A potent enhancer element in the 5′-UTR intron is crucial for transcriptional regulation of the human ubiquitin C gene
A potent enhancer element in the 5′-UTR intron is crucial for transcriptional regulation of the human ubiquitin C gene
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DOI：
10.1016/j.gene.2009.08.013
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发表时间：
2009-12-01
期刊：
影响因子：
3.5
通讯作者：
Magnani, Mauro
中科院分区：
文献类型：
--
作者：
Bianchi, Marzia;Crinelli, Rita;Magnani, Mauro
泛素(Ub)几乎在细胞功能的各个方面都起着至关重要的作用。它由四个基因编码，其中UBC已知在基础和应激条件下都能满足细胞对泛素的需求。为了了解调控UBC基因表达的分子机制，我们对UBC启动子进行了功能鉴定。在-916/+878启动子区51端的缺失分析排除了NT-916/-371在转录调控中的功能重要性，而31个缺失表明内含子去除(NT+65/+876)导致所有报告构建的启动子活性显著降低，无论细胞类型如何。用异源嵌合内含子替换内含子不能恢复启动子的活性，因此可以排除剪接事件本身可能是内含子介导的转录突然发生的原因。凝胶位移分析显示核因子与+137/+766内含子区域结合。携带部分内含子缺失的报告构建体证实，该序列确实是最大转录活性所必需的。基于计算机的分析在内含子序列中发现了潜在的Sp1结合基序，电泳迁移率和超移位分析表明，Sp1和Sp3转录因子在体外都在多个结合位点与UBC内含子相互作用。此外，Sp1和Sp3的异位表达表明这两个转录因子都正向调节UBC启动子的活性。总之，我们的数据突出了非常新的证据，即5‘-UTR内含子在调控UBC基因表达中至关重要，并为Sp1/SP3与内含子增强子结合在UBC转录调控中的关键作用提供了见解。(C)2009爱思唯尔B.V.保留所有权利。
Ubiquitin (Ub) plays a crucial role in almost every aspect of cellular functions. It is encoded by four genes, of which UbC is known to meet cell demand for ubiquitin in both basal and stressful conditions. To understand the molecular mechanisms regulating UbC gene expression, we performed a functional characterization of the UbC promoter. Deletion analyses on the 51 end of the -916/+878 promoter region, excluded the functional importance of nt -916/-371 in the transcriptional regulation of the gene, while 31 deletions revealed that intron removal (nt+65/+876) resulted in a marked reduction of promoter activity in all the reporter constructs, regardless of the cell types. Intron substitution with a heterologous chimeric intron failed to restore promoter activity, thus allowing to exclude that the splicing event, per se, can be responsible for the intron-mediated burst of transcription. Gel shift assays demonstrated nuclear factor binding with the +137/+766 intron region. Reporter constructs carrying partial intron deletions confirmed that this sequence is, indeed, required for maximal transcriptional activity. Computer-based analysis found potential Sp1 binding motifs within the intron sequence and electrophoretic mobility shift and supershift assays demonstrated that both Sp1 and Sp3 transcription factors interact, in vitro, with the UbC intron, at multiple binding sites. Moreover, ectopic expression of Sp1 and Sp3 revealed that both transcription factors positively regulate UbC promoter activity. Collectively, our data highlight the very new evidence that the 5'-UTR intron is crucial in regulating UbC gene expression and provide insights into the pivotal role of Sp1/Sp3 binding to the intronic enhancer in the regulation of UbC transcription. (C) 2009 Elsevier B.V. All rights reserved.