Long-range High-resolution Mapping of Chromatin

描述（由申请人提供）：每一个已知的发生在基因组上的核调控事件似乎都与染色质结构的变化有关。指定转录、复制、DNA修复和重组的调控序列位于结构随这些元件功能调控而变化的区域。本应用程序的目标是设计一种技术，允许在非常大（100 kb或更大）的基因组区域检查染色质结构。重点是绘制化学物质和酶的裂解位点，这些酶的活性已知对染色质结构的变化具有敏感性。这项技术的发展不仅将为寻找新的调控元件提供一个重要的、基本公正的机制，而且还将为增加对染色质结构长期变化的理解提供一个工具。在理解基因调控方面，利用切割位点作图已有先例；例如，dna酶超敏感位点的定位在理解启动子、增强子和LCR功能方面发挥了重要作用。一个系统的、自动化的基因组区域切割位点的图谱，其数量级比以前所检查的要大，将被证明是一个有用的工具，用于鉴定调控元件和形成关于高阶染色质结构调控的假设。

DESCRIPTION (provided by applicant): Every known nuclear regulatory event that occurs on the genome appears to be associated with a change in chromatin structure. Regulatory sequences that specify transcription, replication, DNA repair and recombination are located in regions whose structure changes with regulated function of these elements. The goal of this application is to devise technology that will allow chromatin structure to be examined over very large (100 kb or greater) regions of the genome. The focus is on mapping cleavage sites for chemicals and enzymes whose activity is known to display sensitivity to changes in chromatin structure. Development of this technology will not only provide an important, largely unbiased, mechanism for searching for novel regulatory elements, but will also provide a tool to increase the understanding of long-range changes in chromatin structure. There is precedent for the utility of mapping of cleavage sites in understanding gene regulation; for example the mapping of DNase hypersensitive sites has played a significant role in understanding promoter, enhancer and LCR function. A systematic, automated mapping of cleavage sites over regions of the genome an order of magnitude larger than those examined previously will prove to be a useful tool in both identification of regulatory elements and formation of hypotheses concerning the regulation of higher order chromatin structures. The following Aims will be pursued to develop a technology for long-range high-throughput mapping of cleavage sites: Aim 1 will develop a single tube protocol for mapping cleavage sites in single copy mammalian DNA and will automate that protocol; Aim 2 will apply the technology developed in Aim 1 to mapping large (up to 120 kb) regions of the mouse genome both in an undifferentiated multipotent stem cell line and in a homogeneous differentiated population of cells derived from that stem cell line. Aim 3 will develop bioinformatics tools to interpret the data collected in Aim 2.