In vivo functional significance of direct physical interaction between Period and Cryptochrome in mammalian circadian rhythm generation.

In the current model, the auto-negative feedback action of Period (Per) and Cryptochrome (Cry) on their own transcription is the hallmark mechanism driving cell-autonomous circadian rhythms. Although this model likely makes sense even if Per and Cry undertake this action in a mutually independent manner, many studies have suggested the functional significance of direct physical interaction between Per and Cry. However, even though the interaction is a biochemical process that pertains to the fundamentals of the circadian oscillator, its in vivo contribution to circadian rhythm generation remains undefined. To answer this question, we focused on zinc coordination between Per and Cry, whose contribution to circadian rhythm generation remains undefined. Specifically, we aimed to impair endogenous Per-Cry association by introducing an amino acid substitution to zinc-coordinating residues located at the Per1 and Per2 C-terminal facing Cry in mice. These mice did not show severe impairment in the Per-Cry physical interaction, but rather a shortened period and decreased robustness in circadian rhythms at the tissue-autonomous and whole-body levels. Furthermore, these mice also showed a decrease in Per half-life, suggesting that impaired fine-tuning of Per half-life caused abnormal circadian period and robustness in vivo. We also found a minor but significant impact of a reindeer-specific Per2 mutation located in the Per-Cry interface on circadian rhythms in vivo. These lines of evidence indicate that only partial impairment of the Per-Cry physical interaction produces a substantial effect on circadian period and robustness, supporting the in vivo functional significance of the interaction.