Circadian Proteins Cry and Rev-erb Deepen Cellular Quiescence by Down-regulating Cyclin D and Cdk4,6

The proper balance and transition between cellular quiescence and proliferation are critical to tissue homeostasis, and their deregulations are commonly found in many human diseases, including cancer and aging. Recent studies showed that the reentry of quiescent cells to the cell cycle is subjected to circadian regulation. However, the underlying mechanisms are largely unknown. Here, we report that two circadian proteins, Cryptochrome (Cry) and Rev-erb, deepen cellular quiescence in rat embryonic fibroblasts, resulting in stronger serum stimulation required for cells to exit quiescence and reenter the cell cycle. This finding was opposite from what we expected from the literature. By modeling a library of possible regulatory topologies linking Cry and Rev-erb to a bistable Rb-E2f gene network switch that controls the quiescence-to-proliferation transition and by experimentally testing model predictions, we found Cry and Rev-erb converge to downregulate Cyclin D/Cdk4,6 activity, leading to an ultrasensitive increase of the serum threshold to activate the Rb-E2f bistable switch. Our findings suggest a mechanistic role of circadian proteins in modulating the depth of cellular quiescence, which may have implications in the varying potentials of tissue repair and regeneration at different times of the day.