合酶链法验证 已知靶基因的反应。目标2将侧重于全基因组基因转录图谱的测定 以及响应于传统使用的、化学刺激的p65动力学的细胞间异质性 ScRNAseq.通过将这两个组件结合在一起，Aim 3将直接控制15个不同的P65动态 光遗传输入，并使用scRNAseq评估由此产生的单细胞转录变化。总括而言，我们的目标是 提供一个高效、功能强大的系统，以提供TF动态和 人类细胞中的基因表达与完全控制在细胞工程中的精确控制 通过光遗传学，哺乳动物合成生物学中的精密工具，以及广泛的转录组， 单细胞基因表达谱由scRNAseq提供。