Transcription factor GATA-1 is expressed in four hematopoietic lineages-erythroid cells, megakaryocytes, mast cells, and eosinophils-and is competent to reprogram myb/ets-
transformed chicken myelomonocytic cells into three of these (erythroid, megakaryocytic, and eosinophilic) (1
). Intermediate levels of GATA-1 protein lead to formation of eosinophils, and higher levels to thromboblasts and probably erythroblasts. Gene targeting in mice has revealed essential roles for GATA-1 in both erythroid and megakaryocytic differentiation. In both contexts, the loss of GATA-1 arrests cellular maturation at a relatively late stage after lineage commitment. Overlapping expression of the related factor GATA-2 very likely accounts for lineage commitment in the absence of GATA-1. The extent to which GATA-1 is required for eosinophil or mast cell development or function is unresolved.
Transcription of the GATA-1 gene itself is directed by several cis-regulatory elements. In the sole nonhematopoietic site of expression an upstream promoter (IT) of murine GATA-1 directs Sertoli-cell specific expression (2
). Hematopoietic cells express GATA-1 predominantly from a downstream promoter (IE) and exhibit heterogeneous transcriptional start sites (3
). At least two DNase I hypersensitive regions neighboring, or upstream of, the IE promoter have been identified in hematopoietic cells. The more distal region (termed HSI) is hypersensitive in both erythroid and megakaryocytic cells and encompasses a potent enhancer core of 169 bp that is able to direct expression in these lineages in transgenic mice (8
). Deletion of HSI in the native chromosomal context by gene targeting ablates expression of GATA-1 in megakaryocytes (10
). The expression of GATA-1 in erythroid cells is maintained, presumably due to compensatory cis-elements located elsewhere within the GATA-1 locus.
Other sequences neighboring the IE promoter also exhibit DNase I hypersensitivity in erythroid cells. In vivo DNA footprinting has revealed protein occupancy in a high affinity palindromic (or “double”) GATA-site positioned 500-bp upstream of the heterogeneous start sites (6
). This complex GATA-site binds a single molecule of GATA-1 with an affinity ~7 times that of a single GATA site (6
). In transient transactivation reporter assays the palindromic GATA-site is required for full promoter activity. Chromatin-immunoprecipitation (CHIP) assay demonstrates direct binding of GATA-1 protein to this site in cultured erythroid cells (unpublished data). It has been proposed that the palindromic GATA-site in the GATA-1 promoter mediates positive autoregulation of GATA-1 transcription by GATA-1 itself, and thereby ensures expression of the gene once activated (6
). Indeed, transgenic experiments in zebrafish have shown that GATA-1 expression is dependent on a similar palindromic site in the zebrafish gene promoter and have been interpreted as support for this hypothesis (12
In the studies reported here we sought to determine the in vivo relevance of the palindromic GATA-site by modifying the endogenous GATA-1 locus in mice. Remarkably, mice harboring a deletion of the double GATA-site (ΔdblGATA) are unable to produce eosinophils. Unlike GATA-1-null mice, which die in utero with profound anemia and defective megakaryocyte development, ΔdblGATA mice are viable and fertile. Platelet and mast cell development appear normal, while red cell production is only subtly impaired. Thus, the double GATA-site is not required for activation or maintenance of GATA-1 expression in erythroid cells, megakaryocytes, mast cells, or earlier hematopoietic precursors, but is necessary for eosinophil development. Taken together with evidence that GATA-1 can reprogram myeloid cells to eosinophils, our findings suggest that GATA-1 is required for specification of the eosinophil lineage.