Mixtures of opposing phosphorylations within hexamers precisely time feedback in the cyanobacterial circadian clock

Circadian oscillations are generated by the purified cyanobacterial clock proteins, KaiA, KaiB, and KaiC, through rhythmic interactions that depend on multisite phosphorylation of KaiC. However, the mechanisms that allow these phosphorylation reactions to robustly control the timing of oscillations over a range of protein stoichiometries are not clear. We show that when KaiC hexamers consist of a mixture of differentially phosphorylated subunits, the two phosphorylation sites have opposing effects on the ability of each hexamer to bind to the negative regulator KaiB. We likewise show that the ability of the positive regulator KaiA to act on KaiC depends on the phosphorylation state of the hexamer and that KaiA and KaiB recognize alternative allosteric states of the KaiC ring. Using mathematical models with kinetic parameters taken from experimental data, we find that antagonism of the two KaiC phosphorylation sites generates an ultrasensitive switch in negative feedback strength necessary for stable circadian oscillations over a range of component concentrations. Similar strategies based on opposing modifications may be used to support robustness in other timing systems and in cellular signaling more generally.Circadian clocks are biological timing systems that allow organisms to anticipate and prepare for daily changes in the environment. A hallmark of a circadian oscillator is its ability to drive self-sustained rhythms in gene expression and behavior with a period close to 24 h, even in the absence of environmental cues (1). A general challenge for the biochemical machinery that generates rhythms is to precisely define the duration of the day in the face of perturbations, including fluctuations in the cellular abundance of the molecular components. The importance of maintaining precise circadian timing is underscored by experiments showing that mismatch between the clock period and the rhythms in the external environment results in health problems and fitness defects (2, 3).Although circadian clocks are found across all kingdoms of life, the Kai oscillator from cyanobacteria presents a uniquely powerful model system to study the design principles inherent in the molecular interactions that generate rhythms. A mixture of the purified proteins KaiA, KaiB, and KaiC results in stable oscillations in the phosphorylation state of KaiC in vitro that persist for many days and share many of the properties of circadian clocks in vivo (4–6). In particular, the oscillator can successfully generate near–24-h rhythms over a range of concentrations of the clock proteins both in vivo and in vitro (7–9), so fine-tuning of gene expression is not needed to support a functional clock. Much has been learned about the behavior of the isolated Kai proteins, including the determination of high-resolution crystal structures of all three components (10–12). A critical challenge that remains is to understand how the properties of the Kai proteins are integrated together in the full system to generate precisely timed rhythms.KaiC appears to be the central hub of timing information in the oscillator. Each KaiC molecule consists of two AAA+ family ATPase domains that consume the free energy of ATP hydrolysis to drive oscillations. Like many other members of this family, KaiC forms hexamers, and the enzymatic active sites are formed at the subunit interfaces where nucleotides are bound. The C-terminal, or CII, domain of KaiC has additional phosphotransferase activities that are unusual for the AAA+ family: it can