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The circadian clock is an intracellular timekeeping device that drives daily rhythms in diverse and extensive processes throughout the body. The clock mechanism comprises a core transcription/translation negative feedback loop that is modulated by a complex set of additional interlocking feedback loops. Pharmacological manipulation of the clock may be valuable for treating many maladies including jet lag, shift work and related sleep disorders, various metabolic diseases, and cancer. We review recent identification of small-molecule clock modulators and discuss the biochemical features of the core clock that may be amenable to future drug discovery.

Forward genetics is a powerful approach based on chromosomal mapping of phenotypes and has successfully led to the discovery of many mouse mutations in genes responsible for various phenotypes. Although crossing between genetically remote strains can produce F

Background Excessive daytime sleepiness (EDS) in multiple sclerosis (MS) can be a significant source of disability. Despite this, its prevalence as a patient-reported outcome in this condition has not been well established, and its causes are not well understood. Methods We prospectively assessed EDS as part of an observational study for patients referred for diagnostic neuro-ophthalmological testing. EDS was evaluated by the Epworth Sleepiness Scale (ESS), and visual data were also collected as part of a research protocol. Analysis with patient data was performed following the exclusion of patients with known primary sleep disorders. Results A total of 69 patients with MS were included in the analysis. The mean ESS was 6.5 with a SD of 4.3. ESS ≥ 10 was present in 23% of the cohort even in the presence of minimal mean neurological disability (Patient Determined Disease Steps (PDDS) = 1.5). The ESS score was not associated with age, sex, disease-related disability, retinal nerve fiber layer (RNFL), or optic neuritis (ON), but displayed an association with visual dysfunction. Conclusions There is an increased prevalence of EDS in MS. The increased values of the ESS are not explained by other sleep disorders, suggesting separate mechanisms. Further study of the underlying mechanisms is warranted.

Ultradian rhythms in metabolism and physiology have been described previously in mammals. However, the underlying mechanisms for these rhythms are still elusive. Here, we report the discovery of temperature-sensitive ultradian rhythms in mammalian fibroblasts that are independent of both the cell cycle and the circadian clock. The period in each culture is stable over time but varies in different cultures (ranging from 3 to 24 h). We show that transient, single-cell metabolic pulses are synchronized into stable ultradian rhythms across contacting cells in culture by gap junction–mediated coupling. Coordinated rhythms are also apparent for other metabolic and physiological measures, including plasma membrane potential (Δψ p ), intracellular glutamine, α-ketoglutarate, intracellular adenosine triphosphate (ATP), cytosolic pH, and intracellular calcium. Moreover, these ultradian rhythms require extracellular glutamine, several different ion channels, and the suppression of mitochondrial ATP synthase by α-ketoglutarate, which provides a key feedback mechanism. We hypothesize that cellular coupling and metabolic feedback can be used by cells to balance energy demands for survival.

Polyphasic sleep is the practice of distributing multiple short sleep episodes across the 24-hour day rather than having one major and possibly a minor ("nap") sleep episode each day. While the prevalence of polyphasic sleep is unknown, anecdotal reports suggest attempts to follow this practice are common, particularly among young adults. Polyphasic-sleep advocates claim to thrive on as little as 2 hours of total sleep per day. However, significant concerns have been raised that polyphasic sleep schedules can result in health and safety consequences. We reviewed the literature to identify the impact of polyphasic sleep schedules (excluding nap or siesta schedules) on health, safety, and performance outcomes. Of 40,672 potentially relevant publications, with 2,023 selected for full-text review, 22 relevant papers were retained. We found no evidence supporting benefits from following polyphasic sleep schedules. Based on the current evidence, the consensus opinion is that polyphasic sleep schedules, and the sleep deficiency inherent in those schedules, are associated with a variety of adverse physical health, mental health, and performance outcomes. Striving to adopt a schedule that significantly reduces the amount of sleep per 24 hours and/or fragments sleep into multiple episodes throughout the 24-hour day is not recommended.

In mammals, a set of core clock genes form transcription–translation feedback loops to generate circadian oscillations. We and others recently identified a novel transcript at the Period2 (Per2) locus that is transcribed from the antisense strand of Per2. This transcript, Per2AS, is expressed rhythmically and antiphasic to Per2 mRNA, leading to our hypothesis that Per2AS and Per2 mutually inhibit each other's expression and form a double negative feedback loop. By perturbing the expression of Per2AS, we found that Per2AS transcription, but not transcript, represses Per2. However, Per2 does not repress Per2AS, as Per2 knockdown led to a decrease in the Per2AS level, indicating that Per2AS forms a single negative feedback loop with Per2 and maintains the level of Per2 within the oscillatory range. Per2AS also regulates the amplitude of the circadian clock, and this function cannot be solely explained through its interaction with Per2, as Per2 knockdown does not recapitulate the phenotypes of Per2AS perturbation. Overall, our data indicate that Per2AS is an important regulatory molecule in the mammalian circadian clock machinery. Our work also supports the idea that antisense transcripts of core clock genes constitute a common feature of circadian clocks, as they are found in other organisms.

