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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要动态DNA和染色质修饰的各种细胞和疾病的动态DNA和染色质修饰的分子分析国家导致人类推定的非编码调节元素的全面目录基因组。扰动实验现在对于学习DNA和组蛋白的因果功能至关重要这些基因组元素的修改。但是，现有的操纵基因表达和染色质的工具状态遭受部分或瞬态影响，很大，因此很难传递，并且表现出很高的可变性跨基因座和细胞类型。这些工具目前是从数千天然中的一小部分中汲取的染色质调节复合物。一个更完整的紧凑，高效域的工具箱操纵广泛的染色质途径将改变我们确定因果关系的能力在人基因组中特定的染色质修饰并控制基因的功能表达。在这里，我们建议系统，全面地测量可以与人染色质接口 - 严重缺少的数据集。我们最近的第一个高通量蛋白的开发使它成为可能人类细胞中的域募集分析，能够测量成千上万的效应域的活性同时（Tycko等人，Biorxiv 2020）。使用此系统，我们将从启动子，等待，然后测量转录沉默或激活的幅度和永久性在记者基因座。我们将表征从人类和病毒染色质和基因中绘制的数千个域监管机构。此外，我们将创建正交纳米库库，以募集内源性染色质监管机构。然后，我们将测量这些域的功能代表各种染色质状态。最后，我们将使用遗传筛选和表观基因组映射测定确定支撑这些新型效应子功能的分子网络。因此，我们将创建并共享可以融合到的实验测量，紧凑，高效的域的详细资源 DNA结合蛋白是为了募集所需的染色质调节复合物以作用于基因组元素。同时，这项研究将为所有人类转录和染色质提供详细的功能特性监管领域，是理解疾病影响的未来工作的起点这些调节剂的编码序列中的遗传突变。此外，它将确定新颖的表观基因组和转录效应子域，可以赋予永久表观遗传记忆，并确定赋予一系列染色质状态的域目前无法使用可用的扰动工具访问。这些途径特异性扰动技术对于探测各种染色质状态的功能至关重要目前正在各种基因组基因组和细胞类型中发现和研究的调节元素。