Functional elements DE2A, DE2B, and DElA and the TATA box are required for activity of the chicken αA-Crystallin gene in Transfected lens epithelial cells

αA-crystallin is an abundant soluble protein of the vertebrate eye lens. In addition to the TATA box, four positive cis-regulatory elements of the chicken αA-crystallin gene have been identified by linker scanning mutagenesis, DNase I footprinting, and gel mobility shift experiments. The regulatory elements described here have been named DE2A (at positions -144 to -134), DE2B (at positions -128 to -118), and DE1A (at positions -114 to -103). DE2A and DE2B form a dyad of symmetry between positions -141 and -118 (5′-AGACTGTCAT. . . .AGGTCAGTCT-3′), consistent with the close similarity in the mobility of complexes formed with lens nuclear proteins by these two elements. Mutations in DE2A, DE2B, and DE1A leading to loss of promoter activity using the bacterial chloramphenicol acetyltransferase reporter gene transfected into primary embryonic chicken lens epithelial cells resulted in a corresponding loss in the ability to compete for complex formation with lens nuclear proteins in gel mobility shift assays. Mutation of the αA-CRYBP1-like site (-67/-57), necessary for function of the mouse αA-crystallin promoter, did not affect the activity of the chicken promoter. The DNase I foot-printing and gel mobility shift experiments confirmed the previously noted binding of nuclear proteins to a dyad of symmetry at positions -153 to -140. In contrast to DE2A, DE2B, and DE1A, mutagenesis and gel mobility shift experiments failed to correlate function and protein binding for the -153/-140 dyad. DE2A, DE2B, and DE1A agree well with the regulatory elements αCE1 (-162/-134), αCE3 (-135/-121), and αCE2 (-119/-99) (Matsuo, I., and Yasuda, K. (1992) Nucleic Acids Res. 20, 3701-3712) for this gene. The present results suggest, however, that the lens enhancer activity of αCE1 is due to the sequence -141/ -134, which forms the upper half of the DE2A/DE2B dyad of symmetry, rather than the -153/-140 dyad as previously suspected.