Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1 -: Application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes
Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1 -: Application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes
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DOI：
10.1074/jbc.m414381200
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发表时间：
2005-04-29
影响因子：
4.8
通讯作者：
Mirmira, RG
中科院分区：
文献类型：
--
作者：
Iype, T;Francis, J;Mirmira, RG
同源结构域因子PDX-1调节发育和成熟胰腺中的一系列基因，但目前尚不清楚每个特定基因的调节是通过直接机制(结合启动子元件并激活基础转录机制)还是间接机制(通过调节其他基因)发生的。为了确定PDX-1对胰岛素基因调控的机制，我们对腺病毒介导的针对PDX-1的小干扰RNA导入胰岛素瘤细胞和胰岛以减少内源性PDX-1蛋白的转录进行了动力学分析。用实时逆转录聚合酶链式反应检测长半衰期胰岛素信使核糖核酸(成熟信使核糖核酸)和短半衰期胰岛素前体信使核糖核酸的转录。随着PDX-1水平的逐渐下调，我们观察到前mRNA水平的协调下降(72小时时降至正常水平的约40%)。相比之下，成熟的mRNA水平表现出惊人的小和延迟下降，这表明该物种较长的半衰期低估了PDX-1对胰岛素转录的贡献。染色质免疫沉淀分析显示，胰岛素转录的降低与胰岛素启动子近端的PDX-1和p300的占有率降低有关。虽然RNA聚合酶II在近端启动子的募集没有相应的变化，但它在胰岛素编码区的募集显著减少。我们的结果表明，PDX-1通过与胰岛素启动子近端的转录共激活因子形成复合体来直接调节胰岛素的转录。这种复合体通过基本的转录机制导致伸长的增强。
The homeodomain factor Pdx-1 regulates an array of genes in the developing and mature pancreas, but whether regulation of each specific gene occurs by a direct mechanism ( binding to promoter elements and activating basal transcriptional machinery) or an indirect mechanism ( via regulation of other genes) is unknown. To determine the mechanism underlying regulation of the insulin gene by Pdx-1, we performed a kinetic analysis of insulin transcription following adenovirus-mediated delivery of a small interfering RNA specific for pdx-1 into insulinoma cells and pancreatic islets to diminish endogenous Pdx-1 protein. insulin transcription was assessed by measuring both a long half-life insulin mRNA ( mature mRNA) and a short half-life insulin pre-mRNA species by real-time reverse transcriptase-PCR. Following progressive knock-down of Pdx-1 levels, we observed coordinate decreases in pre-mRNA levels ( to about 40% of normal levels at 72 h). In contrast, mature mRNA levels showed strikingly smaller and delayed declines, suggesting that the longer half-life of this species underestimates the contribution of Pdx-1 to insulin transcription. Chromatin immunoprecipitation assays revealed that the decrease in insulin transcription was associated with decreases in the occupancies of Pdx-1 and p300 at the proximal insulin promoter. Although there was no corresponding change in the recruitment of RNA polymerase II to the proximal promoter, its recruitment to the insulin coding region was significantly reduced. Our results suggest that Pdx-1 directly regulates insulin transcription through formation of a complex with transcriptional coactivators on the proximal insulin promoter. This complex leads to enhancement of elongation by the basal transcriptional machinery.