The long-term goal of this proposal is to understand the molecular mechanisms underlying cell-specific and hypoxia-inducible expression of the human EPO gene. The applicant will continue his previous studies which have characterized cis-acting elements and their associated protein factors which mediate this complex regulation. The PI has shown that DNA sequences located 6-14 kb 5' of the EPO gene are required for its expression in the kidneys of transgenic mice. This region will be analyzed more fully in transgenic mice by utilizing progressively smaller segments of it to drive EPO gene expression. He will also determine whether these regions can function independently of an enhancer located in the 3' flanking region. He will also attempt to identify elements >14 kb upstream of the gene which appear to be necessary for post-natal down-regulation of EPO gene expression in the liver. A hypoxia-inducible factor (HIF-1), that binds to the EPO gene 3' enhancer site, has been purified and partial cDNA clones have been identified. Full-length cDNAs and bacterially expressed fusion proteins for the production of antisera will be obtained. These will be used to establish the kinetics of HIF-1 mRNA and protein expression in normoxic, hypoxic, and post-hypoxic cells. This should permit the PI to establish the molecular mechanisms by which HIF-1 DNA binding activity is induced and EPO gene transcription is activated. An additional factor that binds to the 3' enhancer and collaborates with HIF-1 for EPO transcriptional activation will also be characterized.
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