Search

The aging eye experiences physiological changes that include decreased visual function and increased risk of retinal degeneration. Although there are transcriptomic signatures in the aging retina that correlate with these physiological changes, the gene regulatory mechanisms that contribute to cellular homeostasis during aging remain to be determined. Here, we integrated ATAC-seq and RNA-seq data to identify 57 transcription factors that showed differential activity in aging Drosophila photoreceptors. These 57 age-regulated transcription factors include two circadian regulators, Clock and Cycle, that showed sustained increased activity during aging. When we disrupted the Clock:Cycle complex by expressing a dominant negative version of Clock (ClkDN) in adult photoreceptors, we observed changes in expression of 15-20% of genes including key components of the phototransduction machinery and many eye-specific transcription factors. Using ATAC-seq, we showed that expression of ClkDN in photoreceptors leads to changes in activity of 37 transcription factors and causes a progressive decrease in global levels of chromatin accessibility in photoreceptors. Supporting a key role for Clock-dependent transcription in the eye, expression of ClkDN in photoreceptors also induced light-dependent retinal degeneration and increased oxidative stress, independent of light exposure. Together, our data suggests that the circadian regulators Clock and Cycle act as neuroprotective factors in the aging eye by directing gene regulatory networks that maintain expression of the phototransduction machinery and counteract oxidative stress., Competing Interests: The authors have declared that no competing interests exist.

Cardiac function is highly reliant on mitochondrial oxidative metabolism and quality control. The circadian Clock gene is critically linked to vital physiological processes including mitochondrial fission, fusion and bioenergetics; however, little is known of how the Clock gene regulates these vital processes in the heart. Herein, we identified a putative circadian CLOCK-mitochondrial interactome that gates an adaptive survival response during myocardial ischemia. We show by transcriptome and gene ontology mapping in CLOCK Δ19/Δ19 mouse that Clock transcriptionally coordinates the efficient removal of damaged mitochondria during myocardial ischemia by directly controlling transcription of genes required for mitochondrial fission, fusion and macroautophagy/autophagy. Loss of Clock gene activity impaired mitochondrial turnover resulting in the accumulation of damaged reactive oxygen species (ROS)-producing mitochondria from impaired mitophagy. This coincided with ultrastructural defects to mitochondria and impaired cardiac function. Interestingly, wild type CLOCK but not mutations of CLOCK defective for E-Box binding or interaction with its cognate partner ARNTL/BMAL-1 suppressed mitochondrial damage and cell death during acute hypoxia. Interestingly, the autophagy defect and accumulation of damaged mitochondria in CLOCK-deficient cardiac myocytes were abrogated by restoring autophagy/mitophagy. Inhibition of autophagy by ATG7 knockdown abrogated the cytoprotective effects of CLOCK. Collectively, our results demonstrate that CLOCK regulates an adaptive stress response critical for cell survival by transcriptionally coordinating mitochondrial quality control mechanisms in cardiac myocytes. Interdictions that restore CLOCK activity may prove beneficial in reducing cardiac injury in individuals with disrupted circadian CLOCK. Abbreviations: ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATG14: autophagy related 14; ATG7: autophagy related 7; ATP: adenosine triphosphate; BCA: bovine serum albumin; BECN1: beclin 1, autophagy related; bHLH: basic helix- loop-helix; CLOCK: circadian locomotor output cycles kaput; CMV: cytomegalovirus; COQ5: coenzyme Q5 methyltransferase; CQ: chloroquine; CRY1: cryptochrome 1 (photolyase-like); DNM1L/DRP1: dynamin 1-like; EF: ejection fraction; EM: electron microscopy; FS: fractional shortening; GFP: green fluorescent protein; HPX: hypoxia; i.p.: intraperitoneal; I-R: ischemia-reperfusion; LAD: left anterior descending; LVIDd: left ventricular internal diameter diastolic; LVIDs: left ventricular internal diameter systolic; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN2: mitofusin 2; MI: myocardial infarction; mPTP: mitochondrial permeability transition pore; NDUFA4: Ndufa4, mitochondrial complex associated; NDUFA8: NADH: ubiquinone oxidoreductase subunit A8; NMX: normoxia; OCR: oxygen consumption rate; OPA1: OPA1, mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PBS: phosphate-buffered saline; PER1: period circadian clock 1; PPARGC1A/PGC-1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; qPCR: quantitative real-time PCR; RAB7A: RAB7, member RAS oncogene family; ROS: reactive oxygen species; RT: room temperature; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFAM: transcription factor A, mitochondrial; TFEB: transcription factor EB; TMRM: tetra-methylrhodamine methyl ester perchlorate; WT: wild -type; ZT: zeitgeber time.

Exercise and circadian biology are closely intertwined with physiology and metabolism, yet the functional interaction between circadian clocks and exercise capacity is only partially characterized. Here, we tested different clock mutant mouse models to examine the effect of the circadian clock and clock proteins, namely PERIODs and BMAL1, on exercise capacity. We found that daytime variance in endurance exercise capacity is circadian clock controlled. Unlike wild-type mice, which outperform in the late compared with the early part of their active phase, PERIODs- and BMAL1-null mice do not show daytime variance in exercise capacity. It appears that BMAL1 impairs and PERIODs enhance exercise capacity in a daytime-dependent manner. An analysis of liver and muscle glycogen stores as well as muscle lipid utilization suggested that these daytime effects mostly relate to liver glycogen levels and correspond to the animals' feeding behavior. Furthermore, given that exercise capacity responds to training, we tested the effect of training at different times of the day and found that training in the late compared with the early part of the active phase improves exercise performance. Overall, our findings suggest that clock proteins shape exercise capacity in a daytime-dependent manner through changes in liver glycogen levels, likely due to their effect on animals' feeding behavior., Competing Interests: The authors declare no competing interest.

Aims: Feeding regimens alter circadian rhythms in peripheral tissues, but the mechanism is not understood. We aimed to study whether soluble factors, rather than neuronal-based communication, directly influence circadian rhythms in the liver, in response to a nutritional treatment in type 2 diabetes (T2D) patients., Methods: Cultured hepatocytes were treated with serum of insulin-treated T2D patients following either a three-meal diet (3Mdiet) or six-meal diet (6Mdiet) and the circadian expression of clock and metabolic genes was measured., Results: Serum of the 3Mdiet group led to increased amplitudes and daily mRNA levels of the positive limb of the circadian clock (Clock, Bmal1, Rorα). In parallel, serum of the 3Mdiet group led to the downregulation of the negative limb of the circadian clock (Cry1 and Per1), compared to both baseline and 6Mdiet. In contrast, serum of the 6Mdiet group led to a more distorted expression pattern. The catabolic genes Sirt1 and Ampk were significantly upregulated only by serum of the 3Mdiet group., Conclusions: Our results show that serum of type 2 diabetes patients consuming the 3Mdiet contains soluble factors that reset circadian rhythms leading to an expression pattern similar to that of healthy people. This clock pattern contributes to improved glucose metabolism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Anopheles mating is initiated by the swarming of males at dusk followed by females flying into the swarm. Here, we show that mosquito swarming and mating are coordinately guided by clock genes, light, and temperature. Transcriptome analysis shows up-regulation of the clock genes period ( per ) and timeless ( tim ) in the head of field-caught swarming Anopheles coluzzii males. Knockdown of per and tim expression affects Anopheles gambiae s.s. and Anopheles stephensi male mating in the laboratory, and it reduces male An. coluzzii swarming and mating under semifield conditions. Light and temperature affect mosquito mating, possibly by modulating per and/or tim expression. Moreover, the desaturase gene desat1 is up-regulated and rhythmically expressed in the heads of swarming males and regulates the production of cuticular hydrocarbons, including heptacosane, which stimulates mating activity., (Copyright © 2021, American Association for the Advancement of Science.)

