Jason P. DeBruyne

A clock shock: mouse CLOCK is not required for circadian oscillator function.

The circadian clock mechanism in the mouse is composed of interlocking transcriptional feedback loops. Two transcription factors, CLOCK and BMAL1, are believed to be essential components of the circadian clock. We have used the Cre-LoxP system to generate whole-animal knockouts of CLOCK and evaluated the resultant circadian phenotypes. Surprisingly, CLOCK-deficient mice continue to express robust circadian rhythms in locomotor activity, although they do have altered responses to light. At the molecular and biochemical levels, clock gene mRNA and protein levels in both the master clock in the suprachiasmatic nuclei and a peripheral clock in the liver show alterations in the CLOCK-deficient animals, although the molecular feedback loops continue to function. Our data challenge a central feature of the current mammalian circadian clock model regarding the necessity of CLOCK:BMAL1 heterodimers for clock function.

CLOCK and NPAS2 have overlapping roles in the suprachiasmatic circadian clock

Heterodimers of CLOCK and BMAL1, bHLH-PAS transcription factors, are believed to be the major transcriptional regulators of the circadian clock mechanism in mammals. However, a recent study shows that CLOCK-deficient mice continue to exhibit robust behavioral and molecular rhythms. Here we report that the transcription factor NPAS2 (MOP4) is able to functionally substitute for CLOCK in the master brain clock in mice to regulate circadian rhythmicity.

Peripheral circadian oscillators require CLOCK

Summary In mammals, the circadian system is hierarchical — a brain pacemaker located within the suprachiasmatic nucleus (SCN) is responsible for regulating locomotor activity rhythms and for synchronizing peripheral oscillators [1,2]. Recent genetic evidence in mice indicates that the bHLH transcription factors CLOCK and NPAS2 have partially redundant functions within the SCN [3,4]. To further examine the roles of CLOCK and NPAS2, we generated CLOCK-deficient ( Clock −/− ), NPAS2-deficient ( Npas2 −/− ) and double-mutant ( Clock −/− ;Npas2 −/− ) mice carrying the mPer2 Luciferase reporter gene [5]. We monitored the bioluminescence rhythms of tissue explants in culture and found that while CLOCK or NPAS2 is able to maintain SCN bioluminescence rhythmicity (Supplemental Data) [4], peripheral oscillators are arrhythmic without CLOCK. Thus, there are fundamental differences between the clock machinery of different tissues.

The Polycomb Group Protein EZH2 Is Required for Mammalian Circadian Clock Function

We examined the importance of histone methylation by the polycomb group proteins in the mouse circadian clock mechanism. Endogenous EZH2, a polycomb group enzyme that methylates lysine 27 on histone H3, co-immunoprecipitates with CLOCK and BMAL1 throughout the circadian cycle in liver nuclear extracts. Chromatin immunoprecipitation revealed EZH2 binding and di- and trimethylation of H3K27 on both the Period 1 and Period 2 promoters. A role of EZH2 in cryptochrome-mediated transcriptional repression of the clockwork was supported by overexpression and RNA interference studies. Serum-induced circadian rhythms in NIH 3T3 cells in culture were disrupted by transfection of an RNA interfering sequence targeting EZH2. These results indicate that EZH2 is important for the maintenance of circadian rhythms and extend the activity of the polycomb group proteins to the core clockwork mechanism of mammals.

Casein Kinase 1 Delta Regulates the Pace of the Mammalian Circadian Clock

ABSTRACT Both casein kinase 1 delta (CK1δ) and epsilon (CK1ε) phosphorylate core clock proteins of the mammalian circadian oscillator. To assess the roles of CK1δ and CK1ε in the circadian clock mechanism, we generated mice in which the genes encoding these proteins (Csnk1d and Csnk1e, respectively) could be disrupted using the Cre-loxP system. Cre-mediated excision of the floxed exon 2 from Csnk1d led to in-frame splicing and production of a deletion mutant protein (CK1δΔ2). This product is nonfunctional. Mice homozygous for the allele lacking exon 2 die in the perinatal period, so we generated mice with liver-specific disruption of CK1δ. In livers from these mice, daytime levels of nuclear PER proteins, and PER-CRY-CLOCK complexes were elevated. In vitro, the half-life of PER2 was increased by ∼20%, and the period of PER2::luciferase bioluminescence rhythms was 2 h longer than in controls. Fibroblast cultures from CK1δ-deficient embryos also had long-period rhythms. In contrast, disruption of the gene encoding CK1ε did not alter these circadian endpoints. These results reveal important functional differences between CK1δ and CK1ε: CK1δ plays an unexpectedly important role in maintaining the 24-h circadian cycle length.

Oscillating perceptions: the ups and downs of the CLOCK protein in the mouse circadian system

A functional mouse CLOCK protein has long been thought to be essential for mammalian circadian clockwork function, based mainly on studies of mice bearing a dominant negative, antimorphic mutation in the Clock gene. However, new discoveries using recently developed Clock-null mutant mice have shaken up this view. In this review, I discuss how this recent work impacts and alters the previous view of the role of CLOCK in the mouse circadian clockwork.

Photic resetting and entrainment in CLOCK-deficient mice.

