CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping

Abstract

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ϵ activity to be maintained. In the absence of CRY-mediated 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.

Autonomous clocks that regulate organelle biogenesis, cytoskeletal organization, and intracellular dynamics

“…The mechanism of circadian clocks is a prime example, where complicated maps of transcription-translation feedback loops (TTFLs) have become an indispensable part of this field (reviewed in Takahashi, 2017 ). Although TTFLs appear important in maintaining the robustness of, and sustain prolonged rhythms for, circadian clocks ( Putker et al, 2021 ), several studies have strongly challenged the necessity of TTFLs to run the clocks themselves ( Lipton et al, 2015 ; Nakajima et al, 2005 ; Tomita et al, 2005 ), including the seminal observation of circadian redox rhythms in enucleated mature red blood cells ( O’Neill and Reddy, 2011 ). The unifying feature of these studies is their proposal of alternative mechanisms at the level of proteins, such as the use of autoregulatory post-translational modifications to generate self-sustained feedback loops ( Figure 5 , the shaded green and blue slices).…”

Ultradian rhythms of AKT phosphorylation and gene expression emerge in the absence of the circadian clock components Per1 and Per2

“…Among others, these may include lipid species such as polyphosphoinositides, primarily PIP3, which facilitate phosphorylation and activation of AKT, or proteins such as mTORC2 and RICTOR, as well as downstream AKT effectors such as phosphorylated S6K, S6, and mTOR [ 6 , 25 ]. Another interesting candidate is GSK3β, a target of AKT, which was recently suggested to function also as a cryptochrome-independent component of a cytosolic oscillator [ 28 ]. Furthermore, our bioinformatic analysis hints toward some transcription factor complexes, whose relevant transcripts exhibit about 16-hour rhythmicity.…”

CHRONO and DEC1/DEC2 compensate for lack of CRY1/CRY2 in expression of coherent circadian rhythm but not in generation of circadian oscillation in the neonatal mouse SCN

“…Recently, a post-translational model was proposed for the circadian oscillation of Cry1/Cry2 deficient mice 23 . In this study, in addition to the robust circadian rhythms in PER2::LUC, the temperature compensation and period determination by CK1ε/δ activity were demonstrated in the cultured fibroblasts of Cry1/Cry2 deficient mice.…”

“…Circadian rhythm was detected without the nucleus in the red blood cells in mammals, and in acetabularia, a single cell algae 26 , 27 . A lack of a TTFL of clock genes could be compensated by neural networks and/or other oscillatory mechanisms such as post-translational oscillation 23 and a single molecule oscillation 28 .…”