Circadian rhythms are controlled by a system of negative and positive genetic feedback loops composed of clock genes. Although many genes have been implicated in these feedback loops, it is unclear whether our current list of clock genes is exhaustive. We have recently identified Chrono as a robustly cycling transcript through genome-wide profiling of BMAL1 binding on the E-box. Here, we explore the role of Chrono in cellular timekeeping. Remarkably, endogenous CHRONO occupancy around E-boxes shows a circadian oscillation antiphasic to BMAL1. Overexpression of Chrono leads to suppression of BMAL1-CLOCK activity in a histone deacetylase (HDAC) -dependent manner. In vivo loss-of-function studies of Chrono including Avp neuron-specific knockout (KO) mice display a longer circadian period of locomotor activity. Chrono KO also alters the expression of core clock genes and impairs the response of the circadian clock to stress. CHRONO forms a complex with the glucocorticoid receptor and mediates glucocorticoid response. Our comprehensive study spotlights a previously unrecognized clock component of an unsuspected negative circadian feedback loop that is independent of another negative regulator, Cry2, and that integrates behavioral stress and epigenetic control for efficient metabolic integration of the clock.
Author Summary The circadian clock has a fundamental role in regulating biological temporal rhythms in organisms, and it is tightly controlled by a molecular circuit consisting of positive and negative regulatory feedback loops. Although many of the clock genes comprising this circuit have been identified, there are still some critical components missing. Here, we characterize a circadian gene renamed Chrono (Gm129) and show that it functions as a transcriptional repressor of the negative feedback loop in the mammalian clock. Chrono binds to the regulatory region of clock genes and its occupancy oscillates in a circadian manner. Chrono knockout and Avp-neuron-specific knockout mice display longer circadian periods and altered expression of core clock genes. We show that Chrono-mediated repression involves the suppression of BMAL1-CLOCK activity via an epigenetic mechanism and that it regulates metabolic pathways triggered by behavioral stress. Our study suggests that Chrono functions as a clock repressor and reveals the molecular mechanisms underlying its function.