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REV-ERBα and REV-ERBβ are important components of the mammalian circadian clock and play a crucial role in linking the circadian system to overt daily rhythms in physiology and behavior. Expression of these parologs is driven by the circadian clockwork, and in most tissues, the abundance of REV-ERBα proteins robustly cycles such that they are detected only within a tight interval of 4-6 hours each day, suggesting control of both their synthesis and degradation are tightly controlled. Indeed, several different ubiquitin ligases have been shown to mediate REV-ERBα degradation, but how they interact with REV-ERBα and which lysine residues they ubiquitinate to drive its degradation are unknown. Here, we used a mutagenesis approach to functionally identify both binding and ubiquitination sites within REV-ERBα required for its regulation by the ubiquitin ligases Spsb4 and Siah2. Surprisingly, we found that REV-ERBα mutants with all 20 lysines changed to arginine (K20R) can be efficiently ubiquitinated and degraded in the absence or presence of these E3 ligases, consistent with N-terminal ubiquitination. To explore this, we examined if small deletions at the N-terminus of REV-ERBα will alter its degradation. Interestingly, deletion of amino acid (AA) residues 2 to 9 (delAA2-9) clearly resulted in a less stable REV-ERBα. We found that it was the length (i.e. 8 AA), and not the specific sequence, that confers stability in this region.. Simultaneously, we also mapped the interaction site of the E3 ligase Spsb4 to this same regions, specifically requiring AA4-9 of REV-ERBα. Thus, the first 9 AA of REV-ERBα has two opposing roles in regulating REV-ERBα turnover. Additionally, deleting eight addition additiona AAs (delAA2-17) from REV-ERBα almost prevents its degration. Combined, these results suggest that complex interactions within the first 25AAs that potentially act as a REV-ERBα ‘switch’ that allows a protected/stabilized conformation to accumulate at one time of day, but then rapidly shifts to a destabilized form, to enhance its removal at the end of the daily cycle.

Shift work, performed by approximately 21 million Americans, is irregular or unusual work schedule hours occurring after 6:00 pm. Shift work has been shown to disrupt circadian rhythms and is associated with several adverse health outcomes and chronic diseases such as cancer, gastrointestinal and psychiatric diseases and disorders. It is unclear if shift work influences the complications associated with certain infectious agents, such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility resulting from genital chlamydial infection. We used an Environmental circadian disruption (ECD) model mimicking circadian disruption occurring during shift work, where mice had a 6-h advance in the normal light/dark cycle (LD) every week for a month. Control group mice were housed under normal 12/12 LD cycle. Our hypothesis was that compared to controls, mice that had their circadian rhythms disrupted in this ECD model will have a higher Chlamydia load, more pathology and decreased fertility rate following Chlamydia infection. Results showed that, compared to controls, mice that had their circadian rhythms disrupted (ECD) had higher Chlamydia loads, more tissue alterations or lesions, and lower fertility rate associated with chlamydial infection. Also, infected ECD mice elicited higher proinflammatory cytokines compared to mice under normal 12/12 LD cycle. These results imply that there might be an association between shift work and the increased likelihood of developing more severe disease from Chlamydia infection.

The time-dependent degradation of core circadian clock proteins is essential for the proper functioning of circadian timekeeping mechanisms that drive daily rhythms in gene expression and, ultimately, an organism’s physiology. The ubiquitin proteasome system plays a critical role in regulating the stability of most proteins, including the core clock components. Our laboratory developed a cell-based functional screen to identify ubiquitin ligases that degrade any protein of interest and have started screening for those ligases that degrade circadian clock proteins. This screen identified Spsb4 as a putative novel E3 ligase for RevErbα. In this article, we further investigate the role of Spsb4 and its paralogs in RevErbα stability and circadian rhythmicity. Our results indicate that the paralogs Spsb1 and Spsb4, but not Spsb2 and Spsb3, can interact with and facilitate RevErbα ubiquitination and degradation and regulate circadian clock periodicity.

