Endogenous circadian reporters reveal functional differences of PERIOD paralogs and the significance of PERIOD:CK1 stable interaction.
Endogenous circadian reporters reveal functional differences of PERIOD paralogs and the significance of PERIOD:CK1 stable interaction.
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DOI：
10.1073/pnas.2212255120
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发表时间：
2023-02-07
影响因子：
11.1
通讯作者：
中科院分区：
文献类型：
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基因和环境对生物钟的破坏可能导致包括睡眠和代谢紊乱在内的各种疾病。具有工程化昼夜节律报告器的细胞，特别是生物发光报告器，在理解支持昼夜节律和昼夜节律紊乱的机制方面发挥了重要作用，但由于缺乏可用于人类细胞的内源性昼夜节律报告器而受到限制。为了弥补这一差距，我们在人体时钟细胞模型U-2 OS中开发了两个内源性报告线。使用这些记者结合CRISPR的基因组编辑，我们揭示了与大脑中的主时钟相比，生物钟是如何在外周细胞中受到独特调节的。我们的基于细胞的平台将加速并实现对时钟基因和机制研究中许多突变的扩展测试。生物钟出现故障的不利后果包括短期内睡眠质量下降和表现不佳，长期内则包括代谢性疾病和癌症。然而，我们对昼夜节律紊乱的理解受到分子模型的不完备性和我们缺乏已定义的突变模型的限制。由于开发活体动物模型来研究各种复杂的时钟机制的成本高得令人望而却步，我们在一个经过验证的时钟细胞模型U-2 OS中开发了PER1-LUC和PER2-LUC内源性昼夜节律报告器，在U-2 OS中，基因组可以很容易地操纵，并且可以准确地研究突变的功能后果。当这些细胞中的主要时钟基因被敲除时，与相应的突变小鼠相比，昼夜节律得到了类似的调制，验证了遗传学研究的平台。使用这些报告细胞，我们发现了PER的两个平行序列之间的关键差异。尽管PER1和PER2被认为是多余的，并且两者都可以单独用作起搏器，但它们在稳定性和磷酸化动力学等生化参数上存在显著差异。一直以来，PER1和PER2基因敲除报告细胞的昼夜节律阶段有很大的不同。我们进一步表明，酪蛋白激酶1δ/ε与PER的稳定结合并不是PER磷酸化本身所必需的，而是延迟磷酸化时间的关键。我们的系统可以作为一个有效的平台来研究与致病突变相关的昼夜节律紊乱及其潜在的分子机制。
Genetic and environmental disruption to the circadian clock can lead to diverse diseases including sleep and metabolic disorders. Cells with engineered circadian reporters, especially bioluminescence reporters, have been instrumental in understanding the mechanisms underpinning circadian rhythms and circadian disorders, but have been limited by the lack of endogenous circadian reporters available for human cells. To address this gap, we have developed two endogenous reporter lines in the human clock cell model, U-2 OS. Using these reporters combined with genome editing by CRISPR, we uncovered how the circadian clock is uniquely regulated in peripheral cells compared with the master clock in the brain. Our cell-based platform will accelerate and enable expanded testing of many mutations in clock genes and mechanistic studies. Adverse consequences from having a faulty circadian clock include compromised sleep quality and poor performance in the short-term, and metabolic diseases and cancer in the long-term. However, our understanding of circadian disorders is limited by the incompleteness of our molecular models and our dearth of defined mutant models. Because it would be prohibitively expensive to develop live animal models to study the full range of complicated clock mechanisms, we developed PER1-luc and PER2-luc endogenous circadian reporters in a validated clock cell model, U-2 OS, where the genome can be easily manipulated, and functional consequences of mutations can be accurately studied. When major clock genes were knocked out in these cells, circadian rhythms were modulated similarly compared with corresponding mutant mice, validating the platform for genetics studies. Using these reporter cells, we uncovered critical differences between two paralogs of PER. Although PER1 and PER2 are considered redundant and either one can serve as a pacemaker alone, they were dramatically different in biochemical parameters such as stability and phosphorylation kinetics. Consistently, circadian phase was dramatically different between PER1 and PER2 knockout reporter cells. We further showed that the stable binding of casein kinase1δ/ε to PER is not required for PER phosphorylation itself, but is critical for delayed timing of phosphorylation. Our system can be used as an efficient platform to study circadian disorders associated with pathogenic mutations and their underlying molecular mechanisms.
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影响因子：
14.8
作者：
Koch B;Nijmeijer B;Kueblbeck M;Cai Y;Walther N;Ellenberg J
通讯作者：
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64.5
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64.5
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作者：
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