IDENTIFICATION AND CHARACTERIZATION OF A FUNCTIONAL PROMOTER REGION IN THE HUMAN EOSINOPHIL IL-5 RECEPTOR-ALPHA SUBUNIT GENE

The molecular basis for the commitment of multipotential myeloid proge nitors to the eosinophil lineage, and the transcriptional mechanisms b y which eosinophil-specific genes are subsequently expressed and regul ated during eosinophil development are currently unknown. Interleukin- 5 (IL-5) is a T cell and mast cell-derived cytokine with actions restr icted to the eosinophil and closely related basophil lineages in human s. The high affinity receptor for IL-5 (IL-5R) is composed of an alpha subunit (IL-5R alpha) expressed by the eosinophil lineage, that assoc iates with a beta(c) subunit shared with the receptors for IL-3 and gr anulocyte macrophage colony stimulating factor (GM-CSF). As a prerequi site to studies of the transcriptional regulation of the IL-5R alpha s ubunit gene, we used three different methods, including primer extensi on, 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-5R alpha exon 1. To initially identify t he IL-5R alpha promoter, 3.5 kilobases (kb) and 561 bp of the 5' seque nce flanking the transcriptional start site were subcloned into the pr omoterless pXP2-luciferase vector, Transient transfection of these con structs into an eosinophil-committed HL-60 subline, clone HL-60-C15, i nduced the expression of similar to 240-fold greater luciferase activi ty than the promoterless vector, identifying a strong functionally act ive promoter region within the 561 bp of sequence proximal to the tran scriptional 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 f or promoter activity, a series of 5' deletion mutants of the 561-bp re gion were generated in the pXP2-luciferase vector. Deletion of the reg ion 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-5R alpha 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 s equences for known transcription factor binding sites were not present in the 34-bp region of the promoter required for maximal activity, su ggesting unique myeloid- and possibly eosinophil-specific regulatory e lements, Using electrophoretic mobility shift assays, we have identifi ed a nuclear factor(s) that binds to the 34-bp functional region of th e the promoter and that is expressed in the myeloid and eosinophilic c ell Lines in which the promoter is active, but not in non-myeloid or n on-hematopoietic lines. This functional promoter segment likely serves as the binding site for a myeloid- and possibly eosinophil-specific t ranscription factor(s). Further study of the IL-5R alpha promoter shou ld elucidate unique transcriptional features of this gene whose expres sion is essential to the commitment and differentiation of multipotent ial myeloid progenitors to the eosinophil lineage and to the functiona l activation of the mature cell.