Genital chlamydia infection in women causes complications such as pelvic inflammatory disease and tubal factor infertility, but it is unclear why some women are more susceptible than others. Possible factors, such as time of day of chlamydia infection on chlamydial pathogenesis has not been determined. We hypothesised that infections during the day, will cause increased complications compared to infections at night. Mice placed under normal 12:12 light: dark (LD) cycle were infected intravaginally with Chlamydia muridarum either at zeitgeber time 3, ZT3 and ZT15. Infectivity was monitored by periodic vaginal swabs and chlamydiae isolation. Blood and vaginal washes were collected for host immunologic response assessments. The reproductive tracts of the mice were examined histopathologically, and fertility was determined by embryo enumeration after mating. Mice infected at ZT3 shed significantly more C. muridarum than mice infected at ZT15. This correlated with the increased genital tract pathology observed in mice infected at ZT3. Mice infected at ZT3 were less fertile than mice infected at ZT15. The results suggest that the time of day of infection influences chlamydial pathogenesis, it indicates a possible association between complications from chlamydia infection and host circadian clock, which may lead to a better understanding of chlamydial pathogenesis.

In 1965 Dr. Harry Angelman reported a neurodevelopmental disorder affecting three unrelated children who had similar symptoms: brachycephaly, mental retardation, ataxia, seizures, protruding tongues, and remarkable paroxysms of laughter. Over the past 50 years, the disorder became Angelman’s namesake and symptomology was expanded to include hyper-activity, stereotypies, and severe sleep disturbances. The sleep disorders in many Angelman syndrome (AS) patients are broadly characterized by difficulty falling and staying asleep at night. Some of these patients sleep less than 4 hours a night and, in most cases, do not make up this lost-sleep during the day—leading to the speculation that AS patients may “need” less sleep. Most AS patients also have severely reduced levels of melatonin, a hormone produced by the pineal gland exclusively at night. This nightly pattern of melatonin production is thought to help synchronize internal circadian rhythms and promote nighttime sleep in humans and other diurnal species. It has been proposed that reduced melatonin levels contribute to the sleep problems in AS patients. Indeed, emerging evidence suggests melatonin replacement therapy can improve sleep in many AS patients. However, AS mice show sleep problems that are arguably similar to those in humans despite being on genetic backgrounds that do not make melatonin. This suggests the hypothesis that the change in nighttime melatonin may be a secondary factor rather than the root cause of the sleeping disorder. The goals of this review article are to revisit the sleep and melatonin findings in both AS patients and animal models of AS and discuss what AS may tell us about the underlying mechanisms of, and interplay between, melatonin and sleep.

Circadian clocks enable organisms to predict and align their behaviors and physiologies to constant daily day-night environmental cycle. Because the ubiquitin ligase Siah2 has been identified as a potential regulator of circadian clock function in cultured cells, we have used Siah2-deficient mice to examine its function in vivo. Our experiments demonstrate a striking and unexpected sexually dimorphic effect of Siah2 deficiency on the regulation of rhythmically expressed genes. The absence of Siah2 in females, but not in males, altered the expression of core circadian clock genes and drastically remodeled the rhythmic hepatic transcriptome. Siah2 loss, only in females, increased the expression of 100’s of genes selectively at mid-day, resulting in a ∼70% increase in the number of rhythmically expressed genes, and shifted the expression of 100’s of other genes from a mid-night peak, to a mid-day peak. The combined result is a near inversion of overall rhythmicity in gene expression selectively in Siah2-deficient females. This dramatic reorganization created a substantial misalignment between rhythmic liver functions and feeding/behavioral rhythms, and consequently impaired daily patterns of lipid/lipoprotein metabolism and metabolic responses to high-fat diet. Collectively, our results suggest that Siah2 is part of a female-specific circadian mechanism important for maintaining metabolic homeostasis and may play a key role in the establishing sexual dimorphisms in metabolism. Signficance statement Circadian clocks drive daily rhythms in many aspects of our physiology, optimally aligning functions across the day-night cycle. How circadian clocks drives these rhythms is thought to be due to largely similar transcriptional pathways and mechanisms in males and females, although some rhythms are modulated by sex and growth hormones. In this study, we present data that uncover the surprising existence of a female-specific transcriptional mechanism that is essential for the proper rhythmic control of gene expression in the liver. Disrupting this mechanism substantially impairs the circadian regulation of lipid and cholesterol metabolism selectively in females, impairing their resistance to diet-induced obesity. These results reveal that circadian clocks may be broadly coupled to physiological rhythms using unexpected sex-specific mechanisms.