Circadian clocks keep time via ~ 24 h transcriptional feedback loops. In Drosophila, CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors are feedback loop components whose transcriptional status varies over a circadian cycle. Although changes in the state of activators and repressors has been characterized, how their status is translated to transcriptional activity is not understood. We used mass spectrometry to identify proteins that interact with GFP-tagged CLK (GFP-CLK) in fly heads at different times of day. Many expected and novel interacting proteins were detected, of which several interacted rhythmically and were potential regulators of protein levels, activity or transcriptional output. Genes encoding these proteins were tested to determine if they altered circadian behavior via RNAi knockdown in clock cells. The NIPPED-A protein, a scaffold for the SAGA and Tip60 histone modifying complexes, interacts with GFP-CLK as transcription is activated, and reducing Nipped-A expression lengthens circadian period. RNAi analysis of other SAGA complex components shows that the SAGA histone deubiquitination (DUB) module lengthened period similarly to Nipped-A RNAi knockdown and weakened rhythmicity, whereas reducing Tip60 HAT expression drastically weakened rhythmicity. These results suggest that CLK-CYC binds NIPPED-A early in the day to promote transcription through SAGA DUB and Tip60 HAT activity.

Disruption of circadian rhythms and variations in the FTO gene may interfere with energy homeostasis and play a role in the development of obesity. The current study assessed the association of common polymorphisms in the CLOCK and FTO genes with standardized body mass index scores (BMI z-scores) and their potential modification of the impact of a culinary nutrition and physical activity intervention in school-age children. Anthropometric measurements were collected in 121 children at the baseline and one-year follow-up of a controlled trial of a school-based culinary nutrition and physical activity intervention. Genotypes of the CLOCK polymorphism (rs1801260) and the FTO polymorphism (rs9939609) were obtained from buccal swabs. Linear mixed-effects regression was applied to evaluate the genetic association and adjust for clusters within families and schools. In our participants, obesity affected 6.6% (8/121) of the children at the baseline and 6.4% (7/109) of the children at the follow-up. The associations between the age- and sex-adjusted BMI z-scores and the two polymorphisms did not reach statistically significance. Yet, sex potentially modified the association between rs1801260 and BMI z-scores. In girls, the G allele carriers had a higher BMI z-scores at the baseline and the follow-up. These polymorphisms did not modify the effect of our culinary nutrition and physical activity intervention on BMI z-scores. Sex is a potential modifier for the association between the CLOCK polymorphism, rs1801260, and BMI z-scores in school-age children. Further investigation is warranted to delineate the sex-dependent role of the CLOCK polymorphisms in the development of childhood obesity., Competing Interests: The authors have declared that no competing interests exist.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic β cells that are perturbed in Clock -/- and Bmal1 -/- β-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant β cells, including transcripts encoding Cask ( calcium/calmodulin-dependent serine protein kinase ) and Madd ( MAP kinase-activating death domain ). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd , and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in β-cell function across the sleep/wake cycle., (© 2020 Marcheva et al.; Published by Cold Spring Harbor Laboratory Press.)

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1., Competing Interests: JF, AS, CS, AM, PH, CR, LN, MT, GP, ST, NZ, GL, TH, FT, CP No competing interests declared, (© 2020, Fribourgh et al.)

The mammalian circadian clock, which coordinates various physiological functions, develops gradually during ontogeny. Recently, we have reported the posttranscriptional suppression of CLOCK protein expression as a key mechanism of the emergence of the circadian clock during mouse development. However, whether a common mechanism regulates the development of the human circadian clock remains unclear. In the present study, we show that human induced pluripotent stem cells (iPSCs) have no discernible circadian molecular oscillation. In addition, in vitro differentiation culture of human iPSCs required a longer duration than that required in mouse for the emergence of circadian oscillations. The expression of CLOCK protein in undifferentiated human iPSCs was posttranscriptionally suppressed despite the expression of CLOCK mRNA, which is consistent with our previous observations in mouse embryonic stem cells, iPSCs, and early mouse embryos. These results suggest that CLOCK protein expressions could be posttranscriptionally suppressed in the early developmental stage not only in mice but also in humans.

The circadian oscillator is a complex network of interconnected feedback loops that regulates a wide range of physiological processes. Indeed, variation in clock genes has been implicated in an array of plant environmental adaptations, including growth regulation, photoperiodic control of flowering, and responses to abiotic and biotic stress. Although the clock is buffered against the environment, maintaining roughly 24-h rhythms across a wide range of conditions, it can also be reset by environmental cues such as acute changes in light or temperature. These competing demands may help explain the complexity of the links between the circadian clock network and environmental response pathways. Here, we discuss our current understanding of the clock and its interactions with light and temperature-signaling pathways. We also describe different clock gene alleles that have been implicated in the domestication of important staple crops., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)

The circadian clock generates behavioral rhythms to maximize an organism's physiological efficiency. Light induces the formation of these rhythms by synchronizing cellular clocks. In zebrafish, the circadian clock components Period2 (zPER2) and Cryptochrome1a (zCRY1a) are light-inducible, however their physiological functions are unclear. Here, we investigated the roles of zPER2 and zCRY1a in regulating locomotor activity and behavioral rhythms. zPer2/zCry1a double knockout (DKO) zebrafish displayed defects in total locomotor activity and in forming behavioral rhythms when briefly exposed to light for 3-h. Exposing DKO zebrafish to 12-h light improved behavioral rhythm formation, but not total activity. Our data suggest that the light-inducible circadian clock regulator zCRY2a supports rhythmicity in DKO animals exposed to 12-h light. Single cell imaging analysis revealed that zPER2, zCRY1a, and zCRY2a function in synchronizing cellular clocks. Furthermore, microarray analysis of DKO zebrafish showed aberrant expression of genes involved regulating cellular metabolism, including ATP production. Overall, our results suggest that zPER2, zCRY1a and zCRY2a help to synchronize cellular clocks in a light-dependent manner, thus contributing to behavioral rhythm formation in zebrafish. Further, zPER2 and zCRY1a regulate total physical activity, likely via regulating cellular energy metabolism. Therefore, these circadian clock components regulate the rhythmicity and amount of locomotor behavior.

