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 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.

Keywords: gene regulation; identifiability analysis; sensitivity analysis.

Fig. 1.

Different amplitude effect of small molecule circadian modulators targeting CKI-PER and FBXL3-CRY. (A) Schematic of the core circadian feedback loop, with the targets of small molecule modulators longdaysin (CKI inhibitor) and KL001 (inhibitor of FBXL3-dependent CRY degradation) shown. The size of each protein molecule is representative of relative concentration. (B) Detrended luminescence profiles (first 72 h, mean of two independent replications) obtained from U2OS reporter cells with increasing concentration of longdaysin and KL001. Black profiles indicate control conditions (0 μM); lighter colors indicate higher concentrations of small molecule (from 0.03 to 8 μM). (C) Relative change in period and amplitude of the results shown in B. Error bars indicate SD of two independent experiments.

Fig. 2.

Time series trajectories of the 2,000 bootstrap trials for each model. Shaded regions indicate 95% confidence regions. The data were scaled to have a maximum value of 1, except for protein species, where relative values were important for clock stoichiometry.

Fig. 3.

Bootstrap predictions of circadian actions of FBXL3-CRY and CKI-PER pathways. Violin plots of the relative period sensitivity of parameters associated with potential mechanisms for FBXL3-CRY (A) and CKI-PER (B) activity, in which a box plot is superimposed above a kernel density plot to convey the distribution of sensitivities across 2,000 realizations. The whiskers used extend to the most extreme data point within 1.5× the inner quartile range. A negative or positive period sensitivity indicate that the period of oscillation will increase or decrease when that rate is inhibited, respectively. Distributions that are not different from 0 with 95% confidence are colored red and marked with an asterisk. Descriptions of the parameters shown are listed in Tables 1 and 2.

Fig. 4.

Mechanistic insight into the effects of small molecule modulators KL001 and longdaysin. (A) In silico reproductions of the circadian reporter experiments in Fig. 1 B and C, using the predictions identified in Fig. 3. (B) Comparison of the effects of KL001 and longdaysin. Parameter changes were normalized such that the period change was equal for each pair of perturbations (vdCn: 100% → 23%, vaC1P: 100% → 51%). (C) Comparison of two candidate mechanisms for CKI inhibition. Effects of increasing PER stabilization and nuclear import inhibition on the time profiles of cytoplasmic PER (Upper) and nuclear CRY (Lower). Parameter values were selected such that the amplitudes of cytoplasmic PER are equal at each level. Lighter colors indicate stronger perturbations (vdP: 100% → 22%, vaC1P: 100% → 45%); t = 0 is set to the onset of PER accumulation.

Fig. 5.

Independence of CKI-PER and FBXL3-CRY pathways. (A) Contour plots of period change (Upper) and Per mRNA amplitude (Lower) for simultaneous inhibition of both PER degradation (vdP) and nuclear import (vaC1P). The gray shaded region indicates loss of oscillations. (B) Period and amplitude change contour plots for varying both vdCn (CRY nuclear degradation rate) and vaC1P (PER-CRY nuclear import).

Fig. 6.

Spatiotemporal separation underlies independence of CKI-PER and FBXL3-CRY pathways. Longdaysin, acting through CKI-PER, lengthens the accumulating phase of the circadian cycle, whereas KL001, acting through FBXL3-CRY, lengthens the declining phase.