Shared features of transcription: mutational analysis of the eosinophil/basophil Charcot-Leyden crystal protein gene promoter

The lineage-specific Charcot-Leyden crystal (CLC) protein is found in human eosinophils and basophils where it comprises 7-10% of the cellular protein content, Previous work from our laboratory has identified the motif GGAGA[ A/G] as a powerful enhancer of gene transcription in two eosinophil ribonuc lease genes. To evaluate a potentially larger role for this motif in the tr anscriptional regulation of eosinophil genes, we have isolated 1504 nucleot ides 5' to the transcriptional start site of the gene encoding CLC protein and identified a functionally active promoter that includes three distinct copies of the GGAGAA motif. Destruction of only one of the three motifs by site-directed mutagenesis resulted in loss of promoter activity (73 +/- 6% reduction), suggesting that this core motif is necessary but not sufficient to support enhanced transcriptional activity. Sequence comparisons and sit e-specific mutagenesis has permitted further delineation of this enhancer e lement which, as a result of this work, is now defined as GGAGA[A/G]NNNA. E lectromobility shift assays demonstrated specific binding of nuclear protei n(s) from an eosinophilic clone-15 nuclear extract to this extended motif, Similar analysis of a GATA-1 binding site demonstrated enhancer activity, w ith mutagenesis resulting in a 94 +/- 1.4% reduction in activity, whereas t he AML1 site functioned as a gene silencer.