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首页> 外文期刊>Molecular and Cellular Biology >A CRE/ATF-like site in the upstream regulatory sequence of the human interleukin 1 beta gene is necessary for induction in U937 and THP-1 monocytic cell lines.
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A CRE/ATF-like site in the upstream regulatory sequence of the human interleukin 1 beta gene is necessary for induction in U937 and THP-1 monocytic cell lines.

机译：人白介素1β基因上游调控序列中的CRE / ATF样位点是诱导U937和THP-1单核细胞系所必需的。

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Transfection of U937 and THP-1 cells with a recombinant plasmid, pIL1(4.0kb)-CAT, containing 4 kb of the interleukin 1 beta (IL-1 beta) gene upstream regulatory sequence resulted in inducer-dependent expression of chloramphenicol acetyltransferase activity. Treatment of the transfected cells with various combinations of the inducers lipopolysaccharide, phorbol myristate acetate, and dibutyryl cyclic AMP upregulated the IL-1 beta promoter. In U937 and THP-1 cells, maximum stimulation of both the endogenous IL-1 beta gene and pIL1(4.0kb)-CAT transfectants was observed following treatment with the combination of inducing agents lipopolysaccharide-phorbol myristate acetate-dibutyryl cyclic AMP. This combination of inducing agents was used to identify and study, at the molecular level, some of the regulatory elements necessary for induction of the IL-1 beta gene. A series of 5' deletion derivatives of the upstream regulatory sequence were used in transient transfection assays to identify an 80-bp fragment located between -2720 and -2800 bp upstream of the mRNA start site that was required for induction. Exonuclease III mapping, electrophoretic mobility shift assays (EMSA), and DNA sequence analysis of this region were used to identify a transcription factor binding sequence which contained a potential cyclic AMP response element (CRE/ATF)- and NF-kappa B-like binding site. Site-directed mutagenesis of the CRE/ATF-like site resulted in the loss of binding of a specific factor or factors as determined by EMSA. The loss of binding activity directly correlated with a loss of approximately 75% of promoter activity as determined in transient transfection assays. As determined by EMSA, the factor binding to the CRE/ATF-like site was present in nuclear extracts prepared from both uninduced and induced THP-1 and U937 cells. However, the intensity of the band appeared to be increased when nuclear extracts from induced cells were used. In contrast to the CRE/ATF mutation, which resulted in the loss of promoter activity, mutation of the NF-kappa B-like site resulted in a moderate increase in activity in U937 cells. A similar increase in promoter activity was not observed in THP-1 cells. From these studies, we conclude that a CRE/ATF-like site and a factor or factors interacting with this site are essential for the maximum induction of the IL-1 beta gene in stimulated U937 and THP-1 cells.
机译：用重组质粒pIL1（4.0kb）-CAT转染U937和THP-1细胞，该质粒含有4 kb的白介素1 beta（IL-1 beta）基因上游调控序列，导致氯霉素乙酰转移酶活性的诱导物依赖性表达。用诱导剂脂多糖，肉豆蔻酸乙酸佛波酯和二丁酰基环AMP的各种组合处理转染的细胞上调了IL-1β启动子。在U937和THP-1细胞中，在用诱导剂脂多糖-豆蔻酸豆蔻酸酯乙酸盐-二丁酰环AMP处理后，观察到内源IL-1β基因和pIL1（4.0kb）-CAT转染子均受到最大刺激。诱导剂的这种组合被用于在分子水平上鉴定和研究诱导IL-1β基因所必需的一些调控元件。在瞬时转染测定中使用了上游调节序列的一系列5'缺失衍生物，以鉴定诱导所需的位于mRNA起始位点上游-2720和-2800 bp之间的80 bp片段。使用核酸外切酶III作图，电泳迁移率变动分析（EMSA）和该区域的DNA序列分析来鉴定转录因子结合序列，该序列含有潜在的环状AMP反应元件（CRE / ATF）和NF-κB样结合。现场。 CRE / ATF样位点的定点诱变导致失去一种或多种特定因子的结合，如EMSA所确定。如在瞬时转染测定中所确定的，结合活性的损失与启动子活性的大约75％的损失直接相关。如通过EMSA确定的，与CRE / ATF样位点结合的因子存在于由未诱导的THP-1和诱导的THP-1和U937细胞制备的核提取物中。但是，当使用诱导细胞核提取物时，条带的强度似乎增加了。与导致启动子活性丧失的CRE / ATF突变相反，NF-κB样位点的突变导致U937细胞活性适度增加。在THP-1细胞中未观察到类似的启动子活性增加。从这些研究中，我们得出结论，一个CRE / ATF样位点和一个或多个与该位点相互作用的因子对于在受刺激的U937和THP-1细胞中最大程度地诱导IL-1 beta基因至关重要。

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