Long-range High-resolution Mapping of Chromatin

描述（由申请人提供）：基因组上发生的每个已知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 修复和重组的调节序列位于其结构随着这些元件的调节功能而变化的区域。 此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）的基因组区域中检查染色质结构。 重点是绘制化学物质和酶的裂解位点，已知这些化学物质和酶的活性对染色质结构的变化表现出敏感性。 这项技术的发展不仅将为寻找新的调控元件提供一种重要的、基本公正的机制，而且还将提供一种工具来增加对染色质结构长期变化的理解。 切割位点图谱在理解基因调控方面的应用已有先例；例如，DNase 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。 对基因组区域上的切割位点进行系统的、自动化的绘图，其数量级比之前检查的要大一个数量级，这将被证明是识别调控元件和形成有关高级染色质结构调控的假设的有用工具。 将追求以下目标来开发一种用于切割位点的远程高通量图谱的技术： 目标 1 将开发用于绘制单拷贝哺乳动物 DNA 中的切割位点图谱的单管方案，并使该方案自动化；目标 2 将应用目标 1 中开发的技术来绘制未分化多能干细胞系和源自该干细胞系的同质分化细胞群中小鼠基因组的大区域（高达 120 kb）的图谱。目标 3 将开发生物信息学工具来解释目标 2 中收集的数据。

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.