The hypothalamic suprachiasmatic nucleus (SCN) is the central pacemaker for mammalian circadian rhythms. As such, this ensemble of cell-autonomous neuronal oscillators with divergent periods must maintain coordinated oscillations. To investigate ultrastructural features enabling such synchronization, 805 coronal ultrathin sections of mouse SCN tissue were imaged with electron microscopy and aligned into a volumetric stack, from which selected neurons within the SCN core were reconstructed in silica. We found that clustered SCN core neurons were physically connected to each other via multiple large soma-to-soma plate-like contacts. In some cases, a sliver of a glial process was interleaved. These contacts were large, covering on average ∼21% of apposing neuronal somata. It is possible that contacts may be the electrophysiological substrate for synchronization between SCN neurons. Such plate-like contacts may explain why synchronization of SCN neurons is maintained even when chemical synaptic transmission or electrical synaptic transmission via gap junctions is blocked. Such ephaptic contact-mediated synchronization among nearby neurons may therefore underlie the wave-like oscillations of circadian core clock genes and calcium signals observed in the SCN.SignificanceThree-dimensional reconstruction of SCN tissue via serial electron microscopy revealed a novel structural feature of SCN neurons that may account for inter-neuronal synchronization that persists even when usual mechanisms of neuronal communication are blocked. We found that SCN core neurons are connected by multiple soma-soma contact specializations, ultrastructural elements that could enable synchronization of tightly packed neurons organized in clustered networks. This extensive network of plate-like soma-soma contacts among clustered SCN neurons may provide insight into how ∼20,000 autonomous neuronal oscillators with a broad range of intrinsic periods remain synchronized in the absence of ordinary communication modalities, thereby conferring the resilience required for the SCN to function as the mammalian circadian pacemaker.

Although a wide variety of genetic tools has been developed to study learning and memory, the molecular basis of memory encoding remains incompletely understood. Here, we undertook an unbiased approach to identify novel genes critical for memory encoding. From a large-scale, in vivo mutagenesis screen using contextual fear conditioning, we isolated in mice a mutant, named

Although a wide variety of genetic tools has been developed to study learning and memory, the molecular basis of memory encoding remains incompletely understood. Here, we undertook an unbiased approach to identify novel genes critical for memory encoding. From a large-scale, in vivo mutagenesis screen using contextual fear conditioning, we isolated in mice a mutant, named Clueless , with spatial learning deficits. A causative missense mutation (G434V) was found in the voltage-gated potassium channel, subfamily C member 3 ( Kcnc3) gene in a region that encodes a transmembrane voltage sensor. Generation of a Kcnc3 G434V CRISPR mutant mouse confirmed this mutation as the cause of the learning defects. While G434V had no effect on transcription, translation, or trafficking of the channel, electrophysiological analysis of the G434V mutant channel revealed a complete loss of voltage-gated conductance, a broadening of the action potential, and decreased neuronal firing. Together, our findings have revealed a role for Kcnc3 in learning and memory.

Patients with sleeping sickness, caused by the parasiteTrypanosoma brucei, have disruptions in both sleep timing and sleep architecture. However, the underlying cause of these sleep disturbances is not well understood. Here, we assessed the sleep architecture of male mice infected withT. bruceiand found that infected mice had drastically altered sleep patterns. Interestingly,T. brucei-infected mice also had a reduced homeostatic sleep response to sleep deprivation, a response modulated by the adenosine system. We found that infected mice had a reduced electrophysiological response to an adenosine receptor antagonist and increased adenosine receptor gene expression. Although the mechanism by whichT. bruceiinfection causes these changes remains to be determined, our findings suggest that the symptoms of sleeping sickness may be because of alterations in homeostatic adenosine signaling.SIGNIFICANCE STATEMENTSleeping sickness is a fatal disease that disrupts the circadian clock, causes disordered temperature regulation, and induces sleep disturbance. To examine the neurologic effects of infection in the absence of other symptoms, in this study, we used a mouse model of sleeping sickness in which the acute infection was treated but brain infection remained. Using this model, we evaluated the effects of the sleeping sickness parasite,Trypanosoma brucei, on sleep patterns in mice, under both normal and sleep-deprived conditions. Our findings suggest that signaling of adenosine, a neuromodulator involved in mediating homeostatic sleep drive, may be reduced in infected mice.

Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works http://www.sciencemag.org/about/science-licenses-journal-article-reuseThis is an article distributed under the terms of the Science Journals Default License., Malarial rhythmic fevers are the consequence of the synchronous bursting of red blood cells (RBCs) on completion of the malaria parasite asexual cell cycle. Here, we hypothesized that an intrinsic clock in the parasite Plasmodium chabaudi underlies the 24-hour-based rhythms of RBC bursting in mice. We show that parasite rhythms are flexible and lengthen to match the rhythms of hosts with long circadian periods. We also show that malaria rhythms persist even when host food intake is evenly spread across 24 hours, suggesting that host feeding cues are not required for synchrony. Moreover, we find that the parasite population remains synchronous and rhythmic even in an arrhythmic clock mutant host. Thus, we propose that parasite rhythms are generated by the parasite, possibly to anticipate its circadian environment., F.R.-F. is an Associate, I.K. is a Lab Manager II, and J.S.T. is an Investigator in the Howard Hughes Medical Institute. J.H.A. is supported by NIH NIA F32-AG064886 and NIH T32-HLO9701; E.B.K. by NIH R01-HL128538, K24-HL105664, and P01-AG009975 and the MGH Department of Neurology; and M.M.M. by PTDC/BIA-MOL/30112/2017 and PTDC/MED-IMU/28664/2017. M.M.M. is supported by FCT grants (PTDC/BIA-MOL/30112/2017 and PTDC/MED-IMU/28664/2017) and F.R.-F. by NIH NIGMS 1K99GM132557-01

