The gamma to beta switch in the synthesis of hemoglobins is not well understood. The primary model system for studying this process, the K562 cell line, does not synthesize the beta globin. Recently, the conditions for culture of erythroid progenitors from peripheral blood have been described. Preliminary studies using RNAse protection assays and RNA hybridization indicate that these cells reproduce the gamma to beta switch in culture. An erythroid progenitor culture system has been set up and we are establishing the culture conditions necessary to obtain consistent globin synthesis. Protocols have been established to extract RNA and perform the RNAse protection assay to quantitate the specific globin mRNAs produced. A variety of cell staining techniques have been investigated; the May- Grunwald-Giemsa and benzidine stains are presently in use. We plan to use the erythroid progenitor culture system to study the transcriptional control of switching during erythroid maturation, during the early post-natal period (using cord blood), and in switching mutants such as Hereditary Persistence of Fetal Hemoglobin and certain forms of beta thalassemia. Other planned studies include: studies of the mechanism of transcriptional alteration of globin expression by agents such as hydroxyurea, oxygen, erythropoietin, and iron; studies of the development and terminal differentiation of erythroid progenitor cells; and, ultimately, studies of the proteins involved in stage-specific transcriptional control. This system allows us to reproduce, in culture, the normal terminal differentiation of the erythroid progenitor cell and to correlate this maturation with the production of specific globin mRNA. It will allow the development of studies which are important both clinically and in several areas of basic medical research including: the molecular biology of normal development and maturation, the isolation and study of the proteins involved in the regulation of globin expression, the pharmacology of drugs and chemicals which affect globin transcription and which might be useful in the therapy of sickle cell anemia and beta thalassemia, and studies of the erythropoietin receptor, among others.