phosphorylate and dephosphorylate two residues near the subunit interface, Ser431 and Thr432 (13). KaiC autokinase and autophosphatase activities occur at the same active site (14, 15). In isolation, KaiC has high phosphatase activity, but the enzyme is pushed toward kinase activity by the activator protein KaiA, which interacts directly with the KaiC C-terminal tail (16, 17). Roughly speaking, kinase activity predominates during the day, and phosphatase activity predominates during the night (18). Thus, understanding the feedback mechanisms that generate a precise time delay between these modes is crucial to understanding timing in the oscillator (19).Inactivation of KaiA and a transition from kinase to phosphatase mode occur when KaiB•KaiC complexes form, closing a negative feedback loop by sequestering KaiA in a ternary complex and leaving it unable to act on other KaiC molecules (20, 21). By temporarily removing KaiA molecules from their activating role, this molecular titration mechanism may act to synchronize the activity of all KaiC hexamers in the reaction (20, 22, 23). Phosphorylation and dephosphorylation proceed in a strongly ordered fashion so that in response to a change in KaiA activity, Thr432 is (de)phosphorylated first, followed later by Ser431 (18, 20, 21). It is known that phosphorylated Ser431 is important for allowing the formation of KaiB•KaiC complexes. However, recent work has made it clear that the binding of KaiB involves both KaiC domains—in particular, the slow ATPase activity of the N-terminal CI domain, which is not phosphorylated, is required for KaiB interaction (24, 25).Because of the importance of precisely timing negative feedback via KaiB•KaiC complex formation for generating appropriate rhythms (22), we wanted to understand the role of phosphorylation of the KaiC hexamer in controlling this process. The involvement of both KaiC domains suggests that information about phosphorylation in CII is communicated allosterically through changes in hexamer structure to the CI domain, potentially through ring–ring stacking interactions (24, 26). We therefore hypothesized that the KaiC phosphorylation sites on each subunit might act as allosteric regulators in the context of a hexameric ring so that phosphorylation of one subunit would alter the ability of all other subunits in the ring to engage with KaiA and KaiB, providing a cooperative mechanism to control the timing of these interactions.We conducted a series of biochemical experiments and perturbations to study the effect of altering the status of each phosphorylation site on the KaiC hexamer. To interpret these results, we then developed a mathematical model analogous to classical models of allosteric transitions in multimeric proteins. We constrain the kinetic parameters in this model using experimental measurements of rate constants, allowing us to compare the predictions of the model directly with data. We conclude that maintenance of circadian timing over a range of protein concentrations requires an effectively ultrasensitive switch in each KaiC hexamer from an exclusively KaiA-binding state to a state that can bind to KaiB as phosphorylation proceeds. This effect requires that KaiC hexamers consist of mixtures of differentially phosphorylated subunits, as would be produced by stochastic autophosphorylation of a hexamer. Ultrasensitivity results from opposing effects of phosphorylation on Thr432 and Ser431 in controlling a concerted transition within a given KaiC hexamer. Including this mechanism in the model is necessary to explain the experimentally observed tolerance of the system to altered protein concentrations.     