SummaryCircadian rhythms govern glucose homeostasis, and their dysregulation leads to complex metabolic diseases. Gut microbes also exhibit diurnal rhythms that influence host circadian networks and metabolic processes, yet underlying mechanisms remain elusive. Here, we show hierarchical, bi-directional communication between the liver circadian clock, gut microbes, and glucose homeostasis in mice. The liver clock, but not the forebrain clock, requires gut microbes to drive glucose clearance and gluconeogenesis. Liver clock dysfunctionality expands proportions and abundances of oscillating microbial features by two-fold relative to controls. The liver clock is the primary driver of differential and rhythmic hepatic expression of glucose and fatty acid metabolic pathways. Absent the liver clock, gut microbes provide secondary cues that dampen these rhythms, resulting in reduced utilization of lipids as fuel relative to carbohydrates. Together, the liver clock transduces signals from gut microbes necessary to regulate glucose and lipid metabolism and meet energy demands over 24 hours.HighlightsThe liver circadian clock is autonomous from the central clock in metabolic regulationLiver clock and gut microbes interact to direct hepatic glucose and lipid metabolismReciprocating host-microbe interactions drive rhythmic hepatic transcriptionPerturbed liver Bmal1 results in chaotic downstream oscillators and metabolism

Caloric restriction (CR) prolongs life span, yet the mechanisms by which it does so remain poorly understood. Under CR, mice self-impose chronic cycles of 2-hour feeding and 22-hour fasting, raising the question of if it is calories, fasting, or time of day that is the cause of this increased life span. We show here that 30% CR was sufficient to extend the life span by 10%; however, a daily fasting interval and circadian alignment of feeding acted together to extend life span by 35% in male C57BL/6J mice. These effects were independent of body weight. Aging induced widespread increases in gene expression associated with inflammation and decreases in the expression of genes encoding components of metabolic pathways in liver from ad libitum–fed mice. CR at night ameliorated these aging-related changes. Our results show that circadian interventions promote longevity and provide a perspective to further explore mechanisms of aging.

The circadian system cyclically regulates many physiological and behavioral processes within the day. Desynchronization between physiological and behavioral rhythms increases the risk of developing some, including metabolic, disorders. Here we investigate how the oscillatory nature of metabolic signals, resembling feeding-fasting cycles, sustains the cell-autonomous clock in peripheral tissues. By controlling the timing, period and frequency of glucose and insulin signals via microfluidics, we find a strong effect on Per2::Luc fibroblasts entrainment. We show that the circadian Per2 expression is better sustained via a 24 h period and 12 h:12 h frequency-encoded metabolic stimulation applied for 3 daily cycles, aligned to the cell-autonomous clock, entraining the expression of hundreds of genes mostly belonging to circadian rhythms and cell cycle pathways. On the contrary misaligned feeding-fasting cycles synchronize and amplify the expression of extracellular matrix-associated genes, aligned during the light phase. This study underlines the role of the synchronicity between life-style-associated metabolic signals and peripheral clocks on the circadian entrainment., Chronic desynchronization between physiological and behavioral rhythms has been linked to the onset of metabolic diseases. Here the authors control the cyclic metabolic signals in a microfluidic device to study the effects of the timing, period and frequency of glucose and insulin on the transcriptome of cultured fibroblasts.

Significance Here, we show that expression of key circadian clock genes in the skin is altered by acute inflammation in mice treated topically with the immune activator imiquimod and by chronic inflammation in human psoriatic lesions. We show time-of-day–dependent activation of the interferon pathway, a key pathway involved in the host defense response. Mice lacking circadian rhythms have greater epidermal hyperplasia and more robust activation of the interferon pathway. Furthermore, we show that daytime-restricted feeding shifts the phase of interferon-sensitive gene expression in mouse skin. These findings demonstrate a role for the circadian clock in a major defense pathway in the skin’s response to microbes and cancer and suggest that timing of feeding can affect this response in the skin., The circadian clock coordinates a variety of immune responses with signals from the external environment to promote survival. We investigated the potential reciprocal relationship between the circadian clock and skin inflammation. We treated mice topically with the Toll-like receptor 7 (TLR7) agonist imiquimod (IMQ) to activate IFN-sensitive gene (ISG) pathways and induce psoriasiform inflammation. IMQ transiently altered core clock gene expression, an effect mirrored in human patient psoriatic lesions. In mouse skin 1 d after IMQ treatment, ISGs, including the key ISG transcription factor IFN regulatory factor 7 (Irf7), were more highly induced after treatment during the day than the night. Nuclear localization of phosphorylated-IRF7 was most prominently time-of-day dependent in epidermal leukocytes, suggesting that these cell types play an important role in the diurnal ISG response to IMQ. Mice lacking Bmal1 systemically had exacerbated and arrhythmic ISG/Irf7 expression after IMQ. Furthermore, daytime-restricted feeding, which affects the phase of the skin circadian clock, reverses the diurnal rhythm of IMQ-induced ISG expression in the skin. These results suggest a role for the circadian clock, driven by BMAL1, as a negative regulator of the ISG response, and highlight the finding that feeding time can modulate the skin immune response. Since the IFN response is essential for the antiviral and antitumor effects of TLR activation, these findings are consistent with the time-of-day–dependent variability in the ability to fight microbial pathogens and tumor initiation and offer support for the use of chronotherapy for their treatment.

The mammalian circadian clock has evolved as an adaptation to the 24-h light/darkness cycle on earth. Maintaining cellular activities in synchrony with the activities of the organism (such as eating and sleeping) helps different tissue and organ systems coordinate and optimize their performance. The full extent of the mechanisms by which cells maintain the clock are still under investigation, but involve a core set of clock genes that regulate large networks of gene transcription both by direct transcriptional activation/repression as well as the recruitment of proteins that modify chromatin states more broadly.

