Pharmacology Research Laboratories, Astellas Pharma, Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan.
Nucleic Acids Res. 2010 Dec;38(22):7964-73. doi: 10.1093/nar/gkq678. Epub 2010 Aug 6.
It has been proposed that robust rhythmic gene expression requires clock-controlled elements (CCEs). Transcription of Per1 was reported to be regulated by the E-box and D-box in conventional reporter assays. However, such experiments are inconclusive in terms of how the CCEs and their combinations determine the phase of the Per1 gene. Whereas the phase of Per2 oscillation was found to be the most delayed among the three Period genes, the phase-delaying regions of the Per2 promoter remain to be determined. We therefore investigated the regulatory mechanism of circadian Per1 and Per2 transcription using an in vitro rhythm oscillation-monitoring system. We found that the copy number of the E-box might play an important role in determining the phase of Per1 oscillation. Based on real-time bioluminescence assays with various promoter constructs, we provide evidence that the non-canonical E-box is involved in the phase delay of Per2 oscillation. Transfection experiments confirmed that the non-canonical E-box could be activated by CLOCK/BMAL1. We also show that the D-box in the third conserved segment of the Per2 promoter generated high amplitude. Our experiments demonstrate that the copy number and various combinations of functional CCEs ultimately led to different circadian phases and amplitudes.
有人提出，强大的节律基因表达需要时钟控制元件（CCEs）。在传统的报告基因实验中，报告 Per1 的转录受 E 盒和 D 盒调控。然而，就 CCEs 及其组合如何决定 Per1 基因的相位而言，这些实验的结论并不确定。虽然 Per2 振荡的相位在三个 Period 基因中被发现是最延迟的，但 Per2 启动子的相位延迟区仍有待确定。因此，我们使用体外节律振荡监测系统研究了昼夜节律 Per1 和 Per2 转录的调控机制。我们发现 E 盒的拷贝数可能在决定 Per1 振荡的相位中起重要作用。基于具有各种启动子构建体的实时生物发光测定，我们提供了证据表明非典型 E 盒参与了 Per2 振荡的相位延迟。转染实验证实，非典型 E 盒可以被 CLOCK/BMAL1 激活。我们还表明，Per2 启动子第三保守段中的 D 盒产生高振幅。我们的实验表明，功能 CCEs 的拷贝数和各种组合最终导致了不同的生物钟相位和振幅。
