The long-term goal of this research project is to understand the molecular mechanisms underlying the regulation of human erythropoietin (EPO) gene expression during development. The EPO gene encodes the polypeptide hormone which controls red blood cell production, and the gene is expressed in the fetal liver, adult kidney, and adult liver during stress erythropoiesis. Previous studies of human EPO gene expression in transgenic mice have localized cis-acting DNA regulatory elements which control human EPO gene expression in liver, but not kidney. Larger DNA fragments with more extensive 5'- and 3'-flanking regions will be microinjected into fertilized mouse eggs in order to generate transgenic mice that express the human EPO gene in kidney. The flanking sequences responsible for regulated EPO gene expression in liver and kidney will then each be joined to simian virus 40 early region coding sequences to construct a chimeric gene in which the transforming protein T antigen (Tag) is expressed only in EPO-producing cells of liver and kidney, respectively. Transgenic mice carrying the EPO-Tag transgene will be analyzed and attempts to establish permanent tissue-culture lines of EPO-producing cells will be made. Nuclease-sensitivity studies of the EPO gene will be performed using EPO-producing vs. heterologous cell lines to identify the precise location of DNA sequences controlling EPO gene expression. The importance of these sequences will be confirmed by constructing EPO transgenes for microinjection in which these specific regions have been deleted, and analyzing the resulting pattern of transgene expression in vivo. The trans-acting transcriptional factors which bind to these DNA regulatory elements will be identified using nuclear extracts of EPO-producing cells in gel-shift and DNA footprinting assays. To complement studies of transgenic mice, two human families will be studied in which erythrocytosis is inherited as an autosomal dominant trait and shows co-segregation with restriction fragment length polymorphisms that do not rule out linkage of the phenotype to the EPO gene. EPO gene DNA from affected family members will be amplified by the polymerase chain reaction and analyzed by nucleotide sequence determination to identify potential disease-causing mutations.
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