A high-throughput, comprehensive, and quantitative approach for measuring nucleosome-protein binding

一种用于测量核小体-蛋白质结合的高通量、全面、定量的方法

• 批准号: 10021679
• 负责人: Michael Joseph Buck
• 金额: $ 35.42万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-23 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10021679
• 关键词: Address; Affinity; Apoptosis; Atlases; Behavior; Binding; Binding Proteins; Binding Sites; Biological; Biological Assay; Biological Process; Cell Nucleus; Cell division; Cell physiology; Cells; ChIP-seq; Characteristics; Chromatin; Chromosome Segregation; Consumption; DNA; DNA Binding; DNA Modification Process; DNA Repair; DNA Sequence; DNA biosynthesis; DNA-Binding Proteins; Dependence; Development; Developmental Process; Disease; Distal; Elements; Embryonic Development; Environment; Epigenetic Process; Family; Gene Activation; Genetic Recombination; Goals; Histone H3; Histone H4; Histones; Human; In Vitro; Kinetics; Knowledge; Libraries; Measures; Methodology; Methods; Mission; Modification; Molecular Biology; Nature; Nucleic Acid Regulatory Sequences; Nucleosomes; Organism; PF4 Gene; Positioning Attribute; Post-Translational Protein Processing; Proteins; Public Health; Publications; Reaction; Research; Research Personnel; Role; Rotation; Silicon Dioxide; Site; Squamous cell carcinoma; Stress; TP53 gene; Tail; Tertiary Protein Structure; Testing; Time; Transcriptional Activation; Transcriptional Regulation; United States National Institutes of Health; Variant; cell growth regulation; cell type; cofactor; design; developmental disease; exhaustion; experimental study; genetic regulatory protein; histone modification; in vitro Assay; keratinocyte; next generation sequencing; particle; response; transcription factor

DNA binding to chromosomal DNA is essential for many fundamental biological processes including: transcriptional regulation, DNA replication and repair, recombination, and chromosome segregation. There is a fundamental gap in understanding how transcription factors (TF) bind regulatory regions located in compacted, high-order chromatin. Therefore, there is a fundamental need to determine the mechanistic rules defining TF binding to chromatin. The long-term goal for this project is to define the biological rules dictating TF binding to chromosomal DNA these rules include transcription factor binding site orientation within a nucleosome, DNA and histone modifications, cofactor and cooperative binding, and binding to subnucleosome particles. The overall objective of this application is to develop a high throughput approach to measure the principles of transcription factor binding to nucleosomal DNA. To accomplish this objective a new high-throughput next-generation sequencing assay will be developed to allow the simultaneous and quantitative examination of thousands of different nucleosomes in a single assay. The goal of this application will be accomplished by three specific aims: 1) Formation of a nucleosome library, 2) High-throughput protein-nucleosome binding assays with a library of nucleosomes, and 3) Define TF-nucleosome binding after histone modifications. Under the first aim, in vitro nucleosomes will be generated from thousands of in silica designed and naturally occurring DNA sequences in a single reaction, allowing a TF binding site to occur in all possible nucleosomal orientations with various neighboring sequence context. In the second aim, a new methodology, Pioneer-seq, will be developed where a transcription factor's binding affinity is determined to thousands of nucleosomes within a nucleosome library containing differing sequences, orientations, and variants. In the third aim, Pioneer-seq will be extended to examine how histone tail modifications amend TF-nucleosome binding. Overall, this project will develop a high- throughput quantitative method to determine binding principles for any protein to nucleosomal DNA in the presence or absence of histone modifications. This contribution will be significant because it can be applied to study many biological responses including: cell growth, regulation of cell-division, embryonic development, differentiation, response to environmental stresses, apoptosis and the development of a variety of disease states. In addition, biological principles can be addressed involving cooperative binding, binding to subnucleosomes, TF-histone interactions, and sequence content.

DNA与染色体DNA的结合是许多基本生物过程所必需的 包括：转录调控，DNA复制和修复，重组和染色体 种族隔离在理解转录因子（TF）如何结合方面存在根本性的空白 位于紧密的，高阶染色质中的调节区域。因此，有一个基本的 需要确定定义TF与染色质结合的机制规则。年远景目标 这个项目是为了定义TF与染色体DNA结合的生物学规则 包括核小体、DNA和组蛋白内转录因子结合位点取向 修饰、辅因子和协同结合以及与亚核小体颗粒的结合。的 本申请的总体目标是开发一种高通量方法来测量 转录因子与核小体DNA结合的原理。为了实现这一目标， 将开发高通量的下一代测序分析，以允许同时进行 以及在一次测定中定量检测数千种不同的核小体。目标 本申请的目的将通过三个具体目标来实现：1）形成核小体 2）使用核小体文库的高通量蛋白质-核小体结合测定，和 3)定义组蛋白修饰后TF-核小体结合。在第一个目标下，在体外 核小体将由成千上万的二氧化硅设计和天然存在的DNA产生 在一个单一的反应序列，允许TF结合位点发生在所有可能的核小体 具有各种相邻序列背景的方向。第二个目标是一种新的方法， Pioneer-seq将被开发，其中转录因子的结合亲和力被确定为 在含有不同序列的核小体文库中的数千个核小体， 方向和变体。在第三个目标中，Pioneer-seq将被扩展到研究组蛋白是如何被激活的。 尾部修饰改善TF-核小体结合。总的来说，该项目将开发一个高- 用于确定任何蛋白质与核小体的结合原理的通量定量方法 在存在或不存在组蛋白修饰的情况下的DNA。这一贡献将是巨大的 因为它可以应用于研究许多生物反应，包括：细胞生长， 细胞分裂，胚胎发育，分化，对环境压力的反应， 细胞凋亡和各种疾病状态的发展。此外，生物学原理 可以涉及合作结合，结合亚核小体，TF-组蛋白 相互作用和序列内容。