(taken from application) The research goal of this proposal is to study the molecular and cellular mechanisms involved in the regulation of erythropoiesis mediated by the erythropoietin receptor (EpoR), the expression of which is essential for norma erythroid development. The proposed studies include characterization of nonpromoter-related, cis-acting regulatory elements within the human EpoR gene and identification of trans-acting factors which interact with the regulatory elements and may direct tissue-specific transcriptional control of the gene. The chromatin structure of the gene will be examined in tissue culture cells a well as mouse tissues to identify tissue-and developmental stage-specific changes in the regulatory elements. Recombinant DNA technology will be used in cloning of genomic DNA segments of the EpoR gene relevant to its expression followed by gene transfer/expression studies in tissue culture cells as well a studies of DNA-protein interactions. Another goal of this proposal is to study the signal transduction function of the human EpoR using two human disorders o erythropoiesis as models, familial erythrocytosis and polycythemia vera. The effect of a novel human EpoR mutant causing dominantly-inherited erythrocytosi on the activation of downstream targets of the activated EpoR, such as Jak2, Stat5, Shc and MAP kinase, will be studied to characterize signaling abnormalities associated with Epo hyper-responsiveness of erythroid progenitors. Tissue culture cells expressing the mutant and/or wild type EpoRs and erythroid progenitors from affected individuals will be studied in Epo dos response assays, DNA fragmentation studies, electrophoretic gel mobility shift assays and immunoprecipitation and Western blotting experiments. In another model, purified erythroid progenitors from patients with polycythemia vera wil be studied for the activation kinetics of intracellular signaling molecules that are targets for the activated EpoR, such as Jak2 and Stats, hyperactivity of which may be associated with increased in vitro responsiveness to Epo. This laboratory research will be performed by the principal investigator Dr. Murat O. Arcasoy in conjunction with a didactic program under the guidance of a sponsor, Dr. Bernard G. Forget and an advisory committee. This proposal will allow the candidate to develop the technical expertise, fundamental knowledge and intellectual scientific approach necessary for an independent and productive career in investigative molecular and cellular biology and its application to pathogenesis of hematologic disorders.
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