Department of Genetics & Genome Biology, University of Leicester, Leicester, United Kingdom.
German Cancer Research Center, Heidelberg, Baden-Württemberg, Germany.
PLoS Genet. 2023 Oct 19;19(10):e1011011. doi: 10.1371/journal.pgen.1011011. eCollection 2023 Oct.
Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal (12.4 hour) or other lunar-mediated cycles (15 day, ~29 day), widely expressed in coastal organisms, is unknown. Disrupting circadian clockworks does not appear to affect lunar-based rhythms in several organisms that inhabit the shoreline suggesting a molecular independence of the two cycles. Nevertheless, pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. Here we show that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between these two positive circadian regulators and circatidal behaviour. We therefore performed dsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2 in animals taken from the wild. Epcry2 and Epbmal1 knockdown disrupted Eurydice's circadian phenotypes of chromatophore dispersion, tim mRNA cycling and the circadian modulation of circatidal swimming, as expected. However, circatidal behaviour was particularly sensitive to Epbmal1 knockdown with consistent effects on the power, amplitude and rhythmicity of the circatidal swimming cycle. Thus, three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM (from a previous study), do not appear to be required for the expression of robust circatidal behaviour, in contrast to the positive regulator EpBMAL1. We suggest a neurogenetic model whereby the positive circadian regulators EpBMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator.
陆地动物的生物钟由分子反馈环编码，涉及负调节因子 PERIOD、TIMELSS 或 CRYPTOCHROME2 和正转录因子 CLOCK 和 BMAL1/CYCLE。广泛存在于沿海生物中的约 12.4 小时的潮汐钟或其他 15 天、29 天的月介导周期的分子基础尚不清楚。在几种栖息在海岸线的生物中，破坏生物钟似乎不会影响基于月球的节律，这表明这两个周期的分子是独立的。然而，在哺乳动物和果蝇中靶向 PERIOD 稳定性的酪蛋白激酶 1（CK1）的药理学抑制剂会影响 Eurydice pulchra（Ep），即斑驳海虱的昼夜节律和潮汐钟表型。在这里，我们表明这些 CK1 的药物抑制剂也会影响 EpCLK 和 EpBMAL1 的磷酸化，并破坏果蝇 S2 细胞中 EpCLK-BMAL1 介导的转录，揭示了这两个正昼夜节律调节剂与潮汐钟行为之间的潜在联系。因此，我们在从野外采集的动物中进行了 Epbmal1 以及 Eurydice 中的主要负调节因子 Epcry2 的 dsRNAi 敲低。Epcry2 和 Epbmal1 的敲低破坏了 Eurydice 的色素体分散、tim mRNA 循环和潮汐钟昼夜节律调制的昼夜节律表型，这是意料之中的。然而，潮汐钟行为对 Epbmal1 的敲低特别敏感，一致影响潮汐钟游泳周期的功率、振幅和节律性。因此，三种 Eurydice 负昼夜节律调节因子，除了 EpPER 和 EpTIM（来自之前的研究）外，EpCRY2 似乎不需要表达强大的潮汐钟行为，与正调节因子 EpBMAL1 相反。我们提出了一个神经遗传模型，其中昼夜节律的正调节因子 EpBMAL1-CLK 在 Eurydice 的昼夜钟和潮汐钟机制之间共享，但潮汐钟节律需要一种新的、未知的负调节因子。
