FUNCTIONAL-CHARACTERIZATION OF THE PROMOTER FOR THE GENE ENCODING HUMAN EOSINOPHIL PEROXIDASE

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 cloned 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-pXPS 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.