Ventilator-induced Lung Injury Is Modulated by the Circadian Clock.

Rationale: Mechanical ventilation (MV) is life-saving but may evoke ventilator-induced lung injury (VILI). Objectives: To explore how the circadian clock modulates severity of murine VILI via the core clock component BMAL1 (basic helix-loop-helix ARNT like 1) in myeloid cells. Methods: Myeloid cell BMAL1-deficient (LysM (lysozyme 2 promoter/enhancer driving cre recombinase expression)Bmal1-/-) or wild-type control (LysMBmal1+/+) mice were subjected to 4 hours MV (34 ml/kg body weight) to induce lung injury. Ventilation was initiated at dawn or dusk or in complete darkness (circadian time [CT] 0 or CT12) to determine diurnal and circadian effects. Lung injury was quantified by lung function, pulmonary permeability, blood gas analysis, neutrophil recruitment, inflammatory markers, and histology. Neutrophil activation and oxidative burst were analyzed ex vivo. Measurements and Main Results: In diurnal experiments, mice ventilated at dawn exhibited higher permeability and neutrophil recruitment compared with dusk. Experiments at CT showed deterioration of pulmonary function, worsening of oxygenation, and increased mortality at CT0 compared with CT12. Wild-type neutrophils isolated at dawn showed higher activation and reactive oxygen species production compared with dusk, whereas these day-night differences were dampened in LysMBmal1-/- neutrophils. In LysMBmal1-/- mice, circadian variations in VILI severity were dampened and VILI-induced mortality at CT0 was reduced compared with LysMBmal1+/+ mice. Conclusions: Inflammatory response and lung barrier dysfunction upon MV exhibit diurnal variations, regulated by the circadian clock. LysMBmal1-/- mice are less susceptible to ventilation-induced pathology and lack circadian variation of severity compared with LysMBmal1+/+ mice. Our data suggest that the internal clock in myeloid cells is an important modulator of VILI.

Coupling Controls the Synchrony of Clock Cells in Development and Knockouts.

In mammals, a network of coupled neurons within the hypothalamus coordinates physiological rhythms with daily changes in the environment. In each neuron, delayed negative transcriptional feedbacks generate oscillations, albeit noisy and unreliable ones. Coupling mediated by diffusible neuropeptides lends precision and robustness to circadian rhythms. The double knockout of Cryptochrome Cry turns adult mice arrhythmic. But, remarkably, double knockout neonates continue to show robust oscillation much like wild-type neonates and appear to lose rhythmicity with development. We study quantitatively dispersed neurons and brain slices from wild-type and Cry double knockout mice to understand the links between single cell rhythmicity and intercellular coupling. We quantify oscillator properties of dispersed cells using nonlinear regression and study bifurcations diagrams of network models. We find that varying just three parameters-oscillator strength, strength of coupling, and timing of coupling-can reproduce experimentally observed features. In particular, modeling reveals that minor changes in timing of coupling can destroy synchronization as observed in adult slices from knockout mice.

Timing of neuropeptide coupling determines synchrony and entrainment in the mammalian circadian clock.

Robust synchronization is a critical feature of several systems including the mammalian circadian clock. The master circadian clock in mammals consists of about 20000 'sloppy' neuronal oscillators within the hypothalamus that keep robust time by synchronization driven by inter-neuronal coupling. The complete understanding of this synchronization in the mammalian circadian clock and the mechanisms underlying it remain an open question. Experiments and computational studies have shown that coupling individual oscillators can achieve robust synchrony, despite heterogeneity and different network topologies. But, much less is known regarding the mechanisms and circuits involved in achieving this coupling, due to both system complexity and experimental limitations. Here, we computationally study the coupling mediated by the primary coupling neuropeptide, vasoactive intestinal peptide (VIP) and its canonical receptor, VPAC2R, using the transcriptional elements and generic mode of VIP-VPAC2R signaling. We find that synchrony is only possible if VIP (an inducer of Per expression) is released in-phase with activators of Per expression. Moreover, anti-phasic VIP release suppresses coherent rhythms by moving the network into a desynchronous state. Importantly, experimentally observed rhythms in VPAC2R have little effect on network synchronization, but can improve the amplitude of the SCN network rhythms while narrowing the network entrainment range. We further show that these findings are valid across several computational network models. Thus, we identified a general design principle to achieve robust synchronization: An activating coupling agent, such as VIP, must act in-phase with the activity of core-clock promoters. More generally, the phase of coupling is as critical as the strength of coupling from the viewpoint of synchrony and entrainment.

Relating suborganismal processes to ecotoxicological and population level endpoints using a bioenergetic model.

