Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA
Genes Dev. 2014 Sep 15;28(18):1989-98. doi: 10.1101/gad.249417.114.
The mammalian circadian clock is based on a transcription-translation feedback loop (TTFL) in which CLOCK and BMAL1 proteins act as transcriptional activators of Cryptochrome and Period genes, which encode proteins that repress CLOCK-BMAL1 with a periodicity of ∼ 24 h. In this model, the mechanistic roles of CRY and PER are unclear. Here, we used a controlled targeting system to introduce CRY1 or PER2 into the nuclei of mouse cells with defined circadian genotypes to characterize the functions of CRY and PER. Our data show that CRY is the primary repressor in the TTFL: It binds to CLOCK-BMAL1 at the promoter and inhibits CLOCK-BMAL1-dependent transcription without dissociating the complex ("blocking"-type repression). PER alone has no effect on CLOCK-BMAL1-activated transcription. However, in the presence of CRY, nuclear entry of PER inhibits transcription by displacing CLOCK-BMAL1 from the promoter ("displacement"-type repression). In light of these findings, we propose a new model for the mammalian circadian clock in which the negative arm of the TTFL proceeds by two different mechanisms during the circadian cycle.
哺乳动物的生物钟基于转录-翻译反馈环（TTFL），其中 CLOCK 和 BMAL1 蛋白作为 Cryptochrome 和 Period 基因的转录激活因子，这些基因编码的蛋白质以约 24 小时的周期抑制 CLOCK-BMAL1。在这个模型中，CRY 和 PER 的机械作用尚不清楚。在这里，我们使用受控靶向系统将 CRY1 或 PER2 引入具有明确生物钟基因型的小鼠细胞的核内，以表征 CRY 和 PER 的功能。我们的数据表明，CRY 是 TTFL 中的主要抑制剂：它在启动子处与 CLOCK-BMAL1 结合，抑制 CLOCK-BMAL1 依赖性转录，而不使复合物解离（“阻断”型抑制）。PER 本身对 CLOCK-BMAL1 激活的转录没有影响。然而，在 CRY 的存在下，PER 的核内进入通过将 CLOCK-BMAL1 从启动子上置换出来抑制转录（“置换”型抑制）。鉴于这些发现，我们提出了一个新的哺乳动物生物钟模型，其中 TTFL 的负臂在昼夜节律循环中通过两种不同的机制进行。
