Characterization of Inherited Differences in Transcription of the Human Integrin α2 Gene

Inherited, single-base substitutions are found at only two positions, C−52T and C−92G, within the proximal 5′-regulatory region (within −1096 to +48) of the human integrin α2gene. We recently reported that the T−52substitution results in decreased binding of transcription factor Sp1 to adjacent binding sites, decreased transcription of the α2 gene, and reduced densities of platelet α2β1. In this study, we identify an additional Sp1-binding site at position −107 to −99 and show that the adjacent dimorphic sequence C−92G also influences the rate of gene transcription. In the erythroleukemia cell line Dami, transfected promoter-luciferase constructs bearing the G−92 sequence exhibit roughly a 3-fold decrease in activity relative to the C−92constructs. In transfected CHRF-288-11 megakaryocytic cells, the corresponding activity decreases by 5-fold. DNase I footprinting of the promoter region with Dami nuclear extracts showed a protected segment at −107 to −99 that can be deprotected by coincubation with molar excess of a consensus Sp1 oligonucleotide. Gel mobility shift assays and supershift assays with specific antibodies indicate that Sp1 binds to this region of the α2 gene promoter. Mutation of the Sp1 binding element within −107 to −99 in constructs containing either C−92 or G−92abolishes basal promoter activity and eliminates the binding of Sp1. The G−92 sequence has a gene frequency of 0.15 in a typical Caucasian population, and the presence of this allele correlates with reduced densities of platelet α2β1. The combined substitution G−92/T−52 has an additive influence on gene transcription, resulting in an 8-fold decrease in transfected Dami cells or a 20-fold decrease in transfected CHRF-288-11 cells. In summary, the natural dimorphism C−92G within the proximal 5′-regulatory region of the human integrin α2 gene contributes to the regulation of integrin α2β1 expression on megakaryocytes and blood platelets and must thereby modulate collagen-related platelet function in vivo.