The human beta globin gene domain is flanked by a series of developmentally stable DNase I hypersensitive (HS) sites. Transgenic studies have shown the 5' HS sites to contain elements necessary for high level, regulated expression of globin transgenes. However, the results have been inconclusive concerning the requirement for the 3' HS site. Our investigation of the 3' site (3'HS1) has shown it to be closely linked to scaffold attachment sites, topoisomerase II recognition sequences, several GATA-1 binding sites, and an AP-1/NF-E2 recognition sequence. The combination of structural components and regulatory elements found within 3'HS1 suggest possible regulatory roles for this region. The experiments described in this proposal are designed to determine what role this region plays in the regulation of the beta globin gene domain and how the individual structural components contribute to this regulation. Future attempts at somatic gene therapy as a treatment for sickle cell disease will require a thorough understanding of beta globin regulation.
the specific aims of this proposal are threefold. (1) To identify the subcomponents of 3'HS1. Our preliminary data indicates that 3'HS1 hybridizes to single copy DNA within the mouse genome. I propose to clone and sequence the murine homologue to 3'HS1 and identify those sequences which have been conserved, and presumably important in the regulation of the domain. This approach should complement our transgenic studies and direct the search for important regulatory elements within the region. (2) To determine the biochemical mechanisms for generation of the site. Footprinting experiments will be used to identify DNA/protein interactions within the region. Specific binding sites will be altered by site-directed mutagenesis or deleted in their entirety, incorporated into beta globin transgenes, and the consequences of these changes monitored in transgenic mice and/or MEL cells. Genomic sequences shown to bind nuclear proteins will be used to probe cDNA expression libraries in order to identify the genes which encode the trans-acting nuclear proteins. (3) To determine the role of 3'HS1. Beta globin transgenes containing 3'HS1, either alone or in combination with the activating 5' LCR will be introduced into transgenic mice and MEL cells and their expression monitored by primer extension. Once we establish the function of 3'HS1, deletional analysis of the region will be used to determine the minimum sequences required.
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