The development of an organism form fertilized egg to intact adult is the consequence of differential gene expression; regulation of this process 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 its ontogeny, the human red blood cell activates first an embryonic, then a fetal, and finally an adult globin gene. The long-term goal of this proposal is to elucidate the molecular basis for this """"""""hemoglobin switch"""""""" during development. To this end, the proposed research will comprise an in-depth analysis of the cis- and trans-acting components which specify the regulated expression of mammalian globin genes. A large number of naturally-occurring human mutations result in abnormal globin gene regulation. An understanding of the normal globin gene regulatory mechanisms could therefore contribute significantly to advances in this area of medicine. Initially, the focus of the proposed research will be on the embryonic globin gene epsilon, the earliest of the beta-like globins to be activated. Some comparative studies will also be undertaken using the fetal beta-like globin gene gamma. Three convergent experimental approaches will be pursued, toward an understanding of the mechanism by which this gene is turned on and off in the human embryo. Cis-acting sequences required for tissue- and stage-specific regulation of the human embryonic globin gene will be identified. Two vector systems will be exploited to stably introduce cloned wild type and mutant globin genes into a human embryonic erythroid cell line (K562). The first of these is an episomal vector derived from Epstein-Barr virus sequences which replicates to high copy numbers in human cells. The second is a cosmid vector containing DNase 1-superhypersensitive sites which in vivo are located far upstream of the entire beta-like globin locus. These sequences have been shown to confer high-level, erythroid- specific, position-independent expression on the human adult beta-globin gene both in transgenic mice and in mouse erythroleukemia cells. The transcriptional effects in K562 cells of deletion mutations in the 5'- and 3'-flanking regions, as well as in the coding region, of the human epsilon-globin gene will be analyzed in the context of both vector systems. Erythroid- and stage-specific proteins which bind to epsilon- globin regulatory DNA sequences will be analyzed through a combination of DNase 1 footprinting, dimethyl sulfate protection, methylation interference, and retardation of the electrophoretic mobility of specific DNA fragments following their incubation with embryonic erythroid (K562) cell extracts. These embryonic globin DNA binding proteins will be purified using one or more of the above binding assays, and genes encoding them will be cloned using antibody or oligonucleotide probes. Finally, using a novel genetic assay, other trans-acting regulators of embryonic globin gene expression, which do not themselves bind directly to DNA, will be identified and cloned.
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