A cell-based system that recapitulates the dynamic light-dependent regulation of the vertebrate clock

The primary hallmark of circadian clocks is their ability to entrain to environmental stimuli. The dominant, and therefore most physiologically important, entraining stimulus comes from environmental light cycles. Here we describe the establishment and characterization of a new cell line, designated Z3, which derives from zebrafish embryos and contains an independent, light-entrainable circadian oscillator. Using this system, we show distinct and differential light-dependent gene activation for several central clock components. In particular, activation of Per2 expression is shown to be strictly regulated and dependent on light. Furthermore, we demonstrate that Per1, Per2, and Per3 all have distinct responses to light-dark (LD) cycles and light-pulse treatments. We also show that Clock, Bmal1, and Bmal2 all oscillate under LD and dark-dark conditions with similar kinetics, but only Clock is significantly induced while initiating a light-induced circadian oscillation in Z3 cells that have never been exposed to a LD cycle. Finally, our results suggest that Per2 is responsible for establishing the phase of a circadian rhythm entraining to an alternate LD cycle. These findings not only underscore the complexity by which central clock genes are regulated, but also establishes the Z3 cells as an invaluable system for investigating the links between light-dependent gene activation and the signaling pathways responsible for vertebrate circadian rhythms.     

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.     

Chronic Ethanol Disrupts Circadian Photic Entrainment and Daily Locomotor Activity in the Mouse

Background: Chronic ethanol abuse is associated with disrupted circadian rhythms and sleep. Ethanol administration impairs circadian clock phase‐resetting, suggesting a mode for the disruptive effect of alcohol abuse on circadian timing. Here, we extend previous studies to explore the effects of chronic forced ethanol on photic phase‐resetting, photic entrainment, and daily locomotor activity patterns in C57BL/6J mice. Methods: First, microdialysis was used to characterize the circadian patterns of ethanol uptake in the suprachiasmatic (SCN) circadian clock and correlate this with systemic ethanol levels and episodic drinking of 10 or 15% ethanol. Second, the effects of chronic forced ethanol drinking and withdrawal on photic phase‐delays of the circadian activity rhythm were assessed. Third, the effects of chronic ethanol drinking on entrainment to a weak photic zeitgeber (1 minute of 25 lux intensity light per day) were assessed. This method was used to minimize any masking actions of light that could mask ethanol effects on clock entrainment. Results: Peak ethanol levels in the SCN and periphery occurred during the dark phase and coincided with the time when light normally induces phase‐delays in mice. These delays were dose‐dependently inhibited by chronic ethanol and its withdrawal. Chronic ethanol did not impede re‐entrainment to a shifted light cycle but affected entrainment under the weak photic zeitgeber and disrupted the daily pattern of locomotor activity. Conclusions: These results confirm that chronic ethanol consumption and withdrawal markedly impair circadian clock photic phase‐resetting. Ethanol also disturbs the temporal structure of nighttime locomotor activity and photic entrainment. Collectively, these results suggest a direct action of ethanol on the SCN clock.     

The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B

Developmental and physiological responses are regulated by light throughout the entire life cycle of higher plants. To sense changes in the light environment, plants have developed various photoreceptors, including the red/far-red light-absorbing phytochromes and blue light-absorbing cryptochromes. A wide variety of physiological responses, including most light responses, also are modulated by circadian rhythms that are generated by an endogenous oscillator, the circadian clock. To provide information on local time, circadian clocks are synchronized and entrained by environmental time cues, of which light is among the most important. Light-driven entrainment of the Arabidopsis circadian clock has been shown to be mediated by phytochrome A (phyA), phytochrome B (phyB), and cryptochromes 1 and 2, thus affirming the roles of these photoreceptors as input regulators to the plant circadian clock. Here we show that the expression of PHYB::LUC reporter genes containing the promoter and 5' untranslated region of the tobacco NtPHYB1 or Arabidopsis AtPHYB genes fused to the luciferase (LUC) gene exhibit robust circadian oscillations in transgenic plants. We demonstrate that the abundance of PHYB RNA retains this circadian regulation and use a PHYB::Luc fusion protein to show that the rate of PHYB synthesis is also rhythmic. The abundance of bulk PHYB protein, however, exhibits only weak circadian rhythmicity, if any. These data suggest that photoreceptor gene expression patterns may be significant in the daily regulation of plant physiology and indicate an unexpectedly intimate relationship between the components of the input pathway and the putative circadian clock mechanism in higher plants.     

Pinealectomy interferes with the circadian clock genes expression in white adipose tissue

Melatonin, the main hormone produced by the pineal gland, is secreted in a circadian manner (24‐hr period), and its oscillation influences several circadian biological rhythms, such as the regulation of clock genes expression (chronobiotic effect) and the modulation of several endocrine functions in peripheral tissues. Assuming that the circadian synchronization of clock genes can play a role in the regulation of energy metabolism and it is influenced by melatonin, our study was designed to assess possible alterations as a consequence of melatonin absence on the circadian expression of clock genes in the epididymal adipose tissue of male Wistar rats and the possible metabolic repercussions to this tissue. Our data show that pinealectomy indeed has impacts on molecular events: it abolishes the daily pattern of the expression of Clock, Per2, and Cry1 clock genes and Pparγ expression, significantly increases the amplitude of daily expression of Rev‐erbα, and affects the pattern of and impairs adipokine production, leading to a decrease in leptin levels. However, regarding some metabolic aspects of adipocyte functions, such as its ability to synthesize triacylglycerols from glucose along 24 hr, was not compromised by pinealectomy, although the daily profile of the lipogenic enzymes expression (ATP‐citrate lyase, malic enzyme, fatty acid synthase, and glucose‐6‐phosphate dehydrogenase) was abolished in pinealectomized animals.     