Ecological effects of environmental stressors are commonly evaluated using organismal or suborganismal data, such as standardized toxicity tests that characterize responses of individuals (e.g., mortality and reproduction) and a rapidly growing body of "omics" data. A key challenge for environmental risk assessment is relating such information to population dynamics. One approach uses dynamic energy budget (DEB) models that relate growth and reproduction of individuals to underlying flows of energy and elemental matter. We hypothesize that suborganismal information identifies DEB parameters that are most likely impacted by a particular stressor and that the DEB model can then project suborganismal effects on life history and population endpoints. We formulate and parameterize a model of growth and reproduction for the water flea Daphnia magna. Our model resembles previous generic bioenergetic models, but has explicit representation of discrete molts, an important feature of Daphnia life history. We test its ability to predict six endpoints commonly used in chronic toxicity studies in specified food environments. With just one adjustable parameter, the model successfully predicts growth and reproduction of individuals from a wide array of experiments performed in multiple laboratories using different clones of D. magna raised on different food sources. Fecundity is the most sensitive endpoint, and there is broad correlation between the sensitivities of fecundity and long-run growth rate, as is desirable for the default metric used in chronic toxicity tests. Under some assumptions, we can combine our DEB model with the Euler-Lotka equation to estimate longrun population growth rates at different food levels. A review of Daphnia gene-expression experiments on the effects of contaminant exposure reveals several connections to model parameters, in particular a general trend of increased transcript expression of genes involved in energy assimilation and utilization at concentrations affecting growth and reproduction. The sensitivity of fecundity to many model parameters was consistent with frequent generalized observations of decreased expression of genes involved in reproductive physiology, but interpretation of these observations requires further mechanistic modeling. We thus propose an approach based on generic DEB models incorporating few essential species-specific features for rapid extrapolation of ecotoxicogenomic assays for Daphnia-based population risk assessment.

Quantification of circadian rhythms in mammalian lung tissue snapshot data.

Healthy mammalian cells have a circadian clock, a gene regulatory network that allows them to schedule their physiological processes to optimal times of the day. When healthy cells turn into cancer cells, the circadian clock often becomes cancer specifically disturbed, so there is an interest in the extraction of circadian features from gene expression data of cancer. This is challenging, as clinical gene expression samples of cancer are snapshot-like and the circadian clock is best examined using gene expression time series. In this study, we obtained lists of intersecting circadian genes in public gene expression time series data of lung tissue of mouse and baboon. We base our circadian gene lists on correlations of gene expression levels of circadian genes, which are closely associated to the phase differences between them. Combining circadian gene expression patterns of diurnal and nocturnal species of different ages provides circadian genes that are also important in healthy and cancerous human lung tissue. We tested the quality of the representation of the circadian clock in our gene lists by PCA-based reconstructions of the circadian times of the mouse and baboon samples. Then we assigned potential circadian times to the human lung tissue samples and find an intact circadian clock in the healthy human lung tissue, but an altered, weak clock in the adjacent cancerous lung tissue.

Data integration and analysis for circadian medicine.

Data integration, data sharing, and standardized analyses are important enablers for data-driven medical research. Circadian medicine is an emerging field with a particularly high need for coordinated and systematic collaboration between researchers from different disciplines. Datasets in circadian medicine are multimodal, ranging from molecular circadian profiles and clinical parameters to physiological measurements and data obtained from (wearable) sensors or reported by patients. Uniquely, data spanning both the time dimension and the spatial dimension (across tissues) are needed to obtain a holistic view of the circadian system. The study of human rhythms in the context of circadian medicine has to confront the heterogeneity of clock properties within and across subjects and our inability to repeatedly obtain relevant biosamples from one subject. This requires informatics solutions for integrating and visualizing relevant data types at various temporal resolutions ranging from milliseconds and seconds to minutes and several hours. Associated challenges range from a lack of standards that can be used to represent all required data in a common interoperable form, to challenges related to data storage, to the need to perform transformations for integrated visualizations, and to privacy issues. The downstream analysis of circadian rhythms requires specialized approaches for the identification, characterization, and discrimination of rhythms. We conclude that circadian medicine research provides an ideal environment for developing innovative methods to address challenges related to the collection, integration, visualization, and analysis of multimodal multidimensional biomedical data.

Venn diagram analysis overestimates the extent of circadian rhythm reprogramming.

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.

Inter-layer and inter-subject variability of diurnal gene expression in human skin.

