Knepel W, Jepeal L, Habener J F
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Boston.
J Biol Chem. 1990 May 25;265(15):8725-35.
Interactions of nuclear proteins with cis-control elements are involved in the programmed developmental expression of the islet polypeptide hormone genes. Three transcriptional control elements within 300 base pairs of the 5'-flanking region of the rat glucagon gene interact with regulatory cellular proteins and direct transcription only in glucagon-producing islet cells. Two islet cell-specific enhancer-like elements (G2, G3) act together with the glucagon promoter (including the G1 element), which confers A cell specificity of glucagon gene expression. In the present study, the G3 element was analyzed in detail by protein binding and in vivo and in vitro transcription assays. Mutational analyses showed that the sequence of the G3 element comprises two distinct protein-binding domains: a more upstream domain A (5'-CGCCTGA-3'), and a more downstream domain B (5'GATTGAAGGGTGTA-3'). Binding of proteins to these two domains is mutually exclusive. Domain A, but not domain B, is responsible for both functional protein binding and the enhancement of transcription from the glucagon or thymidine kinase gene promoter chloramphenicol acetyltransferase reporter gene transfected in vivo into glucagon-producing islet cells (InR1-G9) and transcribed in vitro in a HeLa cell-free transcription system. In islet cell extracts, the Southwestern blot technique labeled a protein of 45 kDa binding to domain A within G3. We conclude that although the G3 sequence contains two protein-binding motifs, the organization of the G3 enhancer-like element is not bipartite. The islet cell specificity of the G3 element is conferred by a tissue-specific transcription factor or protein complex interacting with domain A of G3. This protein or protein complex recognizes different DNA sequences and provides promoter as well as enhancer activity because it binds also to the apparently unrelated sequence of the G1 promoter element.
核蛋白与顺式调控元件的相互作用参与胰岛多肽激素基因的程序性发育表达。大鼠胰高血糖素基因5′侧翼区300个碱基对内的三个转录调控元件与调节性细胞蛋白相互作用，并仅在产生胰高血糖素的胰岛细胞中指导转录。两个胰岛细胞特异性增强子样元件（G2、G3）与胰高血糖素启动子（包括G1元件）共同作用，赋予胰高血糖素基因表达的A细胞特异性。在本研究中，通过蛋白质结合以及体内和体外转录分析对G3元件进行了详细分析。突变分析表明，G3元件的序列包含两个不同的蛋白质结合结构域：一个更上游的结构域A（5′-CGCCTGA-3′）和一个更下游的结构域B（5′-GATTGAAGGGTGTA-3′）。蛋白质与这两个结构域的结合是相互排斥的。结构域A而非结构域B负责功能性蛋白质结合以及增强从体内转染到产生胰高血糖素的胰岛细胞（InR1-G9）中并在体外HeLa无细胞转录系统中转录的胰高血糖素或胸苷激酶基因启动子氯霉素乙酰转移酶报告基因的转录。在胰岛细胞提取物中，蛋白质印迹技术标记了一种与G3内结构域A结合的45 kDa蛋白质。我们得出结论，尽管G3序列包含两个蛋白质结合基序，但G3增强子样元件的组织并非二分的。G3元件的胰岛细胞特异性由与G3结构域A相互作用的组织特异性转录因子或蛋白质复合物赋予。这种蛋白质或蛋白质复合物识别不同的DNA序列，并提供启动子以及增强子活性，因为它也与明显不相关的G1启动子元件序列结合。
