The objective of the current proposal is to understand the mechanisms underlying the increased expression of the human Epo gene in response to hypoxia. The specific goal of these studies is to identify and characterize the DNA sequences and nuclear proteins underlying this response. The Hep 3B cell line has been shown to markedly increase Epo production with hypoxia. Using a luciferase reporter system in transfection studies in Hep 3B cells, we have localized a hypoxia- inducible enhancer to a 24 base pair (bp) portion of the Epo gene 3' flanking sequence. In order to further define the sequences responsible for this response, transfection studies will be performed using truncation and point mutation derivatives of this 24 bp fragment. Point mutation fragments to be tested will be designed based on results obtained with truncation mutants and on the presence of homology in the enhancer sequence with known transcription factor binding sites. In order to identify and characterize DNA-protein interactions in the hypoxia-responsive element, gel mobility shift studies will be performed using the 24 bp fragment to probe nuclear protein extracts from normoxic and hypoxic Hep 3B cells, from other cell lines that produce Epo (Hep G2 and RC-3) and from control cell lines which do not make Epo. The specificity of protein binding will be determined by gel shift assays using unlabeled normal and mutant 24 bp enhancer fragments as competitors. In order to define DNA-protein interactions that are specific to hypoxia, nuclear extracts will be used in Deoxyribonuclease I footprint studies of this region of the gene. Since in vitro footprinting studies can only approximate the actual situation in living cells, in vivo footprint studies will also be performed to characterize DNA-protein interactions in this portion of the Epo gene within Hep 3B cells under normoxic and hypoxic conditions. In order to isolate genes encoding the proteins that regulate Epo gene response to hypoxia, an expression cDNA library will be constructed from hypoxic Hep 3B cell mRNA and will be screened with oligonucleotide probes that contain binding sites for the factors that direct this response. Isolation and characterization of these regulatory factors would lead to a better understanding of the mechanisms underlying oxygen-regulated gene expression.
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