Mechanism Of Circadian Oscillations Research Articles

Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines.

Circadian clocks allow organisms to predict environmental changes caused by the rotation of the Earth. Although circadian rhythms are widespread among different taxa, the core components of circadian oscillators are not conserved and differ between bacteria, plants, animals and fungi. Stramenopiles are a large group of organisms in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have investigated cell division and molecular rhythms in Nannochloropsis species. In the four strains tested, cell division occurred principally during the night period under diel conditions; however, these rhythms damped within 2-3days after transfer to constant light. We developed firefly luciferase reporters for the long-term monitoring of invivo transcriptional rhythms in two Nannochlropsis species, Nannochloropsis oceanica CCMP1779 and Nannochloropsis salina CCMP537. The reporter lines express anticipatory behavior under light/dark cycles and free-running bioluminescence rhythms with periods of ~21-31h that damped within ~3-4days under constant light. Using different entrainment regimes, we demonstrate that these rhythms are modulated by a circadian-type oscillator. In addition, the phase of free-running luminescence rhythms can be modulated pharmacologically using a CK1 ε/δ inhibitor, suggesting a role of this kinase in the Nannochloropsis clock. Together with the molecular and genomic tools available for Nannochloropsis species, these reporter lines represent an excellent system for future studies on the molecular mechanisms of stramenopile circadian oscillators.

Clock-Enhancing Small Molecules and Potential Applications in Chronic Diseases and Aging.

Normal physiological functions require a robust biological timer called the circadian clock. When clocks are dysregulated, misaligned, or dampened, pathological consequences ensue, leading to chronic diseases and accelerated aging. An emerging research area is the development of clock-targeting compounds that may serve as drug candidates to correct dysregulated rhythms and hence mitigate disease symptoms and age-related decline. In this review, we first present a concise view of the circadian oscillator, physiological networks, and regulatory mechanisms of circadian amplitude. Given a close association of circadian amplitude dampening and disease progression, clock-enhancing small molecules (CEMs) are of particular interest as candidate chronotherapeutics. A recent proof-of-principle study illustrated that the natural polymethoxylated flavonoid nobiletin directly targets the circadian oscillator and elicits robust metabolic improvements in mice. We describe mood disorders and aging as potential therapeutic targets of CEMs. Future studies of CEMs will shed important insight into the regulation and disease relevance of circadian clocks.

Analysis of Circadian Rhythms in the Basal Filamentous Ascomycete Pyronema confluens.

Many organisms use circadian clocks to adapt to daily changes in the environment. Major insights into the molecular mechanisms of circadian oscillators have been gained through studies of the model organism Neurospora crassa; however, little is known about molecular components of circadian clocks in other fungi. An important part of the N. crassa circadian clock is the frequency (frq) gene, homologs of which can be found in Sordariomycetes, Dothideomycetes, and Leotiomycetes, but not Eurotiomycetes. Recently, we identified a frq homolog in Pyronema confluens, a member of the early-diverging Pezizomycete lineage of filamentous ascomycetes. The P. confluens FRQ shares many conserved domains with the N. crassa FRQ. However, there is no known morphological phenotype showing overt circadian rhythmicity in P. confluens. To investigate whether a molecular clock is present, we analyzed frq transcription in constant darkness, and found circadian oscillation of frq with a peak in the subjective morning. This rhythm was temperature compensated. To identify additional clock-controlled genes, we performed RNA sequencing of two time points (subjective morning and evening). Circadian expression of two morning-specific genes was verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) over a full time course, whereas expression of two putative morning-specific and five putative evening-specific genes could not be verified as circadian. frq expression was synchronized, but not entrained by light. In summary, we have found evidence for two of the three main properties of circadian rhythms (free-running rhythm, temperature compensation) in P. confluens, suggesting that a circadian clock with rhythmically expressed frq is present in this basal filamentous ascomycete.

Cognitive Function and Calcium. Ca2+-dependent regulatory mechanism of circadian clock oscillation and its relevance to neuronal function

Circadian clock generates a variety of biological rhythms such as sleep/wake cycles and blood hormone rhythms. The circadian clock also bolsters daily mental activities. In fact, abnormalities of the circadian rhythms are found in several neurological disorders. The circadian clock has two important functions: (i) a cell-autonomous oscillatory function and (ii) a phase-adjusting function that synchronizes the clock oscillation with environmental cycling conditions such as light/dark cycle. Behavioral rhythms are controlled by the central clock in hypothalamic suprachiasmatic nucleus (SCN). The central clock orchestrates peripheral clocks in the other tissues via neuronal connection and/or actions of humoral factors. The molecular mechanism of the cell-autonomous clock is based on transcriptional feedback regulation of clock genes by their encoded products. Ca2+ is essential for not only the light response of the clock but also the cell autonomous oscillation mechanism. This article provides an overview of recent progress in studies of Ca2+-dependent regulatory mechanism of the molecular clockwork.

