J. Zhang et al., EVIDENCE FOR MULTIPLE PROMOTERS OF THE HUMAN IL-5 RECEPTOR-ALPHA SUBUNIT GENE - A NOVEL 6-BASE PAIR ELEMENT DETERMINES CELL-SPECIFIC PROMOTER FUNCTION, The Journal of immunology, 159(11), 1997, pp. 5412-5421
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.