Differentiation of pluripotential hematopoietic stem cells into mature blood components requires lineage-specific regulation of gene expression. The general aim of this project is to understand transcriptional regulation of erythropoiesis, using globin gene expression as a model. Mammalian alpha-like and beta-like globin genes are organized in cluster, each of which is associated with an upstream regulatory element termed the locus control region (LCR). LCRs enhancers. They are made up of three DNA motifs which bind nuclear regulatory proteins: CACCC motifs binding ubiquitous transcription factors. GATA motifs binding erythroid factor GATA-1 and AP-1 like motifs binding erythroid factor NF-E2. Of these proteins, NF-E2 acts as the primary regulator of LCR enhancer function. It appears to be a dimer of a 45kD tissue-specific basic- leucine zipper (bZIP) protein (p45 NF-E2), and a smaller protein has remained elusive. This investigation is aimed at understanding how NF-E2 mediates LCR enhancer activity. The first step will be to clone p18, using peptide sequences derived from purified protein. Although not essential for further studies, cloning of p 18 will facilitate investigation of the relationship between NF-E3 protein structure and function. Assays of various aspects of LCR activity will be developed using defined components, an employed to examine the functional effects of deliberate mutations of NF-E3. Finally, interactions of p45 NF-E2 and p18 with other partners will be explored, to understand regulation at the level of differential dimerization. Elucidation of the role of NF-E2 in globin gene expression and erythropoiesis will contribute to our overall understanding of the genetic and molecular mechanisms responsible for controlling hematopoietic commitment and differentiation. At the same time, progress will be made towards understanding disorders of erythropoiesis, including thalassemias, congenital anemias, and red cell hypoplasia from HIV infection.
