Nucleosome Positioning

描述(由申请人提供)：该研究计划的长期目标是发展对真核基因调控的量化理解。在真核生物中，基因组DNA被包裹在核小体中，核小体封闭了包裹的DNA的大部分表面并强烈扭曲了它；然而，核小体也通过与其组蛋白尾部结构域的相互作用来招募其他蛋白质。因此，核小体的详细位置在基因调控中具有重要的抑制和促进作用，对于所有真核生物的健康和发育都是必不可少的。在当前的项目期间，我们推进了对核小体沿DNA的位置、支配这些位置的原则以及这些原则如何影响与其他因素的竞争和基因表达的理解。在下一个授权期，我们建议从三个方面扩展这些研究结果。目标1的研究将在全基因组范围内，随着同步的细胞池在细胞周期中的进展，直接和定量地测量核小体因子的竞争；还将在特定的工程转基因中测量，我们不仅将测量核小体的位置和占有率，而且还将测量结合因子的身份、位置和占有率。这些数据将考验我们目前对核小体-因子竞争的理解，以及我们的想法所基于的热力学平衡假说。目标2中的研究试图测量核小体到基底的准确位置。这些信息是为了：了解核小体将或不会与结合在相邻靶点的因子竞争；定义染色质纤维的内在三维结构；建立启动子的详细结构生物学；以及确定真正的核小体-DNA序列偏好。Aim 3的研究将开发和实验测试一个定量预测模型，用于预测给定基因组DNA序列的基因表达输出，以及细胞中活跃的一组转录因子的浓度、结合位置和亲和力。 与公共健康相关：对基因的适当调控对所有生物体的发育和健康至关重要。我们发现了基因调控的一个以前未知的方面，即基因组DNA序列影响自己进入染色体的组织，从而影响各种基本的染色体功能。我们希望，从长远来看，更好地理解这些新信息的性质，以及它如何影响基因表达和DNA复制，将有助于更好地理解疾病过程中的基本机制，并由此产生新的诊断和治疗方法。

DESCRIPTION (provided by applicant): The long-term aim of this research program is to develop a quantitative understanding of eukaryotic gene regulation. In eukaryotes, the genomic DNA is wrapped in nucleosomes, which occlude much of the surface of the wrapped DNA and strongly distort it; yet, nucleosomes also recruit other proteins through interactions with their histone tail domains. Thus, the detailed locations of nucleosomes have important inhibitory and facilitatory roles in gene regulation, and is essential for the health and development of all eukaryotes. In the current project period we advanced the understanding of where nucleosomes are located along the DNA, the principles that govern these locations, and how these influence competition with other factors and gene expression. In the next grant period, we propose to extend these findings in three directions. Studies in Aim 1 will measure nucleosome-factor competition directly and quantitatively, genome-wide, as synchronized pools of cells progress through the cell cycle; and also at specific engineered transgenes, where we will measure not just nucleosome positioning and occupancy but also the identities, locations, and occupancies of bound factors. These data will test our current understanding of nucleosome-factor competition and the thermodynamic equilibrium hypothesis on which our ideas are based. Studies in Aim 2 seek to measure the exact locations of nucleosomes to the basepair. This information is needed in order to: understand where nucleosomes will or will not compete with factors binding at adjacent target sites; to define the intrinsic 3- dimensional structure of the chromatin fiber; to establish the detailed structural biology of promoters; and to define the true nucleosome-DNA sequence preferences. Studies in Aim 3 will develop and experimentally test a quantitative predictive model for predicting the gene expression output given the genomic DNA sequence, and the concentrations, binding sites, and affinities of a set of transcription factors active in the cell. PUBLIC HEALTH RELEVANCE: The proper regulation of genes is essential for the development and health of all organisms. We discovered a previously unrecognized aspect of gene regulation, in which the genomic DNA sequence influences its own organization into chromosomes, and thereby influences diverse essential chromosome functions. Our hope is that a better understanding of the nature of this novel information, and how it influences gene expression and DNA replication will lead, over the long term, to a better understanding of fundamental mechanisms in disease processes, and from there, to new diagnostics and therapeutics.