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CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping

View ORCID ProfileMarrit Putker, David Wong, Estere Seinkmane, Nina Marie Rzechorzek, Aiwei Zeng, Nathaniel P. Hoyle, Johanna E. Chesham, Mathew D. Edwards, Kevin A. Feeney, Robin Fischer, Nicolai Peschel, Ko-Fan Chen, Christopher P. Selby, Aziz Sancar, View ORCID ProfileJohn S. O’Neill
doi: https://doi.org/10.1101/2020.05.14.095968
Marrit Putker
1MRC Laboratory of Molecular Biology, Cambridge, UK
5Hubrecht Institute, Utrecht, the Netherlands
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  • ORCID record for Marrit Putker
David Wong
1MRC Laboratory of Molecular Biology, Cambridge, UK
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Estere Seinkmane
1MRC Laboratory of Molecular Biology, Cambridge, UK
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Nina Marie Rzechorzek
1MRC Laboratory of Molecular Biology, Cambridge, UK
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Aiwei Zeng
1MRC Laboratory of Molecular Biology, Cambridge, UK
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Nathaniel P. Hoyle
1MRC Laboratory of Molecular Biology, Cambridge, UK
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Johanna E. Chesham
1MRC Laboratory of Molecular Biology, Cambridge, UK
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Mathew D. Edwards
1MRC Laboratory of Molecular Biology, Cambridge, UK
6UCL Sainsbury Wellcome Centre for Neural Circuits and Behaviour, London, UK
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Kevin A. Feeney
1MRC Laboratory of Molecular Biology, Cambridge, UK
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Robin Fischer
2Biozentrum Universität, Würzburg, Germany
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Nicolai Peschel
2Biozentrum Universität, Würzburg, Germany
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Ko-Fan Chen
3Institute of Neurology, UCL, London, UK
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Christopher P. Selby
4Dept of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA
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Aziz Sancar
4Dept of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA
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John S. O’Neill
1MRC Laboratory of Molecular Biology, Cambridge, UK
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  • ORCID record for John S. O’Neill
  • For correspondence: oneillj@mrc-lmb.cam.ac.uk
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Abstract

Summary Circadian (approximately daily) rhythms are a pervasive property of mammalian cells, tissues, and behaviour, ensuring physiological and metabolic adaptation to solar time. Models of daily cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of ‘clock proteins’ PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are thus considered essential negative regulators of the oscillation; a function supported by behavioural arrhythmicity of CRY-deficient mice when kept under constant conditions. Challenging this interpretation, however, we find evidence for persistent circadian rhythms in mouse behaviour and cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have a shorter period than wild type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and determination of period by casein kinase 1δ/ε activity to be maintained. In the absence of CRY-mediated transcriptional feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational. Our findings suggest that PER proteins normally act as signalling hubs that transduce timing information to the nucleus, imparting daily rhythms upon the activity of transcriptional effectors.

Highlights

  • ➢ PER/CRY-mediated negative feedback is dispensable for mammalian circadian timekeeping

  • ➢ Circadian variation in PER2 levels persists in the absence of rhythmic Per2 transcription

  • ➢ CK1 and GSK3 are plausible mechanistic components of a ‘cytoscillator’ mechanism

  • ➢ CRY-mediated feedback repression imparts robustness to biological timekeeping

In brief Circadian turnover of mammalian clock protein PERIOD2 persists in the absence of canonical transcriptional feedback repression and rhythmic clock gene activity, demanding a re-evaluation of cellular clock function and evolution.

Competing Interest Statement

The authors have declared no competing interest.

  • List of abbreviations

    CCGs
    Clock controlled genes
    CHX
    Cycloheximide
    CK1
    Casein kinase 1
    CKO
    CRY Knock out
    CPKO
    CRY PER Knock out
    CPS
    Counts per second
    CP 20s
    Counts per 20 seconds
    CRY
    Cryptochrome
    GSK3
    Glycogen synthase kinase 3
    LL>DD
    12h:12h Light:light to dark:dark transition
    MAF
    Mouse adult fibroblast
    MEF
    Mouse embryonic fibroblast
    PER
    Period
    RLU
    Relative light units
    SCN
    Suprachiasmatic nucleus
    SD
    Standard deviation
    SEM
    Standard error of the mean
    Timout
    Timeless knockout
    TTFL
    Transcriptional translational feedback loop
    WCL
    Whole cell lysates
    WT
    Wild type
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    Posted May 15, 2020.
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    CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping
    Marrit Putker, David Wong, Estere Seinkmane, Nina Marie Rzechorzek, Aiwei Zeng, Nathaniel P. Hoyle, Johanna E. Chesham, Mathew D. Edwards, Kevin A. Feeney, Robin Fischer, Nicolai Peschel, Ko-Fan Chen, Christopher P. Selby, Aziz Sancar, John S. O’Neill
    bioRxiv 2020.05.14.095968; doi: https://doi.org/10.1101/2020.05.14.095968
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    CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping
    Marrit Putker, David Wong, Estere Seinkmane, Nina Marie Rzechorzek, Aiwei Zeng, Nathaniel P. Hoyle, Johanna E. Chesham, Mathew D. Edwards, Kevin A. Feeney, Robin Fischer, Nicolai Peschel, Ko-Fan Chen, Christopher P. Selby, Aziz Sancar, John S. O’Neill
    bioRxiv 2020.05.14.095968; doi: https://doi.org/10.1101/2020.05.14.095968

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