The haematopoietic system consists of eight distinct cell lineages that are derived from common precursors in an ongoing process that involves the selective activation of gene expression programmes characteristic of individual cell types. Several transcriptional regulators have been shown to be involved in haematopoietic gene regulation. Phenotypes of c-Myb-, AML-1-, GATA-2- and SCL-1-deficient mice reveal a profound failure of definitive haematopoesis (Mucenski et al., 1991; Tsai et al., 1994; Okuda et al., 1996; Porcher et al., 1996; Robb et al., 1996). In contrast, GATA-1, PU.1, E2A, C/EBPα, C/EBPβ and C/EBPϵ knockouts have more selective phenotypes (Bain et al., 1994; Scott et al., 1994; Zhuang et al., 1994; Screpanti et al., 1995; Tanaka et al., 1995; Fujiwara et al., 1996; McKercher et al., 1996; Yamanaka et al., 1997; Zhang et al., 1997). Thus, it appears that a combination of transcription factors is required for the correct execution of the haematopoietic differentiation programme. Typically, cell type-specific genes are regulated by a combination of these factors characteristic of the lineage. In the non-lymphoid compartment, erythroid-specific genes generally are regulated by combinations of GATA, Ets and Maf family transcription factors and granulocyte/macrophage-specific genes by combinations of AML1/CBFβ, C/EBP, PU.1 and c-Myb. For example, the genes encoding the receptors for the myeloid growth factors granulocyte (G)-, macrophage (M)- and GM-colony-stimulating factor (CSF) all contain binding sites for C/EBP and PU.1 (and, in the case of M-CSF, a receptor for AML-1 also) (Hohaus et al., 1995; Smith et al., 1996; Zhang et al., 1996). The neutrophil elastase (NE) and Mim-1 promoters are activated by C/EBPs in cooperation with Myb (and, for the NE promoter, also PU.1) (Ness et al., 1993; Oelgeschläger et al., 1996). Very little, however, is known about the regulation of eosinophil-specific genes. No eosinophil-specific promoters have been analysed at the molecular level, and no transcription factor knockout has been shown specifically to affect this lineage.
Previously we have shown that the E26 avian erythroleukaemia virus is able selectively to transform multipotent haematopoietic precursors. This virus encodes a tripartite fusion protein between the retroviral Gag protein and two cellular transciption factors c-Myb and c-Ets-1. Cells transformed by this virus spontaneously differentiate along erythroid and thrombocytic lineages and can be induced to differentiate along myelomonocytic and eosinophilic lineages by treatment with phorbol esters or by overexpression of kinase-type oncogenes (Graf et al., 1992; Kraut et al., 1994; Frampton et al., 1995; Rossi et al., 1996).
Using this in vitro differentiation system, we developed a monoclonal antibody, EOS47, which selectively identifies a cell surface antigen expressed by eosinophils but not by cells of any other haematopoietic lineage. Sequence analysis of the EOS47-encoding cDNA identified it as the avian homologue of the human oncofetal antigen, melanotransferrin (McNagny et al., 1996). Within the haematopoietic system, we have shown that the onset of EOS47 expression on normal eosinophils occurs as cells commit to this lineage and precedes the expression of more mature eosinophil markers such as eosinophil peroxidase and the formation of mature granules (McNagny et al., 1996; K.M.McNagny, unpublished). Likewise, it is one of the first genes to be activated when eosinophil differentiation is induced in vitro, either by ectopic GATA-1 expression in chicken myeloblasts or by activation of a conditional C/EBPβ allele in chicken multipotent precursors (Kulessa et al., 1995; Müller et al., 1995). Thus, EOS47 is the earliest and most specific marker of eosinophils described to date and is likely to be regulated by factors directly involved in eosinophil lineage determination.
To approach the molecular basis of eosinophil lineage determination, we therefore analysed the regulation of the EOS47 gene. We report here the identification of its eosinophil-specific promoter and the identification of the promoter elements critical for expression of EOS47 in eosinophils. Our data suggest that an interplay between Ets, C/EBP and GATA factors is required for restricted expression of this protein and probably for proper eosinophil lineage formation.