Mice lacking the CLOCK protein have a relatively subtle circadian phenotype, including a slightly shorter period in constant darkness, differences in phase resetting after 4-hour light pulses in the early and late night, and a variably advanced phase angle of entrainment in a light-dark (LD) cycle. The present series of experiments was conducted to more fully characterize the circadian phenotype of Clock−/− mice under various lighting conditions. A phase-response curve (PRC) to 4-hour light pulses in free-running mice was conducted; the results confirm that Clock−/− mice exhibit very large phase advances after 4-hour light pulses in the late subjective night but have relatively normal responses to light at other phases. The abnormal shape of the PRC to light may explain the tendency of CLOCK-deficient mice to begin activity before lights-out when housed in a 12-hour light:12-hour dark lighting schedule. To assess this relationship further, Clock−/− and wild-type control mice were entrained to skeleton lighting cycles (1L:23D and 1L:10D:1L:12D). Comparing entrainment under the 2 types of skeleton photoperiods revealed that exposure to 1-hour light in the morning leads to a phase advance of activity onset (expressed the following afternoon) in Clock−/− mice but not in the controls. Constant light typically causes an intensity-dependent increase in circadian period in mice, but this did not occur in CLOCK-deficient mice. The failure of Clock−/− mice to respond to the period-lengthening effect of constant light likely results from the increased functional impact of light falling in the phase advance zone of the PRC. Collectively, these experiments reveal that alterations in the response of CLOCK-deficient mice to light in several paradigms are likely due to an imbalance in the shape of the PRC to light.

Maternal Ube3a Loss Disrupts Sleep Homeostasis But Leaves Circadian Rhythmicity Largely Intact.

Individuals with Angelman syndrome (AS) suffer sleep disturbances that severely impair quality of life. Whether these disturbances arise from sleep or circadian clock dysfunction is currently unknown. Here, we explored the mechanistic basis for these sleep disorders in a mouse model of Angelman syndrome (Ube3am−/p+ mice). Genetic deletion of the maternal Ube3a allele practically eliminates UBE3A protein from the brain of Ube3am−/p+ mice, because the paternal allele is epigenetically silenced in most neurons. However, we found that UBE3A protein was present in many neurons of the suprachiasmatic nucleus—the site of the mammalian circadian clock—indicating that Ube3a can be expressed from both parental alleles in this brain region in adult mice. We found that while Ube3am−/p+ mice maintained relatively normal circadian rhythms of behavior and light-resetting, these mice exhibited consolidated locomotor activity and skipped the timed rest period (siesta) present in wild-type (Ube3am+/p+) mice. Electroencephalographic analysis revealed that alterations in sleep regulation were responsible for these overt changes in activity. Specifically, Ube3am−/p+ mice have a markedly reduced capacity to accumulate sleep pressure, both during their active period and in response to forced sleep deprivation. Thus, our data indicate that the siesta is governed by sleep pressure, and that Ube3a is an important regulator of sleep homeostasis. These preclinical findings suggest that therapeutic interventions that target mechanisms of sleep homeostasis may improve sleep quality in individuals with AS. SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of expression of the maternal copy of the UBE3A gene. Individuals with AS have severe sleep dysfunction that affects their cognition and presents challenges to their caregivers. Unfortunately, current treatment strategies have limited efficacy due to a poor understanding of the mechanisms underlying sleep disruptions in AS. Here we demonstrate that abnormal sleep patterns arise from a deficit in accumulation of sleep drive, uncovering the Ube3a gene as a novel genetic regulator of sleep homeostasis. Our findings encourage a re-evaluation of current treatment strategies for sleep dysfunction in AS, and suggest that interventions that promote increased sleep drive may alleviate sleep disturbances in individuals with AS.

The hepatic circadian clock modulates xenobiotic metabolism in mice

The circadian clock generates daily cycles of gene expression that regulate physiological processes. The liver plays an important role in xenobiotic metabolism and also has been shown to possess its own cell-based clock. The liver clock is synchronized by the master clock in the brain, and a portion of rhythmic gene expression can be driven by behavior of the organism as a whole even when the hepatic clock is suppressed. So far, however, there is relatively little evidence indicating whether the liver clock is functionally important in modulating xenobiotic metabolism. Thus, mice lacking circadian clock function in the whole body or specifically in liver were challenged with pentobarbital and acetaminophen, and pentobarbital sleep time (PBST) and acetaminophen toxicity, respectively, was assessed at different times of day in mutant and control mice. The results suggest that the liver clock is essential for rhythmic changes in xenobiotic detoxification. Surprisingly, it seems that the way in which the clock is disrupted determines the rate of xenobiotic metabolism in the liver. CLOCK-deficient mice are remarkably resistant to acetaminophen and exhibit a longer PBST, while PERIOD-deficient mice have a short PBST. These results indicate an essential role of the tissue-intrinsic peripheral circadian oscillator in the liver in regulating xenobiotic metabolism.

Bmal1 function in skeletal muscle regulates sleep

Sleep loss can severely impair the ability to perform, yet the ability to recover from sleep loss is not well understood. Sleep regulatory processes are assumed to lie exclusively within the brain mainly due to the strong behavioral manifestations of sleep. Whole-body knockout of the circadian clock gene Bmal1 in mice affects several aspects of sleep, however, the cells/tissues responsible are unknown. We found that restoring Bmal1 expression in the brains of Bmal1-knockout mice did not rescue Bmal1-dependent sleep phenotypes. Surprisingly, most sleep-amount, but not sleep-timing, phenotypes could be reproduced or rescued by knocking out or restoring BMAL1 exclusively in skeletal muscle, respectively. We also found that overexpression of skeletal-muscle Bmal1 reduced the recovery response to sleep loss. Together, these findings demonstrate that Bmal1 expression in skeletal muscle is both necessary and sufficient to regulate total sleep amount and reveal that critical components of normal sleep regulation occur in muscle. DOI: http://dx.doi.org/10.7554/eLife.26557.001