Therapeutic approaches to hemoglobinopathies such as sickle cell anemia as well as thalassemia include strategies to alter globin gene expression during erythropoiesis. Understanding the developmental switch that determines expression of embryonic, fetal and adult hemoglobins is of particular relevance as in adults with sickle cell anemia or beta-thalassemia, increased fetal hemoglobin levels can be associated with more mild forms of disease. Stimulation by erythropoietin results in the proliferation and differentiation of erythroid progenitors and increased production of transcription factors such as GATA-1 and SCL/Tal-1, further induction of erythropoietin receptor expression, and activation of globin gene expression. The requirement for GATA-1 and SCL/Tal-1 for embryonic hematopoiesis suggests that these factors are also upstream of events leading to differentiation along the erythroid lineage. We use transfection of these transcription factors into erythroid cells to determine their direct effect on erythropoietin receptor expression and globin gene expression. GATA-1 can transactivate the erythropoietin receptor via a GATA-1 binding site in its proximal promoter, suppresses GATA-2 and epsilon globin gene expression in K562 cells with an embryonic erythroid phenotype. In K562 cells, SCL/Tal-1 increases expression of the erythropoietin receptor. The 5 UTR flanking the erythopoietin receptor coding region contains three E-box motifs, potential binding sites for SCL/Tal-1. Reporter gene assays demonstrate that in addition to the GATA-1 binding site in the proximal promoter, these three E-box motifs are important for transcription activation in erythroid cells. Stable clones of erythroleukemia K562 cells containing a SCL/Tal-1 expression vector were isolated and the resultant expression profile assessed incorporating real-time RT-PCR and expression microarray techology. Examination of target genes in the SCL/Tal-1/K562 cells show an increase in expression of GATA-1, beta-globin and carbonic anhydrase I. These data suggest that in addition to activation of the erythropoietin receptor, there was a shift to a mature erythroid phenotype by SCL/Tal-1. As an approach to genetically manipulate globin gene expression, we are using a multiribozyme to target mutant globin transcripts. Ribozymes introduced into K562 cells were able to suppress alpha-globin production. Ribozymes specific for the beta-S globin transcript are now under investigation. Combinations of globin specific ribozymes with vectors targeted to increase gamma-globin production can be of potential use in maintaining alpha/beta-like globin chain balance. - Sickle cell anemia, hemoglobinopathy, globin, ribozyme, therapy, transcription, GATA-1, SCL/Tal-1
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