Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.     

The Impact of HIF1α on the Per2 Circadian Rhythm in Renal Cancer Cell Lines

In mammals, the circadian rhythm central generator consists of interactions among clock genes, including Per1/2/3, Cry1/2, Bmal1, and Clock. Circadian rhythm disruption may lead to increased risk of cancer in humans, and deregulation of clock genes has been implicated in many types of cancers. Among these genes, Per2 is reported to have tumor suppressor properties, but little is known about the correlation between Per2 and HIF, which is the main target of renal cell carcinoma (RCC) therapy. In this study, the rhythmic expression of the Per2 gene was not detectable in renal cancer cell lines, with the exception of Caki-2 cells. In Caki-2 cells, HIF1α increased the amplitude of Per2 oscillation by directly binding to the HIF-binding site located on the Per2 promoter. These results indicate that HIF1α may enhance the amplitude of the Per2 circadian rhythm.     

Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice

Background

Cell proliferation in all rapidly renewing mammalian tissues follows a circadian rhythm that is often disrupted in advanced-stage tumors. Epidemiologic studies have revealed a clear link between disruption of circadian rhythms and cancer development in humans. Mice lacking the circadian genes Period1 and 2 (Per) or Cryptochrome1 and 2 (Cry) are deficient in cell cycle regulation and Per2 mutant mice are cancer-prone. However, it remains unclear how circadian rhythm in cell proliferation is generated in vivo and why disruption of circadian rhythm may lead to tumorigenesis.

Methodology/Principal Findings

Mice lacking Per1 and 2, Cry1 and 2, or one copy of Bmal1, all show increased spontaneous and radiation-induced tumor development. The neoplastic growth of Per-mutant somatic cells is not controlled cell-autonomously but is dependent upon extracellular mitogenic signals. Among the circadian output pathways, the rhythmic sympathetic signaling plays a key role in the central-peripheral timing mechanism that simultaneously activates the cell cycle clock via AP1-controlled Myc induction and p53 via peripheral clock-controlled ATM activation. Jet-lag promptly desynchronizes the central clock-SNS-peripheral clock axis, abolishes the peripheral clock-dependent ATM activation, and activates myc oncogenic potential, leading to tumor development in the same organ systems in wild-type and circadian gene-mutant mice.

Conclusions/Significance

Tumor suppression in vivo is a clock-controlled physiological function. The central circadian clock paces extracellular mitogenic signals that drive peripheral clock-controlled expression of key cell cycle and tumor suppressor genes to generate a circadian rhythm in cell proliferation. Frequent disruption of circadian rhythm is an important tumor promoting factor.     

RNA interference of timeless gene does not disrupt circadian locomotor rhythms in the cricket Gryllus bimaculatus

Molecular studies revealed that autoregulatory negative feedback loops consisting of so-called “clock genes” constitute the circadian clock in Drosophila. However, this hypothesis is not fully supported in other insects and is thus to be examined. In the cricket Gryllus bimaculatus, we have previously shown that period (per) plays an essential role in the rhythm generation. In the present study, we cloned cDNA of the clock gene timeless (tim) and investigated its role in the cricket circadian oscillatory mechanism using RNA interference. Molecular structure of the cricket tim has rather high similarity to those of other insect species. Real-time RT-PCR analysis revealed that tim mRNA showed rhythmic expression in both LD and DD similar to that of per, peaking during the (subjective) night. When injected with tim double-stranded RNA (dstim), tim mRNA levels were significantly reduced and its circadian expression rhythm was eliminated. After the dstim treatment, however, adult crickets showed a clear locomotor rhythm in DD, with a free-running period significantly shorter than that of control crickets injected with Discosoma sp. Red2 (DsRed2) dsRNA. These results suggest that in the cricket, tim plays some role in fine-tuning of the free-running period but may not be essential for oscillation of the circadian clock.     

