Cryptochrome 1 as a state variable of the circadian clockwork of the suprachiasmatic nucleus: Evidence from translational switching

Cryptochrome 1 as a state variable of the circadian clockwork of the suprachiasmatic nucleus: Evidence from translational switching

Edited by Joseph Takahashi, The University of Texas Southwestern Medical Center, Dallas, TX; received March 14, 2022; accepted July 18, 2022

August 17, 2022

119 (34) e2203563119

Significance

Circadian clocks adapt us to our rhythmic world, setting the tempo to our lives. Their disruption (e.g., by shiftwork) therefore carries a severe cost in health. The suprachiasmatic nucleus (SCN) is the principal brain clock of mammals, its time keeping pivoting around a delayed negative feedback loop of gene and protein expression. By using “translational switching” as a means to reversibly control the expression of the negative feedback regulator Cryptochrome 1 (CRY1) in SCN organotypic slices, we show that acute changes in the level of CRY1 define circadian time. We thereby bridge theoretical and biochemical perspectives of the SCN clockwork.

Abstract

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal clock driving circadian rhythms of physiology and behavior that adapt mammals to environmental cycles. Disruption of SCN-dependent rhythms compromises health, and so understanding SCN time keeping will inform management of diseases associated with modern lifestyles. SCN time keeping is a self-sustaining transcriptional/translational delayed feedback loop (TTFL), whereby negative regulators inhibit their own transcription. Formally, the SCN clock is viewed as a limit-cycle oscillator, the simplest being a trajectory of successive phases that progresses through two-dimensional space defined by two state variables mapped along their respective axes. The TTFL motif is readily compatible with limit-cycle models, and in Neurospora and Drosophila the negative regulators Frequency (FRQ) and Period (Per) have been identified as state variables of their respective TTFLs. The identity of state variables of the SCN oscillator is, however, less clear. Experimental identification of state variables requires reversible and temporally specific control over their abundance. Translational switching (ts) provides this, the expression of a protein of interest relying on the provision of a noncanonical amino acid. We show that the negative regulator Cryptochrome 1 (CRY1) fulfills criteria defining a state variable: ts-CRY1 dose-dependently and reversibly suppresses the baseline, amplitude, and period of SCN rhythms, and its acute withdrawal releases the TTFL to oscillate from a defined phase. Its effect also depends on its temporal pattern of expression, although constitutive ts-CRY1 sustained (albeit less stable) oscillations. We conclude that CRY1 has properties of a state variable, but may operate among several state variables within a multidimensional limit cycle.

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.

Acknowledgments

We acknowledge the excellent technical support provided by the Mechanical and Electronic Workshops and the Ares Biomedical Facility of the Laboratory of Molecular Biology. This work was supported by the Biotechnology and Biological Sciences Research Council, UK (Awards BB/P017347/1 and BB/R016658/1 to M.H.H.) and the Medical Research Council (MRC), as part of United Kingdom Research and Innovation (also known as UK Research and Innovation) (MRC File Reference No. MC_U105170643).

Supporting Information

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D. McManus et al., Figure 3: Translational switching of CRY1 expression allows reversible and dose-dependent control of period and amplitude of the TTFL of wild-type SCN. Cry1-Luc pSyn1.Cry1(TAG)::EGFP, 10mM. https://biodare2.ed.ac.uk/experiment/22843. Deposited 25 May 2022.

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D. McManus et al., Figure 3: Translational switching of CRY1 expression allows reversible and dose-dependent control of period and amplitude of the TTFL of wild-type SCN. PER2::LUC pSyn1.Cry1(TAG)::EGFP, Vehicle. https://biodare2.ed.ac.uk/experiment/22836. Deposited 25 May 2022.

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D. McManus et al., Figure 3: Translational switching of CRY1 expression allows reversible and dose-dependent control of period and amplitude of the TTFL of wild-type SCN. PER2::LUC pSyn1.Cry1(TAG)::EGFP, 10mM. https://biodare2.ed.ac.uk/experiment/22839. Deposited 25 May 2022.

72

D. McManus et al., Figure 3: Translational switching of CRY1 expression allows reversible and dose-dependent control of period and amplitude of the TTFL of wild-type SCN. Cry1-Luc pSyn1.Cry1(TAG)::EGFP, Vehicle. https://biodare2.ed.ac.uk/experiment/22840. Deposited 25 May 2022.

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D. McManus et al., Figure 3: Translational switching of CRY1 expression allows reversible and dose-dependent control of period and amplitude of the TTFL of wild-type SCN. Cry1-Luc pSyn1.Cry1(TAG)::EGFP, 10mM. https://biodare2.ed.ac.uk/experiment/22843. Deposited 25 May 2022.


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