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To understand the functional roles that the GATA factors may play during hematopoietic cell differentiation, we examined the expression of GATA factor mRNAs and protein products in various human cell lines. Blot hybridization analyses demonstrated that GATA-1 and GATA-2 mRNAs are expressed abundantly in a set of cell lines established from human myelogenous leukemia cells, but the expression pattern of each factor is distinct. GATA-2 mRNA is expressed in all cell lines tested that express erythroid markers, and, in addition, the mRNA is also expressed in three CD34+ cell lines and two early myeloid cell lines. In contrast, the expression of GATA-1 mRNA showed tight correlation to that of the erythroid/megakaryocytic lineage markers. We also found that the GATA-2 probe identifies two types of mRNA. Structural analysis of genomic DNA clones encoding human GATA-2 coupled with RNA blot analysis demonstrated that there exists an alternative use of polyadenylation consensus sequences in a single exon and this causes the molecular heterogeneity among GATA-2 mRNAs. Through immunochemical and immunohistochemical analyses using anti-GATA-1- and anti-GATA-2- specific antibodies, GATA-2 protein was clearly shown to be present in the nuclei of leukemia-derived early myeloid and CD34+ cell lines, whereas both GATA-1 and GATA-2 proteins are expressed in erythroid/megakaryocytic cell lines. Thus, the expression profile of GATA-2 is consistent with the hypothesis that GATA-2 plays unique roles for the transcriptional activation of genes in cells at an early stage of hematopoietic differentiation and in developing cells of the erythroid and myeloid lineages.

Although the formation of terminally differentiated erythroid cells has been shown to require the presence of a functional GATA-1 gene in vivo, the role of this transcription factor and other members of the GATA family at earlier stages of erythroid differentiation is unclear. In this report, the expression of GATA-1, GATA-2, and GATA-3 has been examined in enriched peripheral blood progenitors before and after culture in a well-characterized liquid culture system. In addition primary leukemic cells as well as several erythroleukemic and nonerythroid cell lines were analyzed for GATA factor expression. The results show that the profile of GATA factor expression in erythroid cells is distinct from that of myeloid or lymphoid lineages. Erythroleukemic cell lines express little or no GATA-3, but high levels of GATA-1 and GATA-2. When they are induced to display the terminal erythroid phenotype, little change in the level of GATA-1 is detected but a significant decline in the levels of GATA-2 is observed commensurate with the degree of maturation achieved by the cells. Enrichment of erythroid progenitors from peripheral blood leads to selection of cells that express both GATA-1 and GATA-2. As the enriched populations are cultured in suspension in the presence of multiple cytokines, the levels of both GATA-1 and GATA-2 initially increase. However, in cultures containing only erythropoietin, which show exclusive erythroid differentiation, the levels of GATA-1 continue to increase, whereas GATA-2 expression declines as erythroid maturation progresses. In contrast, cultures lacking Epo (ie, with interleukin-3 and kit ligand) display limited progression towards both the myeloid and erythroid pathways, and high levels of expression of both GATA-1 and GATA-2 are maintained. Despite the initial upregulation of GATA-1 expression in the latter cultures, terminal erythroid differentiation does not occur in the absence of erythropoietin. These results indicate that GATA-1 upregulation is associated with both the initiation and the maintenance of the erythroid program, but that these two processes appear to be under separate regulatory control. Thus, the dynamic changes in the levels of different GATA factors that occur during primary erythroid differentiation suggest that the levels of these factors may influence the progression to specific hematopoietic pathways.