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Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1 and 2 (Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietin mRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5' erythropoietin promoter. RNAscope® in situ hybridization confirmed the presence of Bmal1 in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy.

The circadian clock modulates key physiological processes in many organisms. This widespread role of circadian rhythms is typically characterized at the molecular level by profiling the transcriptome at multiple time points. Subsequent analysis identifies transcripts with altered rhythms between control and perturbed conditions, that is, are differentially rhythmic (DiffR). Commonly, Venn diagram analysis (VDA) compares lists of rhythmic transcripts to catalog transcripts with rhythms in both conditions, or that have gained or lost rhythms. However, unavoidable errors in rhythmicity detection propagate to the final DiffR classification resulting in overestimated DiffR. We show using artificial experiments on biological data that VDA indeed produces excessive false DiffR hits both in the presence and absence of true DiffR transcripts. We review and benchmark hypothesis testing and model selection approaches that instead compare circadian amplitude and phase of transcripts between the two conditions. These methods identify transcripts that ‘gain’, ‘lose’, ‘change’, or have the ‘same’ rhythms; the third category is missed by VDA. We reanalyzed three studies on the interplay between metabolism and the clock in the mouse liver that used VDA. We found not only fewer DiffR transcripts than originally reported, but VDA overlooked many relevant DiffR transcripts. Our analyses confirmed some and contradicted other conclusions in the original studies and also generated novel insights. Our conclusions equally apply to circadian studies using other omics technologies. We believe that avoiding Venn diagrams and using our convenient r‐package comparerhythms will improve the reliability of analyses in chronobiology., Comparing rhythms with and without an intervention reveals the functioning of the circadian system. High‐throughput studies of clock outputs (transcripts, proteins, etc.) typically compare lists of rhythmic outputs in each condition using Venn diagrams. This approach incorrectly predicts too many altered rhythms, while also overlooking some rhythm changes. Direct comparison of amplitudes and phases using R‐package comparerhythms fixes this problem and reveals limited circadian remodeling due to metabolic changes. image

Local circadian clocks are active in most cells of our body. However, their impact on circadian physiology is still under debate. Mortality by endotoxic (LPS) shock is highly time-of-day dependent and local circadian immune function such as the cytokine burst after LPS challenge has been assumed to be causal for the large differences in survival. Here, we investigate the roles of light and myeloid clocks on mortality by endotoxic shock. Strikingly, mice in constant darkness (DD) show a three-fold increased susceptibility to LPS as compared to mice in light-dark conditions. Mortality by endotoxic shock as a function of circadian time is independent of light-dark cycles as well as myeloid CLOCK or BMAL1 as demonstrated in conditional knockout mice. Unexpectedly, despite the lack of a myeloid clock these mice still show rhythmic patterns of pro- and anti-inflammatory cytokines such as TNFα, MCP-1, IL-18 and IL-10 in peripheral blood as well as time-of-day and site dependent traffic of myeloid cells. We speculate that systemic time-cues are sufficient to orchestrate innate immune response to LPS by driving immune functions such as cell trafficking and cytokine expression.

A defining property of circadian clocks is temperature compensation, characterized by the resilience of circadian free-running periods against changes in environmental temperature. As an underlying mechanism, the balance or critical reaction hypothesis have been proposed. While the former supposes a temperature-dependent balancing of reactions with opposite effects on circadian period, the latter assumes an insensitivity of certain critical period determining regulations upon temperature changes. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions.Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3’-end cleavage and polyadenylation, abolishes circadian temperature compensation in U-2 OS cells. We apply a combination of 3’-End-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3’ UTR length as well as gene and protein expression between wild type and CPSF6 knock-down cells and their dependency on temperature. Analyzing differential responses upon temperature changes in wild type and CPSF6 knockdown cells reveals candidate genes underlying circadian temperature compensation. We identify that eukaryotic translation initiation factor 2 subunit 1 (EIF2S1) is among these candidates. EIF2S1 is known as a master regulator of cellular stress responses that additionally regulates circadian rhythms. We show that knockdown of EIF2S1 furthermore impairs temperature compensation, suggesting that the role of CPSF6 in temperature compensation may be mediated by its regulation of EIF2S1.

In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, we performed a systematic RNAi screen in human cells and identified protein phosphatase 4 (PPP4) with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila. Genetic depletion of PPP4 shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the “kamikaze” properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1δ-mediated inhibition of CLOCK/BMAL1.

Antarctic krill (Euphausia superba) is a high latitude pelagic organism which plays a central role in the Southern Ocean ecosystem. E. superba shows daily and seasonal rhythms in physiology and behaviour, which are synchronized with the environmental cycles of its habitat. Recently, the main components of the krill circadian machinery have been identified and characterized. However, the exact mechanisms through which the endogenous timing system operates the control and regulation of the overt rhythms remains only partially understood. Here we investigate the involvement of the circadian clock in the temporal orchestration of gene expression by using a newly developed version of a krill microarray platform. The analysis of transcriptome data from krill exposed to both light-dark cycles (LD 18:6) and constant darkness (DD), has led to the identification of 1,564 putative clock-controlled genes. A remarkably large proportion of such genes, including several clock components (clock, period, cry2, vrille, and slimb), show oscillatory expression patterns in DD, with a periodicity shorter than 24 hours. Energy-storage pathways appear to be regulated by the endogenous clock in accordance with their ecological relevance in daily energy managing and overwintering. Our results provide the first representation of the krill circadian transcriptome under laboratory, free-running conditions.

Adipose tissue (AT) is a key metabolic organ which functions are rhythmically regulated by an endogenous circadian clock. Feeding is a zeitgeber aligning the clock in AT with the external time but mechanisms of this regulation remain largely unclear. We tested the hypothesis that postprandial changes of the hormone insulin directly entrain circadian clocks in AT and investigated transcriptional-dependent mechanism of this regulation. We analysed gene expression in subcutaneous AT (SAT) of obese subjects collected before and after the hyperinsulinemic-euglycemic clamp (EC) or control saline infusion (SC). The expression of core clock gene PER2, PER3 and NR1D1 in SAT were differentially changed upon insulin and saline infusion suggesting insulin-dependent clock regulation. In human stem cell-derived adipocytes, mouse 3T3-L1 cells and AT explants from mPer2Luc knockin mice, insulin induced a transient increase of the Per2 mRNA and protein expression leading to the phase shift of circadian oscillations and showing similar effects for Per1. Insulin effects were dependent on the region between the -64 and -43 in the Per2 promoter, but not on CRE and E-box elements. Our results demonstrate that insulin directly regulates circadian clocks in AT and isolated adipocytes and thus represent a primary mechanism of feeding-induced AT clock entrainment.

