Sax C M, Cvekl A, Piatigorsky J
Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH, Bethesda, MD 20892, USA.
Gene. 1997 Feb 7;185(2):209-16. doi: 10.1016/s0378-1119(96)00643-9.
Lens preferred-expression of the mouse alpha A-crystallin gene (alpha A-cry) is regulated at the transcriptional level by multiple elements located in the 5' flanking region of the gene. Here we present the first analysis of the functional role of the mouse alpha A-cry +1 region and the protein(s) which bind to it. The -7/+5 region of this promoter exhibits sequence similarity with the consensus upstream stimulating factor (USF) transcription factor binding site. A wild type oligodeoxyribonucleotide (oligo) spanning the mouse alpha A-cry -15/+15 region specifically inhibited the activity of a mouse alpha A-cry promoter-cat gene fusion (p alpha A 111aCAT) in competitive co-transfection studies in the mouse alpha TN4-1 lens cell line, as did an oligo containing the adenovirus 2 major late promoter strong USF binding site. In contrast, an alpha A-cry oligo mutated (-3/+3) within the USF-like binding site did not inhibit p alpha A111aCAT activity. Western blot analysis indicated that alpha TN4-1 cells express USF1. Co-transfection of p alpha A111aCAT and a USF1 cDNA expression vector into alpha TN4-1 cells resulted in a repression of mouse alpha A-cry promoter activity. Electrophoretic mobility shift analyses (EMSA) demonstrated that proteins in an alpha TN4-1 nuclear extract form a single major complex on synthetic oligos spanning the mouse alpha A-cry -15/+15 region. The formation of this complex was inhibited by the presence of unlabeled -15/+15 oligos or an anti-USF1 antibody. In addition, purified USF1 bound to this region, producing a complex similar in size to that observed with alpha TN4-1 nuclear extracts. Taken together, our findings show that USF can bind to the mouse alpha A-cry +1 site, and support the possibility that USF plays a role in promoter activity of this gene. Sequence similarities surrounding the +1 region of the alpha A-cry gene of the mouse, mole rat, hamster, and human, as well as the previously observed utilization of USF by different cry promoters suggest that USF contributes to the high expression of many crys in the ocular lens of diverse species.
小鼠αA-晶体蛋白基因（αA-cry）在晶状体中的优先表达是由位于该基因5'侧翼区域的多个元件在转录水平上调控的。在此，我们首次分析了小鼠αA-cry +1区域及其结合蛋白的功能作用。该启动子的-7 / +5区域与共有上游刺激因子（USF）转录因子结合位点具有序列相似性。在小鼠αTN4-1晶状体细胞系的竞争性共转染研究中，跨越小鼠αA-cry -15 / +15区域的野生型寡脱氧核糖核苷酸（oligo）特异性抑制了小鼠αA-cry启动子 - cat基因融合体（pαA 111aCAT）的活性，含有腺病毒2主要晚期启动子强USF结合位点的oligo也有同样的效果。相反，在类似USF的结合位点内突变（-3 / +3）的αA-cry oligo不抑制pαA111aCAT活性。蛋白质印迹分析表明αTN4-1细胞表达USF1。将pαA111aCAT和USF1 cDNA表达载体共转染到αTN4-1细胞中导致小鼠αA-cry启动子活性受到抑制。电泳迁移率变动分析（EMSA）表明，αTN4-1核提取物中的蛋白质在跨越小鼠αA-cry -15 / +15区域的合成寡核苷酸上形成单一主要复合物。未标记的-15 / +15 oligos或抗USF1抗体的存在会抑制该复合物的形成。此外，纯化的USF1与该区域结合，产生与用αTN4-1核提取物观察到的大小相似的复合物。综上所述，我们的研究结果表明USF可以结合到小鼠αA-cry +1位点，并支持USF在该基因启动子活性中起作用的可能性。小鼠、摩尔大鼠、仓鼠和人类的αA-cry基因+1区域周围的序列相似性，以及先前观察到的不同cry启动子对USF的利用，表明USF有助于多种物种眼晶状体中许多cry的高表达。
