The mammalian suprachiasmatic nucleus (SCN) forms not only the master circadian clock but also a seasonal clock. This neural network of ∼10,000 circadian oscillators encodes season-dependent day-length changes through a largely unknown mechanism. We show that region-intrinsic changes in the SCN fine-tune the degree of network synchrony and reorganize the phase relationship among circadian oscillators to represent day length. We measure oscillations of the clock gene Bmal1, at single-cell and regional levels in cultured SCN explanted from animals raised under short or long days. Coupling estimation using the Kuramoto framework reveals that the network has couplings that can be both phase-attractive (synchronizing) and -repulsive (desynchronizing). The phase gap between the dorsal and ventral regions increases and the overall period of the SCN shortens with longer day length. We find that one of the underlying physiological mechanisms is the modulation of the intracellular chloride concentration, which can adjust the strength and polarity of the ionotropic GABA_A-mediated synaptic input. We show that increasing day-length changes the pattern of chloride transporter expression, yielding more excitatory GABA synaptic input, and that blocking GABA_A signaling or the chloride transporter disrupts the unique phase and period organization induced by the day length. We test the consequences of this tunable GABA coupling in the context of excitation–inhibition balance through detailed realistic modeling. These results indicate that the network encoding of seasonal time is controlled by modulation of intracellular chloride, which determines the phase relationship among and period difference between the dorsal and ventral SCN.

Mammalian circadian clocks have a hierarchical organization, governed by the suprachiasmatic nucleus (SCN) in the hypothalamus. The brain itself contains multiple loci that maintain autonomous circadian rhythmicity, but the contribution of the non-SCN clocks to this hierarchy remains unclear. We examine circadian oscillations of clock gene expression in various brain loci and discovered that in mouse, robust, higher amplitude, relatively faster oscillations occur in the choroid plexus (CP) compared to the SCN. Our computational analysis and modeling show that the CP achieves these properties by synchronization of “twist” circadian oscillators via gap-junctional connections. Using an in vitro tissue coculture model and in vivo targeted deletion of the Bmal1 gene to silence the CP circadian clock, we demonstrate that the CP clock adjusts the SCN clock likely via circulation of cerebrospinal fluid, thus finely tuning behavioral circadian rhythms.

Circadian rhythms are common to most organisms and govern much of homeostasis and physiology. Since a significant fraction of the mammalian genome is controlled by the clock machinery, understanding the genome-wide signaling and epigenetic basis of circadian gene expression is essential. BMAL1 is a critical circadian transcription factor that regulates genes via E-box elements in their promoters. We used multiple high-throughput approaches, including chromatin immunoprecipitation-based systematic analyses and DNA microarrays combined with bioinformatics, to generate genome-wide profiles of BMAL1 target genes. We reveal that, in addition to E-boxes, the CCAATG element contributes to elicit robust circadian expression. BMAL1 occupancy is found in more than 150 sites, including all known clock genes. Importantly, a significant proportion of BMAL1 targets include genes that encode central regulators of metabolic processes. The database generated in this study constitutes a useful resource to decipher the network of circadian gene control and its intimate links with several fundamental physiological functions.

Significance: Each day, over 50 billion synaptic signals, mediated by the neurotransmitter GABA, are sent between neurons in the central circadian pacemaker in the mammalian brain to time and coordinate daily events. Although GABA is the only signaling molecule sent and received by most, if not all of these neurons, its role is not well understood. Past studies have shown paradoxically that GABA can synchronize and desynchronize, as well as excite and inhibit, clock neurons. Through experiments and modeling characterizing the role of GABA in timekeeping, we propose the existence of two types of differentially regulated GABA signaling—fast signaling that regulates neuronal output, and slow signaling that modulates synchrony between neurons—a hypothesis that can explain many previous experimental results.

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. 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.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) is the key enzyme in melatonin synthesis regulated by circadian rhythm. To date, our understanding of the oscillatory mechanism of melatonin has been limited to autoregulatory transcriptional and posttranslational regulations of AANAT mRNA. In this study, we identify three proteins from pineal glands that associate with cis-acting elements within species-specific AANAT 3′ untranslated regions to mediate mRNA degradation. These proteins include heterogeneous nuclear ribonucleoprotein R (hnRNP R), hnRNP Q, and hnRNP L. Their RNA-destabilizing function was determined by RNA interference and overexpression approaches. Expression patterns of these factors in pineal glands display robust circadian rhythm. The enhanced levels detected after midnight correlate with an abrupt decline in AANAT mRNA level. A mathematical model for the AANAT mRNA profile and its experimental evidence with rat pinealocytes indicates that rhythmic AANAT mRNA degradation mediated by hnRNP R, hnRNP Q, and hnRNP L is a key process in the regulation of its circadian oscillation.