Jung Y K, Fricker L D
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461.
Biochimie. 1994;76(3-4):336-45. doi: 10.1016/0300-9084(94)90168-6.
Several of the genes for enzymes involved in peptide hormone processing, such as carboxypeptidase E (CPE), do not contain a TATA box. The region surrounding the major transcription initiation site of the CPE gene has sequence homology with the 'initiator' (Inr) elements of the TATA-less terminal deoxynucleotidyltransferase (TdT) gene, and the adenovirus major late (AdML) and other promoters. To investigate the promoter region of the CPE gene, GH4C1 cells were transiently transfected with constructs containing the luciferase reporter gene attached to various portions of the rat CPE gene (-395 to +45). Positive regulator elements were detected in positions -84 to -12 and +30 to +47. However, the Inr-like element of the CPE gene (-12 to +20) produced detectable luciferase activity in the absence of upstream and downstream sequences. This region of the CPE gene was much more active when expressed in the normal (sense) orientation than when expressed in the antisense orientation. A mutation within the consensus sequence between CPE and other Inr elements was much less active than the wild-type sequence. Interestingly, deletion of the Inr and surrounding sequences produced a large increase in the transcription from upstream sites, suggesting that proteins which bind at, or near, the Inr sequence suppress transcription from other sites. To characterize GH4C1 nuclear proteins which bind to the CPE gene, Southwestern blotting, UV cross-linking, and gel shift analyses were performed. The Southwestern analysis showed that the CPE and AdML Inr sequences labeled several proteins of similar sizes which are distinct from the transcription factor USF; this factor has been previously reported to bind to the AdML Inr sequence. A CPE Inr-binding protein co-purifies with an AdML Inr-binding protein on a CPE Inr affinity column. Gel shift assays showed that with some binding conditions, the proteins that bind to the CPE sequence also bind to the TdT and AdML Inr elements. Taken together, these results indicate that the -12 to +20 region of the CPE gene has the properties of an Inr element which binds some, but not all, of the factors which bind to other Inr elements.
参与肽激素加工的几种酶的基因，如羧肽酶E（CPE），不含TATA框。CPE基因主要转录起始位点周围的区域与无TATA的末端脱氧核苷酸转移酶（TdT）基因、腺病毒主要晚期（AdML）及其他启动子的“起始子”（Inr）元件具有序列同源性。为研究CPE基因的启动子区域，用含有与大鼠CPE基因不同片段（-395至+45）相连的荧光素酶报告基因的构建体瞬时转染GH4C1细胞。在-84至-12和+30至+47位置检测到正调控元件。然而，CPE基因的Inr样元件（-12至+20）在没有上下游序列的情况下产生了可检测的荧光素酶活性。CPE基因的该区域以正常（正义）方向表达时比以反义方向表达时活性高得多。CPE与其他Inr元件之间共有序列内的一个突变比野生型序列活性低得多。有趣的是，Inr及其周围序列的缺失导致上游位点的转录大幅增加，这表明在Inr序列处或其附近结合的蛋白质抑制其他位点的转录。为鉴定与CPE基因结合的GH4C1核蛋白，进行了蛋白质印迹、紫外线交联和凝胶迁移分析。蛋白质印迹分析表明，CPE和AdML Inr序列标记了几种大小相似的蛋白质，这些蛋白质与转录因子USF不同；先前报道该因子可与AdML Inr序列结合。一种CPE Inr结合蛋白在CPE Inr亲和柱上与一种AdML Inr结合蛋白共同纯化。凝胶迁移分析表明，在某些结合条件下，与CPE序列结合的蛋白质也与TdT和AdML Inr元件结合。综上所述，这些结果表明CPE基因的-12至+20区域具有Inr元件的特性，该元件结合一些但不是所有与其他Inr元件结合的因子。
