High-throughput development and characterization of compact tools for transcriptional and chromatin perturbations

用于转录和染色质扰动的紧凑工具的高通量开发和表征

• 批准号: 10632140
• 负责人: MICHAEL C BASSIK
• 金额: $ 99.26万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-09 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10632140
• 关键词: Binding; Biological; Biological Assay; CRISPR interference; CRISPR-mediated transcriptional activation; Catalogs; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA; DNA Binding Domain; DNA Maintenance; DNA Modification Process; DNA Sequence Alteration; DNA-Binding Proteins; Data Set; Development; Disease; Elements; Engineering; Enhancers; Epigenetic Process; Exhibits; Future; Gene Activation; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome; Genomics; Human; Human Genome; Knock-out; Label; Learning; Libraries; Maintenance; Maps; Measures; Memory; Methyltransferase; Molecular; Molecular Profiling; Pathway interactions; Property; Proteins; Regulator Genes; Regulatory Element; Reporter; Reporter Genes; Repressed Memory; Resources; System; Technology; Tertiary Protein Structure; Testing; Time; Transcriptional Activation; Untranslated RNA; Viral; Viral Proteins; Work; Yeasts; cell type; chromatin modification; cofactor; epigenetic memory; epigenetic silencing; epigenome; epigenomics; experimental study; gene therapy; genetic manipulation; genome-wide; genomic locus; high throughput screening; histone modification; human disease; improved; interest; nanobodies; novel; promoter; recruit; screening; synthetic protein; tool; transcription factor

PROJECT SUMMARY Genome-wide molecular profiling of dynamic DNA and chromatin modifications across diverse cell and disease states have resulted in comprehensive catalogs of putative non-coding regulatory elements in the human genome. Perturbation experiments are now critical to learn the causal functions of the DNA & histone modifications at these genomic elements. However, existing tools to manipulate gene expression and chromatin state suffer from partial or transient effects, are large and thus difficult to deliver, and exhibit high variability across loci and cell types. These tools are currently drawn from a tiny fraction of the thousands of natural chromatin regulatory complexes. A more complete toolbox of compact, efficient domains capable of manipulating a broad range of chromatin pathways will transform our ability to determine the causal function of particular chromatin modifications across the human genome and to control gene expression. Here, we propose to systematically and comprehensively measure the gene expression effects of recruiting chromatin regulators and transcription factor protein domains that can interface with human chromatin - a critical missing dataset. This is made possible by our recent development of the first high-throughput protein domain recruitment assay in human cells, capable of measuring activity for tens of thousands of effector domains simultaneously (Tycko et al, bioRxiv 2020). Using this system, we will recruit-and-release effector domains from the promoter, wait, and then measure the magnitude and permanence of transcriptional silencing or activation at a reporter locus. We will characterize thousands of domains drawn from human and viral chromatin and gene regulators. In addition, we will create orthogonal nanobody libraries selected to recruit endogenous chromatin regulators. We will then measure the function of these domains at a panel of endogenous genomic loci chosen to represent diverse chromatin states. Finally, we will use genetic screens and epigenomic mapping assays to determine the molecular networks that underpin the functions of these novel effectors. Therefore, we will create and share a detailed resource of experimentally-measured, compact, efficient domains that can be fused onto DNA-binding proteins in order to recruit desired chromatin regulatory complexes to act upon a genomic element. At the same time, this study will provide detailed functional properties for all human transcriptional and chromatin regulatory domains, serving as a starting point for future work on understanding the disease implications of genetic mutations in the coding sequence of these regulators. Further, it will identify novel epigenome and transcriptional effector domains that can impart permanent epigenetic memory, and identify combinations of domains to impart a range of chromatin states not currently accessible with available perturbation tools. These pathway-specific perturbation technologies will be critical to probe the function of the varied chromatin states at regulatory elements that are currently being discovered and studied across diverse genomic loci and cell types.

项目摘要 不同细胞和疾病中动态DNA和染色质修饰的全基因组分子谱分析 美国已经产生了人类中假定的非编码调节元件的全面目录， 基因组微扰实验现在对于了解DNA和组蛋白的因果功能至关重要 这些基因组元件的修饰。然而，现有的操纵基因表达和染色质的工具， 状态受到部分或瞬时效应影响，很大，因此难以传递，且表现出高可变性 跨基因座和细胞类型。这些工具目前是从成千上万的自然资源中提取的一小部分。 染色质调节复合物。一个更完整的紧凑，高效的域工具箱，能够 操纵广泛的染色质途径将改变我们确定因果关系的能力， 人类基因组中特定染色质修饰的功能以及控制基因 表情 在这里，我们建议系统和全面地测量基因表达的影响， 募集可与人类染色质相互作用的染色质调节因子和转录因子蛋白结构域 - 一个重要的缺失数据集这是我们最近开发的第一个高通量蛋白质 人细胞中的结构域募集测定，能够测量数万个效应结构域的活性 同时（Tycko et al，bioRxiv 2020）。使用这个系统，我们将从 启动子，等待，然后测量转录沉默或激活的程度和持久性 在一个报告者所在地。我们将从人类和病毒的染色质和基因中提取数千个结构域的特征， 监管部门此外，我们还将创建正交纳米抗体库， 监管部门然后，我们将测量这些结构域的功能，在一组内源性基因组位点选择 来代表不同的染色质状态。最后，我们将使用遗传筛选和表观基因组作图分析， 确定支撑这些新型效应器功能的分子网络。因此，我们将创建 并共享实验测量的、紧凑的、高效的域的详细资源， DNA结合蛋白，以募集所需的染色质调节复合物作用于基因组元件。 同时，本研究将提供所有人类转录和染色质的详细功能特性 调控领域，作为未来工作的起点，了解疾病的影响， 这些调节因子编码序列中的基因突变。此外，它将确定新的表观基因组， 转录效应结构域，可以赋予永久的表观遗传记忆，并确定组合 结构域赋予一系列的染色质状态目前无法获得可用的扰动工具。这些 途径特异性微扰技术将是至关重要的，以探测不同染色质状态的功能， 目前正在不同基因组位点和细胞类型中发现和研究的调控元件。