Network Features of the Mammalian Circadian Clock

The mammalian circadian clock is a cell-autonomous system that drives oscillations in behavior and physiology in anticipation of daily environmental change. To assess the robustness of a human molecular clock, we systematically depleted known clock components and observed that circadian oscillations are maintained over a wide range of disruptions. We developed a novel strategy termed Gene Dosage Network Analysis (GDNA) in which small interfering RNA (siRNA)-induced dose-dependent changes in gene expression were used to build gene association networks consistent with known biochemical constraints. The use of multiple doses powered the analysis to uncover several novel network features of the circadian clock, including proportional responses and signal propagation through interacting genetic modules. We also observed several examples where a gene is up-regulated following knockdown of its paralog, suggesting the clock network utilizes active compensatory mechanisms rather than simple redundancy to confer robustness and maintain function. We propose that these network features act in concert as a genetic buffering system to maintain clock function in the face of genetic and environmental perturbation.     

Iron Is Involved in the Maintenance of Circadian Period Length in Arabidopsis

The homeostasis of iron (Fe) in plants is strictly regulated to maintain an optimal level for plant growth and development but not cause oxidative stress. About 30% of arable land is considered Fe deficient because of calcareous soil that renders Fe unavailable to plants. Under Fe-deficient conditions, Arabidopsis (Arabidopsis thaliana) shows retarded growth, disordered chloroplast development, and delayed flowering time. In this study, we explored the possible connection between Fe availability and the circadian clock in growth and development. Circadian period length in Arabidopsis was longer under Fe-deficient conditions, but the lengthened period was not regulated by the canonical Fe-deficiency signaling pathway involving nitric oxide. However, plants with impaired chloroplast function showed long circadian periods. Fe deficiency and impaired chloroplast function combined did not show additive effects on the circadian period, which suggests that plastid-to-nucleus retrograde signaling is involved in the lengthening of circadian period under Fe deficiency. Expression pattern analyses of the central oscillator genes in mutants defective in CIRCADIAN CLOCK ASSOCIATED1/LATE ELONGATED HYPOCOTYL or GIGANTEA demonstrated their requirement for Fe deficiency-induced long circadian period. In conclusion, Fe is involved in maintaining the period length of circadian rhythm, possibly by acting on specific central oscillators through a retrograde signaling pathway.Metals such as iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), molybdenum, and nickel are essential for the various biological processes that govern plant growth and development (Marschner, 1995). For example, Fe is required for DNA synthesis, photosynthesis, nitrogen fixation, hormone synthesis, and electron transport in the respiratory chain (Briat and Lobreaux, 1997). Similarly, Cu is an important component of electron-transfer reactions mediated by proteins such as superoxide dismutase, cytochrome oxidase, and plastocyanin (Clemens, 2001). Zn is a cofactor for many enzymes, and many proteins contain Zn-binding structural domains (Clarke and Berg, 1998). Although only minimal quantities of these micronutrients are required by plants, their limited availability in soils can significantly hinder crop production and affect nutritional quality (Grotz and Guerinot, 2002). In the case of Fe, about 30% of arable land worldwide is considered calcareous, rendering Fe in these soils unavailable to plants (Mori, 1999). Understanding of the fundamental processes involving metal uptake and sequestration has increased in recent years, but how the availability of particular metals interacts with internal signals to govern the growth and development of plants is largely unknown.The daily biological rhythms of many organisms are regulated by a near 24-h circadian clock that is synchronized by environmental changes such as light and temperature (Harmer, 2009; Imaizumi, 2010). The circadian clock regulates diverse aspects of plant growth and development. The operation of the circadian clock in plants can basically be divided into three main parts, input, central oscillator, and output pathways, and each part has its own complex networks. In Arabidopsis (Arabidopsis thaliana), the central oscillator is composed of a network of multiple feedback loops that can be divided into the morning, central, and evening loops (Harmer, 2009). The central feedback loop is composed of the morning-expressed genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the evening-expressed gene TIMING OF CAB EXPRESSION1 (TOC1; Schaffer et al., 1998; Wang and Tobin, 1998; Strayer et al., 2000; Alabadí et al., 2001). Although TOC1 is genetically required for the activation of morning genes (Schaffer et al., 1998; Wang and Tobin, 1998; Strayer et al., 2000), it acts as a repressor and directly regulates the expression of CCA1 and LHY (Gendron et al., 2012; Huang et al., 2012; Pokhilko et al., 2012). In the morning loop, CCA1/LHY form another negative feedback loop with the morning genes PSEUDO-RESPONSE REGULATOR7 (PRR7) and PRR9, with PRR9/PRR7 directly repressing the expression of CCA1 and LHY (Farré et al., 2005; Nakamichi et al., 2010). In the evening loop, TOC1 forms a negative feedback loop with GIGANTEA (GI) by repressing its expression, and GI in turn activates the expression of TOC1 through an unknown component, Y (Huq et al., 2000; Mizoguchi et al., 2005). After receiving input signals in the form of environmental cues, the central oscillator of the Arabidopsis circadian clock generates various rhythmic outputs that control various physiological events (Hotta et al., 2007; de Montaigu et al., 2010).The central oscillator controls a range of important physiological output processes such as flowering, stress and hormone responses, and regulation of nutrient acquisition (Haydon et al., 2011). Although the uptake of nutrition in plants is known to be influenced by light and temperature (Lahti et al., 2005; Baligar et al., 2006), the interaction between nutritional status and the circadian clock is less well studied. The homeostasis of Cu is known to influence the regulation of oscillator genes (Andrés-Colás et al., 2010; Peñarrubia et al., 2010). Arabidopsis under excess Cu or overexpressing Cu transporters COPT1 and COPT3 showed increased Cu accumulation and reduced expression of CCA1, LHY, and circadian clock output genes. Defective developmental phenotypes were also observed in these plants. Spatial and temporal control of Cu homeostasis, therefore, may be important for plant environmental fitness (Andrés-Colás et al., 2010). It has also been reported that disordered circadian rhythm affects Fe homeostasis. Tight regulation of Fe homeostasis to maintain an optimal Fe level in plants has been found to be associated with circadian clock regulators such as TIME FOR COFFEE (TIC) that modulates the expression of the ferritin gene AtFer1 (Duc et al., 2009). The expression of AtFer1 was up-regulated with excess Fe. TIC could repress AtFer1 expression under low-Fe conditions in photoperiodic light and dark cycles (Duc et al., 2009). However, whether Fe status feeds back to regulate the circadian clock is uncertain.Although Fe homeostasis in terms of uptake and translocation has been studied for decades, Fe availability is still an agricultural problem worldwide. Revealing the interplay between Fe homeostasis and internal cues such as modulation of the circadian clock can help increase understanding of their contributions to overall plant development. In this work, we investigated the effect of Fe deficiency on the circadian clock and found that it lengthened the circadian period. Our data suggest that the functional status of chloroplasts under Fe deficiency may play a key role in the lengthened circadian period.     

