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Shared features of transcription: mutational analysis of the eosinophil/basophil Charcot‐Leyden crystal protein gene promoter
Author(s) -
Dyer Kimberly D.,
Rosenberg Helene F.
Publication year - 2000
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.67.5.691
Subject(s) - enhancer , biology , gene , microbiology and biotechnology , promoter , transcription (linguistics) , sequence motif , insertional mutagenesis , transcription factor , regulatory sequence , genetics , gene expression , mutant , linguistics , philosophy
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 ribonuclease genes. To evaluate a potentially larger role for this motif in the transcriptional regulation of eosinophil genes, we have isolated 1504 nucleotides 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 site‐specific mutagenesis has permitted further delineation of this enhancer element which, as a result of this work, is now defined as GGAGA[A/G]NNNA. Electromobility shift assays demonstrated specific binding of nuclear protein(s) from an eosinophilic clone‐15 nuclear extract to this extended motif. Similar analysis of a GATA‐1 binding site demonstrated enhancer activity, with mutagenesis resulting in a 94 ± 1.4% reduction in activity, whereas the AML1 site functioned as a gene silencer. J. Leukoc. Biol. 67: 691–698; 2000.