Ordered changes in histone modifications at the core of the Arabidopsis circadian clock

Circadian clock function in Arabidopsis thaliana relies on a complex network of reciprocal regulations among oscillator components. Here, we demonstrate that chromatin remodeling is a prevalent regulatory mechanism at the core of the clock. The peak-to-trough circadian oscillation is paralleled by the sequential accumulation of H3 acetylation (H3K56ac, K9ac), H3K4 trimethylation (H3K4me3), and H3K4me2. Inhibition of acetylation and H3K4me3 abolishes oscillator gene expression, indicating that both marks are essential for gene activation. Mechanistically, blocking H3K4me3 leads to increased clock-repressor binding, suggesting that H3K4me3 functions as a transition mark modulating the progression from activation to repression. The histone methyltransferase SET DOMAIN GROUP 2/ARABIDOPSIS TRITHORAX RELATED 3 (SDG2/ATXR3) might contribute directly or indirectly to this regulation because oscillator gene expression, H3K4me3 accumulation, and repressor binding are altered in plants misexpressing SDG2/ATXR3. Despite divergences in oscillator components, a chromatin-dependent mechanism of clock gene activation appears to be common to both plant and mammal circadian systems.     

Natural selection against a circadian clock gene mutation in mice

Circadian rhythms with an endogenous period close to or equal to the natural light–dark cycle are considered evolutionarily adaptive (“circadian resonance hypothesis”). Despite remarkable insight into the molecular mechanisms driving circadian cycles, this hypothesis has not been tested under natural conditions for any eukaryotic organism. We tested this hypothesis in mice bearing a short-period mutation in the enzyme casein kinase 1ε (tau mutation), which accelerates free-running circadian cycles. We compared daily activity (feeding) rhythms, survivorship, and reproduction in six replicate populations in outdoor experimental enclosures, established with wild-type, heterozygous, and homozygous mice in a Mendelian ratio. In the release cohort, survival was reduced in the homozygote mutant mice, revealing strong selection against short-period genotypes. Over the course of 14 mo, the relative frequency of the tau allele dropped from initial parity to 20%. Adult survival and recruitment of juveniles into the population contributed approximately equally to the selection for wild-type alleles. The expression of activity during daytime varied throughout the experiment and was significantly increased by the tau mutation. The strong selection against the short-period tau allele observed here contrasts with earlier studies showing absence of selection against a Period 2 (Per2) mutation, which disrupts internal clock function, but does not change period length. These findings are consistent with, and predicted by the theory that resonance of the circadian system plays an important role in individual fitness.Circadian clocks are a ubiquitous feature of life on earth, and serve to maintain synchrony of internal physiology with the external 24-h environment. Colin Pittendrigh, one of the founders of chronobiology, hypothesized that natural selection should favor circadian systems to operate in resonance with the external cycle (1, 2). A prediction from this hypothesis is that individuals exhibiting circadian rhythms with frequencies that are not in close resonance with the 24-h cycle should be selected against in nature. The hypothesis was initially supported by laboratory experiments in fly species that lived longer in a 24-h light–dark (LD) cycle than in non-24-h LD cycles (2–4). Stronger support emerged from dyadic competition experiments in batch cultures of cyanobacteria carrying single gene mutations affecting their circadian period (τ). Strains (either wild type or mutant) with a τ similar to the external LD cycle outcompeted strains with a τ different from the Zeitgeber (5, 6). Whether periods out of resonance with the external cycle entail a real fitness deficit in a natural setting has not been tested in any of these systems.The Ck1ε^tau (hereafter defined as the tau mutation) is a gain-of-function mutation (7) that accelerates the cellular dynamics of the circadian PERIOD protein (8, 9) and affects circadian behavior and physiology (10). It was first detected in Syrian hamsters (Mesocricetus auratus), where it causes τ to shorten with ∼2 h for each copy of the mutant allele (11). In mice, the same mutation shortens the circadian cycle to an almost identical extent (10). As a consequence of the accelerated circadian clockwork, both homozygote tau mice and hamsters are unable to entrain to 24-h LD cycles in the laboratory. Because its frequency deviates considerably from the natural 24-h cycle, the tau mutation provides an excellent model to study effects of deviant circadian periods on fitness in a natural setting. Here we report the consequences of deviant circadian rhythms in six replicate outdoor populations of mice. These populations were established with the release of mice, all born to two heterozygote parents, in identical enclosures, with ∼49% mutant tau alleles in a near Mendelian ratio in each pen. We used s.c. transponders to record each individual’s visits to feeders in each enclosure, which allowed us to quantify the rhythm of feeding activity and to keep track of each individual’s presence—and, hence, monitor lifespan, mortality, and the tau allele frequency in each population.     

Glucocorticoid regulation of the circadian clock modulates glucose homeostasis

Circadian clock genes are regulated by glucocorticoids; however, whether this regulation is a direct or secondary effect and the physiological consequences of this regulation were unknown. Here, we identified glucocorticoid response elements (GREs) at multiple clock genes and showed that 3 were directly regulated by the glucocorticoid receptor. We determined that a GRE within the core clock gene Per2 was continuously occupied during rhythmic expression and essential for glucocorticoid regulation of that gene in vivo. We further demonstrated that mice with a genomic deletion spanning this GRE expressed elevated leptin levels and were protected from glucose intolerance and insulin resistance on glucocorticoid treatment but not from muscle wasting. We conclude that Per2 is an integral component of a particular glucocorticoid regulatory pathway and that glucocorticoid regulation of the peripheral clock is selectively required for some actions of glucocorticoids.     