Light- and temperature-entrainable circadian clock in soybean development

In plants, the spatiotemporal expression of circadian oscillators provides adaptive advantages in diverse species. However, the molecular basis of circadian clock in soybean is not known. In this study, we used soybean hairy roots expression system to monitor endogenous circadian rhythms and the sensitivity of circadian clock to environmental stimuli. We discovered in experiments with constant light and temperature conditions that the promoters of clock genes GmLCLb2 and GmPRR9b1 drive a self-sustained, robust oscillation of about 24-h in soybean hairy roots. Moreover, we demonstrate that circadian clock is entrainable by ambient light/dark or temperature cycles. Specifically, we show that light and cold temperature pulses can induce phase shifts of circadian rhythm, and we found that the magnitude and direction of phase responses depends on the specific time of these two zeitgeber stimuli. We obtained a quadruple mutant lacking the soybean gene GmLCLa1, LCLa2, LCLb1, and LCLb2 using CRISPR, and found that loss-of-function of these four GmLCL orthologs leads to an extreme short-period circadian rhythm and late-flowering phenotype in transgenic soybean. Our study establishes that the morning-phased GmLCLs genes act constitutively to maintain circadian rhythmicity and demonstrates that their absence delays the transition from vegetative growth to reproductive development.     

Effect of Spaceflight on the Circadian Rhythm,Lifespan and Gene Expression of Drosophila melanogaster

Space travelers are reported to experience circadian rhythm disruption during spaceflight. However, how the space environment affects circadian rhythm is yet to be determined. The major focus of this study was to investigate the effect of spaceflight on the Drosophila circadian clock at both the behavioral and molecular level. We used China’s Shenzhou-9 spaceship to carry Drosophila. After 13 days of spaceflight, behavior tests showed that the flies maintained normal locomotor activity rhythm and sleep pattern. The expression level and rhythm of major clock genes were also unaffected. However, expression profiling showed differentially regulated output genes of the circadian clock system between space flown and control flies, suggesting that spaceflight affected the circadian output pathway. We also investigated other physiological effects of spaceflight such as lipid metabolism and lifespan, and searched genes significantly affected by spaceflight using microarray analysis. These results provide new information on the effects of spaceflight on circadian rhythm, lipid metabolism and lifespan. Furthermore, we showed that studying the effect of spaceflight on gene expression using samples collected at different Zeitgeber time could obtain different results, suggesting the importance of appropriate sampling procedures in studies on the effects of spaceflight.     

Pigment‐dispersing factor affects circadian molecular oscillations in the cricket Gryllus bimaculatus

Pigment‐dispersing factor (PDF) is an important neurotransmitter in insect circadian systems. In the cricket Gryllus bimaculatus, it affects nocturnal activity, the free‐running period and photic entrainment. In this study, to investigate whether these effects of PDF occur through a circadian molecular machinery, we measured mRNA levels of clock genes period (per) and timeless (tim) in crickets with pdf expression knocked‐down by pdf RNAi. The pdf RNAi decreased per and tim mRNA levels during the night to reduce the amplitude of their oscillation. The phase of the rhythm advanced by about 4 h in terms of trough and/or peak phases. On the other hand, pdf mRNA levels were little affected by per and tim RNAi treatment. These results suggest that PDF affects the circadian rhythm at least in part through the circadian molecular oscillation while the circadian clock has little effect on the pdf expression.     

In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB

Biochemical circadian oscillation of KaiC phosphorylation, by mixing three Kai proteins and ATP, has been proven to be the central oscillator of the cyanobacterial circadian clock. In vivo, the intracellular levels of KaiB and KaiC oscillate in a circadian fashion. By scrutinizing KaiC phosphorylation rhythm in a wide range of Kai protein concentrations, KaiA and KaiB were found to be “parameter-tuning” and “state-switching” regulators of KaiC phosphorylation rhythm, respectively. Our results also suggest a possible entrainment mechanism of the cellular circadian clock with the circadian variation of intracellular levels of Kai proteins.     