The circadian system of mammals is hierarchically organized. The suprachiasmatic nucleus (SCN) in the hypothalamus is considered the master circadian clock adapting to environmental light–dark cycles and synchronizing subsidiary oscillators in peripheral organs. While being an attractive concept, this has never been convincingly shown in vivo. New findings by Sinturel and colleagues (pp. 329–334) in this issue of Genes & Development now show the requirement of the SCN for temporal orchestration of the periphery in living animals. Surprisingly, this study also found that even in the absence of SCN or extra-SCN clocks, peripheral clocks remain rhythmic, indicating previously controversial circadian oscillator coupling within peripheral tissues.

The circadian clock modulates key physiological processes in many organisms. This widespread role of circadian rhythms is typically characterized at the molecular level by profiling the transcriptome at multiple time points. Subsequent analysis identifies transcripts with altered rhythms between control and perturbed conditions, i.e., are differentially rhythmic (DiffR). Commonly, Venn Diagram analysis (VDA) compares lists of rhythmic transcripts to catalog transcripts with rhythms in both conditions or have gained or lost rhythms. However, unavoidable errors in the rhythmicity detection propagate to the final DiffR classification resulting in overestimated DiffR. We show using artificial experiments constructed from biological data that VDA indeed produces excessive false DiffR hits both in the presence and absence of true DiffR transcripts. We present a hypothesis testing and a model selection approaches in an R-package compareRhythms that instead compare circadian amplitude and phase of transcripts between the two conditions. These methods identify transcripts with ‘gain’, ‘loss’, ‘change’ or have the ‘same’ rhythms; the third category is missed by VDA. We reanalyzed three studies on the interplay between metabolism and the clock in the mouse liver that used VDA. We found not only fewer DiffR transcripts than originally reported, but VDA overlooked many relevant DiffR transcripts. Our analyses confirmed some and contradicted other conclusions in the original studies and also generated novel hypotheses. Our insights also generalize easily to studies using other -omics technologies. We trust that avoiding Venn Diagrams and using our R-package will contribute to improved reproducibility in chronobiology.

Humans and other mammalian species possess an endogenous circadian clock system that has evolved in adaptation to periodically reoccurring environmental changes and drives rhythmic biological functions, as well as behavioural outputs with an approximately 24-hour period. In mammals, body clocks are hierarchically organized, encompassing a so-called pacemaker clock in the hypothalamic suprachiasmatic nucleus (SCN), non-SCN brain and peripheral clocks, as well as cell-autonomous oscillators within virtually every cell type. A functional clock machinery on the molecular level, alignment among body clocks, as well as synchronization between endogenous circadian and exogenous environmental cycles has been shown to be crucial for our health and well-being. Yet, modern life constantly poses widespread challenges to our internal clocks, for example artificial lighting, shift work and trans-meridian travel, potentially leading to circadian disruption or misalignment and the emergence of associated diseases. For instance many of us experience a mismatch between sleep timing on work and free days (social jetlag) in our everyday lives without being aware of health consequences that may arise from such chronic circadian misalignment, Hence, this review provides an overview of the organization and molecular built-up of the mammalian circadian system, its interactions with the outside world, as well as pathologies arising from circadian disruption and misalignment.

The current model of the mammalian circadian oscillator is predominantly based on data from genetics and biochemistry experiments, while the cell biology of circadian clocks is still in its infancy. Here, we describe a new strategy for the efficient generation of knock-in reporter cell lines using CRISPR technology that is particularly useful for lowly or transiently expressed genes, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins, which allowed to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (∼5 hours), and CRY1 levels are much higher (>6 times) compared to PER2 questioning the current model of the circadian oscillator. Our knock-in strategy will allow the generation of additional single, double or triple knock-in cells for circadian clock proteins, which should greatly advance our understanding about the cell biology of circadian clocks.

Background The circadian clock is a fundamental and pervasive biological program that coordinates 24-hour rhythms in physiology, metabolism, and behavior, and it is essential to health. Whereas therapy adapted to time of day is increasingly reported to be highly successful, it needs to be personalized, since internal circadian time is different for each individual. In addition, internal time is not a stable trait, but is influenced by many factors, including genetic predisposition, age, sex, environmental light levels, and season. An easy and convenient diagnostic tool is currently missing. Methods To establish a validated test, we followed a 3-stage biomarker development strategy: (a) using circadian transcriptomics of blood monocytes from 12 individuals in a constant routine protocol combined with machine learning approaches, we identified biomarkers for internal time; and these biomarkers (b) were migrated to a clinically relevant gene expression profiling platform (NanoString) and (c) were externally validated using an independent study with 28 early or late chronotypes. Results We developed a highly accurate and simple assay (BodyTime) to estimate the internal circadian time in humans from a single blood sample. Our assay needs only a small set of blood-based transcript biomarkers and is as accurate as the current gold standard method, dim-light melatonin onset, at smaller monetary, time, and sample-number cost. Conclusion The BodyTime assay provides a new diagnostic tool for personalization of health care according to the patient's circadian clock. Funding This study was supported by the Bundesministerium fur Bildung und Forschung, Germany (FKZ: 13N13160 and 13N13162) and Intellux GmbH, Germany.

The circadian system is composed of coupled endogenous oscillators that allow living beings, including humans, to anticipate and adapt to daily changes in their environment. In mammals, circadian clocks form a hierarchically organized network with a 'master clock' located in the suprachiasmatic nucleus of the hypothalamus, which ensures entrainment of subsidiary oscillators to environmental cycles. Robust rhythmicity of body clocks is indispensable for temporally coordinating organ functions, and the disruption or misalignment of circadian rhythms caused for instance by modern lifestyle is strongly associated with various widespread diseases. This review aims to provide a comprehensive overview of our current knowledge about the molecular architecture and system-level organization of mammalian circadian oscillators. Furthermore, we discuss the regulatory roles of peripheral clocks for cell and organ physiology and their implication in the temporal coordination of metabolism in human health and disease. Finally, we summarize methods for assessing circadian rhythmicity in humans.

A ubiquitous feature of the circadian clock across life forms is its organization as a network of cellular oscillators, with individual cellular oscillators within the network often exhibiting considerable heterogeneity in their intrinsic periods. The interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clock’s entrainability, but our knowledge of mechanisms governing period heterogeneity within circadian clock networks remains largely elusive. In this study, we aimed to explore the principles that underlie intercellular period variation in circadian clock networks (clonal period heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 to 28 h. We report that a single parent clone can produce progeny clones with a wide distribution of circadian periods, and this heterogeneity, in addition to being stochastically driven, has a heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our clone panel, we found that inheritance of expression patterns in at least three clock genes might govern clonal period heterogeneity in circadian clock networks. Furthermore, we provide evidence suggesting that heritable epigenetic variation in gene expression regulation might underlie period heterogeneity., How do genetically identical cells exhibit a different circadian phenotype? This study reveals that a single parent clone can produce progeny with a wide distribution of circadian periods and that this heterogeneity, in addition to being stochastically driven, has a heritable component, likely via heritable epigenetic variation in gene expression regulation.