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding “big data” that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them. Fil: Hughes, Michael E.. Washington University School Of Medicine In St. Louis; Estados Unidos Fil: Abruzzi, Katherine C.. Brandeis University; Estados Unidos Fil: Allada, Ravi. Northwestern University; Estados Unidos Fil: Anafi, Ron. University of Pennsylvania; Estados Unidos Fil: Arpat, Alaaddin Bulak. Universite de Lausanne; Suiza Fil: Asher, Gad. Weizmann Institute Of Science Israel; Israel Fil: Baldi, Pierre. University of California at Irvine; Estados Unidos Fil: de Bekker, Charissa. University Of Central Florida; Estados Unidos Fil: Bell Pedersen, Deborah. Texas A&M University; Estados Unidos Fil: Blau, Justin. University of New York; Estados Unidos Fil: Brown, Steve. Universitat Zurich; Suiza Fil: Ceriani, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Chen, Zheng. University Of Texas Health Science Center At Houston; Estados Unidos Fil: Chiu, Joanna C.. University of California at Davis; Estados Unidos Fil: Cox, Juergen. Max Planck Institute Of Biochemistry; Alemania Fil: Crowell, Alexander M.. Geisel School Of Medicine At Dartmouth; Estados Unidos Fil: DeBruyne, Jason P.. Morehouse School Of Medicine; Estados Unidos Fil: Dijk, Derk-Jan. University Of Surrey; Reino Unido Fil: DiTacchio, Luciano. University Of Kansas Medical Center; Estados Unidos Fil: Doyle, Francis J.. Harvard University; Estados Unidos Fil: Duffield, Giles E.. University of Notre Dame; Estados Unidos Fil: Dunlap, Jay C.. 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Washington University; Estados Unidos Fil: Hastings, Michael H.. The Medical Research Council Laboratory Of Molecular Biology; Reino Unido Fil: Herzel, Hanspeter. Charité Universitätsmedizin Berlin; Alemania Fil: Herzog, Erik D.. Washington University in St. Louis; Estados Unidos Fil: Hoffmann, Christy. Washington University in St. Louis; Estados Unidos Fil: Hong, Christian. Cincinnati Children's Hospital Medical Center; Estados Unidos Fil: Hughey, Jacob J.. Vanderbilt University School Of Medicine; Estados Unidos Fil: Hurley, Jennifer M.. Rensselaer Polytechnic Institute; Estados Unidos Fil: de la Iglesia, Daniel Horacio. University Of Washington; Estados Unidos Fil: Johnson, Carl. Vanderbilt University; Estados Unidos Fil: Kay, Steve A.. University of California at San Diego; Estados Unidos Fil: Koike, Nobuya. Kyoto Prefectural University Of Medicine; Japón Fil: Kornacker, Karl. Ohio State University; Estados Unidos Fil: Kramer, Achim. Charité Universitätsmedizin Berlin; Alemania Fil: Lamia, Katja. The Scripps Research Institute; Estados Unidos Fil: Leise, Tanya. Amherst College; Estados Unidos Fil: Lewis, Scott A.. Washington University; Estados Unidos Fil: Li, Jiajia. Washington University; Estados Unidos Fil: Li, Xiaodong. Wuhan University; China Fil: Liu, Andrew C.. The University Of Memphis; Estados Unidos Fil: Loros, Jennifer J.. Geisel School Of Medicine At Dartmouth; Estados Unidos Fil: Martino, Tami A.. University of Guelph; Canadá Fil: Menet, Jerome S.. Texas A&M University; Estados Unidos Fil: Merrow, Martha. Ludwig Maximilians Universitat; Alemania Fil: Millar, Andrew J.. University of Edinburgh; Reino Unido Fil: Mockler, Todd. Donald Danforth Plant Science Center; Estados Unidos Fil: Naef, Felix. Ecole Polytechnique Federale de Lausanne; Suiza Fil: Nagoshi, Emi. Universidad de Ginebra; Suiza Fil: Nitabach, Michael N.. University of Yale. School of Medicine; Estados Unidos Fil: Olmedo, Maria. Universidad de Sevilla; España Fil: Nusinow, Dmitri A.. Donald Danforth Plant Science Center; Estados Unidos Fil: Ptácek, Louis J.. University of California; Estados Unidos Fil: Rand, David. University of Warwick; Reino Unido Fil: Reddy, Akhilesh B.. The Francis Crick Institute; Reino Unido Fil: Robles, Maria S.. Ludwig Maximilians Universitat; Alemania Fil: Roenneberg, Till. Ludwig Maximilians Universitat; Alemania Fil: Rosbash, Michael. Brandeis University; Estados Unidos Fil: Ruben, Marc D.. Cincinnati Children's Hospital Medical Center; Estados Unidos Fil: Rund, Samuel S.C.. University of Edinburgh; Reino Unido Fil: Sancar, Aziz. University of North Carolina; Estados Unidos Fil: Sassone Corsi, Paolo. University of California at Irvine; Estados Unidos Fil: Sehgal, Amita. University of Pennsylvania; Estados Unidos Fil: Sherrill Mix, Scott. University of Pennsylvania; Estados Unidos Fil: Skene, Debra J.. University Of Surrey; Reino Unido Fil: Storch, Kai Florian. 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An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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 maternalUbe3aallele practically eliminates UBE3A protein from the brain ofUbe3am−/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 thatUbe3acan be expressed from both parental alleles in this brain region in adult mice. We found that whileUbe3am−/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 thatUbe3ais 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 STATEMENTAngelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of expression of the maternal copy of theUBE3Agene. 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 theUbe3agene 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.