Melatonin regulates circadian electroretinogram rhythms in a dose- and time-dependent fashion

The circadian rhythm of the chick electroretinogram (ERG) is regulated by the indoleamine hormone melatonin. To determine if the concentration of melatonin or the time at which it was administered would have differential effects on ERG parameters, we conducted experiments analyzing the effects of melatonin at different times of the day. Circadian rhythms of a- and b-wave implicit times and amplitudes were observed in both light:dark (LD) and in continuous darkness (DD). Intramuscular melatonin administration of 1 mg/kg and 100 ng/kg decreased a- and b-wave amplitudes and increased a- and b-wave implicit times. This effect was significantly greater than that observed for 1 ng/kg melatonin, which had little to no effect over the saline controls. The effect of 1 mg/kg and 100 ng/kg melatonin on a- and b-wave amplitude in LD and on b-wave amplitude in DD was greater during the night (ZT/CT 17) than during the day (ZT/CT 5). The fold change in b-wave implicit time over that of controls was greater during the day (ZT/CT 5) than during the night (ZT/CT 17). These data indicate that melatonin may play a role in regulating a day and night functional shift in the retina, and that it does so via regulation of a retinal clock.     

Hugin+ neurons provide a link between sleep homeostat and circadian clock neurons

Sleep is controlled by homeostatic mechanisms, which drive sleep after wakefulness, and a circadian clock, which confers the 24-h rhythm of sleep. These processes interact with each other to control the timing of sleep in a daily cycle as well as following sleep deprivation. However, the mechanisms by which they interact are poorly understood. We show here that hugin⁺ neurons, previously identified as neurons that function downstream of the clock to regulate rhythms of locomotor activity, are also targets of the sleep homeostat. Sleep deprivation decreases activity of hugin⁺ neurons, likely to suppress circadian-driven activity during recovery sleep, and ablation of hugin⁺ neurons promotes sleep increases generated by activation of the homeostatic sleep locus, the dorsal fan-shaped body (dFB). Also, mutations in peptides produced by the hugin⁺ locus increase recovery sleep following deprivation. Transsynaptic mapping reveals that hugin⁺ neurons feed back onto central clock neurons, which also show decreased activity upon sleep loss, in a Hugin peptide–dependent fashion. We propose that hugin⁺ neurons integrate circadian and sleep signals to modulate circadian circuitry and regulate the timing of sleep.

Sleep is regulated by two processes, circadian and homeostatic (1). The endogenous circadian clock, together with its downstream pathways, is synchronized to external day–night cycles and determines the timing of sleep to produce 24-h rhythms in sleep and wake. The homeostatic process tracks sleep:wake history and generates sleep drive, based on the extent of wakefulness. Overtly, sleep homeostasis can be seen as an increase in sleep duration and depth after prolonged wakefulness. Generally, circadian and homeostatic processes are studied as separate mechanisms that regulate sleep, but they clearly intersect and are coordinated in a daily cycle to promote the onset and maintenance of sleep at night. Following sleep deprivation, the homeostatic system can drive sleep at the wrong time of day, but even under these conditions, interactions between the two systems determine the timing and duration of sleep. However, the neuronal mechanisms by which circadian and homeostatic pathways signal to each other are largely unknown.The functions and regulation of sleep are extensively studied in model organisms, such as Drosophila melanogaster (2). In the Drosophila brain, the circadian clock is expressed in ∼150 clock neurons that are organized into neuroanatomical groups, of which the ventral and dorsal lateral neurons are the most important for driving rhythms of locomotor activity (3, 4). The small ventrolateral neurons (s-LNvs) link to other brain regions through different circuits. One of those circuits connects the s-LNvs to the site of the motor ganglion, the thoracic nerve cord: s-LNvs → DN1s → Dh44⁺ neurons → hugin⁺ neurons→ ventral nerve cord (5). Dh44-expressing neurons in the pars intercerebralis regulate locomotor activity rhythms in part through the signaling of DH44 neuropeptide to hugin-expressing neurons in the subesophageal zone (SEZ) (6, 7). Dh44⁺ and hugin⁺ circadian output neurons do not contain canonical molecular clocks, but display cycling in neuronal activity or peptide release, likely under control of upstream circadian signals (7–9). Links between these neurons and loci regulating sleep homeostasis have not been identified yet.Regulation of sleep homeostasis in flies involves the central complex and mushroom body (10–13). Of particular importance is a group of sleep-promoting neurons labeled by the 23E10-GAL4 driver that projects to the dorsal fan-shaped body (dFB) neuropil in the central complex. These dFB neurons promote sleep when activated (14, 15), and they are required for normal sleep rebound after deprivation (16). 23E10⁺ neurons receive input signals from R5 ellipsoid body neurons, which track sleep need (17).As hugin⁺ neurons are significantly downstream of central clock neurons and close to behavioral outputs, we asked whether they also have a role in sleep. We find that hugin⁺ neurons are dispensable for determining daily sleep amount, but they show decreases in activity following sleep deprivation. They also receive projections from the dFB and counter sleep-promoting effects of 23E10⁺ neurons, such that ablation of hugin⁺ neurons enhances sleep driven by 23E10⁺ cells. Further supporting a role in sleep, mutations in Hugin peptides affect recovery sleep after deprivation and enhance sleep driven by 23E10⁺ cells. hugin⁺ neurons target PDF⁺ s-LNv clock neurons, which also show decreases in intracellular Ca²⁺ levels following sleep deprivation. Thus hugin⁺ neurons serve as an integrations site of signals from both the sleep homeostat and the circadian clock.     