Free Cellular Riboflavin is Involved in Phase Shifting by Light of the Circadian Clock in Neurospora crassa

Phase shifting by light of the circadian conidiation rhythmof the Neurospora crassa strain band, of the riboflavin-deficientdouble mutant band rib2 and of the temperature-sensitive doublemutant band ribl was measured. Fluence response curves of theband strain exhibited two distinct steps, whereas those of bandribl and band rib2 revealed only one step. Maximum phase advancesobserved were 5.5 h in band and 10.4 h in the band rib strains.Sensitivity of band rib2 to light was proportional to the riboflavinconcentration in the growth medium over a 100 fold range. Extracellularflavin in the medium did not sensitize the strains. Riboflavinapplied after exposure to light showed no effect. Light sensitivitycorrelated with the level of cellular riboflavin. Four analogsof riboflavin, none of which can be phosphorylated, increasedthe sensitivity of Neurospora to light. Even at high riboflavinconcentrations in the medium, the sensitivity of the band rib2strain to light was not saturated. In addition, four riboflavinderivatives with bulky substituents at positions 3, 8 or 10of the isoalloxazine nucleus sensitized both strains. From ourdata, we conclude, that a) a cellular flavin controls the sensitivityof Neurospora crassa to light; b) that this flavin compoundis riboflavin; and c) that the active riboflavin is not proteinbound. ⁴Present address: Teikoku Women's University, Department ofHome Economics, 6-173 Touda-cho, Moriguchi-shi, Osaka, 570 Japan. (Received November 27, 1987; Accepted March 15, 1989)     