Context Meal timing affects metabolic homeostasis and body weight, but how composition and timing of meals affect plasma lipidomics in humans is not well studied. Objective We used high throughput shotgun plasma lipidomics to investigate effects of timing of carbohydrate and fat intake on lipid metabolism and its relation to glycemic control. Design 29 nondiabetic men consumed (1) a high-carb test meal (MTT-HC) at 09.00 and a high-fat meal (MTT-HF) at 15.40; or (2) MTT-HF at 09.00 and MTT-HC at 15.40. Blood was sampled before and 180 minutes after completion of each MTT. Subcutaneous adipose tissue (SAT) was collected after overnight fast and both MTTs. Prior to each investigation day, participants consumed a 4-week isocaloric diet of the same composition: (1) high-carb meals until 13.30 and high-fat meals between 16.30 and 22:00 or (2) the inverse order. Results 12 hour daily lipid patterns showed a complex regulation by both the time of day (67.8%) and meal composition (55.4%). A third of lipids showed a diurnal variation in postprandial responses to the same meal with mostly higher responses in the morning than in the afternoon. Triacylglycerols containing shorter and more saturated fatty acids were enriched in the morning. SAT transcripts involved in fatty acid synthesis and desaturation showed no diurnal variation. Diurnal changes of 7 lipid classes were negatively associated with insulin sensitivity, but not with glucose and insulin response or insulin secretion. Conclusions This study identified postprandial plasma lipid profiles as being strongly affected by meal timing and associated with insulin sensitivity.

Circadian clocks are endogenous oscillators essential for orchestrating daily rhythms in physiology, metabolism and behavior. While mouse models have been instrumental to elucidate the molecular mechanism of circadian rhythm generation, our knowledge about the molecular makeup of circadian oscillators in humans is still limited. Here, we used duplex CRISPR/Cas9 technology to generate three cellular models for studying human circadian clocks: CRY1 knockout cells, CRY2 knockout cells as well as CRY1/CRY2 double knockout cells. Duplex CRISPR/Cas9 technology efficiently removed whole exons of CRY genes by using two guide RNAs targeting exon-flanking intron regions of human osteosarcoma cells (U-2 OS). Resulting cell clones did not express CRY proteins and showed short period, low-amplitude rhythms (for CRY1 knockout), long period rhythms (for CRY2 knockout) or were arrhythmic (for CRY1/CRY2 double knockout) similar to circadian phenotypes of cells derived from classical knockout mouse models.

The circadian clock is an endogenous oscillator that controls daily rhythms in metabolism, physiology, and behavior. Although the timekeeping components differ among species, a common design principle is a transcription-translation negative feedback loop. However, it is becoming clear that other mechanisms can contribute to the generation of 24 h rhythms. Peroxiredoxins (Prxs) exhibit 24 h rhythms in their redox state in all kingdoms of life. In mammalian adrenal gland, heart and brown adipose tissue, such rhythms are generated as a result of an inactivating hyperoxidation reaction that is reduced by coordinated import of sulfiredoxin (Srx) into the mitochondria. However, a quantitative description of the Prx/Srx oscillating system is still missing. We investigate the basic principles that generate mitochondrial Prx/Srx rhythms using computational modeling. We observe that the previously described delay in mitochondrial Srx import, in combination with an appropriate separation of fast and slow reactions, is sufficient to generate robust self-sustained relaxation-like oscillations. We find that our conceptual model can be regarded as a series of three consecutive phases and two temporal switches, highlighting the importance of delayed negative feedback and switches in the generation of oscillations. Deutsche Forschungsgemeinschaft

Background & Aims: The mammalian circadian clock controls various aspects of liver metabolism and integrates nutritional signals. Recently, we described Hedgehog (Hh) signaling as a novel regulator of liver lipid metabolism. Herein, we investigated crosstalk between hepatic Hh signaling and circadian rhythm.Methods: Diurnal rhythms of Hh signaling were investigated in liver and hepatocytes from mice with ablation of Smoothened (SAC-KO) and crossbreeds with PER2::LUC reporter mice. By using genome-wide screening, qPCR, immunostaining, ELISA and RNAi experiments in vitro we identified relevant transcriptional regulatory steps. Shotgun lipidomics and metabolic cages were used for analysis of metabolic alterations and behavior.Results: Hh signaling showed diurnal oscillations in liver and hepatocytes in vitro. Correspondingly, the level of Indian Hh, oscillated in serum. Depletion of the clock gene Bmal1 in hepatocytes resulted in significant alterations in the expression of Hh genes. Conversely, SAC-KO mice showed altered expression of clock genes, confirmed by RNAi against Gli1 and Gli3. Genome-wide screening revealed that SAC-KO hepatocytes showed time-dependent alterations in various genes, particularly those associated with lipid metabolism. The clock/hedgehog module further plays a role in rhythmicity of steatosis, and in the response of the liver to a high-fat diet or to differently timed starvation.Conclusions: For the first time, Hh signaling in hepatocytes was found to be time-of-day dependent and to feed back on the circadian clock. Our findings suggest an integrative role of Hh signaling, mediated mainly by GLI factors, in maintaining homeostasis of hepatic lipid metabolism by balancing the circadian clock.Lay summary: The results of our investigation show for the first time that the Hh signaling in hepatocytes is time-of-day dependent, leading to differences not only in transcript levels but also in the amount of Hh ligands in peripheral blood. Conversely, Hh signaling is able to feed back to the circadian clock. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

In mammals, molecular circadian rhythms are generated by autoregulatory transcriptional-translational feedback loops with PERIOD/CRYPTOCHROME containing complexes inhibiting the transcription of their own genes. Although the major circadian oscillator components seem to be identified, an increasing number of additional factors modulating core clock component functions are being discovered. In a systematic screen using short hairpin RNA in human clock reporter cells, we identified FBXL11 (also known as KDM2A), a histone-demethylase, whose gene dosage is crucial for a correct circadian period. Knockdown of FBXL11 leads to period shortening and overexpression to period lengthening. In addition, altering FBXL11 gene dosage modulates clock gene transcript levels, most prominently that of Nr1d1. FBXL11 exercises its role in the mammalian circadian clock by acting as a negative element on CLOCK/BMAL1 and RORα-induced transcription. It binds directly to the promoter regions of CLOCK/BMAL1-regulated genes via a CXXC-type zinc finger motif in a circadian phase-dependent manner; however, the histone-demethylase activity of FBXL11 is not required for transcriptional repression. Therefore, we propose FBXL11 as a novel component of the circadian clock that regulates the circadian gene expression by a so far unknown mechanism.

