From the third week of gestation to the second month of gestation the yolk sac is the major site of erythropoiesis. Embryonic globin chains (epsilon and zeta) are synthesized in the yolk sac. At ten weeks of gestation the site of erythropoiesis switches to the liver and the major globin chains synthesized are alpha and gamma globin. During the perinatal period the site of erythropoiesis switches to be bone marrow and alpha and beta globin are the predominant globin 1, chains synthesized. The developmental switches that occur in globin chain synthesis are used as a model to study spatial and temporal regulation of gene expression. Minimal globin gene promoters contain highly conserved sequences (TATA,CCAAT, CCAAC) which are required for efficient globin gene transcription. These sequences are also present and required for efficient transcription of many non-globin genes. These sequences and transacting factors binding to them play a role in regulating globin gene expression. Because they are highly conserved in all globin gene, it is likely that additional elements are required for spatial and temporal regulation of globin gene expression. Functional studies in which deletion mutants of the epsilon globin gene (an embryonic globin gene) promoter have been transfected and transiently expressed in K562 cells (a cell line that produces embryonic and fetal globin chains) and HeLa cells ( a cell line that does not produce hemoglobin) have identified a region of the epsilon globin gene promoter (epsilon -274 to -392) which is a silencer. This region is upstream of the minimal promoter. Anion exchange chromatography of K562 cell nuclear extract has been used to separate a fraction that inhibits transcription of the epsilon globin gene. A mobility shift assay has identified a factor in this fraction that binds to a region of the epsilon silencer and may be important in regulating temporal and spatial expression of the epsilon globin gene. Additional anion exchange chromatography, gel filtration and affinity chromatography will be conducted in order to purify this and other factors regulating globin gene expression. Once purified the functional activity of these factors will be tested.