Multiple elements in the upstream glucokinase promoter contribute to transcription in insulinoma cells.
Multiple elements in the upstream glucokinase promoter contribute to transcription in insulinoma cells.
复制标题
上游葡萄糖激酶启动子中的多个元件有助于胰岛素瘤细胞中的转录。
DOI：
10.1128/mcb.12.10.4578-4589.1992
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发表时间：
1992
影响因子：
5.3
通讯作者：
Magnuson,MA
中科院分区：
文献类型：
--
作者：
Shelton,KD;Franklin,AJ;Khoor,A;Beechem,J;Magnuson,MA
通过融合基因的转染和DNA-蛋白质相互作用的分析来研究上游葡萄糖激酶启动子的β细胞类型特异性表达。含有1，000 bp的5 '侧翼DNA的构建体在HIT M2.2.2细胞中有效表达，HIT M2.2.2细胞是一种产生胰岛素和葡萄糖激酶的β细胞衍生系，但在NIH 3 T3细胞中不表达，NIH 3 T3细胞是一种异源细胞系。在碱基-1000和-100之间的一系列5'缺失突变（相对于先前指定的+1碱基）中，HIT细胞中的有效表达维持至-280 bp，此后转录以逐步方式降低。通过研究28个以10-bp步长跨越该区域的阻断颠换突变体，确定了在HIT细胞中对转录活性有贡献的-280和-1 bp之间的序列。两个突变使转录降低了10倍或更多，而六个突变使转录降低了3到10倍。发现该启动子的三个突变敏感区域与优先在胰岛β细胞中表达的因子结合。上游启动子元件（upstream promoter elements，UPE）的结合位点共有CAT（T/C）A（C/G）序列。该序列中腺嘌呤和鸟嘌呤残基的甲基化阻止了β细胞因子的结合，位置2、3和5的突变也是如此。对不同细胞系的核提取物进行分析，在HIT M2.2.2和β-TC-3细胞中发现了UPE结合活性，但在AtT-20、NIH 3 T3或HeLa细胞中没有发现;不能排除α-TC-6细胞中UPE结合量大幅降低的可能性。紫外激光交联实验支持该因子的β细胞型表达，并显示其大小为−50 kDa。凝胶迁移率变化竞争实验表明，这种β细胞因子与胰岛素启动子中的类似元件（称为CT盒）结合相同。因此，这些元件（UPE或CT盒）和与它们结合的β细胞因子在决定胰岛β细胞中基因表达中的作用被提出。
β-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 β-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 −280 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 β 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 β-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 β-TC-3 cells but not in AtT-20, NIH 3T3, or HeLa cells; the possibility of a greatly reduced amount in α-TC-6 cells could not be excluded. UV laser cross-linking experiments supported the β-cell type expression of this factor and showed it to be −50 kDa in size. Gel mobility shift competition experiments showed that this β-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 β-cell factor that binds to them, in determining the expression of genes in the β cells of pancreatic islets is suggested.