In this proposal, we aim to identify DNAelements that dictate the organization of the genome into functional chromosomal domains. Large regions of the genome are coordinately replicated during S phase according to a specific spatial and temporal program. These replication domains are likely determined by the overall organization of the genome into highly-ordered structures. In order to further our understanding of nuclear organization and replication timing, this proposal aims to identify replication boundary elements that are responsible for chromatin domain organization. First, potential boundary elements (PBEs) will be identified by changes in replication timing using fluorescence in situ hybridization techniques. As boundary elements are believed to be tethered to nuclear structures by insulator proteins, nuclear matrix attachment and CTCF insulator binding sites will also be mapped to identify PBEs. Next, identified replication PBEs will be assayed for enhancer-blocking and barrier insulator activity using gene expression assays. Finally, a chromosomal interactions with replication domain PBEs will be identified using chromosomeconformation capture technology. These results will yield a better understanding of how replication domains are organized and insight to the mechanisms that regulate replication domain structures. This research will investigate how DNA is duplicated and organized within the cell nucleus. Regulation of chromosome organization is one mechanism that determines if a gene is turned "on" or "off1. Since alterations in gene expression can result in a variety of diseases, studying how DNA is organized within the nucleus will further our understanding of disease formation.