Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms.
Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms.
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DOI：
10.1371/journal.pgen.1000023
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发表时间：
2008-02-29
期刊：
影响因子：
4.5
通讯作者：
Kay SA
中科院分区：
文献类型：
--
作者：
Liu AC;Tran HG;Zhang EE;Priest AA;Welsh DK;Kay SA
哺乳动物生物钟由一个核心的PER/CRY反馈回路以及其他相互关联的回路组成。特别是，由调节Bmal1表达的ROR激活因子和REV - ERB抑制因子组成的ROR/REV/Bmal1回路，被认为可“稳定”核心时钟功能。然而，由于基因缺失的功能冗余和多效性影响，ROR/REV/Bmal1回路的作用尚未得到准确界定。在这项研究中，我们利用基因敲除和RNA干扰相结合的方法检测了细胞自主性昼夜节律振荡，并证明REV - ERBα和β在功能上是冗余的，且对于有节律的Bmal1表达是必需的。相比之下，ROR有助于Bmal1的振幅，但对于Bmal1的节律并非不可或缺。我们提供了直接的体内遗传学证据，表明REV - ERBs也参与Cry1和Rorc表达的组合调控，导致它们相对于Rev - erbα的相位延迟。因此，在基本时钟机制中，REV - ERBs比RORs发挥着更突出的作用。细胞遗传学方法允许对细胞内核心时钟功能的稳健性进行测试。我们发现，REV - ERBα和β功能均缺失的细胞，或那些表达组成型BMAL1的细胞，仍然能够产生并维持正常的Per2节律性。因此，我们的研究结果强调了细胞内时钟机制的弹性，并为生物钟背后的转录拓扑结构提供了重要见解。由于REV - ERB功能以及Bmal1 mRNA/蛋白质循环对于基本时钟功能并非必需，我们提出ROR/REV/Bmal1回路及其组成成分的主要作用是控制时钟输出基因的节律性转录。
植物、真菌、昆虫和哺乳动物的生物钟都具有共同的转录网络结构。在细胞水平上，哺乳动物的生物钟机制由一个核心的Per/Cry负反馈回路以及其他相互关联的回路组成。我们希望通过实验探究相互关联的Bmal1回路对哺乳动物时钟功能的贡献。由于行为节律并不总是反映细胞自主性表型，且易受多效性影响，我们采用基于细胞的遗传学方法，并使用时钟基因表达的生物发光报告基因纵向监测节律。我们发现，在调节Bmal1节律方面，REV - ERB抑制因子比ROR激活因子发挥着更突出的作用。然而，即使Bmal1组成型表达，仍存在显著的节律性，这表明核心回路对Bmal1回路的扰动具有弹性。我们得出结论，虽然相互关联的回路有助于对核心回路进行微调，但其主要功能是为控制局部生理提供离散的时钟基因表达波形。这项研究不仅对跨物种的生物钟生物学，而且对旨在理解遗传网络中复杂相互作用的新兴系统生物学领域都具有重要的普遍意义。
The mammalian circadian clockwork is composed of a core PER/CRY feedback loop and additional interlocking loops. In particular, the ROR/REV/Bmal1 loop, consisting of ROR activators and REV-ERB repressors that regulate Bmal1 expression, is thought to “stabilize” core clock function. However, due to functional redundancy and pleiotropic effects of gene deletions, the role of the ROR/REV/Bmal1 loop has not been accurately defined. In this study, we examined cell-autonomous circadian oscillations using combined gene knockout and RNA interference and demonstrated that REV-ERBα and β are functionally redundant and are required for rhythmic Bmal1 expression. In contrast, the RORs contribute to Bmal1 amplitude but are dispensable for Bmal1 rhythm. We provide direct in vivo genetic evidence that the REV-ERBs also participate in combinatorial regulation of Cry1 and Rorc expression, leading to their phase-delay relative to Rev-erbα. Thus, the REV-ERBs play a more prominent role than the RORs in the basic clock mechanism. The cellular genetic approach permitted testing of the robustness of the intracellular core clock function. We showed that cells deficient in both REV-ERBα and β function, or those expressing constitutive BMAL1, were still able to generate and maintain normal Per2 rhythmicity. Our findings thus underscore the resilience of the intracellular clock mechanism and provide important insights into the transcriptional topologies underlying the circadian clock. Since REV-ERB function and Bmal1 mRNA/protein cycling are not necessary for basic clock function, we propose that the major role of the ROR/REV/Bmal1 loop and its constituents is to control rhythmic transcription of clock output genes. Circadian clocks in plants, fungi, insects, and mammals all share a common transcriptional network architecture. At the cellular level, the mammalian clockwork consists of a core Per/Cry negative feedback loop and additional interlocking loops. We wished to address experimentally the contribution of the interlocking Bmal1 loop to clock function in mammals. Because behavioral rhythms do not always reflect cell-autonomous phenotypes and are subject to pleiotropic effects, we employed cell-based genetic approaches and monitored rhythms longitudinally using bioluminescent reporters of clock gene expression. We showed that REV-ERB repressors play a more prominent role than ROR activators in regulating the Bmal1 rhythm. However, significant rhythmicity remains even with constitutive expression of Bmal1, pointing to the resilience of the core loop to perturbations of the Bmal1 loop. We conclude that while the interlocking loop contributes to fine-tuning of the core loop, its primary function is to provide discrete waveforms of clock gene expression for control of local physiology. This study has important general implications not only for circadian biology across species, but also for the emerging field of systems biology that seeks to understand complex interactions in genetic networks.
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