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: 10276866
• 负责人: MICHAEL C BASSIK
• 金额: $ 99.24万
• 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/10276866
• 关键词: Binding; Biological; Biological Assay; CRISPR interference; Catalogs; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; 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; Lead; Learning; Libraries; Maintenance; Maps; Measures; Memory; Methyltransferase; Molecular; Molecular Profiling; Pathway interactions; Program Development; Property; Proteins; Regulator Genes; Regulatory Element; Reporter; Reporter Genes; Repression; Resources; System; Technology; Tertiary Protein Structure; Testing; Time; Time Study; Transcriptional Activation; Untranslated RNA; Viral; Viral Proteins; Work; Yeasts; cell type; chromatin modification; cofactor; epigenetic memory; epigenetic silencing; epigenome; epigenomics; experimental study; gene therapy; genome-wide; genomic locus; high throughput screening; histone modification; human disease; improved; interest; nanobodies; novel; programs; 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.

项目总结 跨不同细胞和疾病的动态DNA和染色质修饰的全基因组分子图谱 各国已经制定了人类体内假定的非编码调控元件的全面目录 基因组。现在，微扰实验对于了解DNA和组蛋白的因果功能至关重要 这些基因组元素的修饰。然而，现有的操纵基因表达和染色质的工具 国家遭受部分或瞬时影响，规模较大，因此难以交付，并表现出高度的变异性 不同的基因座和细胞类型。这些工具目前是从数以千计的天然工具中提取的一小部分 染色质调节复合体。更完整的紧凑、高效域工具箱，能够 操纵广泛的染色质通路将改变我们确定原因的能力 人类基因组中特定染色质修饰的功能及其对控制基因的作用 表情。 在这里，我们建议系统和全面地衡量基因表达的影响 招募可与人染色质结合的染色质调节因子和转录因子蛋白结构域 -一个关键的缺失数据集。这是因为我们最近开发出了第一个高通量蛋白质 在人类细胞中的结构域招募试验，能够测量数万个效应结构域的活性 同时(Tycko等人，BioRxiv 2020)。使用这个系统，我们将招募和释放效应域 启动子，等待，然后测量转录沉默或激活的幅度和持久性 在一家记者中心。我们将描述从人类和病毒染色质和基因中提取的数千个结构域 监管者。此外，我们将创建正交纳米体库，以招募内源染色质 监管者。然后，我们将在选择的一组内源基因组座位上测量这些结构域的功能 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图谱分析来 确定支撑这些新型效应器功能的分子网络。因此，我们将创造 并共享经过实验测量的、紧凑的、高效的域的详细资源，这些域可以融合到 DNA结合蛋白，以招募所需的染色质调节复合体作用于基因组元件。 同时，这项研究将提供所有人类转录和染色质的详细功能特性 监管领域，作为今后了解以下疾病影响的工作的起点 这些调节子的编码序列发生了基因突变。此外，它还将鉴定新的表观基因组和 转录效应结构域，可以传递永久的表观遗传记忆，并识别 结构域给予一系列染色质状态，目前用现有的扰动工具无法获得。这些 途径特异性扰动技术对于探索不同染色质状态的功能将是至关重要的。 目前在不同的基因组座位和细胞类型中发现和研究的调控元件。