Understanding the molecular regulation of developmental hemoglobin switching would be useful for increasing fetal hemoglobin in adult humans with sickle cell anemia and other hemoglobinopathies, a manipulation likely to alleviate the clinical manifestations of these diseases. The goal of this project is the identification and isolation of 2 n""""""""-acting factors that regulate hemoglobin switching, particularly protein regulatory factors that bind to the 5' noncoding regions of the human beta globin gene. Hypothesis: The observation that K562 cells, and embryonic erythroid cells, have a large amount of ferritin but do not express adult beta globin, whereas adult erythroid cells have opposite characteristics, led me to hypothesize a link between the regulation of iron metabolism and regulation of globin genes, i.e., that a protein involved in the production of ferritin is a repressor of adult beta globin expression in K562 cells. Results: A protein partially purified from K562 nuclear extracts binds the adult beta globin Rsa fragment (-223 to -129) which contains a putative positive control region (5' portion) and (3') possible negative regulatory sequences. Gel shift assays show that the protein binds the 3' portion of the Rsa fragment (-165/-129), a region that contains a DNA version of the consensus hexanucleotide (CAGTGN) of an iron responsive element (IRE), rather than the 5'positive control region (-233/-188). This protein, which is provisionally named locus represser protein, or LRP, is a ferritin-like polypeptide or ferritin subunit as shown by its with and-ferritin antisera and its Ferritin-like physical chacteristics (heat- and proteinase K-resistance), and is prominent in K562 and -ela, but not adult chicken erythroid or MEL, cell nuclear extracts. Pro-ected experiments will further characterize this LRP and elucidate its binding sites. In vivo footprinting will reveal DNA sites that are proteinoccupied in embryonic and adult erythroid cells.
