Klement J F, Cvekl A, Piatigorsky J
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-0006.
J Biol Chem. 1993 Mar 25;268(9):6777-84.
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.
αA-晶体蛋白是脊椎动物眼晶状体中一种丰富的可溶性蛋白质。除TATA框外，通过接头扫描诱变、DNA酶I足迹法和凝胶迁移率变动实验，已鉴定出鸡αA-晶体蛋白基因的四个正向顺式调控元件。这里描述的调控元件已被命名为DE2A（位于-144至-134位）、DE2B（位于-128至-118位）和DE1A（位于-114至-103位）。DE2A和DE2B在-141至-118位之间形成一个对称二聚体（5'-AGACTGTCAT....AGGTCAGTCT-3'），这与这两个元件与晶状体核蛋白形成的复合物在迁移率上的密切相似性一致。在转染到原代鸡胚晶状体上皮细胞中的细菌氯霉素乙酰转移酶报告基因中，DE2A、DE2B和DE1A中的突变导致启动子活性丧失，这在凝胶迁移率变动分析中导致与晶状体核蛋白竞争复合物形成的能力相应丧失。小鼠αA-晶体蛋白启动子功能所必需的αA-CRYBP1样位点（-67/-57）的突变并不影响鸡启动子的活性。DNA酶I足迹法和凝胶迁移率变动实验证实了先前观察到的核蛋白与-153至-140位的对称二聚体结合。与DE2A、DE2B和DE1A相反，诱变和凝胶迁移率变动实验未能将-153/-140对称二聚体的功能与蛋白质结合联系起来。DE2A、DE2B和DE1A与该基因的调控元件αCE1（-162/-134）、αCE3（-135/-121）和αCE2（-119/-99）（松尾，I.，和安田，K.（1992年）核酸研究20，3701-3712）非常吻合。然而，目前的结果表明，αCEI的晶状体增强子活性是由于-141/-134序列，它形成了DE2A/DE2B对称二聚体的上半部分，而不是先前怀疑的-153/-140对称二聚体。
