权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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的结合对于许多基本的生物学过程是必不可少的