The animal circadian timing system interprets environmental time cues and internal metabolic status to orchestrate circadian rhythms of physiology, allowing animals to perform necessary tasks in a time-of-day-dependent manner. Normal progression of circadian rhythms is dependent on the daily cycling of core transcriptional factors that make up cell-autonomous molecular oscillators. In Drosophila , PERIOD (PER), TIMELESS (TIM), CLOCK (CLK), and CYCLE (CYC) are core clock proteins that function in a transcriptional-translational feedback mechanism to regulate the circadian transcriptome. Posttranslational modifications of core clock proteins provide precise temporal control over when they are active as regulators of clock-controlled genes. In particular, phosphorylation is a key regulatory mechanism that dictates the subcellular localization, stability, and transcriptional activity of clock proteins. Previously, casein kinase 1α (CK1α) has been identified as a kinase that phosphorylates mammalian PER1 and modulates its stability, but the mechanisms by which it modulates PER protein stability is still unclear. Using Drosophila as a model, we show that CK1α has an overall function of speeding up PER metabolism and is required to maintain the 24 h period of circadian rhythms. Our results indicate that CK1α collaborates with the key clock kinase DOUBLETIME (DBT) in both the cytoplasm and the nucleus to regulate the timing of PER-dependent repression of the circadian transcriptome. Specifically, we observe that CK1α promotes PER nuclear localization by antagonizing the activity of DBT to inhibit PER nuclear translocation. Furthermore, CK1α enhances DBT-dependent PER phosphorylation and degradation once PER moves into the nucleus. SIGNIFICANCE STATEMENT Circadian clocks are endogenous timers that integrate environmental signals to impose temporal control over organismal physiology over the 24 h day/night cycle. To maintain the 24 h period length of circadian clocks and to ensure that circadian rhythms are in synchrony with the external environment, key proteins that make up the molecular oscillator are extensively regulated by phosphorylation to ensure that they perform proper time-of-day-specific functions. Casein kinase 1α (CK1α) has previously been identified as a kinase that phosphorylates mammalian PERIOD (PER) proteins to promote their degradation, but the mechanism by which it modulates PER stability is unclear. In this study, we characterize the mechanisms by which CK1α interacts with DOUBLETIME (DBT) to achieve the overall function of speeding up PER metabolism and to ensure proper time-keeping., (Copyright © 2018 the authors 0270-6474/18/3810631-13$15.00/0.)

Aims: The sensation of bladder fullness (SBF) is triggered by the release of ATP. Therefore, the aim of this study was to investigate whether time-dependent changes in the levels of stretch-released ATP in mouse primary-cultured urothelial cells (MPCUCs) is regulated by circadian rhythm via clock genes., Methods: MPCUCs were derived from wild-type and Clock mutant mice (Clock Δ19/Δ19 ), presenting a nocturia phenotype. They were cultured in elastic silicone chambers. Stretch-released ATP was quantified every 4 h by ATP photon count. An experiment was also performed to determine whether ATP release correlated with the rhythm of the expression of Piezo1, TRPV4, VNUT, and Connexin26 (Cx26) in MPCUCs regulated by clock genes with circadian rhythms. MPCUCs were treated with carbenoxolone, an inhibitor of gap junction protein; were derived from VNUT-KO mice; or treated with Piezo1-siRNA, TRPV4-siRNA, and Cx26-siRNA., Results: Stretch-released ATP showed time-dependent changes in wild-type mice and correlated with the rhythm of the expression of Piezo1, TRPV4, VNUT, and Cx26. However, these rhythms were disrupted in Clock Δ19/Δ19 mice. Carbenoxolone eliminated the rhythmicity of ATP release in wild-type mice. However, time-dependent ATP release changes were maintained when a single gene was deficient such as VNUT-KO, Piezo1-, TRPV4-, and Cx26-siRNA., Conclusions: ATP release in the bladder urothelium induces SBF and may have a circadian rhythm regulated by the clock genes. In the bladder urothelium, clock gene abnormalities may disrupt circadian ATP release by inducing Piezo1, TRPV4, VNUT, and Cx26. All these genes can trigger nocturia., (© 2018 Wiley Periodicals, Inc.)

Essentially all organisms exhibit recurring patterns of physiology/behavior that oscillate with a period of ~24-h and are synchronized to the solar day. Crustaceans are no exception, with robust circadian rhythms having been documented in many members of this arthropod subphylum. However, little is known about the molecular underpinnings of their circadian rhythmicity. Moreover, the location of the crustacean central clock has not been firmly established, although both the brain and eyestalk ganglia have been hypothesized as loci. The American lobster, Homarus americanus, is known to exhibit multiple circadian rhythms, and immunodetection data suggest that its central clock is located within the eyestalk ganglia rather than in the brain. Here, brain- and eyestalk ganglia-specific transcriptomes were generated and used to assess the presence/absence of transcripts encoding the commonly recognized protein components of arthropod circadian signaling systems in these two regions of the lobster central nervous system. Transcripts encoding putative homologs of the core clock proteins clock, cryptochrome 2, cycle, period and timeless were found in both the brain and eyestalk ganglia assemblies, as were transcripts encoding similar complements of putative clock-associated, clock input pathway and clock output pathway proteins. The presence and identity of transcripts encoding core clock proteins in both regions were confirmed using PCR. These findings suggest that both the brain and eyestalk ganglia possess all of the molecular components needed for the establishment of a circadian signaling system. Whether the brain and eyestalk clocks are independent of one another or represent a single timekeeping system remains to be determined. Interestingly, while most of the proteins deduced from the identified transcripts are shared by both the brain and eyestalk ganglia, assembly-specific isoforms were also identified, e.g., several period variants, suggesting the possibility of region-specific variation in clock function, especially if the brain and eyestalk clocks represent independent oscillators., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Obesity is a major risk factor for the development of illnesses, such as insulin resistance and hypertension, and has become a serious public health problem. Mammals have developed a circadian clock located in the hypothalamic suprachiasmatic nuclei (SCN) that responds to the environmental light-dark cycle. Clocks similar to the one located in the SCN are found in peripheral tissues, such as the kidney, liver, and adipose tissue. The circadian clock regulates metabolism and energy homeostasis in peripheral tissues by mediating activity and/or expression of key metabolic enzymes and transport systems. Knockouts or mutations in clock genes that lead to disruption of cellular rhythmicity have provided evidence to the tight link between the circadian clock and metabolism. In addition, key proteins play a dual role in regulating the core clock mechanism, as well as adipose tissue metabolism, and link circadian rhythms with lipogenesis and lipolysis. Adipose tissues are distinguished as white, brown, and beige (or brite), each with unique metabolic characteristics. Recently, the role of the circadian clock in regulating the differentiation into the different adipose tissues has been investigated. In this review, the role of clock proteins and the downstream signaling pathways in white, brown, and brite adipose tissue function and differentiation will be reviewed. In addition, chronodisruption and metabolic disorders and clinical aspects of circadian adiposity will be addressed.

The circadian clock system drives daily rhythms in physiology, metabolism, and behavior in mammals. Molecular mechanisms of this system consist of multiple clock genes, with Circadian Locomotor Output Cycles Kaput (CLOCK) as a core member that plays an important role in a wide range of behaviors. Alterations in the CLOCK gene are associated with common psychiatric disorders as well as with circadian disturbances comorbidities. This review addresses animal, molecular, and genetic studies evaluating the role of the CLOCK gene on many psychiatric conditions, namely autism spectrum disorder, schizophrenia, attention-deficit/hyperactivity disorder, major depressive disorder, bipolar disorder, anxiety disorder, and substance use disorder. Many animal experiments focusing on the effects of the Clock gene in behavior related to psychiatric conditions have shown consistent biological plausibility and promising findings. In humans, genetic and gene expression studies regarding disorder susceptibility, sleep disturbances related comorbidities, and response to pharmacological treatment, in general, are in agreement with animal studies. However, the number of controversial results is high. Literature suggests that the CLOCK gene exerts important influence on these conditions, and influences the susceptibility to phenotypes of psychiatric disorders., (© 2017 Wiley Periodicals, Inc.)

