Nucleosome Positioning

描述（由申请人提供）：本研究计划的长期目标是发展对真核生物基因调控的定量理解。在真核生物中，基因组DNA被包裹在核小体中，核小体遮挡了包裹DNA的大部分表面并使其严重扭曲；然而，核小体也通过与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，对所有真核生物的健康和发育至关重要。在目前的项目期间，我们进一步了解了核小体沿着DNA的位置，控制这些位置的原则，以及它们如何影响与其他因素和基因表达的竞争。在下一个资助期，我们建议在三个方向扩展这些发现。Aim 1的研究将直接定量地测量核小体因子竞争，全基因组范围内，随着同步细胞池在细胞周期中的进展；在特定的工程转基因中，我们不仅要测量核小体的定位和占用还有结合因子的身份，位置和占用。这些数据将检验我们目前对核小体-因子竞争的理解，以及我们的想法所依据的热力学平衡假设。Aim 2的研究试图测量核小体到碱基对的确切位置。这一信息是必要的，以便：了解核小体将在何处与邻近目标位点的结合因子竞争或不竞争；确定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；并定义真正的核小体- dna序列偏好。Aim 3的研究将开发并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，以及细胞中一组活性转录因子的浓度、结合位点和亲和力。

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.