Feedback Control of Biological Rhythm in Crassulacean Acid Metabolism by CO2-Uptake Signal

The mechanism of endogenous circadian photosynthesis oscillations of plants performing crassulacean acid metabolism (CAM) is investigated in terms of a nonlinear theoretical model. Blasius et al. used throughout continuous time differential equations which adequately reflect the CAM dynamics. The model shows regular endogenous limit cycle oscillations that are stable for a wide range of temperatures in a manner that complies well with experimental data. We have recently presented four types of the feedback controllers for the phase shift of the CAM model of the single cell using the light intensity as an input. In this paper, we deal with the synchronization issues in the coupled cells with light intensity input. We show that the strong coupling causes the unsynchronization of the cells.

Mechanism of the Neurospora circadian clock, a FREQUENCY-centric view.

Circadian clocks are self-sustaining timekeepers found in almost all organisms on earth. The filamentous fungus Neurospora crassa is a preeminent model for eukaryotic circadian clocks. Investigations of the Neurospora circadian clock system have led to elucidation of circadian clock regulatory mechanisms that are common to all eukaryotes. In this work, we will focus on the Neurospora circadian oscillator mechanism with an emphasis on the regulation of the core clock component FREQUENCY.

Structural and biophysical methods to analyze clock function and mechanism.

Structural approaches have provided insight into the mechanisms of circadian clock oscillators. This review focuses upon the myriad structural methods that have been applied to the molecular architecture of cyanobacterial circadian proteins, their interactions with each other, and the mechanism of the KaiABC posttranslational oscillator. X-ray crystallography and solution NMR were deployed to gain an understanding of the three-dimensional structures of the three proteins KaiA, KaiB, and KaiC that make up the inner timer in cyanobacteria. A hybrid structural biology approach including crystallography, electron microscopy, and solution scattering has shed light on the shapes of binary and ternary Kai protein complexes. Structural studies of the cyanobacterial oscillator demonstrate both the strengths and the limitations of the divide-and-conquer strategy. Thus, investigations of complexes involving domains and/or peptides have afforded valuable information into Kai protein interactions. However, high-resolution structural data are still needed at the level of complexes between the 360-kDa KaiC hexamer that forms the heart of the clock and its KaiA and KaiB partners.

Effect of Streptozotocin-induced Diabetes on Clock Gene Expression in Tissues Inside and Outside the Blood-brain Barrier in Rat

The circadian system allows organisms to remain synchronized with rhythmic environmental changes with a 24-h period. The molecular mechanism of circadian oscillations is based on the rhythmic expression of clock genes organized in feedback loops. Alterations in the circadian system contribute to the development of several pathological conditions including diabetes, but the exact mechanisms responsible for such alterations are not known. Therefore, we employed streptozotocin-induced diabetes to elucidate the influence of metabolic changes on clock gene (clock, npas2, per2) expression in peripheral oscillators in tissues inside (frontal cortex, cerebellum) and outside (heart, kidney) the blood-brain barrier. Diabetes was induced by streptozotocin injection. Seventeen days later, sampling was performed during a 24-h cycle. Gene expression was measured using real-time PCR. We observed a phase advance in rhythmic clock gene expression in the heart and kidney of diabetic rats. The study also focused on the possible role of npas2 in locomotor activity regulation in diabetic animals. The most pronounced changes were observed in the frontal cortex, which displayed up-regulation of npas2 expression. A change in locomotor activity was observed in diabetic rats during the dark phase of the 24-h cycle. We suggest that the altered function of the frontal cortex induced by diabetes might contribute to the modified behavior of diabetic rats.