Significance Seasonal migration is dependent on an organism being able to sense and reorient to the Earth’s magnetic field. Cryptochromes (CRYs) have been implicated as light-driven sensors of the Earth’s magnetic field; however, contradictions between in vitro photochemistry and in vivo behavioral studies limit validation of CRYs as magnetosensors. To reconcile these discrepancies, we conducted detailed photochemical and structural studies of a CRY from Columbia livia (pigeon). We present the structure of a photoactive vertebrate CRY that reveals a pathway conserved in migratory organisms that facilitates its function as a magnetosensor., Computational and biochemical studies implicate the blue-light sensor cryptochrome (CRY) as an endogenous light-dependent magnetosensor enabling migratory birds to navigate using the Earth’s magnetic field. Validation of such a mechanism has been hampered by the absence of structures of vertebrate CRYs that have functional photochemistry. Here we present crystal structures of Columba livia (pigeon) CRY4 that reveal evolutionarily conserved modifications to a sequence of Trp residues (Trp-triad) required for CRY photoreduction. In ClCRY4, the Trp-triad chain is extended to include a fourth Trp (W369) and a Tyr (Y319) residue at the protein surface that imparts an unusually high quantum yield of photoreduction. These results are consistent with observations of night migratory behavior in animals at low light levels and could have implications for photochemical pathways allowing magnetosensing.

The essential but enigmatic functions of sleep1,2 must be reflected in molecular changes sensed by the brain's sleep-control systems. In Drosophila, two dozen sleep-inducing neurons3 with projections to the dorsal fan-shaped body (dFB) adjust their electrical output to sleep need4, via the antagonistic regulation of two potassium conductances: the leak channel Sandman imposes silence during waking, whereas augmented A-type currents through Shaker support tonic firing during sleep5. Here, we report that oxidative by-products of mitochondrial electron transport6,7 regulate the activity of dFB neurons through a nicotinamide adenine dinucleotide phosphate (NADPH) cofactor bound to the oxidoreductase domain8,9 of Shaker's K(V)β subunit, Hyperkinetic10,11. Sleep loss elevates mitochondrial reactive oxygen species in dFB neurons, which register this rise by converting Hyperkinetic to the NADP(+)-bound form. The oxidation of the cofactor slows the inactivation of the A-type current and boosts the frequency of action potentials, thereby promoting sleep. Energy metabolism, oxidative stress, and sleep, three processes implicated independently in lifespan, aging, and degenerative disease6,12–14, are thus mechanistically connected. K(V)β substrates8,15,16 or inhibitors capable of altering the NADP(+):NADPH ratio (and hence the neurons' record of sleep debt or waking time) define novel prototypes of sleep-regulatory drugs.

Summary The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in mammals and is entrained by environmental light. However, the molecular basis of the response of the SCN to light is not fully understood. We used RNA/chromatin immunoprecipitation/single-nucleus sequencing with circadian behavioral assays to identify mouse SCN cell types and explore their responses to light. We identified three peptidergic cell types that responded to light in the SCN: arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), and cholecystokinin (CCK). In each cell type, light-responsive subgroups were enriched for expression of neuronal Per-Arnt-Sim (PAS) domain protein 4 (NPAS4) target genes. Further, mice lacking Npas4 had a longer circadian period under constant conditions, a damped phase response curve to light, and reduced light-induced gene expression in the SCN. Our data indicate that NPAS4 is necessary for normal transcriptional responses to light in the SCN and critical for photic phase-shifting of circadian behavior.

Night-migratory songbirds are remarkably proficient navigators1. Flying alone and often over great distances, they use various directional cues including, crucially, a light-dependent magnetic compass2,3. The mechanism of this compass has been suggested to rely on the quantum spin dynamics of photoinduced radical pairs in cryptochrome flavoproteins located in the retinas of the birds4–7. Here we show that the photochemistry of cryptochrome 4 (CRY4) from the night-migratory European robin (Erithacus rubecula) is magnetically sensitive in vitro, and more so than CRY4 from two non-migratory bird species, chicken (Gallus gallus) and pigeon (Columba livia). Site-specific mutations of ErCRY4 reveal the roles of four successive flavin–tryptophan radical pairs in generating magnetic field effects and in stabilizing potential signalling states in a way that could enable sensing and signalling functions to be independently optimized in night-migratory birds. Cryptochrome 4 from the night-migratory European robin displays magnetically sensitive photochemistry in vitro, in which four successive flavin–tryptophan radical pairs generate magnetic-field effects and stabilize potential signalling states.

Sleeping sickness is caused by a eukaryotic unicellular parasite known to infect wild animals, cattle, and humans. It causes a fatal disease that disrupts many rhythmic physiological processes, including daily rhythms of hormonal secretion, temperature regulation, and sleep, all of which are under circadian (24-h) control. In this review, we summarize research on sleeping sickness parasite biology and the impact it has on host health. We also consider the possible evolutionary advantages of sleep and circadian deregulation for the parasite.

In mammals, a set of core clock genes form transcription–translation feedback loops to generate circadian oscillations. We and others recently identified a novel transcript at the Period2 (Per2) locus that is transcribed from the antisense strand of Per2. This transcript, Per2AS, is expressed rhythmically and antiphasic to Per2 mRNA, leading to our hypothesis that Per2AS and Per2 mutually inhibit each other's expression and form a double negative feedback loop. By perturbing the expression of Per2AS, we found that Per2AS transcription, but not transcript, represses Per2. However, Per2 does not repress Per2AS, as Per2 knockdown led to a decrease in the Per2AS level, indicating that Per2AS forms a single negative feedback loop with Per2 and maintains the level of Per2 within the oscillatory range. Per2AS also regulates the amplitude of the circadian clock, and this function cannot be solely explained through its interaction with Per2, as Per2 knockdown does not recapitulate the phenotypes of Per2AS perturbation. Overall, our data indicate that Per2AS is an important regulatory molecule in the mammalian circadian clock machinery. Our work also supports the idea that antisense transcripts of core clock genes constitute a common feature of circadian clocks, as they are found in other organisms.

