HEPATOCYTE NUCLEAR FACTOR 1-ALPHA IS EXPRESSED IN A HAMSTER INSULINOMA LINE AND TRANSACTIVATES THE RAT INSULIN-I GENE
HEPATOCYTE NUCLEAR FACTOR 1-ALPHA IS EXPRESSED IN A HAMSTER INSULINOMA LINE AND TRANSACTIVATES THE RAT INSULIN-I GENE
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DOI：
10.1073/pnas.89.16.7300
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发表时间：
1992-08-15
影响因子：
11.1
通讯作者：
MOSS, LG
中科院分区：
文献类型：
--
作者：
EMENS, LA;LANDERS, DW;MOSS, LG
系统的突变分析以前确定了两个主要的调控元件内的一个minienhancer（-247至-198）的大鼠胰岛素I启动子，是关键的转录活性。Far盒（-241至-232）和FLAT元件（-222至-208）协同上调转录，并且一起足以赋予异源启动子组织特异性和葡萄糖响应性转录活性。对FLAT元件的详细分析进一步揭示，除了与Far box一起介导的正调节活性外，它还是负调节控制的位点。FLAT元件的一部分与肝细胞核因子1-α（HNF 1-α; LF-B1）的共有结合位点具有相当大的序列相似性，HNF 1-α是一种富含肝脏同源结构域的转录因子。在这里，我们表明，HNF 1样网站内的FLAT元件表现出积极的转录活性在HepG 2和HIT细胞和绑定类似的，但可区分的，核蛋白复合物在各自的核提取物。筛选仓鼠胰岛素瘤cDNA文库与PCR衍生的探针，包括大鼠HNF 1-α的DNA结合域导致分离仓鼠HNF 1-α（hHNF 1-α）cDNA同源物。特异性抗血清将HNF 1-α蛋白鉴定为HIT核提取物中特异性FLAT结合复合物的一个组分。hHNF 1-α cDNA在COS细胞中的表达导致含有大鼠胰岛素I minienhancer的多聚化片段的报告构建体的反式激活。因此，HNF 1-α，参与大鼠胰岛素I基因转录调控的DNA结合复合物的一个组成部分，可能在非肝脏以及肝脏基因转录中发挥重要作用。
Systematic mutational analysis previously identified two primary regulatory elements within a minienhancer (-247 to -198) of the rat insulin I promoter that are critical for transcriptional activity. The Far box (-241 to -232) and the FLAT element (-222 to -208) synergistically upregulate transcription and, together, are sufficient to confer tissue-specific and glucose-responsive transcriptional activity on a heterologous promoter. Detailed analysis of the FLAT element further revealed that, in addition to the positive regulatory activity it mediates in tandem with the Far box, it is a site for negative regulatory control. A portion of the FLAT element bears considerable sequence similarity to the consensus binding site for hepatocyte nuclear factor 1-alpha (HNF1-alpha; LF-B1), a liver-enriched homeodomain-containing transcription factor. Here we show that the HNF1-like site within the FLAT element exhibited positive transcriptional activity in both HepG2 and HIT cells and bound similar, but distinguishable, nuclear protein complexes in the respective nuclear extracts. Screening of a hamster insulinoma cDNA library with a PCR-derived probe encompassing the DNA-binding domain of rat HNF1-alpha resulted in isolation of a hamster HNF1-alpha (hHNF1-alpha) cDNA homolog. Specific antiserum identified the HNF1-alpha protein as one component of a specific FLAT-binding complex in HIT nuclear extracts. Expression of the hHNF1-alpha cDNA in COS cells resulted in transactivation of reporter constructs containing multimerized segments of the rat insulin I minienhancer. Thus, HNF1-alpha, one component of a DNA-binding complex involved in transcriptional regulation of the rat insulin I gene, may play a significant role in nonhepatic as well as hepatic gene transcription.