Shelton K D, Franklin A J, Khoor A, Beechem J, Magnuson M A
Department of Molecular Physiology, Vanderbilt University Medical School, Nashville, Tennessee 37232.
Mol Cell Biol. 1992 Oct;12(10):4578-89. doi: 10.1128/mcb.12.10.4578-4589.1992.
beta-cell type-specific expression of the upstream glucokinase promoter was studied by transfection of fusion genes and analysis of DNA-protein interactions. A construct containing 1,000 bp of 5'-flanking DNA was efficiently expressed in HIT M2.2.2 cells, a beta-cell-derived line that makes both insulin and glucokinase, but not in NIH 3T3 cells, a heterologous cell line. In a series of 5' deletion mutations between bases -1000 and -100 (relative to a base previously designated +1), efficient expression in HIT cells was maintained until -280 bp, after which transcription decreased in a stepwise manner. The sequences between -180 and -1 bp contributing to transcriptional activity in HIT cells were identified by studying 28 block transversion mutants that spanned this region in 10-bp steps. Two mutations reduced transcription 10-fold or more, while six reduced transcription between 3- and 10-fold. Three mutationally sensitive regions of this promoter were found to bind to a factor that was expressed preferentially in pancreatic islet beta cells. The binding sites, designated upstream promoter elements (UPEs), shared a consensus sequence of CAT(T/C)A(C/G). Methylation of adenine and guanine residues within this sequence prevented binding of the beta-cell factor, as did mutations at positions 2, 3, and 5. Analysis of nuclear extracts from different cell lines identified UPE-binding activity in HIT M2.2.2 and beta-TC-3 cells but not in AtT-20, NIH 3T3, or HeLa cells; the possibility of a greatly reduced amount in alpha-TC-6 cells could not be excluded. UV laser cross-linking experiments supported the beta-cell type expression of this factor and showed it to be approximately 50 kDa in size. Gel mobility shift competition experiments showed that this beta-cell factor is the same that binds to similar elements, termed CT boxes, in the insulin promoter. Thus, a role for these elements (UPEs or CT boxes), and the beta-cell factor that binds to them, in determining the expression of genes in the beta cells of pancreatic islets is suggested.
通过转染融合基因并分析DNA-蛋白质相互作用，研究了上游葡萄糖激酶启动子的β细胞类型特异性表达。一个含有1000bp 5'侧翼DNA的构建体在HIT M2.2.2细胞（一种能产生胰岛素和葡萄糖激酶的β细胞系）中高效表达，但在异源细胞系NIH 3T3细胞中不表达。在-1000至-100碱基之间（相对于先前指定为+1的碱基）的一系列5'缺失突变中，直到-280bp时HIT细胞中仍保持高效表达，之后转录呈逐步下降。通过研究以10bp步长跨越该区域的28个块颠换突变体，确定了-180至-1bp之间对HIT细胞转录活性有贡献的序列。两个突变使转录降低10倍或更多，而六个突变使转录降低3至10倍。发现该启动子三个对突变敏感的区域与一种在胰岛β细胞中优先表达的因子结合。这些结合位点称为上游启动子元件（UPEs），共有CAT(T/C)A(C/G)的共有序列。该序列内腺嘌呤和鸟嘌呤残基的甲基化以及第2、3和5位的突变均阻止了β细胞因子的结合。对不同细胞系核提取物的分析确定了HIT M2.2.2和β-TC-3细胞中有UPE结合活性，而AtT-20、NIH 3T3或HeLa细胞中没有；不能排除α-TC-6细胞中该活性大量降低的可能性。紫外线激光交联实验支持该因子的β细胞类型表达，并表明其大小约为50kDa。凝胶迁移率变动竞争实验表明，该β细胞因子与胰岛素启动子中类似元件（称为CT盒）结合的因子相同。因此，提示这些元件（UPEs或CT盒）以及与其结合的β细胞因子在决定胰岛β细胞中基因表达方面具有作用。
