The overall goal of this research is to investigate the molecular control of globin gene switching and test the hypothesis that switching is accomplished through two mechanisms, gene silencing and gene competition.
Our specific aims are to: 1) Investigate the control of embryonic globin gene expression. a) Characterize a silencer we have identified in the epsilon gene promoter; b) search for other silencing elements among the sequences flanking the epsilon gene; c) investigate the control of embryonic zeta globin gene expression. 2) Investigate the control of gamma globin gene expression through silencing. a)Characterize a 5' gamma gene silencer we have already identified in transgenic mice b) search for gamma gene silencers located in the flanking region between the Agamma and beta genes. 3) Examine whether temporal expression of globin genes during development is determined by the order of the globin genes along the beta locus, or by their proximity to the LCR or by the prevailing trans acting environment at any given stage of development. 4) Investigate the competition mechanism of globin switching by testing predications of the model that: a) Only one gene can be expressed per beta locus at any given stage of development. b) Disruption of interactions between a gene and the LCR should result in the activation of the gene which is competitively repressed. c) Competition between two genes should be abolished by the availability of multiple LCR's. 50 Investigate the function of LCR. a) Test whether LCR is active in earlier stages of hemopoietic cell development b) determine the role of individual hypersensitive sites in globin gene switching. 6) Produce betaYAC-erythroleukemia cell hybrids and develop these hybrids as a new system for studying the function of LCR and the developmental regulation of human globin genes. It is expected that these studies will advance our understanding of the control of globin genes during development and differentiation. A better understanding of the molecular mechanism of switching will provide insights on the control of fetal hemoglobin in adult cells and may allow development of new approaches for stimulation of fetal hemoglobin in the adult. Such a development will have important consequences for the treatment of patients with sickle cell disease or beta thalassemia syndromes.
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