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. ? ?
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