The molecular basis for commitment of progenitors to the eosinophil lineage and 
mechanisms by which eosinophil-specific genes are expressed and regulated during 
differentiation is unknown. Expression of eosinophil peroxidase (EPO) is 
restricted to the eosinophil lineage. To understand the mechanisms involved in 
transcriptional regulation of EPO gene expression, we clone the region of the 
EPO gene upstream of the transcriptional start site and analyzed the cis-acting 
elements required for EPO promoter activity in an eosinophil-inducible leukemic 
cell line, HL-60-C15. The 5'-flanking region of the EPO gene containing 1.5 
kilobases of sequence upstream of the transcriptional start site was subcloned 
into the promoterless pXP2-luciferase vector. The EPO-pXP2 construct and 5' 
deletion mutants were electroporated into HL-60-C15 cells and luciferase 
reporter activity assessed. The -1.5-kilobase EPO-pXP2 promoter construct 
reproducibly expressed > 120-fold more luciferase activity than did promoterless 
pXP2, and a 12-fold (90%) decrease in promoter activity was obtained when 
sequences between -122 and -45 base pairs (bp) were deleted. The specificity of 
the EPO promoter for the eosinophil lineage was analyzed by transfecting the 
EPO-pXP2 constructs and deletion mutants into HL-60-C15 cells and the parental 
HL-60 line; EPO promoter activity was 8-10-fold less in the HL-60 parental line, 
suggesting lineage specific elements in the -122 to -45 bp region. To further 
characterize regulatory sequences important for promoter activity, we performed 
linker-scanning analysis on the -122 to -45 bp region and identified a number of 
positively and negatively acting elements in the promoter. DNase I footprinting 
was performed with HL-60-C15, HL-60, and HeLa nuclear extracts to identify 
nuclear proteins that may bind to the functional elements; these experiments 
identified three protected regions of the EPO promoter which correspond to the 
functional segments defined by linker-scanning analysis and which contain 
consensus, potential binding sites for Egr-1, H4TF-1, PuF, CTCF, UBP-1, and 
GaEII transcription factors. Further study of EPO promoter regulation should 
elucidate unique transcriptional features of eosinophil gene regulation in 
granulocyte development.