Circadian clocks are molecular timekeeping mechanisms that allow organisms to anticipate daily changes in their environment. The fundamental cellular basis of these clocks is delayed negative feedback gene regulation with PERIOD and CRYPTOCHROME containing protein complexes as main inhibitory elements. For a correct circadian period, it is essential that such clock protein complexes accumulate in the nucleus in a precisely timed manner, a mechanism that is poorly understood. We performed a systematic RNAi-mediated screen in human cells and identified 15 genes associated with the nucleo-cytoplasmic translocation machinery, whose expression is important for circadian clock dynamics. Among them was Transportin 1 (TNPO1), a non-classical nuclear import carrier, whose knockdown and knockout led to short circadian periods. TNPO1 was found in endogenous clock protein complexes and particularly binds to PER1 regulating its (but not PER2’s) nuclear localization. While PER1 is also transported to the nucleus by the classical, Importin β-mediated pathway, TNPO1 depletion slowed down PER1 nuclear import rate as revealed by fluorescence recovery after photobleaching (FRAP) experiments. In addition, we found that TNPO1-mediated nuclear import may constitute a novel input pathway of how cellular redox state signals to the clock, since redox stress increases binding of TNPO1 to PER1 and decreases its nuclear localization. Together, our RNAi screen knocking down import carriers (but also export carriers) results in short and long circadian periods indicating that the regulatory pathways that control the timing of clock protein subcellular localization are far more complex than previously assumed. TNPO1 is one of the novel players essential for normal circadian periods and potentially for redox regulation of the clock., Author summary Circadian clocks are endogenous timekeeping mechanisms allowing organisms to anticipate daily changes in their environment. In mammals, the fundamental mechanism of these clocks is a delayed negative feedback loop, in which timely auto-repression of clock components is essential. This repression occurs at a transcriptional level and requires clock proteins to enter the nucleus in a precisely timed manner, a regulation that is little understood. We performed a systematic genetic screen for factors modulating subcellular localization in oscillating human cells and identified Transportin 1 (TNPO1) as a non-classical carrier protein required for a normal circadian period. The primary target of TNPO1 within the circadian clockwork is PERIOD1, whose nuclear shuttling is modulated by TNPO1. In addition, TNPO1-mediated nuclear import may constitute a novel input pathway of how cellular redox state signals to the clock, since redox stress increases binding of TNPO1 to PER1 and decreases its nuclear localization.

The mammalian circadian clock and the cell cycle are two major biological oscillators whose coupling influences cell fate decisions. In the present study, we use a model-driven experimental approach to investigate the interplay between clock and cell cycle components and the dysregulatory effects of RAS on this coupled system. In particular, we focus on the Ink4a/Arf locus as one of the bridging clock-cell cycle elements. Upon perturbations by the rat sarcoma viral oncogene (RAS), differential effects on the circadian phenotype were observed in wild-type and Ink4a/Arf knock-out mouse embryonic fibroblasts (MEFs), which could be reproduced by our modelling simulations and correlated with opposing cell cycle fate decisions. Interestingly, the observed changes can be attributed to in silico phase shifts in the expression of core-clock elements. A genome-wide analysis revealed a set of differentially expressed genes that form an intricate network with the circadian system with enriched pathways involved in opposing cell cycle phenotypes. In addition, a machine learning approach complemented by cell cycle analysis classified the observed cell cycle fate decisions as dependent on Ink4a/Arf and the oncogene RAS and highlighted a putative fine-tuning role of Bmal1 as an elicitor of such processes, ultimately resulting in increased cell proliferation in the Ink4a/Arf knock-out scenario. This indicates that the dysregulation of the core-clock might work as an enhancer of RAS-mediated regulation of the cell cycle. Our combined in silico and in vitro approach highlights the important role of the circadian clock as an Ink4a/Arf-dependent modulator of oncogene-induced cell fate decisions, reinforcing its function as a tumour-suppressor and the close interplay between the clock and the cell cycle network., Author summary In mammals, the circadian clock controls the punctual regulation of biological processes, which, in turn, affect physiology and behaviour, allowing for the synchronisation of internal time to environmental light-dark cycles. Malfunctions of the circadian clock are associated with pathological phenotypes including cancer. Given the range of molecular time-dependent processes, including metabolism, DNA repair, and the cell cycle, the clock is hypothesised to act as a tumour suppressor. With the help of mathematical modelling and whole-genome analysis combined with machine learning, we investigated the RAS-dependent dysregulation of the circadian clock. We find that the tumour-suppressor Ink4a/Arf acts as a key mediator of RAS oncogene-induced changes in the circadian system, thereby mediating the interplay between the clock and the cell cycle.

The importance of natural gene expression variation for human behavior is undisputed, but its impact on circadian physiology remains mostly unexplored. Using umbilical cord fibroblasts, we have determined by genome-wide association how common genetic variation impacts upon cellular circadian function. Gene set enrichment points to differences in protein catabolism as one major source of clock variation in humans. The two most significant alleles regulated expression of COPS7B, a subunit of the COP9 signalosome. We further show that the signalosome complex is imported into the nucleus in timed fashion to stabilize the essential circadian protein BMAL1, a novel mechanism to oppose its proteasome-mediated degradation. Thus, circadian clock properties depend in part upon a genetically-encoded competition between stabilizing and destabilizing forces, and genetic alterations in these mechanisms provide one explanation for human chronotype.

Most species evolved a circadian clock to adapt to the 24 h period of the solar day. In mammals, these clocks generate endogenous rhythms by regulatory gene networks in almost every cell. A pacemaker, the suprachiasmatic nucleus (SCN) as the master clock, receives environmental input and orchestrates peripheral organs via sympathetic enervation, temperature and humoral factors. However, the mechanisms by which this synchronization is achieved are largely unknown. In order to elucidate paradigms of environmental information transfer within the circadian network, we address the following questions: How is environmental information perceived by different circadian networks? Do different circadian networks vary in their responses to a given signal, and, if so, do the differences depend on inherent circadian properties? Which part of the signal (onset, offset, duration, strength) is relevant for the responses? To address these questions, we combine experimental data from cultured single cells and organotypic slices with mathematical models of circadian oscillators and find that temperature signals have a strong impact on circadian rhythms, depending on the specific circadian properties of the clock cells.

SummaryPeriod (PER) proteins are essential components of the mammalian circadian clock. They form complexes with cryptochromes (CRY), which negatively regulate CLOCK/BMAL1-dependent transactivation of clock and clock-controlled genes. To define the roles of mammalian CRY/PER complexes in the circadian clock, we have determined the crystal structure of a complex comprising the photolyase homology region of mouse CRY1 (mCRY1) and a C-terminal mouse PER2 (mPER2) fragment. mPER2 winds around the helical mCRY1 domain covering the binding sites of FBXL3 and CLOCK/BMAL1, but not the FAD binding pocket. Our structure revealed an unexpected zinc ion in one interface, which stabilizes mCRY1-mPER2 interactions in vivo. We provide evidence that mCRY1/mPER2 complex formation is modulated by an interplay of zinc binding and mCRY1 disulfide bond formation, which may be influenced by the redox state of the cell. Our studies may allow for the development of circadian and metabolic modulators.