The mammalian circadian clock relies on the transcription factor CLOCK:BMAL1 to coordinate the rhythmic expression of 15% of the transcriptome and control the daily regulation of biological functions. The recent characterization of CLOCK:BMAL1 cistrome revealed that although CLOCK:BMAL1 binds synchronously to all of its target genes, its transcriptional output is highly heterogeneous. By performing a meta-analysis of several independent genome-wide datasets, we found that the binding of other transcription factors at CLOCK:BMAL1 enhancers likely contribute to the heterogeneity of CLOCK:BMAL1 transcriptional output. While CLOCK:BMAL1 rhythmic DNA binding promotes rhythmic nucleosome removal, it is not sufficient to generate transcriptionally active enhancers as assessed by H3K27ac signal, RNA Polymerase II recruitment, and eRNA expression. Instead, the transcriptional activity of CLOCK:BMAL1 enhancers appears to rely on the activity of ubiquitously expressed transcription factors, and not tissue-specific transcription factors, recruited at nearby binding sites. The contribution of other transcription factors is exemplified by how fasting, which effects several transcription factors but not CLOCK:BMAL1, either decreases or increases the amplitude of many rhythmically expressed CLOCK:BMAL1 target genes. Together, our analysis suggests that CLOCK:BMAL1 promotes a transcriptionally permissive chromatin landscape that primes its target genes for transcription activation rather than directly activating transcription, and provides a new framework to explain how environmental or pathological conditions can reprogram the rhythmic expression of clock-controlled genes.

The circadian clocks are vital to many organisms for their survival and adaption to the surrounding environment. More and more people are interested in the circadian clock and related researches. One of the key characteristics of this endogenous clock is its periodicity. Mechanisms underlying the mammalian circadian rhythms with ~24 h periodicity involve interlocked transcriptional and translational feedback loops. The circadian clock system in mammals consists of hierarchical structures, with the suprachiasmatic nucleus (SCN) as the central pacemaker and peripheral oscillators in other organs. In spite of the central and peripheral oscillators, the molecular mechanisms are the same within the SCN and peripheral organs. In the past decades, major achievements are accomplished by using forward and reverse genetics, as well as epigenetic approaches. In this review, we recapitulate the history of how clock-related genes were identified, and summarize the main achievements in genetics and epigenetics to understand the molecular underpinnings. We hope it can offer basic knowledge for further researches, a reference for experimental designs aiming to adjust organisms' homeostasis by modulating the clock, and provide a foundation to build interdisciplinary research networks.

In Drosophila, the circadian clock is comprised of transcriptional feedback loops that control rhythmic gene expression responsible for daily rhythms in physiology, metabolism, and behavior. The core feedback loop, which employs CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors to drive rhythmic transcription peaking at dusk, is required for circadian timekeeping and overt behavioral rhythms. CLK-CYC also activates an interlocked feedback loop, which uses the PAR DOMAIN PROTEIN 1ε (PDP1ε) activator and the VRILLE (VRI) repressor to drive rhythmic transcription peaking at dawn. Although Pdp1ε mutants disrupt activity rhythms without eliminating clock function, whether vri is required for clock function and/or output is not known. Using a conditionally inactivatable transgene to rescue vri developmental lethality, we show that clock function persists after vri inactivation but that activity rhythms are abolished. The inactivation of vri disrupts multiple output pathways thought to be important for activity rhythms, including PDF accumulation and arborization rhythms in the small ventrolateral neuron (sLN v ) dorsal projection. These results demonstrate that vri acts as a key regulator of clock output and suggest that the primary function of the interlocked feedback loop in Drosophila is to drive rhythmic transcription required for overt rhythms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Activation of the mineralocorticoid receptor (MR) promotes inflammation, fibrosis, and hypertension. Clinical and experimental studies show that MR antagonists have significant therapeutic benefit for all-cause heart failure; however, blockade of renal MRs limits their widespread use. Identification of key downstream signaling mechanisms for the MR in the cardiovascular system may enable development of targeted MR antagonists with selectivity for pathological MR signaling and lower impact on physiological renal electrolyte handling. One candidate pathway is the circadian clock, the dysregulation of which is associated with cardiovascular diseases. We have previously shown that the circadian gene Per2 is dysregulated in hearts with selective deletion of cardiomyocyte MR. We therefore investigated MR-mediated cardiac inflammation and fibrosis in mice that lack normal regulation and oscillation of the circadian clock in peripheral tissues, that is, CLOCKΔ19 mutant mice. The characteristic cardiac inflammatory/fibrotic response to a deoxycorticosterone (DOC)/salt for 8 weeks was significantly blunted in CLOCKΔ19 mice when compared with wild-type mice, despite a modest increase at "baseline" for fibrosis and macrophage number in CLOCKΔ19 mice. In contrast, cardiac hypertrophy in response to DOC/salt was significantly greater in CLOCKΔ19 vs wild-type mice. Markers for renal inflammation and fibrosis were similarly attenuated in the CLOCKΔ19 mice given DOC/salt. Moreover, increased CLOCK expression in H9c2 cardiac cells enhanced MR-mediated transactivation of Per1, suggesting cooperative signaling between these transcription factors. This study demonstrates that the full development of MR-mediated cardiac inflammation and fibrosis is dependent on intact signaling by the circadian protein CLOCK., (Copyright © 2017 Endocrine Society.)

Circadian clocks are autonomous daily timekeeping mechanisms that allow organisms to adapt to environmental rhythms as well as temporally organize biological functions. Clock-controlled timekeeping involves extensive regulation of rhythmic gene expression. To date, relatively few clock-associated promoter elements have been identified and characterized. In an unbiased search of core clock gene promoters from 12 species of Drosophila, we discovered a 29-bp consensus sequence that we designated as the Clock-Associated Transcriptional Activation Cassette or 'CATAC'. To experimentally address the spatiotemporal expression information associated with this element, we generated constructs with four separate native CATAC elements upstream of a basal promoter driving expression of either the yeast Gal4 or firefly luciferase reporter genes. Reporter assays showed that presence of wild-type, but not mutated CATAC elements, imparted increased expression levels as well as rhythmic regulation. Part of the CATAC consensus sequence resembles the E-box binding site for the core circadian transcription factor CLOCK/CYCLE (CLK/CYC), and CATAC-mediated expression rhythms are lost in the presence of null mutations in either cyc or the gene encoding the CLK/CYC inhibitor, period (per). Nevertheless, our results indicate that CATAC's enhancer function persists in the absence of CLK/CYC. Thus, CATAC represents a novel cis-regulatory element encoding clock-controlled regulation., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Key Message: The early flowering of Lalu was determined to be due to a novel spontaneous eam8 mutation, which resulted in intron retention and the formation of a putative truncated protein. Barley is a staple crop grown over an extensive area in the Qinghai-Tibetan Plateau. Understanding the genetic mechanism for its success in a high altitude is important for crop improvement in marginal environments. Early flowering is a critical adaptive trait that strongly influences reproductive fitness in a short growing season. Loss-of-function mutations at the circadian clock gene EARLY MATURITY 8 (EAM8) promote rapid flowering. In this study, we identified a novel, spontaneous recessive eam8 mutant with an early flowering phenotype in a Tibetan barley landrace Lalu, which is natively grown at a high altitude of approximately 4000 m asl. The co-segregation analysis in a F 2 population derived from the cross Lalu (early flowering) × Diqing 1 (late flowering) confirmed that early flowering of Lalu was determined to be due to an allele at EAM8. The eam8 allele from Lalu carries an A/G alternative splicing mutation at position 3257 in intron 3, designated eam8.l; this alternative splicing event leads to intron retention and a putative truncated protein. Of the 134 sequenced barley accessions, which are primarily native to the Qinghai-Tibet Plateau, three accessions carried this mutation. The eam8.l mutation was likely to have originated in wild barley due to the presence of the Lalu haplotype in H. spontaneum from Tibet. Overall, alternative splicing has contributed to the evolution of the barley circadian clock and in the short-season adaptation of local barley germplasm. The study has also identified a novel donor of early-flowering barley which will be useful for barley improvement.