Adaptive Estimation of Biological Rhythm in Crassulacean Acid Metabolism with Critical Manifold

The mechanism of endogenous circadian photosynthesis oscillations of plants performing crassulacean acid metabolism (CAM) is investigated in terms of a nonlinear theoretical model. Blasius et al. used throughout continuous time differential equations which adequately reflect the CAM dynamics. The model shows regular endogenous limit cycle oscillations that are stable for a wide range of temperatures in a manner that complies well with experimental data. In this paper, we pay attention to the approximation of the fast modes of the CAM dynamics. Using the zero-epsilon approximation of the slow manifold, we derive the critical manifold that is defined by two algebraic nonlinear equations. The critical manifold allows us to give the algebraic estimate of the order of the tonoplast membrane. The dynamic equation of the order of the tonoplast membrane includes the nonlinear function that gives the equilibrium value of the lipid order of tonoplast functions as a hysteresis switch. We identify the nonlinear function with the measurement signals. Using the basis function expansion of the nonlinear and the critical manifold, we propose an adaptive observer to estimate the tonoplast order and the nonlinear function.

Tracking circadian rhythms of bone mineral deposition in murine calvarial organ cultures

Osteoblasts, which orchestrate the deposition of small apatite crystals through the expression of nucleating proteins, have been shown to also express clock genes associated with the circadian signaling pathway. We hypothesized that protein-mediated bone mineralization may be linked to circadian oscillator mechanisms functioning in peripheral bone tissue. In this study, Per1 expression in ex vivo neonatal murine calvaria organ cultures was monitored for 6 days using a Per1-luciferase transgene as a bioluminescent indicator of clock function. Fluctuations in Per1 expression had a period of 25 ± 4 hours (n = 14) with early expression at CT09:59 ± 03:37 (CT = circadian time). We also established the kinetics of mineral deposition in developing bone by using noninvasive Raman microscopy to track mineral accumulation in calvarial tissue. The content and quality of newly deposited mineral was continually examined at the interparietal bone/fontanel boundary for a period of 6 days with 1-hour temporal resolution. Using this approach, mineralization over time exhibited bursts of mineral deposition followed by little or no deposition, which was recurrent with a periodicity of 26.8 ± 9.6 hours. As many as six near-daily mineralization events were observed in the calvaria before deposition ceased. Earliest mineralization events occurred at CT16:51 ± 03:45, which is 6 hours behind Per1 expression. These findings are consistent with the hypothesis that mineralization in developing bone tissue is regulated by a local circadian oscillator mechanism.

Transcriptional corepressor TOPLESS complexes with pseudoresponse regulator proteins and histone deacetylases to regulate circadian transcription

Circadian clocks are ubiquitous molecular time-keeping mechanisms that coordinate physiology and metabolism and provide an adaptive advantage to higher plants. The central oscillator of the plant clock is composed of interlocked feedback loops that involve multiple repressive factors acting throughout the circadian cycle. Pseudo response regulators (PRRs) comprise a five-member family that is essential to the function of the central oscillator. PRR5, PRR7, and PRR9 can bind the promoters of the core clock genes circadian clock associated 1 (CCA1) and late elongated hypocotyl (LHY) to restrict their expression to near dawn, but the mechanism has been unclear. Here we report that members of the plant Groucho/Tup1 corepressor family, topless/topless-related (TPL/TPR), interact with these three PRR proteins at the CCA1 and LHY promoters to repress transcription and alter circadian period. This activity is diminished in the presence of the inhibitor trichostatin A, indicating the requirement of histone deacetylase for full TPL activity. Additionally, a complex of PRR9, TPL, and histone deacetylase 6, can form in vivo, implicating this tripartite association as a central repressor of circadian gene expression. Our findings show that the TPL/TPR corepressor family are components of the central circadian oscillator mechanism and reinforces the role of this family as central to multiple signaling pathways in higher plants.

Clock Gene Variation Is Associated with Breeding Phenology and Maybe under Directional Selection in the Migratory Barn Swallow

BackgroundIn diverse taxa, photoperiodic responses that cause seasonal physiological and behavioural shifts are controlled by genes, including the vertebrate Clock orthologues, that encode for circadian oscillator mechanisms. While the genetic network behind circadian rhythms is well described, relatively few reports exist of the phenological consequences of and selection on Clock genes in the wild. Here, we investigated variation in breeding phenology in relation to Clock genetic diversity in a long-distance migratory bird, the barn swallow (Hirundo rustica).Methodology/Principal FindingsIn a sample of 922 adult barn swallows from a single population breeding in Italy we found one very common (Q7) and three rare (Q5, Q6, Q8) length variants of a functionally significant polyglutamine repeat. Rare (2.9%) Q7/Q8 heterozygous females, but not males, bred significantly later than common (91.5%) Q7/Q7 females, consistent with the expectation that ‘long’ alleles cause late breeding, as observed in a resident population of another bird species. Because breeding date depends on arrival date from migration, present results suggest that the association between breeding date and Clock might be mediated by migration phenology. In addition, fecundity selection appears to be operating against Q7/Q8 because late migrating/breeding swallows have fewer clutches per season, and late breeding has additional negative selection effects via reduced offspring longevity. Genotype frequencies varied marginally non-significantly with age, as Q7/Q8 frequency showed a 4-fold reduction in old individuals. This result suggests negative viability selection against Q7/Q8, possibly mediated by costs of late breeding.Conclusions/SignificanceThis is the first study of migratory birds showing an association between breeding phenology and Clock genotype and suggesting that negative selection occurs on a phenologically deviant genotype. Low polymorphism at Clock may constrain microevolutionary phenological response to changing climate, and may thus contribute to the decline of barn swallow populations.