Role of Sleep Restriction in Daily Rhythms of Expression of Hypothalamic Core Clock Genes in Mice

Lack of sleep time is a menace to modern people, and it leads to chronic diseases and mental illnesses. Circadian processes control sleep, but little is known about how sleep affects the circadian system. Therefore, we performed a 28-day sleep restriction (SR) treatment in mice. Sleep restriction disrupted the clock genes’ circadian rhythm. The circadian rhythms of the Cry1 and Per1/2/3 genes disappeared. The acrophase of the clock genes (Bmal1, Clock, Rev-erbα, and Rorβ) that still had a circadian rhythm was advanced, while the acrophase of negative clock gene Cry2 was delayed. Clock genes’ upstream signals ERK and EIFs also had circadian rhythm disorders. Accompanied by changes in the central oscillator, the plasma output signal (melatonin, corticosterone, IL-6, and TNF-α) had an advanced acrophase. While the melatonin mesor was decreased, the corticosterone, IL-6, and TNF-α mesor was increased. Our results indicated that chronic sleep loss could disrupt the circadian rhythm of the central clock through ERK and EIFs and affect the output signal downstream of the core biological clock.     

A circadian oscillator in Aspergillus spp. regulates daily development and gene expression

We have established the presence of a circadian clock in Aspergillus flavus and Aspergillus nidulans by morphological and molecular assays, respectively. In A. flavus, the clock regulates an easily assayable rhythm in the development of sclerotia, which are large survival structures produced by many fungi. This developmental rhythm exhibits all of the principal clock properties. The rhythm is maintained in constant environmental conditions with a period of 33 h at 30°C, it can be entrained by environmental signals, and it is temperature compensated. This endogenous 33-h period is one of the longest natural circadian rhythms reported for any organism, and this likely contributes to some unique responses of the clock to environmental signals. In A. nidulans, no obvious rhythms in development are apparent. However, a free running and entrainable rhythm in the accumulation of gpdA mRNA (encoding glyceraldehyde-3-phosphate dehydrogenase) is observed, suggesting the presence of a circadian clock in this species. We are unable to identify an Aspergillus ortholog of frequency, a gene required for normal circadian rhythmicity in Neurospora crassa. Together, our data indicate the existence of an Aspergillus circadian clock, which has properties that differ from that of the well-described clock of N. crassa.     

Egg-laying rhythm in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Extensive research has been carried out to understand how circadian clocks regulate various physiological processes in organisms. The discovery of clock genes and the molecular clockwork has helped researchers to understand the possible role of these genes in regulating various metabolic processes. In Drosophila melanogaster, many studies have shown that the basic architecture of circadian clocks is multi-oscillatory. In nature, different neuronal subgroups in the brain of D. melanogaster have been demonstrated to control different circadian behavioural rhythms or different aspects of the same circadian rhythm. Among the circadian phenomena that have been studied so far in Drosophila, the egg-laying rhythm is unique, and relatively less explored. Unlike most other circadian rhythms, the egg-laying rhythm is rhythmic under constant light conditions, and the endogenous or free-running period of the rhythm is greater than those of most other rhythms. Although the clock genes and neurons required for the persistence of adult emergence and activity/rest rhythms have been studied extensively, those underlying the circadian egg-laying rhythm still remain largely unknown. In this review, we discuss our current understanding of the circadian egg-laying rhythm in D. melanogaster, and the possible molecular and physiological mechanisms that control the rhythmic output of the egg-laying process.     

Diurnal Oscillations of Soybean Circadian Clock and Drought Responsive Genes

Rhythms produced by the endogenous circadian clock play a critical role in allowing plants to respond and adapt to the environment. While there is a well-established regulatory link between the circadian clock and responses to abiotic stress in model plants, little is known of the circadian system in crop species like soybean. This study examines how drought impacts diurnal oscillation of both drought responsive and circadian clock genes in soybean. Drought stress induced marked changes in gene expression of several circadian clock-like components, such as LCL1-, GmELF4- and PRR-like genes, which had reduced expression in stressed plants. The same conditions produced a phase advance of expression for the GmTOC1-like, GmLUX-like and GmPRR7-like genes. Similarly, the rhythmic expression pattern of the soybean drought-responsive genes DREB-, bZIP-, GOLS-, RAB18- and Remorin-like changed significantly after plant exposure to drought. In silico analysis of promoter regions of these genes revealed the presence of cis-elements associated both with stress and circadian clock regulation. Furthermore, some soybean genes with upstream ABRE elements were responsive to abscisic acid treatment. Our results indicate that some connection between the drought response and the circadian clock may exist in soybean since (i) drought stress affects gene expression of circadian clock components and (ii) several stress responsive genes display diurnal oscillation in soybeans.