The timing of daily circadian behavior can be highly variable among different individuals, and twin studies have suggested that about half of this variability is environmentally controlled. Similar plasticity can be seen in mice exposed to an altered lighting environment, for example, 22-h instead of 24-h, which stably alters the genetically determined period of circadian behavior for months. The mechanisms mediating these environmental influences are unknown. We found that transient exposure of mice to such lighting stably altered global transcription in the suprachiasmatic nucleus (SCN) of the hypothalamus (the master clock tissue regulating circadian behavior in mammals). In parallel, genome-wide methylation profiling revealed global alterations in promoter DNA methylation in the SCN that correlated with these changes. Behavioral, transcriptional and DNA methylation changes were reversible after prolonged re-entrainment to 24-h d. Notably, infusion of a methyltransferase inhibitor to the SCN suppressed period changes. We conclude that the SCN utilizes DNA methylation as a mechanism to drive circadian clock plasticity.

Antarctic krill (Euphausia superba) is a key species in Southern Ocean ecosystem where it plays a central role in the Antarctic food web. Available information supports the existence of an endogenous timing system in krill enabling it to synchronize metabolism and behavior with an environment characterized by extreme seasonal changes in terms of day length, food availability, and surface ice extent. A screening of our transcriptome database “KrillDB” allowed us to identify the putative orthologues of 20 circadian clock components. Mapping of conserved domains and phylogenetic analyses strongly supported annotations of the identified sequences. Luciferase assays and co-immunoprecipitation experiments allowed us to define the role of the main clock components. Our findings provide an overall picture of the molecular mechanisms underlying the functioning of the endogenous circadian clock in the Antarctic krill and shed light on their evolution throughout crustaceans speciation. Interestingly, the core clock machinery shows both mammalian and insect features that presumably contribute to an evolutionary strategy to cope with polar environment’s challenges. Moreover, despite the extreme variability characterizing the Antarctic seasonal day length, the conserved light mediated degradation of the photoreceptor EsCRY1 suggests a persisting pivotal role of light as a Zeitgeber.

Factors interacting with core circadian clock components are essential to achieve transcriptional feedback necessary for metazoan clocks. Here, we show that all three members of the Drosophila behavior human splicing (DBHS) family of RNA-binding proteins play a role in the mammalian circadian oscillator, abrogating or altering clock function when overexpressed or depleted in cells. Although these proteins are members of so-called nuclear paraspeckles, depletion of paraspeckles themselves via silencing of the structural noncoding RNA (ncRNA) Neat1 did not affect overall clock function, suggesting that paraspeckles are not required for DBHS-mediated circadian effects. Instead, we show that the proteins bound to circadian promoter DNA in a fashion that required the PERIOD (PER) proteins and potently repressed E-box-mediated transcription but not cytomegalovirus (CMV) promoter-mediated transcription when they were exogenously recruited. Nevertheless, mice with one or both copies of these genes deleted show only small changes in period length or clock gene expression in vivo. Data from transient transfections show that each of these proteins can either repress or activate, depending on the context. Taken together, our data suggest that all of the DBHS family members serve overlapping or redundant roles as transcriptional cofactors at circadian clock-regulated genes.

Circadian clocks govern a wide range of cellular and physiological functions in various organisms. Recent evidence suggests distinct functions of local clocks in peripheral mammalian tissues such as immune responses and cell cycle control. However, studying circadian action in peripheral tissues has been limited so far to mouse models, leaving the implication for human systems widely elusive. In particular, circadian rhythms in human skin, which is naturally exposed to strong daytime-dependent changes in the environment, have not been investigated to date on a molecular level. Here, we present a comprehensive analysis of circadian gene expression in human epidermis. Whole-genome microarray analysis of suction-blister epidermis obtained throughout the day revealed a functional circadian clock in epidermal keratinocytes with hundreds of transcripts regulated in a daytime-dependent manner. Among those, we identified a circadian transcription factor, Krüppel-like factor 9 ( Klf9 ), that is substantially up-regulated in a cortisol and differentiation-state-dependent manner. Gain- and loss-of-function experiments showed strong antiproliferative effects of Klf9 . Putative Klf9 target genes include proliferation/differentiation markers that also show circadian expression in vivo, suggesting that Klf9 affects keratinocyte proliferation/differentiation by controlling the expression of target genes in a daytime-dependent manner.

Time of day-dependent variations of immune system parameters are ubiquitous phenomena in immunology. The circadian clock has been attributed with coordinating these variations on multiple levels; however, their molecular basis is little understood. Here, we systematically investigated the link between the circadian clock and rhythmic immune functions. We show that spleen, lymph nodes, and peritoneal macrophages of mice contain intrinsic circadian clockworks that operate autonomously even ex vivo. These clocks regulate circadian rhythms in inflammatory innate immune functions: Isolated spleen cells stimulated with bacterial endotoxin at different circadian times display circadian rhythms in TNF-α and IL-6 secretion. Interestingly, we found that these rhythms are not driven by systemic glucocorticoid variations nor are they due to the detected circadian fluctuation in the cellular constitution of the spleen. Rather, a local circadian clock operative in splenic macrophages likely governs these oscillations as indicated by endotoxin stimulation experiments in rhythmic primary cell cultures. On the molecular level, we show that >8% of the macrophage transcriptome oscillates in a circadian fashion, including many important regulators for pathogen recognition and cytokine secretion. As such, understanding the cross-talk between the circadian clock and the immune system provides insights into the timing mechanism of physiological and pathophysiological immune functions.

Post-translational processes are essential for the generation and dynamics of mammalian circadian rhythms. In particular, phosphorylation of the key circadian protein PER2 precisely controls the period and phase of circadian oscillations. However, the mechanisms underlying that control are poorly understood. Here, we identified in a high-throughput RNAi-based genetic screen casein kinase 2 (CK2) as a PER2-phosphorylating kinase and novel component of the mammalian circadian clock. When CK2 subunits are silenced by RNAi or when CK2 activity is inhibited pharmacologically, circadian rhythms are disrupted. CK2 binds to PER2 in vivo, phosphorylates PER2 specifically at N-terminal residues in vitro, and supports normal nuclear PER2 accumulation. Mutation of CK2 phosphorylation sites decreases PER2 stability and copies CK2 inhibition regarding oscillation dynamics. We propose a new concept of how PER2 phosphorylation and stabilization can set the clock speed in opposite directions, dependent on the phase of action.

Mammals evolved an endogenous timing system to coordinate their physiology and behaviour to the 24h period of the solar day. While it is well accepted that circadian rhythms are generated by intracellular transcriptional feedback loops, it is still debated which network motifs are necessary and sufficient for generating self-sustained oscillations. Here, we systematically explore a data-based circadian oscillator model with multiple negative and positive feedback loops and identify a series of three subsequent inhibitions known as “repressilator” as a core element of the mammalian circadian oscillator. The central role of the repressilator motif is consistent with time-resolved ChIP-seq experiments of circadian clock transcription factors and loss of rhythmicity in core clock gene knockouts., Author Summary Circadian clocks are endogenous oscillators that drive daily rhythms in physiology, metabolism and behavior. The recent years have witnessed enormous progress in our understanding of the mechanistic and genetic basis of these clocks. While mathematical modelling has made important contributions to our current view of the circadian clock network, it is still debated, which network motifs are necessary and sufficient for generating self-sustained oscillations. Exploiting a data-driven mathematical model we here identify the “repressilator” as a key design principal. The central role of the repressilator motif is consistent with time-resolved binding data of circadian clock transcription factors and loss of rhythmicity in core clock gene knockouts.