Reward-related learning, including that associated with drugs of abuse, is largely mediated by the dopaminergic mesolimbic pathway. Mesolimbic neurophysiology and motivated behavior, in turn, are modulated by the circadian timing system which generates ∼24-h rhythms in cellular activity. Both drug taking and seeking and mesolimbic dopaminergic neurotransmission can vary widely over the day. Moreover, circadian clock genes are expressed in ventral tegmental area dopaminergic cells and in mesolimbic target regions where they can directly modulate reward-related neurophysiology and behavior. There also exists a reciprocal influence between drug taking and circadian timing as the administration of drugs of abuse can alter behavioral rhythms and circadian clock gene expression in mesocorticolimbic structures. These interactions suggest that manipulations of the circadian timing system may have some utility in the treatment of substance abuse disorders. Here, the literature on bidirectional interactions between the circadian timing system and drug taking is briefly reviewed, and potential chronotherapeutic considerations for the treatment of addiction are discussed.

The heterodimerized transcription factors CLOCK-BMAL1 regulate the cardiomyocyte circadian rhythms. The L-type calcium currents play important role in the cardiac electrogenesis and arrhythmogenesis. Whether and how the CLOCK-BMAL1 regulate the cardiac L-type calcium channels are yet to be determined. The functions of the L-type calcium channels were evaluated with patch clamping techniques. Recombinant adenoviruses of CLOCK and BMAL1 were used in the expression experiments. We reported that the expressions and functions of CACNA1C (the α-subunit of the L-type calcium channels) showed circadian rhythms, with the peak at zeitgeber time 3 (ZT3). The endocardial action potential durations 90 (APD90) were correspondingly longer at ZT3. The protein levels of the phosphorylated Akt at threonine 308 (pAkt T308) also showed circadian rhythms. Overexpressions of CLOCK-BMAL1 significantly reduced the levels of CACNA1C while increasing the levels of pAkt T308 and pik3r1. Furthermore, the inhibitory effects of CLOCK-BMAL1 on CACNA1C could be abolished by the Akt inhibitor MK2206 or the PDK1 inhibitor GSK2334470. Collectively, our findings suggested that the expressions of the cardiac CACNA1C were under the CLOCK-BMAL1 regulation, probably through the PI3K-Akt signal pathway.

Circadian clocks are composed of transcriptional/translational feedback loops (TTFLs) at the cellular level. In Drosophila TTFLs, the transcription factor dCLOCK (dCLK)/CYCLE (CYC) activates clock target gene expression, which is repressed by the physical interaction with PERIOD (PER). Here, we show that amino acids (AA) 657-707 of dCLK, a region that is homologous to the mouse Clock exon 19-encoded region, is crucial for PER binding and E-box-dependent transactivation in S2 cells. Consistently, in transgenic flies expressing dCLK with an AA657-707 deletion in the Clock (Clk(out)) genetic background (p{dClk-Δ};Clk(out)), oscillation of core clock genes' mRNAs displayed diminished amplitude compared with control flies, and the highly abundant dCLKΔ657-707 showed significantly decreased binding to PER. Behaviorally, the p{dClk-Δ};Clk(out) flies exhibited arrhythmic locomotor behavior in the photic entrainment condition but showed anticipatory activities of temperature transition and improved free-running rhythms in the temperature entrainment condition. Surprisingly, p{dClk-Δ};Clk(out) flies showed pacemaker-neuron-dependent alterations in molecular rhythms; the abundance of dCLK target clock proteins was reduced in ventral lateral neurons (LNvs) but not in dorsal neurons (DNs) in both entrainment conditions. In p{dClk-Δ};Clk(out) flies, however, strong but delayed molecular oscillations in temperature cycle-sensitive pacemaker neurons, such as DN1s and DN2s, were correlated with delayed anticipatory activities of temperature transition. Taken together, our study reveals that the LNv molecular clockwork is more sensitive than the clockwork of DNs to dysregulation of dCLK by AA657-707 deletion. Therefore, we propose that the dCLK/CYC-controlled TTFL operates differently in subsets of pacemaker neurons, which may contribute to their specific functions., Competing Interests: The authors declare no conflict of interest.

The critical regulation of the peripheral circadian gene implicated in osteoarthritis (OA) has been recently recognized; however, the causative role and clinical potential of the peripheral circadian rhythm attributable to such effects remain elusive. The purpose of this study was to elucidate the role of a circadian gene Bmal1 in human cartilage and pathophysiology of osteoarthritis. In our present study, the mRNA and protein levels of circadian rhythm genes, including nicotinamide adenine dinucleotide oxidase (NAD(+)) and sirtuin 1 (Sirt1), in human knee articular cartilage were determined. In OA cartilage, the levels of both Bmal1 and NAD(+) decreased significantly, which resulted in the inhibition of nicotinamide phosphoribosyltransferase activity and Sirt1 expression. Furthermore, the knockdown of Bmal1 was sufficient to decrease the level of NAD(+) and aggravate OA-like gene expression changes under the stimulation of IL-1β. The overexpression of Bmal1 relieved the alteration induced by IL-1β, which was consistent with the effect of the inhibition of Rev-Erbα (known as NR1D1, nuclear receptor subfamily 1, group D). On the other hand, the transfection of Sirt1 small interfering RNA not only resulted in a reduction of the protein expression of Bmal1 and a moderate increase of period 2 (per2) and Rev-Erbα but also further exacerbated the survival of cells and the expression of cartilage matrix-degrading enzymes induced by IL-1β. Overexpression of Sirt1 restored the metabolic imbalance of chondrocytes caused by IL-1β. These observations suggest that Bmal1 is a key clock gene to involve in cartilage homeostasis mediated through sirt1 and that manipulating circadian rhythm gene expression implicates an innovative strategy to develop novel therapeutic agents against cartilage diseases.

