Non-coding cis-element of Period2 is essential for maintaining organismal circadian behaviour and body temperature rhythmicity

Abstract

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive. Cis-element-based transcriptional regulation is believed to be crucial for generating circadian rhythms in behavior and physiology. However, genetic evidence supporting this model is based on mutations in the protein-coding sequences of clock genes. Here, we report generation of mutant mice carrying a mutation only at the E'-box cis-element in the promoter region of the core clock gene Per2. The Per2 E'-box mutation abolishes sustainable molecular clock oscillations and renders circadian locomotor activity and body temperature rhythms unstable. Without the E'-box, Per2 messenger RNA and protein expression remain at mid-to-high levels. Our work delineates the Per2 E'-box as a critical nodal element for keeping sustainable cell-autonomous circadian oscillation and reveals the extent of the impact of the non-coding cis-element in daily maintenance of animal locomotor activity and body temperature rhythmicity.

Fig. 1

Generation of mice carrying a targeted mutation at Per2 E′-box. a Genome modification strategy using the piggyBac transposon. The CACGTT Per2 E′-box was mutated to GCTAGT. Top line, genome architecture of the mouse Per2 gene; E1, exon 1; E2, exon 2; Blue, wildtype E′-box; Red, mutated E′-box; Green, piggyBac transposon carrying a neomycin-resistant gene (PB-Neo cassette) inserted at a genomic TTAA site (+600 to +603); Gray bars, Southern blot probes. Numbering shows the position relative to the putative transcription start site (+1). b Interbreeding scheme. Mice heterozygous for the targeted allele (+/PB-Neo) were intercrossed with ROSA26-PBase mice. The resultant mutant mice without the marker cassette (+/m) were backcrossed into the C57BL/6J strain. c Southern blot and PCR analysis showing the insertion (+/PB-Neo) and excision (+/m and +/m*) of the piggyBac transposon. *, a reintegrated PB-Neo fragment. d Genomic DNA sequences of WT (+/+) and Per2 E′-box mutant (+/m and m/m) mice, illustrating the precise mutation of the E′-box and seamless excision of the PB-Neo cassette from the TTAA site. Heterozygous (+/m) mouse sequences exhibit dual signals for CACGTT and GCTAGT at the E′-box. e ChIP values for WT (+/+) and homozygous mutant (m/m) mouse liver sampled at 4 h intervals for 24 h on the first day in DD. Values are means ± s.e.m. of three technical replicates. Source data for (c) and (e) are provided as a Source Data file

Fig. 2

Per2 E′-box is essential for maintaining cell-autonomous circadian oscillations. a Temporal profiles of PER2 protein expression in Per2E′^+/+ and Per2E′^m/m fibroblasts. Representative immunoblots and normalized densitometry values (n = 3, mean ± s.e.m.) are shown. b mRNA profiling of clock genes in Per2E′^+/+ and Per2E′^m/m fibroblasts (n = 2, for each data point). For Per2, both intron and exon RNA were analyzed. The data are presented as the mean ± variation. Source data are provided as a Source Data file

Fig. 3

Per2 E′-box is essential for sustainable Bmal1 oscillations in SCN and lung explants. a Bmal1-Eluc bioluminescence traces of ex vivo SCN cultures from Per2E′^+/+ (n = 5), Per2E′^m/m (n = 5), Per2E′^m/m; Per1^+/− (n = 5), and Per1^+/− (n = 3) mice. Averaged de-trended data are shown. b FFT amplitude of (a). *P < 0.05, **P < 0.001, one-way ANOVA, Bonferroni post hoc test. c Daily max-to-min variations of (a). The data are presented as the mean ± s.e.m. d Bmal1-Eluc bioluminescence traces of lung tissue explant cultures from Per2E′^+/+ (n = 5), Per2E′^m/m (n = 5), Per2E′^m/m; Per1^+/− (n = 5), and Per1^+/− (n = 3) mice. Averaged de-trended data are shown. e FFT amplitude of (d). *P < 0.05, **P < 0.001, one-way ANOVA, Bonferroni post hoc test. f Daily max-to-min variations of (d). The data are presented as the mean ± s.e.m. Source data for (c) and (f) are provided as a Source Data file

Fig. 4

a Destabilized circadian locomotor activity and body temperature rhythms of Per2 E′-box mutant mice. Representative locomotor activity records of Per2E′^+/+ and Per2E′^m/m mice under light/dark (LD) followed by constant light (LL). The graph shows changes in FFT power in LL. **P < 0.01, two-way repeated-measures ANOVA, Bonferroni post hoc test (Per2E′^+/+, n = 7; Per2E′^m/m, n = 9). b Representative body temperature records of Per2E′^+/+ and Per2E′^m/m mice in LL. The mean temperature of the entire data series was calculated, and the data points above the mean are plotted. Rayleigh plots show phase distribution of elevated body temperature on days 7–21 (n = 3, each genotype). Data from independent animals are color-coded. Arrow length reflects the r-value of each distribution. *P < 0.05, two-tailed unpaired t-test. c Representative records of locomotor activity (left) and plots of activity onset (middle) of Per2E′^+/+ and Per2E′^m/m mice before and after an 8-h phase advance in LD cycles. The PS₅₀ values represent the time required for 50% phase-shift (right). ***P < 0.0001, two-tailed unpaired t-test (Per2E′^+/+, n = 7; Per2E′^m/m, n = 9). d Representative locomotor activity records (left), activity onset (middle), and magnitude of phase-shift (right) of Per2E′^+/+ and Per2E′^m/m mice subjected to an 8-h phase advance on day 1 and released to DD. ***P < 0.0001, two-tailed unpaired t-test (Per2E′^+/+, n = 7; Per2E′^m/m, n = 11). The data are the mean ± s.e.m. Source data are provided as a Source Data file