The skin is the largest human organ with a circadian clock that regulates its function. Although circadian rhythms in specific functions are known, rhythms in the proximal clock output, gene expression, in human skin have not been thoroughly explored. This work reports 24 h gene expression rhythms in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep-wake schedules. 10% of the expressed genes showed such diurnal rhythms at the population level, of which only a third differed between the two layers. Amplitude and phases of diurnal gene expression varied more across subjects than layers, with amplitude being more variable than phases. Expression amplitudes in the epidermis were larger and more subject-variable, while they were smaller and more consistent in the dermis. Core clock gene expression was similar across layers at the population-level, but were heterogeneous in their variability across subjects. We also identified small sets of biomarkers for internal clock phase in each layer, which consisted of layer-specific non-core clock genes. This work provides a valuable resource to advance our understanding of human skin and presents a novel methodology to quantify sources of variability in human circadian rhythms.

Stochastic growth reduces population fluctuations in Daphnia-algal systems.

Deterministic, size-structured models are widely used to describe consumer-resource interactions. Such models typically ignore potentially large random variability in juvenile development rates. We present simple representations of this variability and show five approaches to calculating the model parameters for Daphnia pulex interacting with its algal food. Using our parameterized models of growth variability, we investigate the robustness of a recently proposed stabilizing mechanism for Daphnia populations. Growth rate variability increases the range of enrichments over which small-amplitude cycles or quasi-cycles occur, thus increasing the plausibility that the underlying mechanism contributes to the prevalence of small-amplitude cycles in the field and in experiments. More generally, our approach allows us to relate commonly available information on variance of development times to population stability.

Susceptibility rhythm to bacterial endotoxin in myeloid clock-knockout mice.

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 threefold 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.

Circadian rhythms in septic shock patients.

BACKGROUND: Despite the intensive efforts to improve the diagnosis and therapy of sepsis over the last decade, the mortality of septic shock remains high and causes substantial socioeconomical burden of disease. The function of immune cells is time-of-day-dependent and is regulated by several circadian clock genes. This study aims to investigate whether the rhythmicity of clock gene expression is altered in patients with septic shock. METHODS: This prospective pilot study was performed at the university hospital Charité-Universitätsmedizin Berlin, Department of Anesthesiology and Operative Intensive Care Medicine (CCM, CVK). We included 20 patients with septic shock between May 2014 and January 2018, from whom blood was drawn every 4 h over a 24-h period to isolate CD14-positive monocytes and to measure the expression of 17 clock and clock-associated genes. Of these patients, 3 whose samples expressed fewer than 8 clock genes were excluded from the final analysis. A rhythmicity score SP was calculated, which comprises values between -1 (arrhythmic) and 1 (rhythmic), and expression data were compared to data of a healthy study population additionally. RESULTS: 77% of the measured clock genes showed inconclusive rhythms, i.e., neither rhythmic nor arrhythmic. The clock genes NR1D1, NR1D2 and CRY2 were the most rhythmic, while CLOCK and ARNTL were the least rhythmic. Overall, the rhythmicity scores for septic shock patients were significantly (p < 0.0001) lower (0.23 ± 0.26) compared to the control group (12 healthy young men, 0.70 ± 0.18). In addition, the expression of clock genes CRY1, NR1D1, NR1D2, DBP, and PER2 was suppressed in septic shock patients and CRY2 was significantly upregulated compared to controls. CONCLUSION: Molecular rhythms in immune cells of septic shock patients were substantially altered and decreased compared to healthy young men. The decrease in rhythmicity was clock gene-dependent. The loss of rhythmicity and down-regulation of clock gene expression might be caused by sepsis and might further deteriorate immune responses and organ injury, but further studies are necessary to understand underlying pathophysiological mechanisms. Trail registration Clinical trial registered with www.ClinicalTrials.gov (NCT02044575) on 24 January 2014.

Effects of Daytime Dry Fasting on Hydration, Glucose Metabolism and Circadian Phase: A Prospective Exploratory Cohort Study in Bahá'í Volunteers.

