This is a proposal to investigate mechanisms that regulate gene expression and chromatin structure in differentiated mammalian cells. Using a novel chromosome shuttle system that allows for efficient modification of human chromosomal genes by homologous recombination, we will define and characterize regulatory elements that control gene activity and chromatin structure in different cell types. Three main areas of research will be pursued. First, we will continue to define the long-range chromatin organization of the human serpin gene cluster at 14q32.1 by using various nuclease-accessibility tests to monitor the chromatin configuration of the locus in different cell types. We will also extend this analysis to include recently identified non-serpin chromatin domains just proximal and distal to the serpin locus. These maps provide essential information for the rational design of locus modification experiments. Second, recombination-proficient microcell hybrids containing human chromosome 14 will be used to modify the serpin locus specifically in order to define regulatory elements both within and between individual chromatin domains. Modified chromosomes carrying mutant serpin alleles will then be transferred to expressing and non-expressing mammalian cells, and the effects of the modifications on gene activity and chromatin structure will be assessed. Third, the functions of individual regulatory elements will be analyzed in various heterologous tests. These will include the construction of regulated mini-domains at ectopic chromosomal sites, transgenesis experiments, and position-effect assays in Drosophila. The overall goal of these experiments will be a comprehensive definition and analysis of regulatory elements in individual chromatin domains.
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