Tuning the period of the mammalian circadian clock: additive and independent effects of CK1εTau and Fbxl3Afh mutations on mouse circadian behavior and molecular pacemaking

Abstract

Circadian pacemaking in the suprachiasmatic nucleus (SCN) revolves around a transcriptional/posttranslational feedback loop in which period (Per) and cryptochrome (Cry) genes are negatively regulated by their protein products. Genetically specified differences in this oscillator underlie sleep and metabolic disorders, and dictate diurnal/nocturnal preference. A critical goal, therefore, is to identify mechanisms that generate circadian phenotypic diversity, through both single gene effects and gene interactions. The individual stabilities of PER or CRY proteins determine pacemaker period, and PER/CRY complexes have been proposed to afford mutual stabilization, although how PER and CRY proteins with contrasting stabilities interact is unknown. We therefore examined interactions between two mutations in male mice: Fbxl3(Afh), which lengthens period by stabilizing CRY, and Csnk1ε(tm1Asil) (CK1ε(Tau)), which destabilizes PER, thereby accelerating the clock. By intercrossing these mutants, we show that the stabilities of CRY and PER are independently regulated, contrary to the expectation of mutual stabilization. Segregation of wild-type and mutant alleles generated a spectrum of periods for rest-activity behavior and SCN bioluminescence rhythms. The mutations exerted independent, additive effects on circadian period, biased toward shorter periods determined by CK1ε(Tau). Notably, Fbxl3(Afh) extended the duration of the nadir of the PER2-driven bioluminescence rhythm but CK1ε(Tau) reversed this, indicating that despite maintained CRY expression, CK1ε(Tau) truncated the interval of negative feedback. These results argue for independent, additive biochemical actions of PER and CRY in circadian control, and complement genome-wide epistatic analyses, seeking to decipher the multigenic control of circadian pacemaking.

Figure 1.

Independent control over PER and CRY protein stabilities by CK1ε^Tau and Fbxl3^Afh alleles. a, Western blots for CRY1 in single representative SCN slices carrying either wild-type, CK1ε^Tau, or Fbxl3^Afh alleles, treated with CHX at CT12 and harvested immediately or after 12 h. Right, Blots from CRY2 and CRY1 null mice confirm specificity of antiserum; bottom panels, actin loading controls. b, Group data (mean ± SEM, n = 3–4 SCNs per observation) reveal significant (ANOVA, p < 0.01) reduction in rate of CRY degradation in Afh mutant, with (diamond) and without (triangle) Tau mutation compared to wild type (circle). c, Group data (mean ± SEM, n = 6–8 SCNs per genotype) for PER2 half-life across genotypes. *p < 0.05 significant versus wild-type control, ⁺⁺⁺ p < 0.001 versus Afh mutant.

Figure 2.

Tuning of the period of circadian behavioral rhythms in vivo by additive and independent effects of CK1ε^Tau and Fbxl3^Afh. a, Representative actograms of mice carrying wild-type, CK1ε^Tau and Fbxl3^Afh alleles, initially held on LD and transferred to DD after 12 d. b, Group data (mean ± SEM, n = 6–11, N = 69) to illustrate individual and combined effects of CK1ε^Tau and Fbxl3^Afh alleles on circadian period of rest/activity cycle. Closed circles, Wild type; open circles, CK1ε^Tau/+; closed triangles, CK1ε^Tau/Tau. c, Predicted and observed effects of CK1ε^Tau and Fbxl3^Afh on circadian period of behavioral rhythms. The square symbol represents wild types, and the closed circles are the data from single gene mutants (“predicted” periods for these groups are the actual observed group mean). Open circles represent double mutant groups in which the predicted period was calculated on the basis of the single allele effects. The dotted regression line is the fit derived only from SCNs with complex genotypes; the solid regression line is calculated for the total population.

Figure 3.

Tuning of the period of molecular pacemaking in the SCN by additive and independent effects of CK1ε^Tau and Fbxl3^Afh. a, Representative recordings of bioluminescence rhythms from individual SCN slices: wild type (solid black), CK1ε^Tau/+::Fbxl3 ^Afh/+ (solid gray), and CK1ε^Tau/Tau::Fbxl3^Afh/Afh (dashed gray). b, Group data (mean ± SEM, n = 6–20, N = 95) illustrating individual and combined effects of CK1ε^Tau and Fbxl3^Afh on circadian period of SCN bioluminescence rhythms. Two-way ANOVA revealed significant (p < 0.001) effects of both mutations and an interaction. Closed circles, Wild type; open circles, CK1ε^Tau/+; closed triangles CK1ε^Tau/Tau. c, Relationship between predicted and observed effects of CK1ε^Tau and Fbxl3^Afh alleles on SCN circadian period. The symbols are the same as in Figure 2 c. The dotted regression line is derived only from SCNs with complex genotypes.

Figure 4.

Circadian waveform for PER-driven bioluminescence from wild-type and single and double mutant SCN slices. a, Normalized plots of bioluminescence curves from wild-type (solid black), Afh homozygous (dashed black), Tau homozygous (solid gray), and double homozygous (dashed gray) SCNs, aligned by peak expression. Curves represent mean (±SEM) of 5 slices per genotype. b, Group data (mean ± SEM, n = 5 per genotype) for the duration of the interval of basal bioluminescence (duration of curve below level corresponding to 20% above nadir) from wild-type and single and double mutant SCN slices. *p < 0.05, **p < 0.01, ***p < 0.001 versus wild type, ⁺⁺⁺ p < 0.001 versus Afh mutant (ANOVA and post hoc Bonferroni).