The regulation of metabolism by circadian systems is believed to be a key reason for the extensive representation of circadian rhythms within the tree of life. Despite this, surprisingly little work has focused on the link between metabolism and the clock in Neurospora, a key model system in circadian research. The analysis that has been performed has focused on the unidirectional control from the clock to metabolism and largely ignored the feedback from metabolism on the clock. Recent efforts to understand these links have broken new ground, revealing bidirectional control from the clock to metabolism and vise-versa, showing just how strongly interconnected these two cellular systems can be in fungi. This review describes both well understood and emerging links between the clock and metabolic output of fungi as well as the role that metabolism plays in influencing the rhythm set by the clock., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Recent studies have led to an expansion of potential factors capable of stimulating obesity. Increasing evidence indicates that environmental factors, including disturbance of circadian rhythms, also contribute to its etiology., Objectives: To determine the effects of altered circadian rhythms on adipogenesis and to better understand how circadian and adipogenic regulatory pathways are linked, zebrafish larvae were exposed to various light/dark cycles or hypercaloric feeding (HCF)., Methods: Clock and adipogenic gene expression was quantitative real time PCR. Adipogenesis was characterized using coherent anti-Stokes Raman scattering microscopy (CARS) and whole-mount lipid composition was analyzed by gas chromatography. The clock protein Rev-erbα and the adipogenesis-regulating protein Pparγ were localized by immunohistochemistry., Results: Zebrafish larvae exposed to continuous light (LL) had a sevenfold higher prevalence of adipocytes compared with control fish under a 14 h light and 10 h dark cycle. It was also significantly higher compared with that in HCF larvae with control light/dark cycle, which showed a 5.5-fold increase compared with control animals. Although total fatty acid content was unaffected, adipocyte lipid composition was altered in LL zebrafish. In contrast, shifting the onset and duration of the light periods did not affect adipogenesis or total fatty acid content. Gene expression analysis revealed effects of LL and HCF on circadian cyclicity, with increased expression of the clock gene period2 and altered circadian rev-erbα expression in LL larvae. Immunostaining revealed for the first time that Rev-erbα and Pparγ colocalize in adipocytes, which together with the gene expression analysis suggests interplay between Rev-erbα and Ppar isoforms., Conclusions: The amount of light, but not shifted light/dark cycles, affected adipogenesis and lipid composition, possibly due to increased period2 expression, which, in turn, enhances Rev-erbα-regulated gene expression. As the pparβδ promoter includes three Rev-erbα binding sites, we hypothesize that pparβδ may be a direct target that ultimately activates Pparγ.

Background: Circadian rhythm disruption is a prevalent feature of modern day society that is associated with an increase in pro-inflammatory diseases, and there is a clear need for a better understanding of the mechanism(s) underlying this phenomenon. We have previously demonstrated that both environmental and genetic circadian rhythm disruption causes intestinal hyperpermeability and exacerbates alcohol-induced intestinal hyperpermeability and liver pathology. The intestinal microbiota can influence intestinal barrier integrity and impact immune system function; thus, in this study, we sought to determine whether genetic alteration of the core circadian clock gene, Clock, altered the intestinal microbiota community., Methods: Male Clock(Δ19) -mutant mice (mice homozygous for a dominant-negative-mutant allele) or littermate wild-type mice were fed 1 of 3 experimental diets: (i) a standard chow diet, (ii) an alcohol-containing diet, or (iii) an alcohol-control diet in which the alcohol calories were replaced with dextrose. Stool microbiota was assessed with 16S ribosomal RNA gene amplicon sequencing., Results: The fecal microbial community of Clock-mutant mice had lower taxonomic diversity, relative to wild-type mice, and the Clock(Δ19) mutation was associated with intestinal dysbiosis when mice were fed either the alcohol-containing or the control diet. We found that alcohol consumption significantly altered the intestinal microbiota in both wild-type and Clock-mutant mice., Conclusions: Our data support a model by which circadian rhythm disruption by the Clock(Δ19) mutation perturbs normal intestinal microbial communities, and this trend was exacerbated in the context of a secondary dietary intestinal stressor., (Copyright © 2016 by the Research Society on Alcoholism.)

There are several reports suggesting that the pathophysiology of psoriasis may be associated with aberrant circadian rhythms. However, the mechanistic link between psoriasis and the circadian time-keeping system, "the circadian clock," remains unclear. This study determined whether the core circadian gene, Clock, had a regulatory role in the development of psoriasis. For this purpose, we compared the development of psoriasis-like skin inflammation induced by the Toll-like receptor 7 ligand imiquimod (IMQ) between wild-type mice and mice with a loss-of-function mutation of Clock. We also compared the development of IMQ-induced dermatitis between wild-type mice and mice with a loss-of-function mutation of Period2 (Per2), another key circadian gene that inhibits CLOCK activity. We found that Clock mutation ameliorated IMQ-induced dermatitis, whereas the Per2 mutation exaggerated IMQ-induced dermatitis, when compared with wild-type mice associated with decreased or increased IL-23 receptor (IL-23R) expression in γ/δ+ T cells, respectively. In addition, CLOCK directly bound to the promoter of IL-23R in γ/δ+ T cells, and IL-23R expression in the mouse skin was under circadian control. These findings suggest that Clock is a novel regulator of psoriasis-like skin inflammation in mice via direct modulation of IL-23R expression in γ/δ+ T cells, establishing a mechanistic link between psoriasis and the circadian clock.

The circadian rhythm regulates blood pressure and maintains fluid and electrolyte homeostasis with central and peripheral clock. However, the role of circadian rhythm in the pathogenesis of tubulointerstitial fibrosis remains unclear. Here, we found that the amplitudes of circadian rhythm oscillation in kidneys significantly increased after unilateral ureteral obstruction. In mice that are deficient in the circadian gene Clock, renal fibrosis and renal parenchymal damage were significantly worse after ureteral obstruction. CLOCK-deficient mice showed increased synthesis of collagen, increased oxidative stress, and greater transforming growth factor-β (TGF-β) expression. TGF-β mRNA expression oscillated with the circadian rhythms under the control of CLOCK-BMAL1 heterodimers. The expression of cyclooxygenase 2 was significantly higher in kidneys from CLOCK-deficient mice with ureteral obstruction. Treatment with a cyclooxygenase 2 inhibitor celecoxib significantly improved renal fibrosis in CLOCK-deficient mice. Taken together, these data establish the importance of the circadian rhythm in tubulointerstitial fibrosis and suggest CLOCK/TGF-β signaling as a novel therapeutic target of cyclooxygenase inhibition., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Disrupted daily or circadian rhythms of lung function and inflammatory responses are common features of chronic airway diseases. At the molecular level these circadian rhythms depend on the activity of an autoregulatory feedback loop oscillator of clock gene transcription factors, including the BMAL1:CLOCK activator complex and the repressors PERIOD and CRYPTOCHROME. The key nuclear receptors and transcription factors REV-ERBα and RORα regulate Bmal1 expression and provide stability to the oscillator. Circadian clock dysfunction is implicated in both immune and inflammatory responses to environmental, inflammatory, and infectious agents. Molecular clock function is altered by exposomes, tobacco smoke, lipopolysaccharide, hyperoxia, allergens, bleomycin, as well as bacterial and viral infections. The deacetylase Sirtuin 1 (SIRT1) regulates the timing of the clock through acetylation of BMAL1 and PER2 and controls the clock-dependent functions, which can also be affected by environmental stressors. Environmental agents and redox modulation may alter the levels of REV-ERBα and RORα in lung tissue in association with a heightened DNA damage response, cellular senescence, and inflammation. A reciprocal relationship exists between the molecular clock and immune/inflammatory responses in the lungs. Molecular clock function in lung cells may be used as a biomarker of disease severity and exacerbations or for assessing the efficacy of chronotherapy for disease management. Here, we provide a comprehensive overview of clock-controlled cellular and molecular functions in the lungs and highlight the repercussions of clock disruption on the pathophysiology of chronic airway diseases and their exacerbations. Furthermore, we highlight the potential for the molecular clock as a novel chronopharmacological target for the management of lung pathophysiology., (Copyright © 2015 the American Physiological Society.)

