Recent discoveries in our laboratory offer new insights into normal erythroid biology and beta-thalassemia. The high- level production of hemoglobin that occurs during erythroid maturation is tightly coordinated so as to minimize toxicities caused by accumulation of individual alpha- and beta- globin subunits, which tend to precipitate in cells. Prior studies of normal and beta-thalassemic erythroid precursors predict that compensatory mechanisms exist to neutralize free alpha-globin. To learn more about the control of hemoglobin production, we isolated RNA transcripts that are induced by the essential transcription factor GATA-1, a global regulator of erythropoiesis. We identified Erythroid Differentiation Related Factor (EDRF), a small, abundant highly erythroid-specific protein that is strongly upregulated during terminal erythroid maturation and appears to be a direct GATA-1 target gene. We determined that alpha-globin is a specific EDRF binding partner in two independent protein interaction screens. EDRF interacts with free alpha-globin but not with beta-globin or hemoglobin A (alpha2beta2). Moreover, EDRF markedly inhibits precipitation of free alpha-globin in solution and in mammalian cells. Our findings raise the possibility that EDRF acts as a chaperone protein to prevent precipitation and subsequent toxicity of free alpha-globin in erythroid cells. Now that we have established a physical and functional connection between EDRF and alpha-globin in vitro and in heterologous cells, we will study the significance of this association in normal erythropoiesis. Structure-function analyses in Aim 1 will define the domains that are required for physical and functional interactions between EDRF and alpha-globin. In Aim 2, we will assess the biological role of EDRF and its association with alpha-globin in established cell lines and in primary erythroid cells derived from in vitro culture of EDRF gene-targeted embryonic stem (ES) cells. To this end, we have developed EDRF heterozygous and homozygous-null ES cells. In Aim 3, we will determine the hematopoietic consequences of altered EDRF expression in mice. By genetically manipulating EDRF and free alpha-globin levels, we will determine how their relative stoichiometry affects viability and differentiation of erythroid cells. Specifically, we will establish whether EDRF-null animals exhibit excessive alpha- globin precipitation in erythroid precursors, and whether altered EDRF gene expression affects the severity of beta-thalassemia, a disorder that is distinguished by alpha-globin precipitation. Our studies to characterize a highly expressed erythroid specific protein that prevents aggregation of free alpha-globin are important for understanding how hemoglobin chain balance is modulated by non-globin proteins during normal erythropoiesis and might provide a novel approach to alleviate the deleterious effects of excessive alpha-globin in beta-thalassemia.