The protein kinase CK2 is involved in regulation of circadian rhythms in Arabidopsis

A wide range of processes in plants, including expression of certain genes, is regulated by endogenous circadian rhythms. The circadian clock-associated 1 (CCA1) and the late elongated hypocotyl (LHY) proteins have been shown to be closely associated with clock function in Arabidopsis thaliana. The protein kinase CK2 can interact with and phosphorylate CCA1, but its role in the regulation of the circadian clock remains unknown. Here we show that plants overexpressing CKB3, a regulatory subunit of CK2, display increased CK2 activity and shorter periods of rhythmic expression of CCA1 and LHY. CK2 is also able to interact with and phosphorylate LHY in vitro. Additionally, overexpression of CKB3 shortened the periods of four known circadian clock-controlled genes with different phase angles, demonstrating that many clock outputs are affected. This overexpression also reduced phytochrome induction of an Lhcb gene. Finally, we found that the photoperiodic flowering response, which is influenced by circadian rhythms, was diminished in the transgenic lines, and that the plants flowered earlier on both long-day and short-day photoperiods. These data demonstrate that CK2 is involved in regulation of the circadian clock in Arabidopsis.     

Minireview: Entrainment of the suprachiasmatic clockwork in diurnal and nocturnal mammals

Daily rhythmicity, including timing of wakefulness and hormone secretion, is mainly controlled by a master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN clockwork involves various clock genes, with specific temporal patterns of expression that are similar in nocturnal and diurnal species (e.g. the clock gene Per1 in the SCN peaks at midday in both categories). Timing of sensitivity to light is roughly similar, during nighttime, in diurnal and nocturnal species. Molecular mechanisms of photic resetting are also comparable in both species categories. By contrast, in animals housed in constant light, exposure to darkness can reset the SCN clock, mostly during the resting period, i.e. at opposite circadian times between diurnal and nocturnal species. Nonphotic stimuli, such as scheduled voluntary exercise, food shortage, exogenous melatonin, or serotonergic receptor activation, are also capable of shifting the master clock and/or modulating photic synchronization. Comparison between day- and night-active species allows classifications of nonphotic cues in two, arousal-independent and arousal-dependent, families of factors. Arousal-independent factors, such as melatonin (always secreted during nighttime, independently of daily activity pattern) or gamma-aminobutyric acid (GABA), have shifting effects at the same circadian times in both nocturnal and diurnal rodents. By contrast, arousal-dependent factors, such as serotonin (its cerebral levels follow activity pattern), induce phase shifts only during resting and have opposite modulating effects on photic resetting between diurnal and nocturnal species. Contrary to light and arousal-independent nonphotic cues, arousal-dependent nonphotic stimuli provide synchronizing feedback signals to the SCN clock in circadian antiphase between nocturnal and diurnal animals.     

A zinc knuckle protein that negatively controls morning-specific growth in Arabidopsis thaliana

Growth in plants is modulated by a complex interplay between internal signals and external cues. Although traditional mutagenesis has been a successful approach for the identification of growth regulatory genes, it is likely that many genes involved in growth control remain to be discovered. In this study, we used the phenotypic variation between Bay-0 and Shahdara, two natural strains (accessions) of Arabidopsis thaliana, to map quantitative trait loci (QTL) affecting light- and temperature-regulated growth of the embryonic stem (hypocotyl). Using heterogeneous inbred families (HIFs), the gene underlying one QTL, LIGHT5, was identified as a tandem zinc knuckle/PLU3 domain encoding gene (At5g43630; TZP), which carries a premature stop codon in Bay-0. Hypocotyl growth assays in monochromatic light and microarray analysis demonstrate that TZP controls blue light associated growth in a time-of-day fashion by regulating genes involved in growth, such as peroxidase and cell wall synthesis genes. TZP expression is phased by the circadian clock and light/dark cycles to the beginning of the day, the time of maximal growth in A. thaliana in short-day conditions. Based on its domain structure and localization in the nucleus, we propose that TZP acts downstream of the circadian clock and photoreceptor signaling pathways to directly control genes responsible for growth. The identification of TZP thus provides new insight into how daily synchronization of growth pathways plays a critical role in growth regulation.     

Light signaling to the zebrafish circadian clock by Cryptochrome 1a

Zebrafish tissues and cells have the unusual feature of not only containing a circadian clock, but also being directly light-responsive. Several zebrafish genes are induced by light, but little is known about their role in clock resetting or the mechanism by which this might occur. Here we show that Cryptochrome 1a (Cry1a) plays a key role in light entrainment of the zebrafish clock. Intensity and phase response curves reveal a strong correlation between light induction of Cry1a and clock resetting. Overexpression studies show that Cry1a acts as a potent repressor of clock function and mimics the effect of constant light to "stop" the circadian oscillator. Yeast two-hybrid analysis demonstrates that the Cry1a protein interacts directly with specific regions of core clock components, CLOCK and BMAL, blocking their ability to fully dimerize and transactivate downstream targets, providing a likely mechanism for clock resetting. A comparison of entrainment of zebrafish cells to complete versus skeleton photoperiods reveals that clock phase is identical under these two conditions. However, the amplitude of the core clock oscillation is much higher on a complete photoperiod, as are the levels of light-induced Cry1a. We believe that Cry1a acts on the core clock machinery in both a continuous and discrete fashion, leading not only to entrainment, but also to the establishment of a high-amplitude rhythm and even stopping of the clock under long photoperiods.