REGULATION OF FETAL HEMOGLOBIN SYNTHESIS

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The clinical course in patients with sickle cell disease is more mild when Hb F levels are elevated. Trials are ongoing to evaluate effects of HU on stimulating Hb F synthesis and modifying clinical expression of disease. A detailed molecular understanding of the switch from fetal to adult globins together with the ability to manipulate this switch holds promise for many of the severe human hemoglobinopathies and thalassemia syndromes. General goals of this proposal are 1) to define hematopoietic progenitor response to prolonged HU treatment; and 2) to define the molecular basis for elevated expression of fetal globins, in a non-deletional form of BPFH. Specific aims include the following: (1) Numbers of BFU-Es in peripheral blood, cells per colony, percentage of BFU-Es in S phase and gamma/beta globin mRNA ratios will be monitored before and after HU treatment in patients with sickle cell disease expressing various Hb F levels in order to test the hypothesis that BFU-Es in peripheral blood decrease when Hb F levels are pharmacologically increased. (2) The role of the LCR and A-gamma 3' enhancer in potentiating relative promoter-strength differences between the -175 non-deletional form of HPFH and wild type gamma promoter will be evaluated using transient expression assays in a variety of erythroid and nonerythroid environments. Proteins binding to the area of the -175 bp change and/or directly interacting with GATA- 1 will be isolated and characterized. Attempts will be made to express these proteins and GATA- 1 in an effort to transactivate the -175 HPFH promoter in a non-erythroid environment. (3) We will attempt to activate the fetal globin program in lymphocytes from this HPFH patient. Transient heterokaryons will be made by fusing MEL cells expressing an adult globin program with transformed lymphocytes from normals and the patient with HPFH. Timed footprints in vivo will be done after fusion to monitor for differences in protein binding to the LCR, 3' enhancer and immediate 5' flank of the fetal globin genes. Expression and difference or subtractive cDNA libraries will be made from MEL cells either expressing only adult or embryonic and adult programs in an effort to identify cDNAs critical to activation of the human fetal globin program in transient heterokaryons.