The long-term objective of our research is to understand the tissue specific and developmental regulation of globin gene expression at the molecular level. We are particularly interested in describing the basis for the switch from fetal to adult hemoglobin. It is hoped that this information will not only provide a more detailed understanding of gene expression in mammalian cells but will also be helpful in developing ways to prevent or reverse the HbF to HbA switch in individuals who have altered or deficient beta globin chain production. Our approach is to identify nucleotide sequences in or flanking the globin genes which bind factors and to test the importance of these regions in the regulation of globin gene expression. We have identified a factor which binds to a region in the 5'flanking sequence of the human gamma globin gene. This region has been identified by gel electrophoresis band-retardation assays and by DNase I footprinting. We plan to determine the importance of this region in expression by introducing nucleotide changes into the binding sequence by site specific mutagenesis and comparing binding activity and gene expression levels using in vitro systems. The factors will be purified using a combination of standard protein fractionation methods and DNA affinity procedures. We have also identified erythroid specific factors which bind to defined regions of the mouse beta maj globin gene. The involvement of these sequences in expression will be determined by introducing the binding regions into the corresponding position of the human gamma globin gene and/or flanking this gene and determining if the gamma globin gene is now expressed as an adult gene in transgenic mice. The gamma globin gene has been shown to be expressed only in the embryonic erythroid compartment of such animals. Site specific mutagenesis of the DNA regions which bind factors will also be performed and the effect these alterations have on protein binding and expression will be determined. Once specific factors have been identified, they will be purified and their role in globin gene expression defined.
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