Circadian transcriptome studies identified a novel transcript at the Period2 (Per2) locus, which we named Per2AS. Per2AS is a long non-coding RNA transcribed from the antisense strand of Per2, and is expressed rhythmically and anti-phasic to Per2 mRNA. Previously, we mathematically tested the hypothesis that Per2AS and Per2 mutually inhibit each other’s expression by forming a double negative feedback loop, and found that Per2AS expands the oscillatory domain. In this study, we have experimentally tested this prediction by perturbing the expression of Per2AS in mouse fibroblasts. We found that Per2AS represses Per2 pre-transcriptionally in cis and regulates the amplitude of the circadian clock, but not period or phase. Unexpectedly, we also found that Per2 positively regulates Per2AS post-transcriptionally, indicating that Per2AS and Per2 form a single negative feedback loop. Because knock-down of Per2 does not recapitulate the phenotypes of Per2AS perturbation and Per2AS also activates Bmal1 in trans, we propose that Per2AS regulates the amplitude of the circadian clock without producing a protein by rewiring the molecular clock circuit.

Circadian oscillations are generated via transcriptional-translational negative feedback loops. However, individual cells from fibroblast cell lines have heterogeneous rhythms, oscillating independently and with different period lengths. Here we showed that heterogeneity in circadian period is heritable and used a multi-omics approach to investigate underlying mechanisms. By examining large-scale phenotype-associated gene expression profiles in hundreds of mouse clonal cell lines, we identified and validated multiple novel candidate genes involved in circadian period determination in the absence of significant genomic variants. We also discovered differentially co-expressed gene networks that were functionally associated with period length. We further demonstrated that global differential DNA methylation bidirectionally regulated these same gene networks. Interestingly, we found that depletion of DNMT1 and DNMT3A had opposite effects on circadian period, suggesting non-redundant roles in circadian gene regulation. Together, our findings identify novel gene candidates involved in periodicity, and reveal DNA methylation as an important regulator of circadian periodicity.

Significance Our findings have revealed a previously unrecognized link between circadian oscillations and intercellular variation and provide experimental evidence that stochastic transcriptional noise contributes significantly to cell-autonomous circadian periodicity. Interestingly, in separate studies, aging and cancer have been associated with increased transcriptional noise and less robust circadian rhythms. Here, we establish a direct association between transcriptional noise and circadian period. These findings may provide additional directions for researchers in the aging and cancer fields. Furthermore, circadian period may also be used as an indicator of variance in heterogeneity research and drug screening for noise control., Nongenetic cellular heterogeneity is associated with aging and disease. However, the origins of cell-to-cell variability are complex and the individual contributions of different factors to total phenotypic variance are still unclear. Here, we took advantage of clear phenotypic heterogeneity of circadian oscillations in clonal cell populations to investigate the underlying mechanisms of cell-to-cell variability. Using a fully automated tracking and analysis pipeline, we examined circadian period length in thousands of single cells and hundreds of clonal cell lines and found that longer circadian period is associated with increased intercellular heterogeneity. Based on our experimental results, we then estimated the contributions of heritable and nonheritable factors to this variation in circadian period length using a variance partitioning model. We found that nonheritable noise predominantly drives intercellular circadian period variation in clonal cell lines, thereby revealing a previously unrecognized link between circadian oscillations and intercellular heterogeneity. Moreover, administration of a noise-enhancing drug reversibly increased both period length and variance. These findings suggest that circadian period may be used as an indicator of cellular noise and drug screening for noise control.

Neuronal activity and gene expression in response to the loss of sleep can provide a window into the enigma of sleep function. Sleep loss is associated with brain differential gene expression, an increase in pyramidal cell mEPSC frequency and amplitude, and a characteristic rebound and resolution of slow wave sleep-slow wave activity (SWS-SWA). However, the molecular mechanism(s) mediating the sleep loss response are not well understood. We show that sleep-loss regulates MEF2C phosphorylation, a key mechanism regulating MEF2C transcriptional activity, and that MEF2C function in postnatal excitatory forebrain neurons is required for the biological events in response to sleep loss. These include altered gene expression, the increase and recovery of synaptic strength, and the rebound and resolution of SWS-SWA, which implicate MEF2C as an essential regulator of sleep function.One Sentence SummaryMEF2C is critical to the response to sleep loss.

Environmental light cycles entrain circadian feeding behaviors in animals that produce rhythms in exposure to foodborne bacteria. Here, we show that the intestinal microbiota generates diurnal rhythms in innate immunity that synchronize with feeding rhythms to anticipate microbial exposure. Rhythmic expression of antimicrobial proteins was driven by daily rhythms in epithelial attachment by segmented filamentous bacteria (SFB), members of the mouse intestinal microbiota. Rhythmic SFB attachment was driven by the circadian clock through control of feeding rhythms. Mechanistically, rhythmic SFB attachment activated an immunological circuit involving group 3 innate lymphoid cells. This circuit triggered oscillations in epithelial STAT3 expression and activation that produced rhythmic antimicrobial protein expression and caused resistance to Salmonella Typhimurium infection to vary across the day-night cycle. Thus, host feeding rhythms synchronize with the microbiota to promote rhythms in intestinal innate immunity that anticipate exogenous microbial exposure.

