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

基本信息

批准号：
10240593
负责人：
Michael Joseph Buck
金额：
$ 35.41万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
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/10240593
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 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 environmental stressor exhaustion experimental study genetic regulatory protein histone modification in vitro Assay keratinocyte next generation sequencing particle response transcription factor

项目摘要

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结合对于许多基本生物学过程至关重要包括：转录调控，DNA复制和修复，重组和染色体隔离。理解转录因子（TF）如何结合存在根本差距位于紧凑的高阶染色质中的调节区域。因此，有一个基本需要确定定义与染色质结合的机械规则。长期目标该项目是定义决定TF与染色体DNA结合的生物学规则包括核小体，DNA和组蛋白中的转录因子结合位点方向修饰，辅因子和合作结合，并与亚核小体颗粒结合。这该应用程序的总体目的是开发一种高吞吐量方法来测量转录因子与核小体DNA结合的原理。实现这一目标将开发高通量的下一代测序测定法，以同时允许在单个测定中对数千个不同核小体的定量检查。目标该应用将通过三个特定目的来完成：1）形成核小体图书馆，2）用核小体库和 3）定义组蛋白修饰后定义TF核体结合。在第一个目标下，体外核小体将是由数千个在设计和自然存在的DNA中产生的单个反应中的序列，允许在所有可能的核小体中发生TF结合位点具有各种相邻序列上下文的方向。在第二个目标中，一种新方法，先锋seq将在确定转录因子的结合亲和力的情况下开发核小体库中的数千个核小体，其中包含不同的序列，方向和变体。在第三个目的中，先锋seq将扩展以检查组蛋白的方式尾部修饰修正了TF核体结合。总体而言，该项目将发展高吞吐量定量方法以确定任何蛋白质与核小体的结合原理在存在或不存在组蛋白修饰的情况下DNA。这项贡献将是重要的因为它可以应用于研究许多生物学反应，包括：细胞生长，调节细胞分割，胚胎发育，分化，对环境应力的反应，凋亡和各种疾病状态的发展。另外，生物学原理可以解决涉及合作结合，与亚核小体，TF-histone结合的解决相互作用和序列内容。