Background: Religiously motivated Bahá'í fasting (BF) is a form of intermittent dry fasting celebrated by abstaining from food and drinks during daylight hours every year in March for 19 consecutive days. Aim: To test the safety and effects of BF on hydration, metabolism, and the circadian clock. Methods: Thirty-four healthy Bahá'í volunteers (15 women) participated in this prospective, exploratory cohort study. Laboratory examinations were carried out in four study visits: before fasting (V0), in the third week of fasting (V1) as well as 3 weeks (V3) and 3 months (V4) after fasting. Data collection included blood and urine samples, anthropometric measurements and bioelectrical impedance analysis. At V0 and V1, 24- and 12-hour urine and serum osmolality were measured. At V0-V2, alterations in the circadian clock phase were monitored in 16 participants. Our study was augmented by an additional survey with 144 healthy Bahá'í volunteers filling out questionnaires and with subgroups attending metabolic measurements (n = 11) and qualitative interviews (n = 13), the results of which will be published separately. Results: Exploratory data analysis revealed that serum osmolality (n = 34, p < 0.001) and 24-hour urine osmolality (n = 34, p = 0.003) decreased during daytime fasting but remained largely within the physiological range and returned to pre-fasting levels during night hours. BMI (body mass index), total body fat mass, and resting metabolic rate decreased during fasting (n = 34, p < 0.001), while body cell mass and body water appeared unchanged. The circadian phase estimated by transcript biomarkers of blood monocytes advanced by 1.1 h (n = 16, p < 0.005) during fasting and returned to pre-fasting values 3 weeks after fasting. Most observed changes were not detectable anymore 3 months after fasting. Conclusions: Results indicate that BF (Bahá'í fasting) is safe, has no negative effects on hydration, can improve fat metabolism and can cause transient phase shifts of circadian rhythms. Trial Registration:https://www.clinicaltrials.gov/, identifier: NCT03443739.

Regulation of Rest, Rather Than Activity, Underlies Day-Night Activity Differences in Mice.

The suprachiasmatic nucleus (SCN), which serves as the central pacemaker in mammals, regulates the 24-h rhythm in behavioral activity. However, it is currently unclear whether and how bouts of activity and rest are regulated within the 24-h cycle (i.e., over ultradian time scales). Therefore, we used passive infrared sensors to measure temporal behavior in mice housed under either a light-dark (LD) cycle or continuous darkness (DD). We found that a probabilistic Markov model captures the ultradian changes in the behavioral state over a 24-h cycle. In this model, the animal's behavioral state in the next time interval is determined solely by the animal's current behavioral state and by the "toss" of a proverbial "biased coin." We found that the bias of this "coin" is regulated by light input and by the phase of the clock. Moreover, the bias of this "coin" for an animal is related to the average length of rest and activity bouts in that animal. In LD conditions, the average length of rest bouts was greater during the day compared to during the night, whereas the average length of activity bouts was greater during the night compared to during the day. Importantly, we also found that day-night changes in the rest bout lengths were significantly greater than day-night changes in the activity bout lengths. Finally, in DD conditions, the activity and rest bouts also differed between subjective night and subjective day, albeit to a lesser extent compared to LD conditions. The ultradian regulation represented by the model does not result in ultradian rhythms, although some weak ultradian rhythms are present in the data. The persistent differences in bout length over the circadian cycle following loss of the external LD cycle indicate that the central pacemaker plays a role in regulating rest and activity bouts on an ultradian time scale.

Amplitude Effects Allow Short Jet Lags and Large Seasonal Phase Shifts in Minimal Clock Models.

Mathematical models of varying complexity have helped shed light on different aspects of circadian clock function. In this work, we question whether minimal clock models (Goodwin models) are sufficient to reproduce essential phenotypes of the clock: a small phase response curve (PRC), fast jet lag, and seasonal phase shifts. Instead of building a single best model, we take an approach where we study the properties of a set of models satisfying certain constraints; here, a 1h-pulse PRC with a range of 3h and clock periods between 22h and 26h is designed. Surprisingly, almost all these randomly parameterized models showed a 4h change in phase of entrainment between long and short days and jet lag durations of three to seven days in advance and delay. Moreover, intrinsic clock period influenced jet lag duration and entrainment amplitude and phase. Fast jet lag was realized in this model by means of an interesting amplitude effect: the association between clock amplitude and clock period termed "twist." This twist allows amplitude changes to speed up and slow down clocks enabling faster shifts. These findings were robust to the addition of positive feedback to the model. In summary, the known design principles of rhythm generation - negative feedback, long delay, and switch-like inhibition (we review these in detail) - are sufficient to reproduce the essential clock phenotypes. Furthermore, amplitudes play a role in determining clock properties and must be always considered, although they are difficult to measure.

Conceptual Models of Entrainment, Jet Lag, and Seasonality.

Understanding entrainment of circadian rhythms is a central goal of chronobiology. Many factors, such as period, amplitude, Zeitgeber strength, and daylength, govern entrainment ranges and phases of entrainment. We have tested whether simple amplitude-phase models can provide insight into the control of entrainment phases. Using global optimization, we derived conceptual models with just three free parameters (period, amplitude, and relaxation rate) that reproduce known phenotypic features of vertebrate clocks: phase response curves (PRCs) with relatively small phase shifts, fast re-entrainment after jet lag, and seasonal variability to track light onset or offset. Since optimization found multiple sets of model parameters, we could study this model ensemble to gain insight into the underlying design principles. We found complex associations between model parameters and entrainment features. Arnold onions of representative models visualize strong dependencies of entrainment on periods, relative Zeitgeber strength, and photoperiods. Our results support the use of oscillator theory as a framework for understanding the entrainment of circadian clocks.