Transcriptional regulation of numerous interferon-regulated genes, including Toll-like receptor 3 (Tlr3), which encodes an innate immune sensor of viral double-stranded RNA, depends on the interferon regulatory factor 1 (IRF1) and IRF2 transcription factors. We detected specific abrogation of macrophage responses to polyinosinic-polycytidylic acid (poly(I:C)) resulting from three independent N-ethyl-N-nitrosourea–induced mutations in host cell factor C2 (Hcfc2). Hcfc2 mutations compromised survival during influenza virus and herpes simplex virus 1 infections. HCFC2 promoted the binding of IRF1 and IRF2 to the Tlr3 promoter, without which inflammatory cytokine and type I IFN responses to the double-stranded RNA analogue poly(I:C) are reduced in mouse macrophages. HCFC2 was also necessary for the transcription of a large subset of other IRF2-dependent interferon-regulated genes. Deleterious mutations of Hcfc2 may therefore increase susceptibility to diverse infectious diseases.

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.. Geisel School Of Medicine At Dartmouth; Estados Unidos Fil: Eckel Mahan, Kristin. University Of Texas Medical School At Houston; Estados Unidos Fil: Esser, Karyn A.. University Of Florida College Of Medicine; Estados Unidos Fil: FitzGerald, Garret A.. University of Pennsylvania; Estados Unidos Fil: Forger, Daniel B.. University Of Michigan; Estados Unidos Fil: Francey, Lauren J.. Cincinnati Children's Hospital Medical Center; Estados Unidos Fil: Fu, Ying Hui. University of California; Estados Unidos Fil: Gachon, Frédéric. Nestlé Institute Of Health Sciences; Suiza Fil: Gatfield, David. Universite de Lausanne; Suiza Fil: de Goede, Paul. University Of Amsterdam; Países Bajos Fil: Golden, Susan S.. University of California at San Diego; Estados Unidos Fil: Green, Carla. University of Texas; Estados Unidos Fil: Harer, John. University of Duke; Estados Unidos Fil: Harmer, Stacey. University of California at Davis; Estados Unidos Fil: Haspel, Jeff. 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. Mcgill University, Douglas Mental Health University Institute; Canadá Fil: Takahashi, Joseph S.. Ut Southwestern Medical Center; Estados Unidos Fil: Ueda, Hiroki R.. The University of Tokyo; Japón Fil: Wang, Han. Soochow University; China Fil: Weitz, Charles. Harvard Medical School; Estados Unidos Fil: Westermark, P. O.. Leibniz Institute For Farm Animal Biology; Alemania Fil: Wijnen, Herman. University of Southampton; Reino Unido Fil: Xu, Ying. Soochow University; China Fil: Wu, Gang. Cincinnati Children's Hospital Medical Center; Estados Unidos Fil: Yoo, Seung Hee. University of Texas; Estados Unidos Fil: Young, Michael. The Rockefeller University; Estados Unidos Fil: Zhang, Eric Erquan. National Institute Of Biological Sciences, Beijing; China Fil: Zielinski, Tomasz. University of Edinburgh; Reino Unido Fil: Hogenesch, John B.. Cincinnati Children's Hospital Medical Center; Estados Unidos

Fumarylacetoacetate hydrolase (FAH) is the last enzyme in tyrosine catabolism, and mutations in the FAH gene are associated with hereditary tyrosinemia type I (HT1 or TYRSN1) in humans. In a behavioral screen of N -ethyl- N -nitrosourea mutagenized mice we identified a mutant line which we named “ swingshift ” ( swst , MGI:3611216) with a nonsynonymous point mutation (N68S) in Fah that caused age-dependent disruption of sleep–wake patterns. Mice homozygous for the mutation had an earlier onset of activity (several hours before lights off) and a reduction in total activity and body weight when compared with wild-type or heterozygous mice. Despite abnormal behavioral entrainment to light–dark cycles, there were no differences in the period or phase of the central clock in mutant mice, indicating a defect downstream of the suprachiasmatic nucleus. Interestingly, these behavioral phenotypes became milder as the mice grew older and were completely rescued by the administration of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione], an inhibitor of 4-hydroxyphenylpyruvate dioxygenase, which is upstream of FAH. Mechanistically, the swst mutation had no effect on the enzymatic activity of FAH, but rather promoted the degradation of the mutant protein. This led to reduced FAH protein levels and enzymatic activity in the liver and kidney (but not the brain or fibroblasts) of homozygous mice. In addition, plasma tyrosine—but not methionine, phenylalanine, or succinylacetone—increased in homozygous mice, suggesting that swst mutants provide a model of mild, chronic HT1.

Circadian clocks are endogenous oscillators that control 24-h physiological and behavioral processes. The central circadian clock exerts control over myriad aspects of mammalian physiology, including the regulation of sleep, metabolism, and the immune system. Here, we review advances in understanding the genetic regulation of sleep through the circadian system, as well as the impact of dysregulated gene expression on metabolic function. We also review recent studies that have begun to unravel the circadian clock’s role in controlling the cardiovascular and nervous systems, gut microbiota, cancer, and aging. Such circadian control of these systems relies, in part, on transcriptional regulation, with recent evidence for genome-wide regulation of the clock through circadian chromosome organization. These novel insights into the genomic regulation of human physiology provide opportunities for the discovery of improved treatment strategies and new understanding of the biological underpinnings of human disease.

Dear Colleagues,The UT Southwestern community continues to mourn the loss of Dr. Beth Levine – a renowned physician-scientist, dedicated and caring educator, mentor, respected colleague and friend....

