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

基本信息

批准号：
10538121
负责人：
Leandra Caywood
金额：
$ 3.38万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
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/10538121
关键词：
Address Affect Autoimmune Diseases Biomedical Engineering Bypass Cell Fate Control Cell Line Cells Chemicals 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 RELA gene Regulation Research Reverse Transcription Signal Transduction Stimulus Stretching Sum System TNF gene Techniques Technology Therapeutic Transcriptional Regulation Tumor Suppression Validation Work base biological systems cellular engineering differential expression environmental stressor experience extracellular genome-wide interest optogenetics p65 programs promoter response single-cell RNA sequencing synthetic biology tool transcription factor transcriptome transcriptomics transmission process whole genome

项目摘要

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.

项目概要尽管信号成分和基因数量有限，但细胞必须能够清楚地对大量输入信号（例如环境压力）做出反应，并执行不同的基因细胞准确地接收、传输、过滤和作用这些信号以触发表达程序至关重要。适当的下游基因反应程序，因为失调和异常信号传导具有重要意义对免疫紊乱和癌症等疾病的发生或进展的影响。具体来说，单个转录因子（TF）可以通过改变其转录因子来响应多种输入信号。核定位动力学来激活特定的启动子然而，转录动力学的研究是。由于技术障碍，例如细胞间变异性、多效性，仅限于很大程度上相关的关系实验扰动、掩盖异质性的表达平均值以及基于荧光的影响限制可测量目标基因数量的技术。这项工作的中心假设是 TF 动态及其下游基因表达调控之间的功能联系可以通过综合使用仔细表征的程序来识别和调整程序具有单细胞 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。调节数百个基因，与免疫反应和癌症密切相关，将作为我们系统的型号 TF。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基础上我们实验室开发的将基于光遗传学的 TF 易位与 scRNAseq 相结合的工具。强大、严格控制的系统，并检查 NF-κB 直接扰动对全基因组的影响将实现三个主要目标：目标 1 将导致开发一个完全的细胞。可控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，定位动力学优化，并通过逆转录定量聚合酶链进行验证目标 2 将侧重于确定全基因组基因转录组谱。以及细胞间异质性响应传统使用的化学刺激 p65 动力学 scRNAseq 通过结合这两个组件，Aim 3 将直接控制具有 15 个不同的 p65 动力学。光遗传学输入并使用 scRNAseq 评估由此产生的单细胞转录组变化总而言之，我们的目标是提供一个高效的、功能性的关系系统，以在 TF 动态和人体细胞中的基因表达以及通过利用完全控制来精确控制细胞工程通过光遗传学、哺乳动物合成生物学中的精确工具和转录组范围进行 TF 易位， scRNAseq 提供的单细胞基因表达谱。