Identification and characterization of a functional promoter region in the human eosinophil IL-5 receptor α subunit gene

The molecular basis for the commitment of multipotential myeloid progenitors to the eosinophil lineage, and the transcriptional mechanisms by which eosinophil-specific genes are subsequently expressed and regulated during eosinophil development are currently unknown. Interleukin-5 (IL-5) is a T cell and mast cell-derived cytokine with actions restricted to the eosinophil and closely related basophil lineages in humans. The high affinity receptor for IL-5 (IL-5R) is composed of an α subunit (IL-5Rα) expressed by the eosinophil lineage, that associates with a β(c) subunit shared with the receptors for IL-3 and granulocyte-macrophage colony stimulating factor (GM- CSF). As a prerequisite to studies of the transcriptional regulation of the IL-5Rα subunit gene, we used three different methods, including primer extension, RNase protection, and 5'-RACE to precisely map the transcriptional start site to a position 15 base pairs (bp) upstream of the 5' end of the published sequence of IL-5Rα exon 1. To initially identify the IL-5Rα promoter, 3.5 kilobases (kb) and 561 bp of the 5' sequence flanking the transcriptional start site were subcloned into the promoterless pXP2- luciferase vector. Transient transfection of these constructs into an eosinophil-committed HL-60 subline, clone HL-60-C15, induced the expression of ~240-fold greater luciferase activity than the promoterless vector, identifying a strong functionally active promoter region within the 561 bp of sequence proximal to the transcriptional start site and with activity equivalent to pXP2 constructs containing the entire 3.5 kb of upstream sequence. To more precisely localize the cis-acting regulatory elements in this region important for promoter activity, a series of 5' deletion mutants of the 561-bp region were generated in the pXP2-luciferase vector. Deletion of the region between bp -432 and -398 reduced promoter activity by more than 80% in the HL-60-C15 cell line. Further analyses of the activity of the IL- 5Rα promoter constructs in various other eosinophil, myeloid, and non- myeloid cell lines indicated that the promoter was relatively myeloid and eosinophil lineage-specific in its expression. Consensus sequences for known transcription factor binding sites were not present in the 34-bp region of the promoter required for maximal activity, suggesting unique myeloid- and possibly eosinophil-specific regulatory elements. Using electrophoretic mobility shift assays, we have identified a nuclear factor(s) that binds to the 34-bp functional region of the promoter and that is expressed in the myeloid and eosinophilic cell lines in which the promoter is active, but not in non-myeloid or non-hematopoietic lines. This functional promoter segment likely serves as the binding site for a myeloid- and possibly eosinophil- specific transcription factor(s). Further study of the IL-5Rα promoter should elucidate unique transcriptional features of this gene whose expression is essential to the commitment and differentiation of multipotential myeloid progenitors to the eosinophil lineage and to the functional activation of the mature cell.