The nature of the regulatory information that directs the development of an organism from the fertilized egg to an intact adult is one of the central problems in biology. The mammalian globin gene family is an ideal model system for the study of gene regulation: globin genes are expressed only in erythroid cells, and individual globin genes are expressed only during restricted stages of development. During development, the human red blood cell activates first an embryonic, then a fetal, and finally an adult gene. The long-term goal of this proposal is to elucidate the molecular basis of this """"""""hemoglobin switching,"""""""" through molecular, biochemical, and genetic analyses. The focus on the proposed research is the human embryonic beta-like globin epsilon, the earliest of the beta-like globin to be activated. The investigator has defined cluster of positive (PRE) and negative (NRE) cis- acting regulatory elements upstream of this gene. Two of the elements (epsilon-PRE II and V) interact synergistically to confer stage-specific expression on a minimal promoter. Binding of a nuclear factor from embryonic erythroid cells to a novel, conserved sequence motif within epsilon-PRE II is required for epsilon-PRE II function, strongly implicating this factor in the developmental regulation of epsilon gene expression. Interestingly, a post- transcriptionally modified epsilon -PRE II binding activity is observed in adult erythroid cells. The investigator proposes here to purify the epsilon- PRE II binding factor from embryonic erythroid cells and to isolate the corresponding cDNA(s), as necessary steps in understanding the mechanism by which proteins bound at epsilon-PRE's II and V cooperate to direct stage- specific expression in the embryo. Accordingly, a more detailed characterization of the function and protein binding properties of epsilon-PRE V will be undertaken. The role played by epsilon-PRE's and NRE's in the temporal control of the human epsilon-globin gene will be rigorously examined in vivo, in a transgenic mouse system. The long- term goal of these studies will be to determine the nature of the temporal information that specifies the timing of gene switching during development. The proposed research has a number of potential applications to human medicine. Many acquired and genetic abnormalities influence the pattern of hemoglobin production during human development. Not only the level of production but also the timing of specific development switches may be perturbed in a variety of ways. Interestingly, embryonic and fetal, but not adult, globin genes have been found to be activated in virtually all human erythroleukemia cell lines, even though little or no expression of these genes is detected after birth in normal erythroid cells. The deregulation expression of these genes in human erythroleukemia may reflect a defect in normal gene silencing, or aberrant activation, or both. An understanding of normal and abnormal globin gene control mechanisms is likely to have important medical implications, not only for the origins of hematopoietic malignancies, but also for the design of rational treatment strategies for a variety of other erythroid cell disorders.
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