Regulated degradation of circadian clock proteins is a crucial step for rhythm generation per se but also for establishing a normal circadian period. Here, the authors show that the F-box protein beta-transducin repeat containing protein 1 (β-TrCP1) as part of the E3 ubiquitin ligase complex is an essential component of the mammalian circadian oscillator. Down-regulation of endogenous β-TrCP1 as well as expression of a dominant-negative form both result in lengthening of the circadian period in oscillating fibroblasts. These phenotypes are due to an impaired degradation of PERIOD (PER) proteins, since expression of β-TrCP interaction-deficient PER2 variants—but not wild-type PER2—results in a dramatic stabilization of PER2 protein as well as in the disruption of circadian rhythmicity. Mathematical modeling conceptualizes the authors' findings and suggests that loss of sustained rhythmicity in cells with eliminated β-TrCP-mediated PER2 degradation is due to excessive nuclear repression, a prediction they verified experimentally.

At the core of the mammalian circadian clock is a feedback loop in which the heterodimeric transcription factor CLOCK-Brain, Muscle Arnt-like-1 (BMAL1) drives expression of its negative regulators, periods (PERs) and cryptochromes (CRYs). Here, we provide evidence that CLOCK-Interacting Protein, Circadian (CIPC) is an additional negative-feedback regulator of the circadian clock. CIPC exhibits circadian regulation in multiple tissues, and it is a potent and specific inhibitor of CLOCK-BMAL1 activity that functions independently of CRYs. CIPC-CLOCK protein complexes are present in vivo, and depletion of endogenous CIPC shortens the circadian period length. CIPC is unrelated to known proteins and has no recognizable homologues outside vertebrates. Our results suggest that negative feedback in the mammalian circadian clock is divided into distinct pathways, and that the addition of new genes has contributed to the complexity of vertebrate clocks.

Odor discrimination behavior displays circadian fluctuations in mice, indicating that mammalian olfactory function is under control of the circadian system. This is further supported by the facts that odor discrimination rhythms depend on the presence of clock genes and that olfactory tissues contain autonomous circadian clocks. However, the molecular link between circadian function and olfactory processing is still unknown. To elucidate the molecular mechanisms underlying this link, we focused on the olfactory epithelium (OE), the primary target of odors and the site of the initial events in olfactory processing. We asked whether olfactory sensory neurons (OSNs) within the OE possess an autonomous circadian clock and whether olfactory pathways are under circadian control. Employing clock gene–driven bioluminescence reporter assays and time-dependent immunohistochemistry on OE samples, we found robust circadian rhythms of core clock genes and their proteins in OSNs, suggesting that the OE indeed contains an autonomous circadian clock. Furthermore, we performed a circadian transcriptome analysis and identified several OSN-specific components that are under circadian control, including those with putative roles in circadian olfactory processing, such as KIRREL2—an established factor involved in short-term OSN activation. The spatiotemporal expression patterns of our candidate proteins suggest that they are involved in short-term anabolic processes to rhythmically prepare the cell for peak performances and to promote circadian function of OSNs.

Background The circadian clock coordinates numerous metabolic processes to adapt physiological responses to light-dark and feeding regimens and is itself regulated by metabolic cues. The implication of the circadian clock in the regulation of energy balance and body weight is widely studied in rodents but not in humans. Here we investigated (1) whether the expression of clock genes in human adipose tissue is changed by weight loss and (2) whether these alterations are associated with metabolic parameters. Subjects/methods Subcutaneous adipose tissue (SAT) samples were collected before and after 8 weeks of weight loss on an 800 kcal per day hypocaloric diet (plus 200 g per day vegetables) at the same time of the day. Fifty overweight subjects who lost at least 8% weight after 8 weeks were selected for the study. The expression of 10 clock genes and key metabolic and inflammatory genes in adipose tissue was determined by quantitative real-time PCR. Results The expression of core clock genes PER2 and NR1D1 was increased after the weight loss. Correlations of PERIOD expression with body mass index (BMI) and serum total, high-density lipoprotein and low-density lipoprotein (LDL) cholesterol levels and of NR1D1 expression with total and LDL cholesterol were found that became non-significant after correction for multiple testing. Clock gene expression levels and their weight loss-induced changes tightly correlated with each other and with genes involved in fat metabolism (FASN, CPT1A, LPL, PPARG, PGC1A, ADIPOQ), energy metabolism (SIRT1), autophagy (LC3A, LC3B) and inflammatory response (NFKB1, NFKBIA, NLRP3, EMR1). Conclusion Clock gene expression in human SAT is regulated by body weight changes and associated with BMI, serum cholesterol levels and the expression of metabolic and inflammatory genes. Our data confirm the tight crosstalk between molecular clock and metabolic and inflammatory pathways involved in adapting adipose tissue metabolism to changes of the energy intake in humans.

CONTEXT: The circadian clock coordinates numerous metabolic processes with light-dark and feeding regimens. However, in humans it is unknown whether dietary patterns influence circadian rhythms. OBJECTIVE: We examined the effects of switching from a high-carbohydrate, low-fat diet to a low-carbohydrate, high fat (LC/HFD) isocaloric diet on the central and peripheral circadian clocks in humans. DESIGN: Diurnal patterns of salivary cortisol and gene expression were analyzed in blood monocytes of 29 nonobese healthy subjects before and 1 and 6 weeks after the dietary switch. For this, we established a method of rhythm prediction by 3-time point data. RESULTS: The centrally driven cortisol rhythm showed a phase delay 1 and 6 weeks after the dietary switch to a LC/HFD as well as an amplitude increase. The dietary switch altered diurnal oscillations of core clock genes (PER1, PER2, PER3, and TEF) and inflammatory genes (CD14, CD180, NFKBIA, and IL1B). The LC/HFD also affected the expression of nonoscillating genes contributing to energy metabolism (SIRT1) and fat metabolism (ACOX3 and IDH3A). Expression of clock genes but not of salivary cortisol in monocytes tightly correlated with levels of blood lipids and with expression of metabolic and inflammatory genes. CONCLUSIONS: Our results suggest that the modulation of the dietary fat and carbohydrate content alters the function of the central and peripheral circadian clocks in humans.

