The human ankyrin-1 gene is selectively transcribed in erythroid cell lines despite the presence of a housekeeping-like promoter

To begin to study the sequence variations identified in the 5' flanking gen omic DNA of the ankyrin gene in ankyrin-deficient hereditary spherocytosis patients end to provide additional insight into our understanding of the re gulation of genes encoding erythrocyte membrane proteins, we have identifie d end characterized the erythroid promoter of the human ankyrin-1 gene. Thi s compact promoter has characteristics of a housekeeping gene promoter, inc luding very high G+C content and enzyme restriction sites characteristic of an HTF-island, no TATA, InR, or CCAAT consensus sequences, and multiple tr anscription initiation sites. In vitro DNAsel footprinting analyses reveale d binding sites for GATA-1, CACCC-binding, and CGCCC-binding proteins. Tran sfection of ankyrin promoter/reporter plasmids into tissue culture cell lin es yielded expression in erythroid, but not muscle, neural, or HeLa cells. Electrophoretic mobility shift assays, including competition and antibody s upershift experiments, demonstrated binding of GATA-1, BKLF, and Sp1 to cor e ankyrin promoter sequences, In transfection assays, mutation of the Sp1 s ite had no effect on reporter gene expression, mutation of the CACCC site d ecreased expression by half, and mutation of the GATA-1 site completely abo lished activity. The ankyrin gene erythroid promoter was transactivated in heterologous cells by forced expression of GATA-1 and to a lesser degree BK LF. (C) 2000 by The American Society of Hematology.