Functional elements DE2A, DE2B, and DE1A and the TATA box are required for activity of the chicken alpha A-crystallin gene in transfected lens epithelial cells.
alpha A-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 alpha A-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 alpha A-CRYBP1-like site (-67/-57), necessary for function of the mouse alpha A-crystallin promoter, did not affect the activity of the chicken promoter. The DNase I footprinting 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 alpha CE1 (-162/-134), alpha CE3 (-135/-121), and alpha CE2 (-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 alpha CE1 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.
Klement
J F
JF
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-0006.
Cvekl
A
A
Piatigorsky
J
J
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D003459:Crystallins / Q000235:genetics*; D004247:DNA; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005786:Gene Expression Regulation; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012045:Regulatory Sequences, Nucleic Acid; D016385:TATA Box; D014158:Transcription, Genetic; D014162:Transfection
D003459:Crystallins; D004247:DNA