The suprachiasmatic nucleus (SCN) acts as a master pacemaker driving circadian behavior and physiology. Although the SCN is small, it is composed of many cell types, making it difficult to study the roles of particular cells. Here we have developed bioluminescent circadian reporter mice that are Cre-dependent, allowing the circadian properties of genetically-defined populations of cells to be studied in real time. Using a Color-Switch PER2::LUCIFERASE reporter that switches from red PER2::LUCIFERASE to green PER2::LUCIFERASE upon Cre recombination, we assessed circadian rhythms in two of the major classes of peptidergic neurons in the SCN: AVP (arginine vasopressin) and VIP (vasoactive intestinal polypeptide). Surprisingly, we find that circadian function in AVP neurons, not VIP neurons, is essential for autonomous network synchrony of the SCN and stability of circadian rhythmicity.

Chronic fatigue is a debilitating disorder with widespread consequences, but effective treatment strategies are lacking. Novel genetic mouse models of fatigue may prove invaluable for studying its underlying physiological mechanisms and for testing treatments and interventions. In a screen of voluntary wheel-running behavior in N-ethyl-N-nitrosourea mutagenized C57BL/6J mice, we discovered two lines with low body weights and aberrant wheel-running patterns suggestive of a fatigue phenotype. Affected progeny from these lines had lower daily activity levels and exhibited low amplitude circadian rhythm alterations. Their aberrant behavior was characterized by frequent interruptions and periods of inactivity throughout the dark phase of the light-dark cycle and increased levels of activity during the rest or light phase. Expression of the behavioral phenotypes in offspring of strategic crosses was consistent with a recessive inheritance pattern. Mapping of phenotypic abnormalities showed linkage with a single locus on chromosome 1, and whole exome sequencing identified a single point mutation in the Slc2a4 gene encoding the GLUT4 insulin-responsive glucose transporter. The single nucleotide change (A-T, which we named "twiggy") was in the distal end of exon 10 and resulted in a premature stop (Y440*). Additional metabolic phenotyping confirmed that these mice recapitulate phenotypes found in GLUT4 knockout mice. However, to the best of our knowledge, this is the first time a mutation in this gene has been shown to result in extensive changes in general behavioral patterns. These findings suggest that GLUT4 may be involved in circadian behavioral abnormalities and could provide insights into fatigue in humans.

Nocturnin (NOCT) belongs to the Mg2+ dependent Exonucleases, Endonucleases, Phosphatase (EEP) family of enzymes that exhibit various functions in vitro and in vivo. NOCT is known to function as a deadenylase, cleaving poly-A tails from mRNA (messenger RNA) transcripts. Previously, we reported a role for NOCT in regulating bone marrow stromal cell differentiation through its interactions with PPARγ. In this study, we characterized the skeletal and adipose tissue phenotype when we globally overexpressed Noct in vivo. After 12 weeks of Noct overexpression, transgenic male mice had lower fat mass compared to controls, with no significant differences in the skeleton. Based on the presence of a mitochondrial target sequence in NOCT, we determined that mouse NOCT protein localizes to the mitochondria; subsequently, we found that NOCT overexpression led to a significant increase in the preadipocytes ability to utilize oxidative phosphorylation for ATP (adenosine triphosphate) generation. In summary, the effects of NOCT on adipocytes are likely through its novel role as a mediator of mitochondrial function.

Mammalian circadian clocks are driven by a transcription/translation feedback loop composed of positive regulators (CLOCK/BMAL1) and repressors (CRYPTOCHROME 1/2 (CRY1/2) and PER1/2). To understand the structural principles of regulation, we used evolutionary sequence analysis to identify co-evolving residues within the CRY/PHL protein family. Here we report the identification of an ancestral secondary cofactor-binding pocket as an interface in repressive CRYs, mediating regulation through direct interaction with CLOCK and BMAL1. Mutations weakening binding between CLOCK/BMAL1 and CRY1 lead to acceleration of the clock, suggesting that subtle sequence divergences at this site can modulate clock function. Divergence between CRY1 and CRY2 at this site results in distinct periodic output. Weaker interactions between CRY2 and CLOCK/BMAL1 at this pocket are strengthened by co-expression of PER2, suggesting that PER expression limits the length of the repressive phase in CRY2-driven rhythms. Overall, this work provides a model for the mechanism and evolutionary variation of clock regulatory mechanisms., The molecular mechanisms that define the periodicity or rate of the circadian clock are not well understood. Here the authors use a multidisciplinary approach and identify a mechanism for period regulation that depends on the affinity of the core clock proteins for one another.

Historically, work on peripheral circadian clocks has been focused on organs and tissues that have prominent metabolic functions, such as the liver, fat, and muscle. In recent years, skin has emerged as a model for studying circadian clock regulation of cell proliferation, stem cell functions, tissue regeneration, aging, and carcinogenesis. Morphologically, skin is complex, containing multiple cell types and structures, and there is evidence for a functional circadian clock in most, if not all, of its cell types. Despite the complexity, skin stem cell populations are well defined, experimentally tractable, and exhibit prominent daily cell proliferation cycles. Hair follicle stem cells also participate in recurrent, long-lasting cycles of regeneration: the hair growth cycles. Among other advantages of skin is a broad repertoire of available genetic tools enabling the creation of cell type–specific circadian mutants. Also, due to the accessibility of skin, in vivo imaging techniques can be readily applied to study the circadian clock and its outputs in real time, even at the single-cell level. Skin provides the first line of defense against many environmental and stress factors that exhibit dramatic diurnal variations such as solar ultraviolet (UV) radiation and temperature. Studies have already linked the circadian clock to the control of UVB-induced DNA damage and skin cancers. Due to the important role that skin plays in the defense against microorganisms, it also represents a promising model system to further explore the role of the clock in the regulation of the body’s immune functions. To that end, recent studies have already linked the circadian clock to psoriasis, one of the most common immune-mediated skin disorders. Skin also provides opportunities to interrogate the clock regulation of tissue metabolism in the context of stem cells and regeneration. Furthermore, many animal species feature prominent seasonal hair molt cycles, offering an attractive model for investigating the role of the clock in seasonal organismal behaviors.

