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首页> 外文期刊>Life sciences >Analysis of the interactions of Nrf-2, PMF-1, and CSN-7 with the 5'-flanking sequence of the mouse 4E-BP1 gene.
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Analysis of the interactions of Nrf-2, PMF-1, and CSN-7 with the 5'-flanking sequence of the mouse 4E-BP1 gene.

机译：Nrf-2，PMF-1和CSN-7与小鼠4E-BP1基因5'侧翼序列的相互作用分析。

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Nuclear factor erythroid 2-related factor 2 (Nrf-2) binds to a specific polyamine responsive element (PRE) in the promoter region of the spermidine-spermine acetyltransferase (SSAT) gene, a key component of the polyamine catabolic pathway. Regulation of SSAT gene transcription requires the additional interaction of Nrf-2 with polyamine modulated factor 1 (PMF-1). Likewise, transcription of the eukaryotic initiation factor 4E binding protein 1 (4E-BP1) gene is regulated in a polyamine-dependent manner, but the actual mechanism has not previously been determined. Analysis of the 5'-flanking sequence of the murine 4E-BP1 gene indicated the presence of several potential PRE sites, which might be involved in regulating its transcription. Our goal in this research was to determine potential interactions between Nrf-2, PMF-1, the human homologue of the Arabidopsis signalosome complex (CSN-7), and these potential PRE sites. Four PCR fragments containing regions with considerable homology (78%) to the human PREwere generated from the 5'-flanking sequence of the mouse 4E-BP1 gene and the fragments were used in electrophoretic gel mobility shift and supershift assays. Purified Nrf-2 interacted with all four of these fragments, and similar gel shifts were observed with both cytoplasmic and nuclear fractions of NIH-3T3 cells. However, polyamine depletion with difluoromethylornithine (DFMO) eliminated the gel shift. Supershift assays indicated that the shift was due to the binding of Nrf-2, and the binding was competitive with a known Nrf-2 binding sequence. Purified PMF-1 did not bind any of the PCR fragments alone, but when added with Nrf-2, decreased the magnitude of the gel shift for one of the fragments (PRE located at -2060 relative to the transcription start site). CSN-7 did not interact with the sequences, nor did it inhibit protein/DNA interaction. These data indicate a possible mechanism by which polyamines enhance the binding of a Nrf-2/PMF-1 complex to the 5'-flanking region of the 4E-BP1 gene. Since polyamines increase expression of the 4E-BP1 gene, it seems likely that formation of this complex is involved in its transcriptional regulation.
机译：核因子类胡萝卜素2相关因子2（Nrf-2）与亚精胺-亚精胺乙酰转移酶（SSAT）基因（多胺分解代谢途径的关键部分）的启动子区域中的特定多胺响应元件（PRE）结合。 SSAT基因转录的调控需要Nrf-2与多胺调节因子1（PMF-1）的额外相互作用。同样，真核生物起始因子4E结合蛋白1（4E-BP1）基因的转录以多胺依赖性方式调节，但实际机制尚未得到确定。鼠4E-BP1基因5'侧翼序列的分析表明存在几个潜在的PRE位点，可能参与调节其转录。我们在这项研究中的目标是确定Nrf-2，PMF-1，拟南芥信号体复合物（CSN-7）的人类同源物与这些潜在的PRE位点之间的潜在相互作用。从小鼠4E-BP1基因的5'侧翼序列生成了四个与人类PRE同源性相当（78％）的区域的PCR片段，并将这些片段用于电泳凝胶迁移率迁移和超迁移测定。纯化的Nrf-2与所有这四个片段相互作用，并且NIH-3T3细胞的细胞质和细胞核部分均观察到相似的凝胶变化。但是，用二氟甲基鸟氨酸（DFMO）消耗多胺可消除凝胶位移。超位移测定法表明该位移是由于Nrf-2的结合引起的，并且该结合与已知的Nrf-2结合序列竞争。纯化的PMF-1不能单独结合任何PCR片段，但是当与Nrf-2结合时，可降低其中一个片段的凝胶位移幅度（相对于转录起始位点，PRE位于-2060）。 CSN-7不与序列相互作用，也不抑制蛋白质/ DNA相互作用。这些数据表明多胺增强Nrf-2 / PMF-1复合物与4E-BP1基因5'侧翼区域结合的可能机制。由于多胺增加了4E-BP1基因的表达，这种复合物的形成似乎与其转录调控有关。

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