No evidence for a phase delay in human circadian rhythms after a single morning melatonin administration

Although there is good consensus that a single administration of melatonin in the early evening can phase advance human circadian rhythms, the evidence for phase delay shifts to a single melatonin stimulus given in the early morning is sparse. We therefore carried out a double-blind randomized-order placebo-controlled study under modified constant routine (CR) conditions (58 hr bedrest under approximately 8 lux with sleep 23:00-07:00 hr) in nine healthy young men. A single (pharmacological) dose of melatonin (5 mg p.o.) or a placebo was administered at 07:00 hr on the first morning. Core body temperature (CBT) and heart rate (HR) were continuously recorded, and saliva was collected half-hourly for assay of melatonin. Neither the timing of the mid-range crossing times of temperature (MRCT) and HR rhythms, nor dim light melatonin onset (DLMOn) or offset (DLMOff) were phase shifted the day after melatonin administration compared with placebo. The only change was an altered wave form of the CBT rhythm: longer duration of higher-than-average temperature after melatonin administration. Under the same modified CR conditions we have previously demonstrated a clear phase advance of the above circadian rhythms following a single administration of 5 mg melatonin in the evening. This study's failure to find significant delays to a single administration does not negate other positive findings with multiple doses, which may be necessary for a 'weak zeitgeber'.     

Effect of light treatment on sleep and circadian rhythms in demented nursing home patients

OBJECTIVES: To determine whether fragmented sleep in nursing home patients would improve with increased exposure to bright light. DESIGN: Randomized controlled trial. SETTING: Two San Diego-area nursing homes. PARTICIPANTS: Seventy-seven (58 women, 19 men) nursing home residents participated. Mean age +/- standard deviation was 85.7 +/- 7.3 (range 60-100) and mean Mini-Mental State Examination was 12.8 +/- 8.8 (range 0-30). INTERVENTIONS: Participants were assigned to one of four treatments: evening bright light, morning bright light, daytime sleep restriction, or evening dim red light. MEASUREMENTS: Improvement in nighttime sleep quality, daytime alertness, and circadian activity rhythm parameters. RESULTS: There were no improvements in nighttime sleep or daytime alertness in any of the treatment groups. Morning bright light delayed the peak of the activity rhythm (acrophase) and increased the mean activity level (mesor). In addition, subjects in the morning bright light group had improved activity rhythmicity during the 10 days of treatment. CONCLUSION: Increasing exposure to morning bright light delayed the acrophase of the activity rhythm and made the circadian rhythm more robust. These changes have the potential to be clinically beneficial because it may be easier to provide nursing care to patients whose circadian activity patterns are more socially acceptable.     

