Spatiotemporal separation of PER and CRY posttranslational regulation in the mammalian circadian clock

Abstract

Posttranslational regulation of clock proteins is an essential part of mammalian circadian rhythms, conferring sensitivity to metabolic state and offering promising targets for pharmacological control. Two such regulators, casein kinase 1 (CKI) and F-box and leucinerich repeat protein 3 (FBXL3), modulate the stability of closely linked core clock proteins period (PER) and cryptochrome (CRY), respectively. Inhibition of either CKI or FBXL3 leads to longer periods, and their effects are independent despite targeting proteins with similar roles in clock function. A mechanistic understanding of this independence, however, has remained elusive. Our analysis of cellular circadian clock gene reporters further differentiated between the actions of CKI and FBXL3 by revealing opposite amplitude responses from each manipulation. To understand the functional relationship between the CKI-PER and FBXL3-CRY pathways, we generated robust mechanistic predictions by applying a bootstrap uncertainty analysis to multiple mathematical circadian models. Our results indicate that CKI primarily regulates the accumulating phase of the PER-CRY repressive complex by controlling the nuclear import rate, whereas FBXL3 separately regulates the duration of transcriptional repression in the nucleus. Dynamic simulations confirmed that this spatiotemporal separation is able to reproduce the independence of the two regulators in period regulation, as well as their opposite amplitude effect. As a result, this study provides further insight into the molecular clock machinery responsible for maintaining robust circadian rhythms.identifiability analysis | gene regulation | sensitivity analysis C ircadian rhythms are autonomous, near-24-h oscillations that coordinate daily changes in physiology and metabolism. Because circadian and metabolic regulators are tightly integrated, circadian disruptions often manifest in metabolic disease (1). Recent efforts have therefore sought to gain a mechanistic understanding of these pathways, such that the metabolic burdens imposed by a 24-h society might be mitigated. Posttranslational regulators, which play key roles in connecting circadian and metabolic processes, serve as likely targets for future therapeutics, demonstrated by the wealth of available circadian-active small molecules (2, 3).Oscillations in circadian gene transcription are generated through a time-delayed transcription-translation negative-feedback loop. In mammals, transcription factors CLOCK and BMAL1 promote transcription of E box-containing genes Period (Per) and Cryptochrome (Cry) (Fig. 1A). PER and CRY protein products form heterodimers to accumulate in the nucleus, in which PER is stoichiometrically limiting (4), and subsequently close the negative feedback loop by inhibiting CLOCK-BMAL1-promoted gene expression. Although steady-state endpoint assays have shown the possibility of nuclear entry of CRY without PER (5-7), experiments from Per1 −/− Per2 −/− mice demonstrated that PER proteins are required for the timely nuclear accumulation of ...

In Vivo Role of Phosphorylation of Cryptochrome 2 in the Mouse Circadian Clock

“…The overaccumulated CRY2-PER2 complex should need a longer time to be cleared from the nucleus, leading to the lengthening of the circadian period. This model is supported by a previous simulation-based study indicating that an increase of PER2 nuclear transport rather than its stability has a period-lengthening effect (31). Here, we demonstrate that Ser557 phosphorylation of CRY2 is critical not only for the slow accumulation of nuclear CRY2 protein in the increasing phase (ZT10 to -18) but also for suppressing the overaccumulation of CRY2 protein at peak timing (ZT22) (Fig.…”

“…CRY2 regulates the cellular distribution of PER2. It is well established that PER proteins accumulate in the nucleus during the nighttime when E-box-mediated transcription is inhibited (29,30), and its nuclear transport is a critical period-determining step (31). In the liver of CRY2(S557A) knock-in mice, nuclear PER2 levels were remarkably increased, whereas total PER2 levels were largely unaffected ( Fig.…”

Regulating the Suprachiasmatic Nucleus (SCN) Circadian Clockwork: Interplay between Cell-Autonomous and Circuit-Level Mechanisms

“…Thus, a general model has been proposed that CKI primarily regulates the accumulating phase of the PER-CRY repressive complex by controlling the nuclear import rate, whereas FBXL3/FBXL21 separately regulate the duration of transcriptional repression in the nucleus (St John et al 2014). Given the dramatic individual effects of PER and CRY stability on SCN and behavioral periodicity, the question arises of how do they interact?…”

Unique Aspects of Cryptochrome in Chronobiology and Metabolism, Pancreaticβ-Cell Dysfunction, and Regeneration: Research into Cysteine414-Alanine Mutant CRY1

“…For regulating circadian periods and/or metabolism by the ability to modulate CRY molecular structures, various chemicals have been created which bind to CRYs and which affect them. One such chemical is KL001 [28]. Reportedly, FBXL3 binds to CRY2 by occupying CRY2's binding pocket to FAD [29].…”