Analysis of theDrosophila ClockPromoter Reveals Heterogeneity in Expression between Subgroups of Central Oscillator Cells and Identifies a Novel Enhancer Region

The CLOCK-CYCLE (CLK-CYC) heterodimer lies at the heart of the circadian oscillator mechanism in Drosophila, yet little is known about the identity of transcription factors that regulate the expression of Clk and/or cyc. Here, the authors have used a transgenic approach to isolate regions of the Clk locus that are necessary for expression in central oscillator neurons in the adult fly brain. This analysis shows that central clock cells can be subdivided into 2 distinct groups based on Clk gene regulation. Expression in the lateral neuron (LN), dorsal neuron 1 anterior (DN1a) and 2 (DN2) clusters requires cis-elements located in a 122 base-pair (bp) region (-206 to -84) of the Clk promoter. Expression in the remaining dorsal neurons, 1 posterior (DN1p) and 3 (DN3) and the lateral posterior neurons (LPN), requires regulatory elements located in the -856 to -206 region. In addition, expression in photoreceptors of the compound eye is enhanced by cis-elements located in a 3rd region of the Clk locus (-1982 to -856). This region also enhances expression in nonoscillator cells in the brain including the Kenyon cells, but expression in these neurons is suppressed by regulatory sites located further upstream of -1982. The authors' analysis reveals clear heterogeneity in Clk gene expression in the adult brain and provides a necessary focus to isolate novel transcription factors that bind at the Clk locus to regulate expression in different oscillator neuron subgroups. These results also suggest that the DN1a/DN2 neurons may have more molecular commonality with the LNs than they do with the DN1p/DN3/LPN neurons. Finally, this analysis has generated new transgenic lines that will enable genes to be misexpressed in subgroups of central oscillator cells that have previously been resistant to discrete genetic manipulation. Hence, these lines provide important new tools to facilitate a more complete dissection of the neural network that regulates output rhythms in physiology and behavior.

Mechanism of Robust Circadian Oscillation of KaiC Phosphorylation In Vitro

By incubating the mixture of three cyanobacterial proteins, KaiA, KaiB, and KaiC, with ATP in vitro, T. Kondo and his colleagues in recent work reconstituted the robust circadian rhythm of the phosphorylation level of KaiC. This finding indicates that protein-protein interactions and the associated hydrolysis of ATP suffice to generate the circadian rhythm. Several theoretical models have been proposed to explain the rhythm generated in this “protein-only” system, but the clear criterion to discern different possible mechanisms was not known. In this article, we discuss a model based on two basic assumptions: the assumption of the allosteric transition of a KaiC hexamer and the assumption of the monomer exchange between KaiC hexamers. The model shows a stable rhythmic oscillation of the phosphorylation level of KaiC, which is robust against changes in concentration of Kai proteins. We show that this robustness gives a clue to distinguish different possible mechanisms. We also discuss the robustness of oscillation against the change in the system size. Behaviors of the system with the cellular or subcellular size should shed light on the role of the protein-protein interactions in in vivo circadian oscillation.