PERIOD (PER) proteins are central components within the mammalian circadian oscillator, and are believed to form a negative feedback complex that inhibits their own transcription at a particular circadian phase. Phosphorylation of PER proteins regulates their stability as well as their subcellular localization. In a systematic screen, we have identified 21 phosphorylated residues of mPER2 including Ser 659, which is mutated in patients suffering from familial advanced sleep phase syndrome (FASPS). When expressing FASPS-mutated mPER2 in oscillating fibroblasts, we can phenocopy the short period and advanced phase of FASPS patients’ behavior. We show that phosphorylation at Ser 659 results in nuclear retention and stabilization of mPER2, whereas phosphorylation at other sites leads to mPER2 degradation. To conceptualize our findings, we use mathematical modeling and predict that differential PER phosphorylation events can result in opposite period phenotypes. Indeed, interference with specific aspects of mPER2 phosphorylation leads to either short or long periods in oscillating fibroblasts. This concept explains not only the FASPS phenotype, but also the effect of the tau mutation in hamster as well as the doubletime mutants (dbtS and dbtL) in Drosophila.

To adapt the timing of processes regulated by the circadian clock to seasonally varying photoperiods, the phase relation between the circadian clock and dusk or dawn (“phase of entrainment”) must be tightly adjusted. The authors use a mathematical model of the molecular mammalian circadian oscillator to investigate the influence of the free-running period (•) and the shape of the PRC on the phase of entrainment. They find that a phase-dependent sensitivity (“gating”) of light-induced period gene transcription enables a constant phase relation to dusk or dawn under different photoperiods. Depending on the freerunning period • and on the shaping of the PRC by gating, the model circadian oscillator tracks either light onset or light offset under different photoperiods. The study indicates that the phase of entrainment of oscillating cells can be systematically controlled by regulating both gating and the free-running period τ.

The circadian clock in the suprachiasmatic nucleus (SCN) is thought to drive daily rhythms of behavior by secreting factors that act locally within the hypothalamus. In a systematic screen, we identified transforming growth factor-α (TGF-α) as a likely SCN inhibitor of locomotion. TGF-α is expressed rhythmically in the SCN, and when infused into the third ventricle it reversibly inhibited locomotor activity and disrupted circadian sleep-wake cycles. These actions are mediated by epidermal growth factor (EGF) receptors on neurons in the hypothalamic subparaventricular zone. Mice with a hypomorphic EGF receptor mutation exhibited excessive daytime locomotor activity and failed to suppress activity when exposed to light. These results implicate EGF receptor signaling in the daily control of locomotor activity, and identify a neural circuit in the hypothalamus that likely mediates the regulation of behavior both by the SCN and the retina.

Circadian rhythms are essential to the temporal regulation of molecular processes in living systems and as such to life itself. Deregulation of these rhythms leads to failures in biological processes and eventually to the manifestation of pathological phenotypes including cancer. To address the questions as to what are the elicitors of a disrupted clock in cancer, we applied a systems biology approach to correlate experimental, bioinformatics and modelling data from several cell line models for colorectal and skin cancer. We found strong and weak circadian oscillators within the same type of cancer and identified a set of genes, which allows the discrimination between the two oscillator-types. Among those genes are IFNGR2, PITX2, RFWD2, PPARγ, LOXL2, Rab6 and SPARC, all involved in cancer-related pathways. Using a bioinformatics approach, we extended the core-clock network and present its interconnection to the discriminative set of genes. Interestingly, such gene signatures link the clock to oncogenic pathways like the RAS/MAPK pathway. To investigate the potential impact of the RAS/MAPK pathway - a major driver of colorectal carcinogenesis - on the circadian clock, we used a computational model which predicted that perturbation of BMAL1-mediated transcription can generate the circadian phenotypes similar to those observed in metastatic cell lines. Using an inducible RAS expression system, we show that overexpression of RAS disrupts the circadian clock and leads to an increase of the circadian period while RAS inhibition causes a shortening of period length, as predicted by our mathematical simulations. Together, our data demonstrate that perturbations induced by a single oncogene are sufficient to deregulate the mammalian circadian clock., Author Summary Living systems possess an endogenous time-generating system – the circadian clock - accountable for a 24 hours oscillation in the expression of about 10% of all genes. In mammals, disruption of oscillations is associated to several diseases including cancer. In this manuscript, we address the following question: what are the elicitors of a disrupted clock in cancer? We applied a systems biology approach to correlate experimental, bioinformatics and modelling data and could thereby identify key genes which discriminate strong and weak oscillators among cancer cell lines. Most of the discriminative genes play important roles in cell cycle regulation, DNA repair, immune system and metabolism and are involved in oncogenic pathways such as the RAS/MAPK. To investigate the potential impact of the Ras oncogene in the circadian clock we generated experimental models harbouring conditionally active Ras oncogenes. We put forward a direct correlation between the perturbation of Ras oncogene and an effect in the expression of clock genes, found by means of mathematical simulations and validated experimentally. Our study shows that perturbations of a single oncogene are sufficient to deregulate the mammalian circadian clock and opens new ways in which the circadian clock can influence disease and possibly play a role in therapy.

Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK), the master transcription factor driving cyclical gene expression and the rate-limiting component in the Drosophila circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a Drosophila tissue culture system, we identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A) rescues the arrythmicity of Clk out flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression., Author Summary Circadian clocks are synchronized to local time by daily cycles in light-dark and temperature. Although light is generally thought to be the most dominant entraining cue in nature, daily cycles in temperature are sufficient to synchronize clocks in a large range of organisms. In Drosophila, dCLOCK is a master circadian transcription factor that drives cyclical gene expression and is likely the rate-limiting component in the transcriptional/translational feedback loops that underlie the timekeeping mechanism. dCLOCK undergoes temporal changes in phosphorylation throughout a day, which is also observed for mammalian CLOCK. However, the role of CLOCK phosphorylation at the organismal level is still unclear. Using mass-spectrometry, we identified more than a dozen phosphorylation sites on dCLOCK. Blocking global phosphorylation of dCLOCK by mutating phospho-acceptor sites to alanine increases its abundance and transcriptional activity, leading to higher peak values and amplitudes in the mRNA rhythms of core clock genes, which likely explains the accelerated clock speed. Surprisingly, the clock-controlled daily activity rhythm fails to maintain synchrony with daily temperature cycles, although there is no observable defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the effective strengths of entraining modalities by adjusting clock amplitude.