Circadian timekeeping systems drive oscillatory gene expression to regulate essential cellular and physiological processes. When the systems are perturbed, pathological consequences ensue and disease risks rise. A growing number of small-molecule modulators have been reported to target circadian systems. Such small molecules, identified via high-throughput screening or derivatized from known scaffolds, have shown promise as drug candidates to improve biological timing and physiological outputs in disease models. In this review, we first briefly describe the circadian system, including the core oscillator and the cellular networks. Research progress on clock-modulating small molecules is presented, focusing on development strategies and biological efficacies. We highlight the therapeutic potential of small molecules in clock-related pathologies, including jet lag and shiftwork; various chronic diseases, particularly metabolic disease; and aging. Emerging opportunities to identify and exploit clock modulators as novel therapeutic agents are discussed.

The molecular mechanisms underlying human brain evolution are not fully understood; however, previous work suggested that expression of the transcription factor CLOCK in the human cortex might be relevant to human cognition and disease. In this study, we investigated this novel transcriptional role for CLOCK in human neurons by performing chromatin immunoprecipitation sequencing for endogenous CLOCK in adult neocortices and RNA sequencing following CLOCK knockdown in differentiated human neurons in vitro. These data suggested that CLOCK regulates the expression of genes involved in neuronal migration, and a functional assay showed that CLOCK knockdown increased neuronal migratory distance. Furthermore, dysregulation of CLOCK disrupts coexpressed networks of genes implicated in neuropsychiatric disorders, and the expression of these networks is driven by hub genes with human-specific patterns of expression. These data support a role for CLOCK-regulated transcriptional cascades involved in human brain evolution and function.

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, eLife digest We spend nearly one third of our lives asleep. Sleep plays a critical role in human health and is regulated by multiple brain regions. Genes are some of the factors that control sleep. Recent studies have shown that mice in which a gene called Bmal1 had been completely removed, sleep more than mice that still have the gene. These Bmal1-deficient mice also respond differently to sleep loss. However, until now, it was not known which tissues and cells that carry active (or ‘expressed’) Bmal1 are involved in regulating sleep. To find out if Bmal1 activity in the brain is sufficient to recover from sleep loss, Ehlen, Brager et al. compared genetically modified mice that either expressed Bmal1 only in the brain, or only in the muscle tissue that covers the skeleton. After the mice were kept awake for six hours, their sleep was monitored by measuring electrical signals on the surface of the skull. Contrary to what they expected, Ehlen et al. found that mice with Bmal1 expressed in the skeletal muscle were able to have a normal sleep pattern, while mice with Bmal1 expressed in the brain had an abnormal sleep pattern. Further experiments show that removing Bmal1 from the skeletal muscle of mice, but allowing the gene to be expressed in other tissues, produced sleeping patterns that were similar to those seen in mice that were completely missing the Bmal1 gene. These results indicate that Bmal1 in skeletal muscle is important to help regulate sleep, and that the signal for sleepiness does not only originate from the brain. This is the first study to show that skeletal muscle can regulate sleep. The next step will be to identify the specific signal the muscle uses to trigger the brain to sleep. Understanding the mechanisms that regulate sleep may help to develop new treatments for sleep disorders. DOI: http://dx.doi.org/10.7554/eLife.26557.002

In a screen of voluntary wheel-running behavior designed to identify genetic mouse models of chronic fatigue in ENU mutagenized C57BL/6J mice, we discovered two lines that showed aberrant wheel-running patterns. These lines both stem from a single original founder identified as a low body-weight candidate in a recessive screen. Progeny from both of these lines showed the abnormal wheel-running behavior, with affected mice showing significantly lower daily activity levels than unaffected mice. They also exhibited low amplitude circadian rhythms, consisting of lower activity levels during the normal active phase, and increased levels of activity during the rest or light phase, but only a modest alteration in free-running period. Their activity is not consolidated into longer bouts, but is frequently interrupted with periods of inactivity throughout the dark phase of the light-dark (LD) cycle. As seen with the low body weight, expression of the behavioral phenotypes in offspring of strategic crosses was consistent with a recessive heritance pattern. Mapping of these phenotypic abnormalities showed linkage to a single locus on chromosome 11, and whole exome sequencing (WES) identified a single point mutation in theSlc2a4gene encoding the GLUT4 insulin-responsive glucose transporter. The single nucleotide change (A to T) was found in the distal end of exon 10, and results in a premature stop (Y440*). To our knowledge, this is the first time a mutation in this gene has been shown to result in extensive changes in general behavioral patterns.SIGNIFICANCE STATEMENTChronic fatigue is a debilitating and devastating disorder with widespread consequences for both the patient and the persons around them, but effective treatment strategies are lacking. The identification of novel genetic mouse models of chronic fatigue may prove invaluable for the study of its underlying physiological mechanisms and for the testing of treatments and interventions. A novel mutation inSlc2a4(GLUT4) was identified in a forward mutagenesis screen because affected mice showed abnormal daily patterns and levels of wheel running consistent with chronic fatigue. This new mouse model may shed light on the pathophysiology of chronic fatigue.