Circadian Genes Are Expressed during Early Development in Xenopus laevis

BackgroundCircadian oscillators are endogenous time-keeping mechanisms that drive twenty four hour rhythmic changes in gene expression, metabolism, hormone levels, and physical activity. We have examined the developmental expression of genes known to regulate circadian rhythms in order to better understand the ontogeny of the circadian clock in a vertebrate.Methodology/Principal FindingsIn this study, genes known to function together in part of the core circadian oscillator mechanism (xPeriod1, xPeriod2, and xBmal1) as well as a rhythmic, clock-controlled gene (xNocturnin) were analyzed using in situ hybridization in embryos from neurula to late tailbud stages. Each transcript was present in the developing nervous system in the brain, eye, olfactory pit, otic vesicle and at lower levels in the spinal cord. These genes were also expressed in the developing somites and heart, but at different developmental times in peripheral tissues (pronephros, cement gland, and posterior mesoderm). No difference was observed in transcript levels or localization when similarly staged embryos maintained in cyclic light were compared at two times of day (dawn and dusk) by in situ hybridization. Quantitation of xBmal1 expression in embryonic eyes was also performed using qRT-PCR. Eyes were isolated at dawn, midday, dusk, and midnight (cylic light). No difference in expression level between time-points was found in stage 31 eyes (p = 0.176) but stage 40 eyes showed significantly increased levels of xBmal1 expression at midnight (RQ = 1.98+/−0.094) when compared to dawn (RQ = 1+/−0.133; p = 0.0004).Conclusions/SignificanceWe hypothesize that when circadian genes are not co-expressed in the same tissue during development that it may indicate pleiotropic functions of these genes that are separate from the timing of circadian rhythm. Our results show that all circadian genes analyzed thus far are present during early brain and eye development, but rhythmic gene expression in the eye is not observed until after stage 31 of development.

Inducible and Reversible Clock Gene Expression in Brain Using the tTA System for the Study of Circadian Behavior

The mechanism of circadian oscillations in mammals is cell autonomous and is generated by a set of genes that form a transcriptional autoregulatory feedback loop. While these “clock genes” are well conserved among animals, their specific functions remain to be fully understood and their roles in central versus peripheral circadian oscillators remain to be defined. We utilized the in vivo inducible tetracycline-controlled transactivator (tTA) system to regulate Clock gene expression conditionally in a tissue-specific and temporally controlled manner. Through the use of Secretogranin II to drive tTA expression, suprachiasmatic nucleus– and brain-directed expression of a tetO::ClockΔ19 dominant-negative transgene lengthened the period of circadian locomotor rhythms in mice, whereas overexpression of a tetO::Clockwt wild-type transgene shortened the period. Low doses (10 μg/ml) of doxycycline (Dox) in the drinking water efficiently inactivated the tTA protein to silence the tetO transgenes and caused the circadian periodicity to return to a wild-type state. Importantly, low, but not high, doses of Dox were completely reversible and led to a rapid reactivation of the tetO transgenes. The rapid time course of tTA-regulated transgene expression demonstrates that the CLOCK protein is an excellent indicator for the kinetics of Dox-dependent induction/repression in the brain. Interestingly, the daily readout of circadian period in this system provides a real-time readout of the tTA transactivation state in vivo. In summary, the tTA system can manipulate circadian clock gene expression in a tissue-specific, conditional, and reversible manner in the central nervous system. The specific methods developed here should have general applicability for the study of brain and behavior in the mouse.

The Rhythms of Life: Circadian Output Pathways in Neurospora

Research in Neurospora crassa pioneered the isolation of clock-controlled genes (ccgs), and more than 180 ccgs have been identified that function in various aspects of the fungal life cycle. Many clock-controlled genes are associated with damage repair, stress responses, intermediary metabolism, protein synthesis, and development. The expression of most of these genes peaks just before dawn and appears to prepare the cells for the desiccation, mutagenesis, and stress caused by sunlight. Progress on characterization of the output signaling pathways from the circadian oscillator mechanism to the ccgs is discussed. The authors also review evidence suggesting that, similar to other clock model organisms, a connection exists between the redox state of the cell and the Neurospora clock. The authors speculate that the clock system may sense not only light but also the redox potential of the cell through one of the PAS domains of the core clock components WC-1 or WC-2.

Persistence of a behavioral food-anticipatory circadian rhythm following dorsomedial hypothalamic ablation in rats

Circadian rhythms of behavior in rodents are regulated by a system of circadian oscillators, including a master light-entrainable pacemaker in the suprachiasmatic nucleus that mediates synchrony to the day-night cycle, and food-entrainable oscillators located elsewhere that generate rhythms of food-anticipatory activity (FAA) synchronized to daily feeding schedules. Despite progress in elucidating neural and molecular mechanisms of circadian oscillators, localization of food-entrainable oscillators driving FAA remains an enduring problem. Recent evidence suggests that the dorsomedial hypothalamic nucleus (DMH) may function as a final common output for behavioral rhythms and may be critical for the expression of FAA (Gooley JJ, Schomer A, and Saper CB. Nat Neurosci 9: 398-407, 2006). To determine whether the reported loss of FAA by DMH lesions is specific to one behavioral measure or generalizes to other measures, rats received large radiofrequency lesions aimed at the DMH and were recorded in cages with movement sensors. Total and partial DMH ablation was associated with a significant attenuation of light-dark-entrained activity rhythms during ad libitum food access, because of a selective reduction in nocturnal activity. When food was restricted to a single 3-h daily meal in the middle of the lights-on period, all DMH and intact rats exhibited significant FAA. The rhythm of FAA persisted during a 48-h food deprivation test and reappeared during a 72-h deprivation test after ad libitum food access. The DMH is not the site of oscillators or entrainment pathways necessary for all manifestations of FAA, but may participate on the output side of this circadian function.