Essentially all biological processes depend on protein–protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (∼24-hour) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes, PPI dynamics on this timescale are largely unknown. Here, we identified 109 novel PPIs among circadian clock proteins via a yeast-two-hybrid approach. Among them, the interaction of protein phosphatase 1 and CLOCK/BMAL1 was found to result in BMAL1 destabilization. We constructed a dynamic circadian PPI network predicting the PPI timing using circadian expression data. Systematic circadian phenotyping (RNAi and overexpression) suggests a crucial role for components involved in dynamic interactions. Systems analysis of a global dynamic network in liver revealed that interacting proteins are expressed at similar times likely to restrict regulatory interactions to specific phases. Moreover, we predict that circadian PPIs dynamically connect many important cellular processes (signal transduction, cell cycle, etc.) contributing to temporal organization of cellular physiology in an unprecedented manner., Author Summary Circadian clocks are endogenous oscillators that drive daily rhythms in physiology, metabolism, and behavior. In mammals, circadian rhythms are generated within nearly every cell; and, although dysfunction of circadian clocks is associated with various diseases (including diabetes and cancer), the molecular mechanisms linking the clock machinery with output pathways are little understood. Since essentially all biological processes depend on protein–protein interactions, we investigated here on a systems-wide level how time-of-day-specific protein–protein interactions contribute to the temporal organization of cellular physiology. We constructed a circadian interactome using experimentally generated protein–protein interaction data and made this network dynamic by the incorporation of time-of-day-dependent expression data. Interestingly, systematic genetic network perturbation (RNAi and overexpression) suggests a crucial role for circadian components involved in dynamic interactions. Systems analysis of a global network revealed that interacting proteins are in the liver significantly more expressed at similar daytimes likely to restrict regulatory interactions to specific circadian phases within cells. Overall, circadian protein–protein interactions are predicted to dynamically connect important cellular processes (signal transduction, cell cycle, etc.) using—very often—protein modules with components co-expressed in time, shedding new light on the daily organization of cellular physiology.

Many physiological and behavioral processes such as sleep and wakefulness, hormone secretion, and olfactory sensitivity exhibit a 24-h rhythmicity that persists in constant conditions with a period close to (circa) 24 h. These circadian rhythms are driven by a network of endogenous clocks residing in various tissues, including the olfactory system, and are synchronized to the outside world by environmental time cues such as light, temperature, and food. In addition to having these well-known zeitgebers of circadian clocks, most environments consist of a multitude of odors that report, for example, the availability of food or the presence of predators—and often, they do so in a time-of-day-dependent manner. Considering the evolutionary significance of odors for various fitness-related behaviors such as mate choice, predator avoidance, and foraging strategies, we asked whether odors—similar to light, temperature, or food—might act as a circadian time cue able to influence circadian locomotor behavior in mammals. Administering individual air flow, periodically saturated with an artificial odor mix, to running wheel-equipped mouse cages, we found that rhythmic odor administrations significantly lengthened the period of circadian activity rhythms. Additionally, odor cues led to partial reemergence of circadian locomotor rhythmicity in suprachiasmatic nuclei (SCN)–lesioned mice, suggesting that the SCN as the central circadian pacemaker are not immediately required for odor-mediated effects on circadian behavior. However, odor-based modulation of circadian behavior did not occur in clock mutant ( cry1−/−/ cry2−/−) mice, indicating an odor-mediated mechanism that involves extra-SCN canonical clocks, such as the olfactory clock itself. Our results indicate not only that odor stimuli can act as a circadian time cue modulating circadian behavior but also that odor effects are even more pronounced in the absence of the SCN but nevertheless require the presence of a functional canonical clock.

The endogenous circadian timing system has evolved to synchronize an organism to periodically recurring environmental conditions. Those external time cues are called Zeitgebers. When entrained by a Zeitgeber, the intrinsic oscillator adopts a fixed phase relation ψ to the Zeitgeber. Here, we systematically study how the phase of entrainment depends on clock and Zeitgeber properties. We combine numerical simulations of amplitude-phase models with predictions from analytically tractable models. In this way we derive relations between the phase of entrainment ψ to the mismatch between the endogenous and Zeitgeber period, the Zeitgeber strength, and the range of entrainment. A core result is the "180° rule" asserting that the phase ψ varies over a range of about 180° within the entrainment range. The 180° rule implies that clocks with a narrow entrainment range ("strong oscillators") exhibit quite flexible entrainment phases. We argue that this high sensitivity of the entrainment phase contributes to the wide range of human chronotypes.

Your alarm clock rings in the morning—it's time to get up, but you feel like it's the middle of the night. If this is happening regularly, your circadian clock may not be adjusted to your social life. Circadian clocks are endogenous oscillators that coordinate not only our sleep-wake behavior with the environmental 24-hour light-dark cycle but also a myriad of rhythmic physiological and metabolic processes. A set of so-called clock genes comprises a regulatory network that generates self-sustained molecular ~24-hour rhythms of gene expression. For eukaryotic clocks, the prevailing conceptual model proposes a negative transcriptional-translational feedback loop ( 1 ). On page 518 of this issue, Larrondo et al. ( 2 ) challenge the molecular basis of this view.

The circadian clock is accountable for the regulation of internal rhythms in most living organisms. It allows the anticipation of environmental changes during the day and a better adaptation of physiological processes. In mammals the main clock is located in the suprachiasmatic nucleus (SCN) and synchronizes secondary clocks throughout the body. Its molecular constituents form an intracellular network which dictates circadian time and regulates clock-controlled genes. These clock-controlled genes are involved in crucial biological processes including metabolism and cell cycle regulation. Its malfunction can lead to disruption of biological rhythms and cause severe damage to the organism. The detailed mechanisms that govern the circadian system are not yet completely understood. Mathematical models can be of great help to exploit the mechanism of the circadian circuitry. We built a mathematical model for the core clock system using available data on phases and amplitudes of clock components obtained from an extensive literature search. This model was used to answer complex questions for example: how does the degradation rate of Per affect the period of the system and what is the role of the ROR/Bmal/REV-ERB (RBR) loop? Our findings indicate that an increase in the RNA degradation rate of the clock gene Period (Per) can contribute to increase or decrease of the period - a consequence of a non-monotonic effect of Per transcript stability on the circadian period identified by our model. Furthermore, we provide theoretical evidence for a potential role of the RBR loop as an independent oscillator. We carried out overexpression experiments on members of the RBR loop which lead to loss of oscillations consistent with our predictions. These findings challenge the role of the RBR loop as a merely auxiliary loop and might change our view of the clock molecular circuitry and of the function of the nuclear receptors (REV-ERB and ROR) as a putative driving force of molecular oscillations., Author Summary Most organisms have evolved an internal clock which allows them to anticipate and react to the light/dark daily rhythm and is able to generate oscillation with a circa 24 hour rhythm. A molecular network involving feedback loops is responsible for the rhythm generation. A large number of clock-controlled genes pass on time messages and control several biological processes. In spite of its medical importance (role in cancer, sleep disorders, diabetes and others) the mechanism of action of the circadian clock and the role of its constituent's feedback loops remains partially unknown. Using a mathematical model, we were able to bring insight in open circadian biology questions. Firstly, increasing the mRNA degradation rate of Per can contribute to increase or decrease of the period which might explain contradictory experimental findings. Secondly, our data points to a more relevant role of the ROR/Bmal/REV-ERB loop. In particular, that this loop can be an oscillator on its own. We provide experimental evidence that overexpression of members of the ROR/Bmal/REV-ERB lead to loss of Bmal reporter mRNA oscillations. The fact that REV-ERB and ROR are nuclear receptors and therefore important regulators in many cellular processes might have important implications for molecular biology and medicine.