Regulated degradation of proteins by the proteasome is often critical to their function in dynamic cellular pathways. The molecular clock underlying mammalian circadian rhythms relies on the rhythmic expression and degradation of its core components. However, because the tools available for identifying the mechanisms underlying the degradation of a specific protein are limited, the mechanisms regulating clock protein degradation are only beginning to be elucidated. Here we describe a cell-based functional screening approach designed to quickly identify the ubiquitin E3 ligases that induce the degradation of potentially any protein of interest. We screened the nuclear hormone receptor RevErbα (Nr1d1), a key constituent of the mammalian circadian clock, for E3 ligases that regulate its stability and found Seven in absentia2 (Siah2) to be a key regulator of RevErbα stability. Previously implicated in hypoxia signaling, Siah2 overexpression destabilizes RevErbα/β, and siRNA depletion of Siah2 stabilizes endogenous RevErbα. Moreover, Siah2 depletion delays circadian degradation of RevErbα and lengthens period length. These results demonstrate the utility of functional screening approaches for identifying regulators of protein stability and reveal Siah2 as a previously unidentified circadian clockwork regulator that mediates circadian RevErbα turnover.

As part of an ongoing program to identify genes involved in maintaining circadian rhythms of zebrafish, 6,500 mutagenized genomes were screened for dominant mutants affecting circadian locomotor activity. Molecular analysis of one of these mutant lines, Clk1(dg3), revealed an I254N mutation in the PAS domain of the Clock1 protein. This isoleucine is tightly conserved in the Clock genes of several different species, and the I254N was not seen in any of the wild-type zebrafish population tested. Analysis of circadian activity rhythms as well as melatonin rhythms in homozygotes revealed the biological clock runs with a shortened period. The effect of this Clock1 mutation was characterized in vitro using a cell culture system where it appears to enhance the transactivation ability of the I254N Clock1 protein compared with that of the normal gene product.

