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 the existence of a second promoter for the human IL-5Ralpha gene. Initially, a genomic region (P2) 5' upstream of human IL-5Ralpha exon 2 was 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 nuclease protection assays. Transfection of eosinophilic HL-60 cells with reporter gene constructs in which either P1 or P2 was linked to the bacterial chloramphenicol acetyltransferase (CAT) gene resulted in CAT expression; little or no CAT expression occurred in other myeloid and nonmyeloid cell lines. Deletion studies showed that a 66-bp region, ranging from -31 to +35, was sufficient to promote CAT expression in eosinophilic HL-60 cells. Analysis of linker-scanning mutants identified a novel 6-bp element (5' CTAATT 3') spanning -19 to -14 that was essential for P2 promoter activity. In electrophoretic mobility shift assays, a P2 region from -31 to +1 containing the unique 6-bp element, when used as a probe, formed a complex with a protein(s) that was found only in the eosinophilic cell line. This binding activity was lost upon replacement 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 cell-specific transcription factor(s). Together, these data suggest an important role for P2 promoter in the regulation of eosinophil-specific expression of the human IL-5 receptor alpha gene.
Zhang
J
J
Department of Allergy, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA. ji.zhang@spcorp.com
Kuvelkar
R
R
Cheewatrakoolpong
B
B
Williams
S
S
Egan
R W
RW
Billah
M M
MM
D001483:Base Sequence; D003001:Cloning, Molecular; D004804:Eosinophils / Q000737:chemistry; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D018123:Receptors, Interleukin / Q000235:genetics*; D053649:Receptors, Interleukin-5
D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5