Experiments comparing the tissue-specific expression of transgenes vs. the intact chromosomal locus demonstrate clearly that chromosomal gene regulation is more complex than previously thought. For example, sequences that appear to be dispensable in assays involving transgenes are required for expression at the natural site. This proposal describes the use of a novel recombination-proficient hybrid cell system, developed by our laboratory, to investigate the requirement of cis elements in the tissue-specific regulation of the human serpin gene cluster. Specifically, microcell hybrids will be used to modify DNA sequences in the genomic region separating the a1AT and CBG genes. Modified chromosomes will be transferred to three different recipient cell types: hepatic cells, which express both genes; fibroblasts, which express neither; and macrophages, which express alpha1AT but not CBG. Gene expression patterns and chromatin structures of the various mutant alleles will be assessed in the three hybrid cell types. Thus, regulatory elements that control chromatin structure within this large genomic region will be identified. The concordant and differential regulation of expression may depend upon the relaxation and strengthening, respectively, of a boundary element that separates the two genes. Thus, these experiments should provide important new information concerning mechanisms that regulate the structure and function of individual chromatin domains.
