Real-Time Analysis of the Circadian Oscillation of the Rev-Erbβ Promoter
Real-Time Analysis of the Circadian Oscillation of the Rev-Erbβ Promoter
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DOI：
10.5551/jat.14381
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发表时间：
2013-01-01
影响因子：
4.4
通讯作者：
Ikeda, Masaaki
中科院分区：
文献类型：
--
作者：
Yang, Fang;Inoue, Ikuo;Ikeda, Masaaki
目的：Rev-Erb β 基因在昼夜节律、脂质和葡萄糖代谢以及多种疾病中发挥着至关重要的作用。 Rev-Erb beta产生并决定昼夜节律振荡相位的转录调控分子机制尚不清楚。方法：通过荧光素酶报告基因检测、实时生物发光监测分析和电泳迁移率变化分析对Rev-Erb beta启动子进行分析。结果：荧光素酶报告基因检测表明，只有5'区和外显子1具有明显的启动子活性。实时生物发光监测分析表明，E1、E2、E3、D 盒对于维持 Rev-Erb β 振荡幅度非常重要。根据 EMSA 结果，REV-ERB beta 与 Bmal1 启动子区域的 RORE 结合并抑制 Bmal1 启动子活性。结论：我们提供了直接证据，表明位于第一个内含子的三个 E 盒和一个 D 盒对于 Rev-Erb beta 表达中的昼夜节律振荡阶段至关重要，并且其转录起始位点上游的序列充当不受昼夜节律调节的启动子。我们还发现 E1 盒影响 Rev-Erb beta 振荡相位。我们的结果为 Rev-Erb beta 在昼夜节律系统中的作用提供了新的见解。
Aim: The Rev-Erb beta gene plays crucial roles in circadian rhythm, lipid and glucose metabolism, and several diseases. The molecular mechanisms of the transcriptional regulation of Rev-Erb beta that generate and determine the phase of the circadian oscillation remain unclear.Methods: We analyzed the Rev-Erb beta promoter by luciferase reporter assays, real-time bioluminescence monitoring assays and electrophoretic mobility shift assays.Results: Luciferase reporter assays indicated that only the 5' region and exon 1 have obvious promoter activity. Real-time bioluminescence monitoring assays revealed that E1, E2, E3, D boxes are important for maintenance of the amplitude of Rev-Erb beta oscillation. Based on EMSA results, REV-ERB beta binds ROREs in the Bmal1 promoter region and inhibits Bmal1 promoter activity. Conclusion: We provide direct evidence that three E-boxes and one D-box located in the first intron are crucial for the phase of circadian oscillation in Rev-Erb beta expression and that the sequences upstream from its transcription start site function as a promoter with no circadian regulation. We also found that the E1 box affects the Rev-Erb beta oscillation phase. Our results offer new insight into the role of Rev-Erb beta in the circadian rhythm system.