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

Significance Maintaining robust circadian rhythms has been linked to longevity and metabolic health. Because these rhythms are disturbed by factors such as jet lag, shift work, and high-fat diets, there is interest in developing pharmacological control strategies to modulate circadian function. The design of therapeutic strategies is currently limited by the lack of a clear mechanistic understanding of interactions between posttranslational regulators, as efficient control of clock behavior will likely require several simultaneous modulations. Here we show fundamentally different clock responses from the manipulation of two clock regulators previously thought to act via similar mechanisms. Using mathematical modeling, we provide a mechanistic interpretation for the relationship between these two regulators, lending insight into circadian regulation and potential pharmacological control. 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 leucine-rich 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.