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 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.
Handen
J S
JS
Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
Rosenberg
H F
HF
D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D047208:Eosinophil-Derived Neurotoxin; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011485:Protein Binding; D011506:Proteins / Q000235:genetics*; D012260:Ribonucleases; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011506:Proteins; D014157:Transcription Factors; D004247:DNA; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases; C479221:RNASE2 protein, human