Oscillatory Mechanisms and Functional Differentiation of Circadian Clocks

Circadian rhythms govern behavior, physiology and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and mammals. The core molecular mechanism underlying circadian oscillation in Drosophila is comprised of two interlocked feedback transcriptional loops: a period (per)/ timeless (tim) loop as a negative loop, and a Clock (Clk)/cycle (cyc) loop as a positive one. The per/tim loop is initiated by CLK-CYC heterodimer which binds to the E-box sequences (CACGTG) in per and tim promoters. The per and tim gene are activated, and their products form a heterodimer (PER-TIM) in cytoplasm. The heterodimer then moves into the nucleus and inhibits CLK-CYC activity, there by repressing per/tim own transcriptions. The Clk/cyc positive loop is also initiated by CLK-CYC. CLK-CYC activates the expression of vrille (vri) and PAR domain protein 1 (Pdp1) both of which also have the E-box in the promoter region. While VRI acts as a negative regulator of Clk, Pdp1, which accumulates in a delayed fasion compared to VRI, activates the Clk transcription. Although the framework of molecular mechanism of circadian oscillation is extensively elucidated, there are many issues unsolved concerning to the basic features of circadian rhythm. For example, so far we don’t have the definitive explanation why the period of circadian rhythm is about 24 hours. Additionally, the molecular mechanism of temperature compensation of period, which is one of the most remarkable features of circadian rhythm, is still remained as absolute mystery. Also, we still don’t know the exact number of output genes whose expressions are controled by the feedback loops. To study how gene expression is globally regulated by the clock mechanism, we used a high-density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions and among them expression of 115 genes was still cycling in constant darkness, i.e. under a free-running condition. Unexpectedly, expression of a large number of genes cycled only under constant darkness. We are now doing behavior screenings for core clock genes by RNAi method. This project is the collaborate work with Hiroki Ueda (RIKEN CDB), Ryu Ueda, Kuniaki Takahashi (NIG), Kaoru Saigo (Univ. Tokyo) and Teiichi Tanimura (Kyushu Univ.).

Modeling the mammalian circadian clock: Sensitivity analysis and multiplicity of oscillatory mechanisms

We extend the study of a computational model recently proposed for the mammalian circadian clock (Proc. Natl Acad. Sci. USA 100 (2003) 7051). The model, based on the intertwined positive and negative regulatory loops involving the Per, Cry, Bmal1, and Clock genes, can give rise to sustained circadian oscillations in conditions of continuous darkness. These limit cycle oscillations correspond to circadian rhythms autonomously generated by suprachiasmatic nuclei and by some peripheral tissues. By using different sets of parameter values producing circadian oscillations, we compare the effect of the various parameters and show that both the occurrence and the period of the oscillations are generally most sensitive to parameters related to synthesis or degradation of Bmal1 mRNA and BMAL1 protein. The mechanism of circadian oscillations relies on the formation of an inactive complex between PER and CRY and the activators CLOCK and BMAL1 that enhance Per and Cry expression. Bifurcation diagrams and computer simulations nevertheless indicate the possible existence of a second source of oscillatory behavior. Thus, sustained oscillations might arise from the sole negative autoregulation of Bmal1 expression. This second oscillatory mechanism may not be functional in physiological conditions, and its period need not necessarily be circadian. When incorporating the light-induced expression of the Per gene, the model accounts for entrainment of the oscillations by light–dark (LD) cycles. Long-term suppression of circadian oscillations by a single light pulse can occur in the model when a stable steady state coexists with a stable limit cycle. The phase of the oscillations upon entrainment in LD critically depends on the parameters that govern the level of CRY protein. Small changes in the parameters governing CRY levels can shift the peak in Per mRNA from the L to the D phase, or can prevent entrainment. The results are discussed in relation to physiological disorders of the sleep–wake cycle linked to perturbations of the human circadian clock, such as the familial advanced sleep phase syndrome or the non-24h sleep–wake syndrome.