Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109.
Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109.
Proc Natl Acad Sci U S A. 2021 Jul 13;118(28). doi: 10.1073/pnas.2019756118.
Circadian clocks regulate ∼24-h oscillations in gene expression, behavior, and physiology. While the genetic and molecular mechanisms of circadian rhythms are well characterized, what remains poorly understood are the intracellular dynamics of circadian clock components and how they affect circadian rhythms. Here, we elucidate how spatiotemporal organization and dynamics of core clock proteins and genes affect circadian rhythms in clock neurons. Using high-resolution imaging and DNA-fluorescence in situ hybridization techniques, we demonstrate that clock proteins (PERIOD and CLOCK) are organized into a few discrete foci at the nuclear envelope during the circadian repression phase and play an important role in the subnuclear localization of core clock genes to control circadian rhythms. Specifically, we show that core clock genes, and , are positioned close to the nuclear periphery by the PERIOD protein specifically during the repression phase, suggesting that subnuclear localization of core clock genes might play a key role in their rhythmic gene expression. Finally, we show that loss of Lamin B receptor, a nuclear envelope protein, leads to disruption of PER foci and gene peripheral localization and results in circadian rhythm defects. These results demonstrate that clock proteins play a hitherto unexpected role in the subnuclear reorganization of core clock genes to control circadian rhythms, revealing how clocks function at the subcellular level. Our results further suggest that clock protein foci might regulate dynamic clustering and spatial reorganization of clock-regulated genes over the repression phase to control circadian rhythms in behavior and physiology.
生物钟调节基因表达、行为和生理的约 24 小时振荡。虽然昼夜节律的遗传和分子机制已经很好地描述，但生物钟成分的细胞内动力学以及它们如何影响昼夜节律仍然知之甚少。在这里，我们阐明了核心生物钟蛋白和基因的时空组织和动力学如何影响时钟神经元中的昼夜节律。使用高分辨率成像和 DNA 荧光原位杂交技术，我们证明了 PERIOD 和 CLOCK 等时钟蛋白在昼夜节律抑制阶段在核膜上形成少数离散焦点，并在核心时钟基因的亚核定位中发挥重要作用，以控制昼夜节律。具体来说，我们表明核心时钟基因、 和 是通过 PERIOD 蛋白在抑制阶段特别靠近核周定位的，这表明核心时钟基因的亚核定位可能在其节律性基因表达中发挥关键作用。最后，我们表明核膜蛋白 Lamin B 受体的缺失会导致 PER 焦点和 基因外周定位的破坏，并导致昼夜节律缺陷。这些结果表明，时钟蛋白在核心时钟基因的亚核重排中发挥了迄今为止意想不到的作用，以控制昼夜节律，揭示了时钟在亚细胞水平上的功能。我们的结果进一步表明，时钟蛋白焦点可能调节时钟调节基因在抑制阶段的动态聚类和空间重排，以控制行为和生理中的昼夜节律。
