Induction of dystrophin Dp71 expression during neuronal differentiation: opposite roles of Sp1 and AP2α in Dp71 promoter activity
Induction of dystrophin Dp71 expression during neuronal differentiation: opposite roles of Sp1 and AP2α in Dp71 promoter activity
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DOI：
10.1111/j.1471-4159.2009.06467.x
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发表时间：
2010-01-01
影响因子：
4.7
通讯作者：
Cisneros, Bulmaro
中科院分区：
文献类型：
--
作者：
Luz Morales-Lazaro, Sara;Gonzalez-Ramirez, Ricardo;Cisneros, Bulmaro
在这项研究中，我们利用N1E-115细胞系，描述了在神经元分化过程中调节Dp71表达的分子机制。我们证明了Dp71的表达在camp介导的这些细胞的神经元分化中上调，并且这种诱导是在启动子水平上控制的。Dp71启动子的功能缺失分析显示，包含Sp1和AP2结合位点的5'侧159 bp DNA片段对于该无tata启动子的基础表达以及在神经元分化过程中的诱导是必要和充分的。电泳迁移率转移和染色质免疫沉淀分析显示Sp1和AP2 α与Dp71基础启动子内各自的DNA元件结合。总的来说，Sp1和AP2结合位点的诱变实验、Sp1和AP2 α的过表达以及Sp1和AP2 α基因表达的敲低实验表明，Dp71的基础表达受Sp1和AP2 α的共同作用控制，它们分别作为激活因子和抑制因子。此外，我们证明分化细胞中Dp71表达的诱导是Sp1维持正调控的结果，也是AP2 α结合缺失的结果，AP2 α结合最终将启动子从抑制中释放出来。
In this study, we delineated the molecular mechanisms that modulate Dp71 expression during neuronal differentiation, using the N1E-115 cell line. We demonstrated that Dp71 expression is up-regulated in response to cAMP-mediated neuronal differentiation of these cells, and that this induction is controlled at promoter level. Functional deletion analysis of the Dp71 promoter revealed that a 5'-flanking 159-bp DNA fragment that contains Sp1 and AP2 binding sites is necessary and sufficient for basal expression of this TATA-less promoter, as well as for its induction during neuronal differentiation. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that Sp1 and AP2 alpha bind to their respective DNA elements within the Dp71 basal promoter. Overall, mutagenesis assays on the Sp1 and AP2 binding sites, over-expression of Sp1 and AP2 alpha, as well as knock-down experiments on Sp1 and AP2 alpha gene expression established that Dp71 basal expression is controlled by the combined action of Sp1 and AP2 alpha, which act as activator and repressor, respectively. Furthermore, we demonstrated that induction of Dp71 expression in differentiated cells is the result of the maintenance of positive regulation exerted by Sp1, as well as of the loss of AP2 alpha binding, which ultimately releases the promoter from repression.