High Resolution Mapping of Functional Elements in the Yeast Genome

描述(由申请人提供)： 真核DNA被包装成染色质，这个染色质有一个明确的组织。染色质是由核小体组成的，其在DNA上的位置决定了基因调控元件的可及性，最终决定了基因的表达水平。核小体在基因上以规则的重复间隔出现。这些阵列通过许多机制受到高度调控，包括：翻译后修饰，伴侣促进的沉积和驱逐，以及染色质重塑复合体促进的重新定位。我们建议在基因组范围内加深对核小体阵列的理解。在目标1中，我们将使用我们新开发的超高分辨率芯片-Exo分析来确定整个基因组中组蛋白标记沿着核小体阵列的亚核小体组织。在目标2中，我们将使用CHIP-EXO定位染色质重构体的核小体和亚核小体组织。它们在特定的核小体上的位置以及它们在核小体表面的方向应该能让我们深入了解它们在基因组规模上是如何发挥作用的。在目标3中，我们将使用我们最近开发的正确定位的核小体阵列的全基因组重组来探索染色质重构体导向的阵列形成的生化机制。这些目标的完成有望提供对核小体阵列的基本原理及其在调控基因表达中的作用的理解。

DESCRIPTION (provided by applicant): Eukaryotic DNA is packaged into chromatin, and this chromatin has a well-defined organization. Chromatin is composed of nucleosome building blocks, whose positioning along the DNA dictates the accessibility of gene regulatory elements, and ultimately the expression levels of genes. Nucleosomes occur in regular repeating intervals on genes. These arrays are highly regulated through many mechanisms including: post- translational modifications, deposition and eviction that are facilitated by chaperones, and re-positioning facilitated by chromatin remodeling complexes. We propose to further our understanding of nucleosomal arrays on a genomic scale. In aim 1, we will use our newly developed ultra-high resolution ChIP-exo assay to determine the subnucleosomal organization of histone marks along nucleosomal arrays across a genome. In aim 2, we will map the nucleosomal and subnucleosomal organization of chromatin remodelers using ChIP-exo. Their placement at specific nucleosomes, and their orientation on the nucleosome surface should provide insight into how they function on a genomic scale. In aim 3, we will use our recently developed genome-wide reconstitution of properly positioned nucleosomal arrays to probe biochemical mechanisms of chromatin remodeler-directed array formation. Completion of these aims is expected to provide an understanding of fundamental principles of nucleosomal array and their role in regulating gene expression.