Sequences Downstream of the Erythroid Promoter Are Required for High Level Expression of the Human α-Spectrin Gene

Abstract α-Spectrin is a membrane protein critical for the flexibility and stability of the erythrocyte. We are attempting to identify and characterize the molecular mechanisms controlling the erythroid-specific expression of the α-spectrin gene. Previously, we demonstrated that the core promoter of the human α-spectrin gene directed low levels of erythroid-specific expression only in the early stages of erythroid differentiation. We have now identified a region 3′ of the core promoter that contains a DNase I hypersensitive site and directs high level, erythroid-specific expression in reporter gene/transfection assays. In vitro DNase I footprinting and electrophoretic mobility shift assays identified two functional GATA-1 sites in this region. Both GATA-1 sites were required for full activity, suggesting that elements binding to each site interact in a combinatorial manner. This region did not demonstrate enhancer activity in any orientation or position relative to either the α-spectrin core promoter or the thymidine kinase promoter in reporter gene assays. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of this region and occupancy by GATA-1 and CBP (cAMP-response element-binding protein (CREB)-binding protein). These results demonstrate that a region 3′ of the α-spectrin core promoter contains a GATA-1-dependent positive regulatory element that is required in its proper genomic orientation. This is an excellent candidate region for mutations associated with decreased α-spectrin gene expression in patients with hereditary spherocytosis and hereditary pyropoikilocytosis.