Mutations or deletions of the maternal allele of the UBE3A gene cause Angelman syndrome (AS), a severe neurodevelopmental disorder. The paternal UBE3A/Ube3a allele becomes epigenetically silenced in most neurons during postnatal development in humans and mice; hence, loss of the maternal allele largely eliminates neuronal expression of UBE3A protein. However, recent studies suggest that paternal Ube3a may escape silencing in certain neuron populations, allowing for persistent expression of paternal UBE3A protein. Here we extend evidence in AS model mice (Ube3am–/p+) of paternal UBE3A expression within the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Paternal UBE3A-positive cells in the SCN show partial colocalization with the neuropeptide arginine vasopressin (AVP) and clock proteins (PER2 and BMAL1), supporting that paternal UBE3A expression in the SCN is often of neuronal origin. Paternal UBE3A also partially colocalizes with a marker of neural progenitors, SOX2, implying that relaxed or incomplete imprinting of paternal Ube3a reflects an overall immature molecular phenotype. Our findings highlight the complexity of Ube3a imprinting in the brain and illuminate a subpopulation of SCN neurons as a focal point for future studies aimed at understanding the mechanisms of Ube3a imprinting.

Widespread use of zebrafish (Danio rerio) in genetic analysis of embryonic development has led to rapid advances in the technology required to generate, map and clone mutated genes. To identify genes involved in the generation and regulation of vertebrate circadian rhythmicity, we screened for dominant mutations that affect the circadian periodicity of larval zebrafish locomotor behavior. In a screen of 6,500 genomes, we recovered 8 homozygous viable, semi-dominant mutants, and describe one of them here. The circadian period of the lager and lime (lag(dg2)) mutant is shortened by 0.7 h in heterozygotes,and 1.3 h in homozygotes. This mutation also shortens the period of the melatonin production rhythm measured from cultured pineal glands, indicating that the mutant gene product affects circadian rhythmicity at the tissue level, as well as at the behavioral level. This mutation also alters the sensitivity of pineal circadian period to temperature, but does not affect phase shifting responses to light. Linkage mapping with microsatellite markers indicates that the lag mutation is on chromosome 7. A zebrafish homolog of period1(per1) is the only known clock gene homolog that maps near the lag locus. However, all sequence variants found in per1 cDNA from lag(dg2) mutants are also present in wild type lines, and we were unable to detect any defect in per1 mRNA splicing, so this mutation may identify a novel clock gene.

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.

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.

As part of an effort to characterize the circadian system of the zebrafish, we examined the circadian regulation of locomotor activity in adult males and females. Gross locomotor activity was measured using infrared movement detectors. The effects of light, dark, and temperature on the amplitude, phase, and free-running periods of locomotor rhythms were determined. When zebrafish were maintained in a 12-h light:12 h dark cycle at 25 degrees C, 86% of the fish were most active during the light phase of the cycle. The phases of free-running rhythms measured after transfer of fish from light cycles to constant conditions indicate that this diurnal activity profile reflects entrained circadian rhythmicity. When animals were maintained in constant conditions, the proportion that expressed significant circadian rhythmicity depended on ambient temperature. At 21 degrees C, 73% of the animals were rhythmic in constant darkness, and 65% were rhythmic in constant light. Fewer (28-59%) were rhythmic at 18 degrees, 25 degrees, and 28.5 degrees C. The free-running period of rhythmic animals was not affected by temperature within this range. The average period was shorter in constant light (LL; 12 lx) than in constant darkness (DD) in all but one experiment, and the difference was statistically significant for animals held at 21 degrees C. These data indicate that zebrafish locomotor activity is regulated by a circadian clock that is temperature compensated. Because rhythmicity is most robust at 21 degrees C, this would be the optimal temperature for future studies of the physiological basis of zebrafish behavioral rhythms.

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.