Ambient temperature response establishes ELF3 as a required component of the core Arabidopsis circadian clock

Circadian clocks synchronize internal processes with environmental cycles to ensure optimal timing of biological events on daily and seasonal time scales. External light and temperature cues set the core molecular oscillator to local conditions. In Arabidopsis, EARLY FLOWERING 3 (ELF3) is thought to act as an evening-specific repressor of light signals to the clock, thus serving a zeitnehmer function. Circadian rhythms were examined in completely dark-grown, or etiolated, null elf3-1 seedlings, with the clock entrained by thermocycles, to evaluate whether the elf3 mutant phenotype was light-dependent. Circadian rhythms were absent from etiolated elf3-1 seedlings after exposure to temperature cycles, and this mutant failed to exhibit classic indicators of entrainment by temperature cues, consistent with global clock dysfunction or strong perturbation of temperature signaling in this background. Warm temperature pulses failed to elicit acute induction of temperature-responsive genes in elf3-1. In fact, warm temperature-responsive genes remained in a constitutively “ON” state because of clock dysfunction and, therefore, were insensitive to temperature signals in the normal time of day-specific manner. These results show ELF3 is broadly required for circadian clock function regardless of light conditions, where ELF3 activity is needed by the core oscillator to allow progression from day to night during either light or temperature entrainment. Furthermore, robust circadian rhythms appear to be a prerequisite for etiolated seedlings to respond correctly to temperature signals.     

Classification of circadian pain rhythms and pain characteristics in chronic pain patients: An observational study

This study aimed to perform cluster analysis in patients with chronic pain to extract groups with similar circadian rhythms and compare neuropathic pain and psychological factors among these groups to identify differences in pain-related outcomes. A total of 63 community-dwellers with pain lasting at least 3 months and Numerical Rating Scale scores of ≥2 were recruited from 3 medical institutions. Their pain circadian rhythms were evaluated over 7 days by measuring pain intensity at 6-time points per day using a 10-cm visual analog scale. Cluster analysis was performed using 6 variables with standardized visual analog scale values at 6-time points for individual participants to extract groups with similar pain circadian rhythms. The results of the Neuropathic Pain Symptom Inventory and psychological evaluations in each group were compared using the Kruskal–Wallis test. The results revealed 3 clusters with different circadian rhythms of pain. The total and evoked pain subscale Neuropathic Pain Symptom Inventory scores differed among the 3 clusters. The results suggest that a thorough understanding of circadian pain rhythms in chronic pain patients may facilitate the performance of activities of daily living and physical exercise from the perspective of pain management.     

Influence of the pineal gland and circadian rhythms in circulating levels of thyroid hormones of male hamsters

Thyroxin (T4) and triiodothyronine (T3) were measured by radioimmunoassay in serum of hamsters sacrificed at 4-hr intervals throughout the daily light-dark cycle (14L/10D). Both T4 and T3 concentrations increased significantly during the L period of the daily cycle and decreased during the D period of the cycle; A.M. versus P.M. differences in free thyroxin indices (FTI) were also studied using the T4 and T3 uptake assays of Nuclear Medical Laboratories (Dallas, Texas). The free thyroxin index was significantly greater in serum samples of hamsters sacrificed at 7 P.M. than at 7 A.M. (lights on at 6:30 A.M.). Serum taken at 7 P.M. had less unsaturated binding sites than serum taken at 7 A.M. No significant A.M. versus P.M. differences in free thyroxin index were found in blind hamsters, although blind hamsters had significantly lower T4 and FTI than controls. Placing melatonin in the drinking water at a dose of 80 μg/ml did not significantly influence hormone levels. The greatest difference in hormone concentrations between control and blinded hamsters was found in P.M. samples. Blind hamsters had FTIs that were 48% of P.M. controls. Pinealectomy prevented the effects of blinding on T4 levels and FTIs.