INTRONIC ENHANCER ACTIVITY OF THE EOSINOPHIL-DERIVED NEUROTOXIN (RNS2) AND EOSINOPHIL CATIONIC PROTEIN (RNS3) GENES IS MEDIATED BY AN NFAT-1 CONSENSUS BINDING SEQUENCE

The eosinophil derived neurotoxin (EDN) and eosinophil cationic protei n (ECP) are both small, cationic ribonuclease toxins that are stored i n and secreted by activated human eosinophilic leukocytes. We have pre viously shown that optimal expression of the EDN gene is dependent on an interaction between an intronic enhancer element or elements and th e 5' promoter region. Here we present evidence demonstrating that the gene encoding ECP is regulated in an analogous fashion and that an int ronic enhancer element functioning in both genes is a consensus bindin g sequence for the transcription factor NFAT-1. Our initial results de monstrate that one or more nuclear proteins isolated from human promye locytic leukemia (HL-60) cells bind specifically at this consensus sit e (5'-GGAGAA-3') within the intron of the EDN gene and that disruption of this sequence reduced the characteristic 20-30-fold increase in re porter gene activity observed with the tandem EDN promoter/exon 1/intr on construct to background levels. The NFAT-1 consensus site in the EC P 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 furth er enhancement of the reporter gene activity supported by the ECP prom oter/exon 1/intron construct. Interestingly, no ''supershift'' was obs erved in gel shift assays performed in the presence of anti NFAT-1 ant iserum, suggesting that a nuclear protein other than NFAT-1 may be act ing at this consensus site.