No abstract

The eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are both small, cationic ribonuclease toxins that are stored in and secreted by activated human eosinophilic leukocytes. We have previously shown that optimal expression of the EDN gene is dependent on an interaction between an intronic enhancer element or elements and the 5 promoter region. Here we present evidence demonstrating that the gene encoding ECP is regulated in an analogous fashion and that an intronic enhancer element functioning in both genes is a consensus binding sequence for the transcription factor NFAT-1. Our initial results demonstrate that one or more nuclear proteins isolated from human promyelocytic leukemia (HL-60) cells bind specifically at this consensus site (5-GGAGAA-3) within the intron of the EDN gene and that disruption of this sequence reduced the characteristic 20 -30-fold increase in reporter gene activity observed with the tandem EDN promoter/ exon 1/intron construct to background levels. The NFAT-1 consensus site in the ECP gene differs from that found in the EDN gene by a single nucleotide (5-GGAGAG-3); the conversion of the 3 G to an A resulted in a further enhancement of the reporter gene activity supported by the ECP promoter/exon 1/intron construct. Interestingly, no "supershift" was observed in gel shift assays performed in the presence of anti-NFAT-1 antiserum, suggesting that a nuclear protein other than NFAT-1 may be acting at this consensus site. Eosinophil-derived neurotoxin (EDN1

The eosinophil-derived neurotoxin (EDN/RNS2) is a member of the mammalian ribonuclease gene family and is one of four proteins found in the large specific granules of human eosinophilic leukocytes. The gene encoding EDN consists of two exons, including a noncoding exon 1, separated by a single intron from the coding sequence in exon 2. We have identified a functional promoter of the EDN gene and shown that optimal expression depends on interaction between the promoter and one or more sequence elements found in the single intron. Cells of the clone 15 eosinophilic variant of the human promyelocytic HL-60 cell line were transfected with constructs that included the promoter region of the EDN gene alone, promoter with exon 1, and promoter with both exon 1 and the intron positioned 5 to the chloramphenicol acetyltransferase (CAT) reporter gene (constructs referred to as PrCAT, PrExCAT, and PrExInCAT, respectively). Although reporter gene activity from either PrCAT or PrExCAT was only 2-3 fold higher than baseline (CAT alone), inclusion of the single intron (PrExInCAT) resulted in a 28-fold increase in reporter gene activity in uninduced clone 15 cells, and an 80-fold in activity when clone 15 cells were induced to differentiate toward eosinophils with butyric acid. The intron-mediated enhancer activity was reproduced in other human hematopoietic cell lines (K562, Jurkat, U937, and HL-60), but was not found in human 293 kidney cells, suggesting that the function of the enhancer element(s) may be tissue-specific. A significant portion of the observed enhancer activity resides in the first 60 base pairs the the intron, which includes consensus binding sites for both AP-1 and NF-ATp transcription factors, and a 15-base pair segment that is identical to a sequence found in the promoter of the gene encoding the neutrophil granule protein, lactoferrin. The noncoding exon 1/single intron/coding exon 2 genomic structure is a common feature among the mammalian ribonucleases; this finding suggests the possibility of a conserved mechanism of regulation in this gene family.