Long-range High-resolution Mapping of Chromatin

描述(由申请人提供)：基因组上发生的每一个已知的核调控事件似乎都与染色质结构的变化有关。特定转录、复制、DNA修复和重组的调控序列位于其结构随这些元件的调控功能而变化的区域。这项应用的目标是设计一种技术，允许在基因组的很大(100kb或更大)区域内检查染色质结构。重点是绘制化学物质和酶的裂解位点，这些化学物质和酶的活性已知对染色质结构的变化敏感。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、基本上没有偏见的机制，而且还将提供一个工具，以增加对染色质结构长期变化的理解。裂解位点的定位在理解基因调控方面是有先例的；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。系统地、自动地绘制基因组区域上比以前所检查的大一个数量级的切割位点的图谱，将被证明是识别调控元件和形成关于高阶染色质结构调控的假说的有用工具。 将致力于开发一种用于远距离高通量绘制切割位点图谱的技术：Aim 1将开发用于测绘单拷贝哺乳动物DNA中切割位点的单管协议，并将使该协议自动化；Aim 2将应用在Aim 1中开发的技术来绘制小鼠基因组的大区域(最多120kb)，这既包括在未分化的多潜能干细胞系中，也包括在从该干细胞系衍生的同质分化的细胞群体中。AIM 3将开发生物信息学工具来解释在AIM 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.