Molecular Medicine Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan.
FEBS Lett. 2011 Jul 21;585(14):2217-22. doi: 10.1016/j.febslet.2011.05.038. Epub 2011 May 27.
The phenotypes of mice carrying clock gene mutations have been critical to understanding the mammalian clock function. However, behavior does not necessarily reflect cell-autonomous clock phenotypes, because of the hierarchical dominance of the central clock. We performed cell-based siRNA knockdown and cDNA overexpression and monitored rhythm using bioluminescent reporters of clock genes. We found that knockdown of DBP, D-box positive regulator, in our model led to a short-period phenotype, whereas overexpressing of DBP produced a long-period rhythm when compared to controls. Furthermore, knockdown and overexpressing of E4BP4, D-box negative regulator, led to an opposite effect of DBP. Our experiments demonstrated that D-box regulators play a crucial role in determining the period length of Per1 and Per2 promoter-driven circadian rhythms in Rat-1 fibroblasts.
携带时钟基因突变的小鼠表型对于理解哺乳动物时钟功能至关重要。然而，由于中枢时钟的层级优势，行为并不一定反映细胞自主的时钟表型。我们进行了基于细胞的 siRNA 敲低和 cDNA 过表达，并使用时钟基因的生物发光报告基因监测节律。我们发现，与对照组相比，我们模型中 DBP（D 框正调控因子）的敲低导致短周期表型，而过表达 DBP 则产生长周期节律。此外，E4BP4（D 框负调控因子）的敲低和过表达导致 DBP 的相反效应。我们的实验表明，D 框调控因子在决定 Rat-1 成纤维细胞中 Per1 和 Per2 启动子驱动的生物钟节律的周期长度方面起着关键作用。
