Journal: bioRxiv

Article Title: The reindeer circadian clock is rhythmic and temperature-compensated but shows evidence of weak coupling between the secondary and core molecular clock loops

doi: 10.1101/2024.04.24.590752

Figure Lengend Snippet: Endogenous and temperature-compensated clock gene expression in mouse and reindeer fibroblast culture. (a) Representative luminescence data of mouse and reindeer fibroblasts under 37°C ambient temperature. Expression of the Bmal1 :luc reporter (black) is in approximate antiphase to the expression of Per2:luc reporter (grey) in both mouse and reindeer fibroblast cultures. Bioluminescence was measured in counts per minute (cpm). Phase angle difference (Ψ) between Bmal1 :luc and Per2:luc for the first day after DEX synchronisation (from hour 24 to hour 48) are plotted as circular plots. Data is normalized for the respective free-running period (τ). (b) Half-life times of Bmal1 :luc expression under different ambient temperatures. Half-life times were calculated by damped sine wave analysis. (c) Periods of Bmal1 :luc expression under different ambient temperatures. Periods have been determined by damped sine wave analysis. (d) Q10 values of Bmal1 :luc in mouse and reindeer fibroblast cultures. Q10 values are based on periods as shown in panel c. Both Q10 values are within the acceptable range for temperature-compensation (0.8-1.4) and do not differ significantly from each other (ns).

Article Snippet: The Per2:luc reporter vector (pLV6-Per2-luc) consists of the backbone of the Bmal1:luc reporter vector (pLV6-Bmal-luc) and a murine Per2 promoter with adjacent luciferase sequence which is contained in the pGL3 basic E2 vector (Addgene 48747, a gift from Joseph Takahashi).

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: The reindeer circadian clock is rhythmic and temperature-compensated but shows evidence of weak coupling between the secondary and core molecular clock loops

doi: 10.1101/2024.04.24.590752

Figure Lengend Snippet: Temperature entrainment of clock genes expression in mouse and reindeer fibroblast cultures. Expression of Per2:luc and Bmal1:luc in mouse (a-c) and reindeer fibroblasts (d-f) under constant and cycling ambient temperature (Ta). Two sets of cultures were DEX-synchronized 12h apart from each other. Bioluminescence was measured in counts per minute (cpm). For reindeer, bioluminescence data after the temperature cycle were plotted in zoomed-in plots beside the corresponding graphs. Times of peak expressions (centre of gravity) were determined with the CircWave programme for each rhythm to track phase relationships and responses before, during and after the temperature cycle (c, f). Phase relationships between the initially 12h-apart synchronised cell cultures of the same gene were calculated after the temperature cycle and tested with an unpaired t-test (asterisk indicates p< 0.05, ns denotes p> 0.05). (g, h) Relative gene expressions of Bmal1 and Per2 in mouse and reindeer fibroblasts under one temperature cycle. Gene expression data was measured by qPCR with species-specific primers.

Article Snippet: The Per2:luc reporter vector (pLV6-Per2-luc) consists of the backbone of the Bmal1:luc reporter vector (pLV6-Bmal-luc) and a murine Per2 promoter with adjacent luciferase sequence which is contained in the pGL3 basic E2 vector (Addgene 48747, a gift from Joseph Takahashi).

Techniques: Expressing, Gene Expression

Journal: bioRxiv

Article Title: The reindeer circadian clock is rhythmic and temperature-compensated but shows evidence of weak coupling between the secondary and core molecular clock loops

doi: 10.1101/2024.04.24.590752

Figure Lengend Snippet: In mouse fibroblasts, the Bmal1:luc reporter entrains gradually to an ambient temperature cycle eventually resuming its approximate antiphase relationship with Per2 expression, suggesting that the Bmal1 -specific temperature effect is dominantly mediated via the circadian clock. In reindeer fibroblasts, Bmal1 is acutely responding to temperature cycles and moves into phase with Per2 expression. We suggest that a weak coupling between circadian loops reduces the effect of temperature via the RORE elements on the Bmal1 promoter, instead an unknown RORE-independent pathway drives Bmal1 expression during temperature cycles in reindeer fibroblasts. Supporting this model: when we experimentally decoupled the circadian clock from the Bmal1:luc reporter by mutations of the ROREs we rendered the promoter-reporter acutely temperature-sensitive which led to a reindeer-like phase response under temperature cycles.

Article Snippet: The Per2:luc reporter vector (pLV6-Per2-luc) consists of the backbone of the Bmal1:luc reporter vector (pLV6-Bmal-luc) and a murine Per2 promoter with adjacent luciferase sequence which is contained in the pGL3 basic E2 vector (Addgene 48747, a gift from Joseph Takahashi).

Techniques: Expressing