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

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)如何结合方面存在着根本性的差距 调节区位于紧密的、高阶染色质中。因此，有一个根本的 需要确定定义转铁蛋白与染色质结合的机制规则。的长期目标是 这个项目是定义生物规则，指示转铁蛋白结合到染色体DNA这些规则 包括核小体、DNA和组蛋白内转录因子结合位点定位 修饰、辅因子和协同结合，以及与亚核小体颗粒的结合。这个 这个应用程序的总体目标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。为了实现这一目标，一个新的 高通量的下一代测序分析将被开发出来，以允许同时 以及在一次化验中对数千个不同的核小体进行定量检查。目标是 这一应用将通过三个具体目标来实现：1)形成核小体 文库，2)与核小体文库的高通量蛋白质-核小体结合分析，以及 3)确定组蛋白修饰后的Tf-核小体结合。在第一个目标下，在体外 核小体将由数以千计的硅胶设计和自然产生的DNA产生 单个反应中的序列，允许在所有可能的核小体中出现TF结合位点 具有各种相邻序列上下文的定向。在第二个目标中，一种新的方法， 将开发先锋序列，其中转录因子的结合亲和力确定为 包含不同序列的核小体文库中的数千个核小体， 方向和变种。在第三个目标中，先锋-SEQ将被扩展到研究组蛋白如何 尾部修饰增加了Tf-核小体的结合。总体而言，该项目将发展一个高- 确定任何蛋白质与核小体结合原理的吞吐量定量方法 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。 因为它可以用于研究许多生物反应，包括：细胞生长，调节 细胞分裂，胚胎发育，分化，对环境压力的反应， 细胞凋亡与多种疾病状态的发展。此外，生物学原理 可涉及协同结合、与亚核小体的结合、Tf-组蛋白 交互和序列内容。

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.