Colour As a Signal for Entraining the Mammalian Circadian Clock

Twilight is characterised by changes in both quantity (“irradiance”) and quality (“colour”) of light. Animals use the variation in irradiance to adjust their internal circadian clocks, aligning their behaviour and physiology with the solar cycle. However, it is currently unknown whether changes in colour also contribute to this entrainment process. Using environmental measurements, we show here that mammalian blue–yellow colour discrimination provides a more reliable method of tracking twilight progression than simply measuring irradiance. We next use electrophysiological recordings to demonstrate that neurons in the mouse suprachiasmatic circadian clock display the cone-dependent spectral opponency required to make use of this information. Thus, our data show that some clock neurons are highly sensitive to changes in spectral composition occurring over twilight and that this input dictates their response to changes in irradiance. Finally, using mice housed under photoperiods with simulated dawn/dusk transitions, we confirm that spectral changes occurring during twilight are required for appropriate circadian alignment under natural conditions. Together, these data reveal a new sensory mechanism for telling time of day that would be available to any mammalian species capable of chromatic vision.     

Protein Kinase C Differentially Regulates Entrainment of the Mammalian Circadian Clock

The environmental day-night cycle provides the principal synchronizing signal for behavioral activity in most mammals. Light information is relayed to the master circadian pacemaker, the suprachiasmatic nucleus (SCN), via synaptic transmission from the retina directly to the SCN, where a predominately glutamate-driven cellular signaling pathway is able to reset biochemical, physiological, and behavioral activities. In the present study, we aimed to decipher the key roles played by protein kinase C (PKC) in regulating light-induced behavioral resetting under both a temporal and intensity-dependent manner; in addition, we also investigate PKC contributions to advancing and delaying re-entrainment paradigms. Our findings show that during the early night PKC acts in a temporal manner, where PKC inhibition selectively attenuates light-induced behavioral resetting in response to subsaturating and saturating light intensities. Declines in light response were also evident upon PKC inhibition during the late night, but restricted to bright light stimuli. The positive regulatory actions of PKC were further demonstrated in response to an 8-h delayed re-entrainment paradigm where inhibition of PKC resulted in slower re-entrainment. Further, analysis of both classic and novel PKC isozymes present within the SCN showed significant circadian variation in the mRNA expression of PKCα, indicating possible isozyme-specific mediators in photic signaling. Our data provide evidence of a PKC contribution to both acute light-induced clock resetting, which is intensity and time of day dependent, and a functional role in circadian photoentrainment. (Author correspondence: g.lall@kent.ac.uk)     

Phase Delaying the Human Circadian Clock with Blue-Enriched Polychromatic Light

The human circadian system is maximally sensitive to short-wavelength (blue) light. In a previous study we found no difference between the magnitude of phase advances produced by bright white versus bright blue-enriched light using light boxes in a practical protocol that could be used in the real world. Since the spectral sensitivity of the circadian system may vary with a circadian rhythm, we tested whether the results of our recent phase-advancing study hold true for phase delays. In a within-subjects counterbalanced design, this study tested whether bright blue-enriched polychromatic light (17000 K, 4000 lux) could produce larger phase delays than bright white light (4100 K, 5000 lux) of equal photon density (4.2×10¹⁵ photons/cm²/sec). Healthy young subjects (n?=?13) received a 2 h phase delaying light pulse before bedtime combined with a gradually delaying sleep/dark schedule on each of 4 consecutive treatment days. On the first treatment day the light pulse began 3 h after the dim light melatonin onset (DLMO). An 8 h sleep episode began at the end of the light pulse. Light treatment and the sleep schedule were delayed 2 h on each subsequent treatment day. A circadian phase assessment was conducted before and after the series of light treatment days to determine the time of the DLMO and DLMOff. Phase delays in the blue-enriched and white conditions were not significantly different (DLMO: ?4.45±2.02 versus ?4.48±1.97 h; DLMOff: ?3.90±1.97 versus ?4.35±2.39 h, respectively). These results indicate that at light levels commonly used for circadian phase shifting, blue-enriched polychromatic light is no more effective than the white polychromatic lamps of a lower correlated color temperature (CCT) for phase delaying the circadian clock. (Author correspondence: ceastman@rush.edu)     

The Circadian Clock Gene Period1 Connects the Molecular Clock to Neural Activity in the Suprachiasmatic Nucleus