The circadian clock mechanism in animals involves a transcriptional feedback loop in which the bHLH-PAS proteins CLOCK and BMAL1 form a transcriptional activator complex to activate the transcription of the Period and Cryptochrome genes, which in turn feed back to repress their own transcription. In the mouse liver, CLOCK and BMAL1 interact with the regulatory regions of thousands of genes, which are both cyclically and constitutively expressed. The circadian transcription in the liver is clustered in phase and this is accompanied by circadian occupancy of RNA polymerase II recruitment and initiation. These changes also lead to circadian fluctuations in histone H3 lysine4 trimethylation (H3K4me3) as well as H3 lysine9 acetylation (H3K9ac) and H3 lysine27 acetylation (H3K27ac). Thus, the circadian clock regulates global transcriptional poise and chromatin state by regulation of RNA polymerase II., (© 2015 John Wiley & Sons Ltd.)

Most internationally travelled researchers will have encountered jetlag. If not, working odd hours makes most of us feel somehow dysfunctional. How can all this be linked to circadian rhythms and circadian clocks? In this review, we define circadian clocks, their composition and underlying molecular mechanisms. We describe and discuss recent crystal structures of Drosophila and mammalian core clock components and the enormous impact they had on the understanding of circadian clock mechanisms. Finally, we highlight the importance of circadian clocks for the daily regulation of human/mammalian physiology and show connections to overall fitness, health and disease., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Circadian rhythms are daily endogenous oscillations of behavior, metabolism, and physiology. At a molecular level, these oscillations are generated by transcriptional-translational feedback loops composed of core clock genes. In turn, core clock genes drive the rhythmic accumulation of downstream outputs-termed clock-controlled genes (CCGs)-whose rhythmic translation and function ultimately underlie daily oscillations at a cellular and organismal level. Given the circadian clock's profound influence on human health and behavior, considerable efforts have been made to systematically identify CCGs. The recent development of next-generation sequencing has dramatically expanded our ability to study the expression, processing, and stability of rhythmically expressed mRNAs. Nevertheless, like any new technology, there are many technical issues to be addressed. Here, we discuss considerations for studying circadian rhythms using genome scale transcriptional profiling, with a particular emphasis on RNA sequencing. We make a number of practical recommendations-including the choice of sampling density, read depth, alignment algorithms, read-depth normalization, and cycling detection algorithms-based on computational simulations and our experience from previous studies. We believe that these results will be of interest to the circadian field and help investigators design experiments to derive most values from these large and complex data sets., (© 2015 Elsevier Inc. All rights reserved.)

Night follows day and as a consequence, organisms have evolved molecular machineries that allow them to anticipate and respond to the many changes that accompany these transitions. Circadian clocks are precise yet plastic pacemakers that allow the temporal organization of a plethora of biological process. Circadian clocks are widespread across the tree of life and while their exact molecular components differ among phyla, they tend to share common design principles. In this review, we discuss the circadian system of the filamentous fungus Neurospora crassa. Historically, this fungus has served a key role in the genetic and molecular dissection of circadian clocks, aiding in their detailed mechanistic understanding. Recent studies have provided new insights into the daily molecular dynamics that constitute the Neurospora circadian oscillator, some of which have questioned traditional paradigms describing timekeeping mechanisms in eukaryotes. In addition, recent reports support the idea of a dynamic network of transcription factors underlying the rhythmicity of thousands of genes in Neurospora, many of which oscillate only under specific conditions. Besides Neurospora, which harbors the best characterized circadian system among filamentous fungi, the recent characterization of the circadian system of the plant-pathogenic fungus Botrytis cinerea has provided additional insights into the physiological impact of the clock and potential additional functions of clock proteins in fungi. Finally, we speculate on the presence of FRQ or FRQ-like proteins in diverse fungal lineages., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The circadian clock plays an integral role in the regulation of physiological processes, including the regulation of blood pressure. However, deregulation of the clock can lead to pathophysiological states including hypertension. Recent work has implicated the circadian clock genes in the regulation of processes in the heart, kidney, vasculature, and the metabolic organs, which are all critical in the regulation of the blood pressure. The goal of this review is to provide an introduction and general overview into the role of circadian clock genes in the regulation of blood pressure with a focus on their deregulation in the etiology of hypertension. This review will focus on the core circadian clock genes CLOCK, BMAL1, Per, and Cry.

Circadian clocks are fundamental machinery in organisms ranging from archaea to humans. Disruption of the circadian system is associated with premature aging in mice, but the molecular basis underlying this phenomenon is still unclear. In this study, we found that telomerase activity exhibits endogenous circadian rhythmicity in humans and mice. Human and mouse TERT mRNA expression oscillates with circadian rhythms and are under the control of CLOCK-BMAL1 heterodimers. CLOCK deficiency in mice causes loss of rhythmic telomerase activities, TERT mRNA oscillation, and shortened telomere length. Physicians with regular work schedules have circadian oscillation of telomerase activity while emergency physicians working in shifts lose the circadian rhythms of telomerase activity. These findings identify the circadian rhythm as a mechanism underlying telomere and telomerase activity control that serve as interconnections between circadian systems and aging., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Clock genes have been implicated in several disorders, such as schizophrenia, bipolar disorder, autism spectrum disorders, and drug dependence. However, few studies to date have examined the role of clock genes in fear-related behaviors. The authors used mice with the ClockΔ19 mutation to assess the involvement of this gene in contextual fear conditioning. Male wild-type (WT) and ClockΔ19 mutant mice underwent a single session of contextual fear conditioning (12 min, 4 unsignaled shocks), followed by daily 12-min retention trials. There were no differences between mutant and WT mice in the acquisition of contextual fear, and WT and mutant mice demonstrated similar freezing during the first retention session. However, extinction of contextual fear was accelerated in mutant mice across the remaining retention sessions, as compared to WT mice, suggesting a role for Clock in extinction following aversive learning. Because the ClockΔ19 mutation has previously been demonstrated to result in an increase in dopamine signaling, the authors confirmed the role of dopamine in extinction learning using preretention session administration of a low dose of the dopamine transport reuptake inhibitor modafinil (0.75 mg/kg), which resulted in decreased freezing across retention sessions. These findings are consistent with an emerging portrayal of the importance of Clock genes in noncircadian functions, as well as the important role of dopamine in extinction learning.

Circadian clocks are endogenous oscillators driving daily rhythms in physiology. The cell-autonomous clock is governed by an interlocked network of transcriptional feedback loops. Hundreds of clock-controlled genes (CCGs) regulate tissue specific functions. Transcriptome studies reveal that different organs (e.g. liver, heart, adrenal gland) feature substantially varying sets of CCGs with different peak phase distributions. To study the phase variability of CCGs in mammalian peripheral tissues, we develop a core clock model for mouse liver and adrenal gland based on expression profiles and known cis-regulatory sites. 'Modulation factors' associated with E-boxes, ROR-elements, and D-boxes can explain variable rhythms of CCGs, which is demonstrated for differential regulation of cytochromes P450 and 12 h harmonics. By varying model parameters we explore how tissue-specific peak phase distributions can be generated. The central role of E-boxes and ROR-elements is confirmed by analysing ChIP-seq data of BMAL1 and REV-ERB transcription factors.

