Yin Yang 1 intronic binding sequences and splicing elicit intron-mediated enhancement of ubiquitin C gene expression.
Yin Yang 1 intronic binding sequences and splicing elicit intron-mediated enhancement of ubiquitin C gene expression.
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DOI：
10.1371/journal.pone.0065932
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Magnani M
中科院分区：
文献类型：
--
作者：
Bianchi M;Crinelli R;Giacomini E;Carloni E;Radici L;Magnani M
在许多生物体中，内含子影响包含它们的基因的表达。我们的前期研究发现，人泛素C（ubiquitin C，UbC）基因的5′-UTR内含子在体外能够与阴阳1（Yin Yang 1，YY 1）反式作用因子结合，并能促进报告基因的表达。在这项工作中，我们证明，完整的YY 1结合序列所需的最大启动子活性和YY 1沉默导致荧光素酶mRNA水平下调。然而，YY 1基序不能增强基因表达时，内含子被移动上游的近端启动子，排除典型的增强子假说，并支持上下文依赖的行动，如内含子介导的增强（IME）。然而，在含有UbC内含子的不可剪接版本的构建体中几乎没有看到表达，表明剪接对于启动子活性是必需的。此外，YY 1结合位点的诱变和YY 1敲低负面影响UbC内含子从内源性和报告基因转录物中去除。因此，YY 1顺式元件和蛋白因子对剪接效率的调节可能是YY 1控制UbC启动子活性的机制的一部分。我们的数据突出了IME中序列特异性DNA结合因子参与的第一个证据。
In a number of organisms, introns affect expression of the gene in which they are contained. Our previous studies revealed that the 5′-UTR intron of human ubiquitin C (UbC) gene is responsible for the boost of reporter gene expression and is able to bind, in vitro, Yin Yang 1 (YY1) trans-acting factor. In this work, we demonstrate that intact YY1 binding sequences are required for maximal promoter activity and YY1 silencing causes downregulation of luciferase mRNA levels. However, YY1 motifs fail to enhance gene expression when the intron is moved upstream of the proximal promoter, excluding the typical enhancer hypothesis and supporting a context-dependent action, like intron-mediated enhancement (IME). Yet, almost no expression is seen in the construct containing an unspliceable version of UbC intron, indicating that splicing is essential for promoter activity. Moreover, mutagenesis of YY1 binding sites and YY1 knockdown negatively affect UbC intron removal from both endogenous and reporter transcripts. Modulation of splicing efficiency by YY1 cis-elements and protein factor may thus be part of the mechanism(s) by which YY1 controls UbC promoter activity. Our data highlight the first evidence of the involvement of a sequence-specific DNA binding factor in IME.
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