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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 1136-1143
The human ankyrin-1 gene is selectively transcribed in erythroid
cell lines despite the presence of a housekeeping-like promoter
Patrick G. Gallagher,
Marc Romana,
William T. Tse,
Samuel E. Lux, and
Bernard G. Forget
From the Departments of Pediatrics, Internal Medicine and Genetics,
Yale University School of Medicine, New Haven, CT, and Division of
Hematology/Oncology, Children's Hospital, Boston, MA.
To begin to study the sequence variations identified in the 5'
flanking genomic DNA of the ankyrin gene in ankyrin-deficient hereditary spherocytosis patients and to provide additional insight into our understanding of the regulation of genes encoding erythrocyte membrane proteins, we have identified and characterized the erythroid promoter of the human ankyrin-1 gene. This compact promoter has characteristics of a housekeeping gene promoter, including very high
G+C content and enzyme restriction sites characteristic of an
HTF-island, no TATA, InR, or CCAAT consensus sequences, and multiple
transcription initiation sites. In vitro DNAseI footprinting analyses
revealed binding sites for GATA-1, CACCC-binding, and CGCCC-binding
proteins. Transfection of ankyrin promoter/reporter plasmids into
tissue culture cell lines yielded expression in erythroid, but not
muscle, neural, or HeLa cells. Electrophoretic mobility shift assays,
including competition and antibody supershift experiments, demonstrated
binding of GATA-1, BKLF, and Sp1 to core ankyrin promoter sequences. In
transfection assays, mutation of the Sp1 site had no effect on reporter
gene expression, mutation of the CACCC site decreased expression by
half, and mutation of the GATA-1 site completely abolished activity.
The ankyrin gene erythroid promoter was transactivated in heterologous
cells by forced expression of GATA-1 and to a lesser degree BKLF.
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