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 the disease. The goal of this project is the identification and isolation of trans-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 us to hypothesize a link between the regulation of iron metabolism and regulation of globin genes - particularly that a subunit of ferritin (or a structurally-related protein) is a repressor of adult beta globin expression in K562 cells and mediates its effect by binding to the DNA and/or associated proteins of one or more of the 5' control regions of the beta globin gene. Results: H and L ferritins and a partially purified ferritin-like protein from K562 cell nuclear extracts bind to the 5' beta Rsa fragment (-223 to - 129) whereas transferrin and apotransferrin do not. The binding of the K562 ferritin preparations increases with the amount of protein added. Gel shift experiments with anti-ferritin antisera show that ferritin subunits or a ferritin-like protein(s) present in all K562 nuclear extracts is responsible for this binding. Gel shifts with nuclear extracts, anti- ferritin antiserum, and oligonucleotides have narrowed the binding site to a 37bp stretch (-165/-129) within a putative negative regulatory region. Interestingly, this region contains a DNA version of the consensus hexanucleotide (CAGTGN) of an iron responsive element (IRE). Competition gel shifts with mutated oligonucleotides and DNase-I footprinting are in progress to further localize the binding site, and the binding protein is also being further purified.