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首页> 外文期刊>Molecular and Cellular Biology >Interaction of a liver-specific factor with an enhancer 4.8 kilobases upstream of the phosphoenolpyruvate carboxykinase gene.
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Interaction of a liver-specific factor with an enhancer 4.8 kilobases upstream of the phosphoenolpyruvate carboxykinase gene.

机译：肝特异性因子与磷酸烯醇丙酮酸羧激酶基因上游4.8 kb增强子的相互作用。

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摘要

We have previously identified a series of five DNase-I hypersensitive (HS) sites within and around the rat phosphoenolpyruvate carboxykinase (PEPCK) gene. The far upstream region has now been sequenced, and the tissue-specific HS site has been mapped more precisely at 4,800 base pairs upstream of the transcription start site of the PEPCK gene. DNA fragments that include the HS site were cloned upstream of various promoters to test whether these regions modulate transcription of the chloramphenicol acetyltransferase reporter gene. Chloramphenicol acetyltransferase activity was enhanced when the DNA fragment encompassing the upstream HS site was linked to various lengths of the PEPCK promoter or to the heterologous simian virus 40 promoter. This upstream region in conjunction with the proximal promoter, which may contain a tissue-specific element, conferred maximum activation in H4IIE hepatoma cells, which express the endogenous PEPCK gene. When these experiments were performed in XC cells, in which the gene is not expressed, transcriptional activation by the upstream element was still significant. Evidence of a specific protein-DNA interaction, using DNA mobility shift and DNase I footprinting assays, was obtained only when using H4IIE cell nuclear extracts. Competition assay showed that the interacting factor may be similar or identical to the liver-specific factor HNF3. We suggest that this protein factor binds to DNA within the HS site and interacts with the proximal promoter region to control tissue-specific high-level expression of the PEPCK gene.
机译：我们先前已经确定了大鼠磷酸烯醇丙酮酸羧激酶（PEPCK）基因内和周围的一系列五个DNase-I超敏（HS）位点。现在已对远上游区域进行了测序，并且组织特异性HS位点已更精确地定位在PEPCK基因转录起始位点上游的4,800个碱基对处。将包含HS位点的DNA片段克隆到各种启动子的上游，以测试这些区域是否调节了氯霉素乙酰转移酶报道基因的转录。当将包含上游HS位点的DNA片段连接到各种长度的PEPCK启动子或异源猿猴病毒40启动子时，氯霉素乙酰转移酶的活性会增强。该上游区域与可能包含组织特异性元件的近端启动子结合，在表达内源性PEPCK基因的H4IIE肝癌细胞中赋予了最大的激活。当在不表达基因的XC细胞中进行这些实验时，上游元件的转录激活仍然很重要。仅当使用H4IIE细胞核提取物时，才能使用DNA迁移率变化和DNase I足迹测定法获得特定蛋白质与DNA相互作用的证据。竞争测定表明，相互作用因子可能与肝脏特异性因子HNF3相似或相同。我们建议该蛋白因子与HS位点内的DNA结合并与近端启动子区域相互作用，以控制PEPCK基因的组织特异性高水平表达。

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