Circadian rhythms are rhythms in gene expression, metabolism, physiology, and behavior that persist in constant environmental conditions with a cycle length near 24 h. In mammals, the circadian timing system is hierarchical. The primary pacemaker regulating circadian behavioral rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Most cell types express circadian clock genes and will express rhythmicity in vitro. In vivo, the SCN entrains peripheral oscillators through a complex set of physiological and hormonal rhythms (31, 32, 36). At the molecular level, circadian oscillations are governed by a cell-autonomous negative-feedback loop in which transcription factors drive the expression of their own negative regulators, leading to oscillation between periods of transcriptional activation and repression (reviewed in references 32 and 36). The bHLH-PAS containing transcription factors CLOCK or NPAS2 form heterodimers with BMAL1. These heterodimers binds to E-box elements within regulatory regions of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes to stimulate their transcription. Approximately 12 h after transcriptional activation, PER and CRY proteins reach concentrations sufficient to form repressor complexes that inhibit the activity of the CLOCK/NPAS2:BMAL1 heterodimer, reducing the transcription of Per and Cry genes and subsequently relieving PER/CRY-mediated negative feedback. E-box-mediated expression of other transcription factors, including members of the DBP/HLF/TEF and nuclear orphan receptor families (e.g., Rev-Erbα and ROR-A), provides a mechanism for clock control of genes with diverse promoters and with gene expression peaks occurring at a variety of phases. Posttranslational modifications of circadian clock proteins play a well-established role in the regulation of circadian cycle length. In both flies and mammals, phosphorylation of PER proteins by casein kinase 1 (CK1) proteins is thought to play a key step in determining the speed of the circadian clock (reviewed in reference 5). Mammalian cell culture studies indicate that the phosphorylation of PER proteins by CK1 epsilon (CK1ɛ) regulates their subcellular localization, likely affects their transcription repression capability, and promotes their degradation through a proteasomal pathway dependent upon the F-box proteins β-TrCP1 and β-TrCP2 (12, 29, 34, 35). Interference with β-TrCP1 activity lengthens circadian period in oscillating fibroblasts (27). The CK1 inhibitor IC261 and the proteasome inhibitors MG132 and lactacystin also lengthen period in fibroblasts (12). CRY proteins are also subjected to phosphorylation and degradation cycles that regulate circadian period. The F-box protein FBXL3 plays a key role in regulating CRY1 stability; mutations inactivating this gene increase circadian cycle length (6, 16, 30). Collectively, these studies indicate that the duration of activity of the PER/CRY repressor complex, regulated primarily by the stability of PER and CRY proteins, dictates the cycle length of the molecular oscillator (15). Genetic studies also support an important role for casein kinase action on PER proteins in regulating circadian period. A mutation in the Syrian hamster CK1ɛ gene, tau, shortens the circadian period of behavioral rhythms. Biochemically, the tau mutation (CK1ɛtau, a T178C substitution) differentially affects the activity of the kinase protein, reducing general kinase activity while increasing activity at specific residues of the PER proteins (14, 23). The tau mutation is a gain-of-function mutation with respect to circadian substrates, resulting in decreased PER stability and a reduction in circadian period length in tau mutant hamsters and mice (14, 24). In humans, familial advanced sleep phase syndrome (FASPS) is a circadian-based sleep disorder, in which affected individuals have a short circadian period and an advanced phase of the sleep-wake cycle. One study identified a FASPS pedigree with a mutation in human PER2 (hPER2; S662G mutation); this mutation prevents a priming phosphorylation, thus preventing CK1-mediated phosphorylation (33). A second study identified a dominant mutation within the kinase domain of CK1δ in a family with FASPS (38). Modeling this mutation in mice and flies revealed alterations in period length (38). In the circadian field, much of the attention on mammalian casein kinases has focused on CK1ɛ. The few studies examining CK1δ suggest that it plays a role similar to CK1ɛ. For example, CK1δ, like CK1ɛ, phosphorylates PER proteins, reducing their stability in vitro (7, 38), and both CK1δ and CK1ɛ are present in PER/CRY repressor complexes in vivo (21). Despite these similarities, the role of CK1δ in the molecular clockwork is not well understood. We report here results demonstrating important differences between CK1δ and CK1ɛ in the regulation of circadian cycle length, based on studies utilizing mice in which these genes have been inactivated.

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.

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.

CRY1 is essential for normal circadian clock function, but its transcriptional regulation by the clock has not been considered an important feature for its function. However, reporting in Cell, Ukai-Tadenuma et al. (2011) now show that rhythmic Cry1 expression in the early night is critical for clock function.

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.

SummaryThe 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.