The neural activity patterns of suprachiasmatic nucleus (SCN) neurons are dynamically regulated throughout the circadian cycle with highest levels of spontaneous action potentials during the day. These rhythms in electrical activity are critical for the function of the circadian timing system and yet the mechanisms by which the molecular clockwork drives changes in the membrane are not well understood. In this study, we sought to examine how the clock gene Period1 (Per1) regulates the electrical activity in the mouse SCN by transiently and selectively decreasing levels of PER1 through use of an antisense oligodeoxynucleotide. We found that this treatment effectively reduced SCN neural activity. Direct current injection to restore the normal membrane potential partially, but not completely, returned firing rate to normal levels. The antisense treatment also reduced baseline [Ca²⁺]i levels as measured by Fura2 imaging technique. Whole cell patch clamp recording techniques were used to examine which specific potassium currents were altered by the treatment. These recordings revealed that the large conductance [Ca²⁺]i-activated potassium currents were reduced in antisense-treated neurons and that blocking this current mimicked the effects of the anti-sense on SCN firing rate. These results indicate that the circadian clock gene Per1 alters firing rate in SCN neurons and raise the possibility that the large conductance [Ca²⁺]i-activated channel is one of the targets.     

Phase Shifting of the Circadian Clock by Diethylstilbestrol and Related Compounds in Neurospora crassa

Phase shifts of the circadian conidiation rhythm in Neurospora crassa were induced by 3-hour treatments of mycelia in liquid medium with diethylstilbestrol (DES), dienestrol (DIE), hexestrol (HEX), diethylstilbestroldipropionate (DESP), and dienestroldiacetate (DIEA). Over a 24-hour period beginning 24 hours after the transition from light to constant dark, maximum phase shifts occurred about 36 hours. DES was the most effective of the drugs tested, giving 10-hour phase advances at 20 micromolar. DIE and HEX caused similar phase shifts as DES at 40 micromolar. The two derivatives of the last, DESP and DIEA, were much less effective in shifting phase; only a few hours of phase advance result from treatments at 80 micromolar concentrations.

The activity of isolated plasma membrane ATPase was inhibited by DES and partially by HEX, but not by DIE, DESP, or DIEA. O₂ consumption of the mycelia was inhibited equally by DES, DIE, and HEX, while DIEA and DESP had little effect. Phase-shifts by DES cannot be interpreted as evidence that plasma membrane ATPase is a component of the circadian clock.

     

Mammalian TIMELESS Is Required for ATM-dependent CHK2 Activation and G2/M Checkpoint Control

Timeless (Tim), a core circadian clock gene in Drosophila, is retained in mammals but has no apparent mammalian circadian clock function. Mammalian TIM is essential for ATR-dependent Chk1 activation and S-phase arrest. We report that TIM is likewise essential for ATM-dependent Chk2-mediated signaling of doxorubicin-induced DNA double strand breaks. TIM depletion attenuates doxorubicin-induced G₂/M cell cycle arrest and sensitizes cancer cells to doxorubicin-induced cytotoxicity. TIM is, thereby, a potential novel anticancer drug target whose inhibition may enhance the therapeutic cytotoxicity of agents that activate DNA damage pathways as part of their mechanism.     

Assembly of a Comprehensive Regulatory Network for the Mammalian Circadian Clock: A Bioinformatics Approach

By regulating the timing of cellular processes, the circadian clock provides a way to adapt physiology and behaviour to the geophysical time. In mammals, a light-entrainable master clock located in the suprachiasmatic nucleus (SCN) controls peripheral clocks that are present in virtually every body cell. Defective circadian timing is associated with several pathologies such as cancer and metabolic and sleep disorders. To better understand the circadian regulation of cellular processes, we developed a bioinformatics pipeline encompassing the analysis of high-throughput data sets and the exploitation of published knowledge by text-mining. We identified 118 novel potential clock-regulated genes and integrated them into an existing high-quality circadian network, generating the to-date most comprehensive network of circadian regulated genes (NCRG). To validate particular elements in our network, we assessed publicly available ChIP-seq data for BMAL1, REV-ERBα/β and RORα/γ proteins and found strong evidence for circadian regulation of Elavl1, Nme1, Dhx6, Med1 and Rbbp7 all of which are involved in the regulation of tumourigenesis. Furthermore, we identified Ncl and Ddx6, as targets of RORγ and REV-ERBα, β, respectively. Most interestingly, these genes were also reported to be involved in miRNA regulation; in particular, NCL regulates several miRNAs, all involved in cancer aggressiveness. Thus, NCL represents a novel potential link via which the circadian clock, and specifically RORγ, regulates the expression of miRNAs, with particular consequences in breast cancer progression. Our findings bring us one step forward towards a mechanistic understanding of mammalian circadian regulation, and provide further evidence of the influence of circadian deregulation in cancer.