EVIDENCE FOR MULTIPLE PROMOTERS OF THE HUMAN IL-5 RECEPTOR-ALPHA SUBUNIT GENE - A NOVEL 6-BASE PAIR ELEMENT DETERMINES CELL-SPECIFIC PROMOTER FUNCTION

In addition to a previously characterized promoter (P1), we now show t he existence of a second promoter for the human IL-5R alpha gene, Init ially, a genomic region (P2) 5' upstream of human IL-5R alpha exon 2 w as cloned by an inverted PCR. The transcriptional start site was then mapped to a deoxycytidine (C) residue within P2 by analyzing cellular mRNA with both the 5' rapid amplification of cDNA end-PCR and S1 nucle ase protection assays, Transfection of eosinophilic HL-60 cells with r eporter gene constructs in which either P1 or P2 was linked to the bac terial chloramphenicol acetyltransferase (CAT) gene resulted in CAT ex pression; little or no CAT expression occurred in other myeloid and no nmyeloid cell lines, Deletion studies showed that a 66-bp region, rang ing from -31 to +35, was sufficient to promote CAT expression in eosin ophilic HL-60 cells, Analysis of linker-scanning mutants identified a novel 6-bp element (5' CTAATT 3') spanning -19 to -14 that was essenti al for P2 promoter activity, In electrophoretic mobility shift assays, a P2 region from -31 to +1 containing the unique 6-bp element, when u sed as a prober formed a complex with a protein(s) that was found only in the eosinophilic cell line, This binding activity was lost upon re placement of the 6-bp element with a 6-bp linker, suggesting that this element likely serves as the binding site for an eosinophilic HL-60 c ell-specific transcription factor(s), Together, these data suggest an important role for P2 promoter in the regulation of eosinophil-specifi c expression of the human IL-5 receptor alpha gene.