Unlabelled: The protein deacetylase, sirtuin 1 (SIRT1), involved in regulating hepatic insulin sensitivity, shows circadian oscillation and regulates the circadian clock. Recent studies show that circadian misalignment leads to insulin resistance (IR); however, the underlying mechanisms are largely unknown. Here, we show that CLOCK and brain and muscle ARNT-like protein 1 (BMAL1), two core circadian transcription factors, are correlated with hepatic insulin sensitivity. Knockdown of CLOCK or BMAL1 induces hepatic IR, whereas their ectopic expression attenuates hepatic IR. Moreover, circadian change of insulin sensitivity is impaired in Clock mutant, liver-specific Bmal1 knockout (KO) or Sirt1 KO mice, and CLOCK and BMAL1 are required for hepatic circadian expression of SIRT1. Further studies show that CLOCK/BMAL1 binds to the SIRT1 promoter to enhance its expression and regulates hepatic insulin sensitivity by SIRT1. In addition, constant darkness-induced circadian misalignment in mice decreases hepatic BMAL1 and SIRT1 levels and induces IR, which can be dramatically reversed by resveratrol., Conclusion: These findings offer new insights for coordination of the circadian clock and metabolism in hepatocytes by circadian regulation of hepatic insulin sensitivity via CLOCK/BMAL1-dependent SIRT1 expression and provide a potential application of resveratrol for combating circadian misalignment-induced metabolic disorders., (© 2014 by the American Association for the Study of Liver Diseases.)

In mammalian circadian clockwork, the CLOCK-BMAL1 complex binds to DNA enhancers of target genes and drives circadian oscillation of transcription. Here we identified 7,978 CLOCK-binding sites in mouse liver by chromatin immunoprecipitation-sequencing (ChIP-Seq), and a newly developed bioinformatics method, motif centrality analysis of ChIP-Seq (MOCCS), revealed a genome-wide distribution of previously unappreciated noncanonical E-boxes targeted by CLOCK. In vitro promoter assays showed that CACGNG, CACGTT, and CATG(T/C)G are functional CLOCK-binding motifs. Furthermore, we extensively revealed rhythmically expressed genes by poly(A)-tailed RNA-Seq and identified 1,629 CLOCK target genes within 11,926 genes expressed in the liver. Our analysis also revealed rhythmically expressed genes that have no apparent CLOCK-binding site, indicating the importance of indirect transcriptional and posttranscriptional regulations. Indirect transcriptional regulation is represented by rhythmic expression of CLOCK-regulated transcription factors, such as Krüppel-like factors (KLFs). Indirect posttranscriptional regulation involves rhythmic microRNAs that were identified by small-RNA-Seq. Collectively, CLOCK-dependent direct transactivation through multiple E-boxes and indirect regulations polyphonically orchestrate dynamic circadian outputs.

Mice with a mutation in the Clock gene (ClockΔ19) have been identified as a model of mania; however, the mechanisms that underlie this phenotype, and the changes in the brain that are necessary for lithium's effectiveness on these mice remain unclear. Here, we find that cholecystokinin (Cck) is a direct transcriptional target of CLOCK and levels of Cck are reduced in the ventral tegmental area (VTA) of ClockΔ19 mice. Selective knockdown of Cck expression via RNA interference in the VTA of wild-type mice produces a manic-like phenotype. Moreover, chronic treatment with lithium restores Cck expression to near wild-type and this increase is necessary for the therapeutic actions of lithium. The decrease in Cck expression in the ClockΔ19 mice appears to be due to a lack of interaction with the histone methyltransferase, MLL1, resulting in decreased histone H3K4me3 and gene transcription, an effect reversed by lithium. Human postmortem tissue from bipolar subjects reveals a similar increase in Cck expression in the VTA with mood stabilizer treatment. These studies identify a key role for Cck in the development and treatment of mania, and describe some of the molecular mechanisms by which lithium may act as an effective antimanic agent.

Research into the molecular mechanisms of eukaryotic circadian clocks has proceeded at an electrifying pace. In this review, we discuss advances in our understanding of the structures of central molecular players in the timing oscillators of fungi, insects, and mammals. A series of clock protein structures demonstrate that the PAS (Per/Arnt/Sim) domain has been used with great variation to formulate the transcriptional activators and repressors of the clock. We discuss how posttranslational modifications and external cues, such as light, affect the conformation and function of core clock components. Recent breakthroughs have also revealed novel interactions among clock proteins and new partners that couple the clock to metabolic and developmental pathways. Overall, a picture of clock function has emerged wherein conserved motifs and structural platforms have been elaborated into a highly dynamic collection of interacting molecules that undergo orchestrated changes in chemical structure, conformational state, and partners.

Circadian rhythmicity is an important component of physiological processes which provides them with a 24-hour temporal organization and adjustment to cyclical changes in the environment. Circadian rhythms are controlled by a network of endogenous clocks, comprising the main clock in the suprachiasmatic nuclei of the hypothalamus and many secondary clocks in the brain and peripheral tissues. All aspects of energy metabolism, from food intake to intracellular signaling pathways, are strongly influenced by circadian rhythmicity. In turn, meal timing is an efficient synchronizer (time-giver) to set the phase of the peripheral clocks, while the suprachiasmatic clock is synchronized by ambient light. In certain nutritional conditions (i.e., low- or high-calory diets), metabolic factors remaining to be identified modulate the functioning of the suprachiasmatic clock. Animal models of obesity and diabetes show circadian alterations. Conversely, when circadian rhythmicity is disturbed, either due to genetically defective circadian clocks, or to circadian desynchronization (chronic light exposure or repeated meals at odd times of the cycle), lipid and glucose metabolism is deregulated. The metabolic impact of circadian desynchronization justifies the development of preventive or therapeutic strategies that could rely, among others, on dietary interventions combining timed meals and specific composition., (© Société de Biologie, 2015.)

Living organisms use biological clocks to maintain their internal temporal order and anticipate daily environmental changes. In Drosophila, circadian regulation of locomotor behavior is controlled by ∼150 neurons; among them, neurons expressing the PIGMENT DISPERSING FACTOR (PDF) set the period of locomotor behavior under free-running conditions. To date, it remains unclear how individual circadian clusters integrate their activity to assemble a distinctive behavioral output. Here we show that the BONE MORPHOGENETIC PROTEIN (BMP) signaling pathway plays a crucial role in setting the circadian period in PDF neurons in the adult brain. Acute deregulation of BMP signaling causes period lengthening through regulation of dClock transcription, providing evidence for a novel function of this pathway in the adult brain. We propose that coherence in the circadian network arises from integration in PDF neurons of both the pace of the cell-autonomous molecular clock and information derived from circadian-relevant neurons through release of BMP ligands., Competing Interests: The authors have declared that no competing interests exist.

Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator-like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus. Mice lacking the clock gene repressors period circadian clock 1 (Per1) and period circadian clock 2 (Per2) had no observed astrogliosis. Bmal1 deletion caused the degeneration of synaptic terminals and impaired cortical functional connectivity, as well as neuronal oxidative damage and impaired expression of several redox defense genes. Targeted deletion of Bmal1 in neurons and glia caused similar neuropathology, despite the retention of intact circadian behavioral and sleep-wake rhythms. Reduction of Bmal1 expression promoted neuronal death in primary cultures and in mice treated with a chemical inducer of oxidative injury and striatal neurodegeneration. Our findings indicate that BMAL1 in a complex with CLOCK or NPAS2 regulates cerebral redox homeostasis and connects impaired clock gene function to neurodegeneration.

Circadian rhythms govern nearly every physiological process in our brains and bodies. At the most basic level, the molecular clockwork in each cell interacts with metabolic cycles to influence the redox state, allowing for increased cellular activity at specific times of day. In this issue of the JCI, Musiek et al. show that genetic disruptions in the positive arm of the molecular clock can lead to severe astrogliosis, which likely occurs through disruptions in output genes that keep oxidative stress in check. This study demonstrates the importance of proper circadian protein function in the maintenance of neuronal integrity.