This application seeks competitive renewal of a project that has focused upon regulation of the erythropoietin gene in a tissue culture model Hep3B cells. During the past 5 years, the Investigators have shown that the induction of erythropoietin by a synthesis by hypoxia involves a 100 fold increase in steady state mRNA levels, due largely to enhanced transcription. A heme protein is a primary mediator of the induction. This group and others have also implicated two DNA sequence elements flanking the structural gene for Epo. One lies in a 117 bp minimal promoter, and the second in a 40 bp minimal enhancer 3' to the polyadenylation site. Both must interact in order to mediate increased transcription. Each site contains hexanucleotide consensus sequences that are involved in the binding of the nuclear receptor family (eg thyroid receptor) of transcription factors. Preliminary data included in this application suggests that the orphan receptor HNF-4 is likely to be involved, and that it may interact with another factor called HIF-1. This proposal requests 5-year's support to continue, refine, and extend this preliminary progress. The first Specific Aim will focus on continued characterization of the cis-acting DNA sequence elements and the transacting proteins that interact with the Epo gene to promote efficient transcription. These are standard experiments involving the use of reporter genes, putative promoter and enhancer elements, and mutagenized versions of elements found to have functional activity. The second Specific Aim will explore the potential role of nuclear receptors in Epo gene regulation, with a particular focus on HNF-4. The rationale for these studies is that the critical flanking regions of the genes contain recognition sites for this family of proteins, and that these sites bind to proteins, at least as defined by gel retardation assays. The third Specific Aim will utilize the two hybrid system to clone genes encoding proteins in Hep-3B cells that bind to HNF-4. The Investigators postulate that identification of HNF-4 binding partners should elucidate additional steps in the pathway between the heme protein sensor, and the elements binding directly to the Epo gene. The fourth Specific Aim will continue attempts to identify and characterize the heme-binding protein that mediates the initial steps in oxygen sensing. Finally, Specific Aim 5 will be an exploration of genetic defects in erythropoietin regulation in four patients who have been identified to have lifelong erythrocytosis and elevated levels of plasma Epo. Epo mRNA production by cells derived from these patients, and DNA sequence analysis of genomic DNA from these individuals will be performed.
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