Beyond spikes: Multiscale computational analysis of in vivo long-term recordings in the cockroach circadian clock.

The circadian clock of the nocturnal Madeira cockroach is located in the accessory medulla, a small nonretinotopic neuropil in the brain's visual system. The clock comprises about 240 neurons that control rhythms in physiology and behavior such as sleep-wake cycles. The clock neurons contain an abundant number of partly colocalized neuropeptides, among them pigment-dispersing factor (PDF), the insects' most important circadian coupling signal that controls sleep-wake rhythms. We performed long-term loose-patch clamp recordings under 12:12-hr light-dark cycles in the cockroach clock in vivo. A wide range of timescales, from milliseconds to seconds, were found in spike and field potential patterns. We developed a framework of wavelet transform-based methods to detect these multiscale electrical events. We analyzed frequencies and patterns of events with interesting dynamic features, such as mixed-mode oscillations reminiscent of sharp-wave ripples. Oscillations in the beta/gamma frequency range (20-40 Hz) were observed to rise at dawn, when PDF is released, peaking just before the onset of locomotor activity of the nocturnal cockroach. We expect that in vivo electrophysiological recordings combined with neuropeptide/antagonist applications and behavioral analysis will determine whether specific patterns of electrical activity recorded in the network of the cockroach circadian clock are causally related to neuropeptide-dependent control of behavior.

Ultradian Rhythms in the Transcriptome of Neurospora crassa.

In many organisms, the circadian clock drives rhythms in the transcription of clock-controlled genes that can be either circadian (∼24-hr period) or ultradian (<24-hr period). Ultradian rhythms with periods that are a fraction of 24 hr are termed harmonics. Several harmonic transcripts were discovered in the mouse liver, but their functional significance remains unclear. Using a model-based analysis, we report for the first time ∼7-hr third harmonic transcripts in Neurospora crassa, a well-established fungal circadian model organism. Several third harmonic genes are regulated by female fertility 7 (FF-7), whose transcript itself is third harmonic. The knockout of circadian output regulator CSP1 superimposes circadian rhythms on the third harmonic genes, whereas the knockout of stress response regulator MSN1 converts third harmonic rhythms to second harmonic rhythms. The 460 ∼7-hr genes are co-regulated in two anti-phasic groups in multiple genotypes and include kinases, chromatin remodelers, and homologs of harmonic genes in the mouse liver.

High-accuracy determination of internal circadian time from a single blood sample.

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 für Bildung und Forschung, Germany (FKZ: 13N13160 and 13N13162) and Intellux GmbH, Germany.

Mining for novel candidate clock genes in the circadian regulatory network.

BACKGROUND: Most physiological processes in mammals are temporally regulated by means of a master circadian clock in the brain and peripheral oscillators in most other tissues. A transcriptional-translation feedback network of clock genes produces near 24 h oscillations in clock gene and protein expression. Here, we aim to identify novel additions to the clock network using a meta-analysis of public chromatin immunoprecipitation sequencing (ChIP-seq), proteomics and protein-protein interaction data starting from a published list of 1000 genes with robust transcriptional rhythms and circadian phenotypes of knockdowns. RESULTS: We identified 20 candidate genes including nine known clock genes that received significantly high scores and were also robust to the relative weights assigned to different data types. Our scoring was consistent with the original ranking of the 1000 genes, but also provided novel complementary insights. Candidate genes were enriched for genes expressed in a circadian manner in multiple tissues with regulation driven mainly by transcription factors BMAL1 and REV-ERB α,ß. Moreover, peak transcription of candidate genes was remarkably consistent across tissues. While peaks of the 1000 genes were distributed uniformly throughout the day, candidate gene peaks were strongly concentrated around dusk. Finally, we showed that binding of specific transcription factors to a gene promoter was predictive of peak transcription at a certain time of day and discuss combinatorial phase regulation. CONCLUSIONS: Combining complementary publicly-available data targeting different levels of regulation within the circadian network, we filtered the original list and found 11 novel robust candidate clock genes. Using the criteria of circadian proteomic expression, circadian expression in multiple tissues and independent gene knockdown data, we propose six genes (Por, Mtss1, Dgat2, Pim3, Ppp1r3b, Upp2) involved in metabolism and cancer for further experimental investigation. The availability of public high-throughput databases makes such meta-analysis a promising approach to test consistency between sources and tap their entire potential.