Several lines of clinical and experimental evidence suggest that elevated levels of fetal hemoglobin (HbF) may improve the clinical course of individuals with sickle disease and b-thalassemia. A number of cytotoxic drugs have shown to enhance gamma-globin synthesis (and HbF levels) in animals and patients with hemoglobinopathies, although the mechanism of action of these is not known. The K562 human erythroleukemia cell line shows constitutively low levels of embryonic and fetal hemoglobin, and can be reversibly induced to increase gamma-globin gene expression in response to hydroxyurea and other agents. We are therefore using the K562 and human erythroid progenitor cells as a model system to understand the mechanism of induction of globin gene transcription. Both cells have been grown in the presence of 25mM hemin and 100mM hydroxyurea. By gel shift analysis, we show different mobility shift patterns at -226 to -134 of the g-gene promoter in the hemin and HU treated cells. This suggests the possibility of a novel binding activity underlying the hemin and hydroxyurea effect. The status of in vivo protein-DNA interactions in the promoter region of the Ggamma-gene was investigated by a new ligation-mediated polymerase chain reaction (LMPCR), using N-ethyl,N- nitrosourea(ENU). We find that ENU acts efficiently on suspension cells and can detect protein-DNA interactions not revealed by the commonly used dimethyl sulfate (DMS) method. In vivo footprinting results suggest that both CCAAT sites are activated in gamma-globin gene transcription, octamer binding site is specific for hemin induction, and -50 and-200 regions have major roles in gamma-globin transcriptional control. The protection pattern in the -200 region strongly suggests that repressor molecules bind in adult stage and activators in untreated and hemin treated K562 cells. Incubation in hydroxyurea inhibits binding at this region. It is hoped that further identification, characterization and purification of these putative binding proteins would not only extend the current knowledge of the molecular basis of the fetal to adult """"""""switch"""""""", but also suggest novel pharmacological approach to the reversal of this switch in several clinically significant hemoglobinopathies.
