REGULATION OF ZETA GLOBIN GENE EXPRESSION

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The broad, long-term objectives of this project are to determine the molecular basis for regulation of globin gene expression, and to extend these studies to the dissection of pathways involved in hematopoietic commitment during development. The specific aims of this project are to: 1. Test the hypothesis that development regulation of zeta-globin expression is due to differences between embryonic and non-embryonic erythroid cells in protein-DNA-interactions occurring within the proximal zeta-globin promoter, 2. Test the functional significance of the above protein-DNA interactions within the proximal zeta-globin promoter by introducing altered promoters into both erythroid cell lines and transgenic mice, 3. Identify the features of a novel upstream positive regulatory region that are responsible for high-level zeta-globin expression in the embryonic erythroid cell line K562, 4. Characterize a trans-acting factor important for regulation of zeta- globin expression by isolating a cDNA clone using expression cloning strategies and/or purifying the factor to homogeneity. The health-relatedness of this project is threefold. First, the study of zeta-globin gene regulation will contribute to overall understanding of regulation of globin expression in normal individuals and thalassemic patients. Second, since mechanisms that regulate zeta-globin expression are distinct from those used by other globulin genes, novel regulatory elements will be identified that can be adapted for somatic gene therapy expression constructs. Third, since zeta-globin is one of the first hematopoietic genes expressed during development, understanding its regulation will contribute to better understanding of normal hematopoiesis and abnormal hematopoietic conditions such as leukemia, in which inappropriate embryonic gene expression occurs. The research design and methods include characterization of protein-DNA interactions within the zeta-globin promoter by electrophoretic mobility shift assays, DNAaseI footprinting, and protein-DNA cross-linking studies, functional analysis of regulatory elements by testing the effects of site-directed mutations on zeta-globin promoter function in leukemic cell lines, transgenic mice, and embryoid bodies, and molecular cloning and/or purification of DNA-binding activities responsible for developmental regulation of zeta-globin expression.