Operational mechanisms of the plant circadian clock in stress

Circadian clocks are endogenous timing mechanisms that control molecular, cellular, physiological, and behavioral rhythms in all organisms from unicellulars to humans. Circadian rhythms influence many aspects of insect biology, finetuning life functions to the light and temperature cycles associated with the solar day. Genetic studies in the fruit fly Drosophila melanogaster have led to the cloning and characterization of several genes involved in the mechanism of the circadian clock. Periodic transcription and translation of these clock genes form the basis of a molecular feedback loop that has a "circa" 24-hour period. Rhythmic expression of clock genes in specific brain neurons appears to control behavioral rhythms in adult flies. However, clock genes are also expressed in other tissues, both within and outside of the nervous system. These observations prompted chronobiologists to investigate whether nonneural tissues possess intrinsic circadian clocks, what role they may be playing, and what the relationships are between clocks in the nervous system and those in peripheral tissues. Answers to those questions are providing important insights into the overall organization of the circadian system in insects.

BackgroundCircadian rhythms, observed across all domains of life, enable organisms to anticipate and prepare for diel changes in environmental conditions. In bacteria, a circadian clock mechanism has only been characterized in cyanobacteria to date. These clocks regulate cyclical patterns of gene expression and metabolism which contribute to the success of cyanobacteria in their natural environments. The potential impact of self-generated circadian rhythms in other bacterial and microbial populations has motivated extensive research to identify novel circadian clocks.Main textDaily oscillations in microbial community composition and function have been observed in ocean ecosystems and in symbioses. These oscillations are influenced by abiotic factors such as light and the availability of nutrients. In the ocean ecosystems and in some marine symbioses, oscillations are largely controlled by light-dark cycles. In gut systems, the influx of nutrients after host feeding drastically alters the composition and function of the gut microbiota. Conversely, the gut microbiota can influence the host circadian rhythm by a variety of mechanisms including through interacting with the host immune system. The intricate and complex relationship between the microbiota and their host makes it challenging to disentangle host behaviors from bacterial circadian rhythms and clock mechanisms that might govern the daily oscillations observed in these microbial populations.ConclusionsWhile the ability to anticipate the cyclical behaviors of their host would likely be enhanced by a self-sustained circadian rhythm, more evidence and further studies are needed to confirm whether host-associated heterotrophic bacteria possess such systems. In addition, the mechanisms by which heterotrophic bacteria might respond to diel cycles in environmental conditions has yet to be uncovered.

Maintenance of circadian alignment between an organism and its environment is essential to ensure metabolic homeostasis. Synchrony is achieved by cell autonomous circadian clocks. Despite a growing appreciation of the integral relation between clocks and metabolism, little is known regarding the direct influence of a peripheral clock on cellular responses to fatty acids. To address this important issue, we utilized a genetic model of disrupted clock function specifically in cardiomyocytes in vivo (termed cardiomyocyte clock mutant (CCM)). CCM mice exhibited altered myocardial response to chronic high fat feeding at the levels of the transcriptome and lipidome as well as metabolic fluxes, providing evidence that the cardiomyocyte clock regulates myocardial triglyceride metabolism. Time-of-day-dependent oscillations in myocardial triglyceride levels, net triglyceride synthesis, and lipolysis were markedly attenuated in CCM hearts. Analysis of key proteins influencing triglyceride turnover suggest that the cardiomyocyte clock inactivates hormone-sensitive lipase during the active/awake phase both at transcriptional and post-translational (via AMP-activated protein kinase) levels. Consistent with increased net triglyceride synthesis during the end of the active/awake phase, high fat feeding at this time resulted in marked cardiac steatosis. These data provide evidence for direct regulation of triglyceride turnover by a peripheral clock and reveal a potential mechanistic explanation for accelerated metabolic pathologies after prevalent circadian misalignment in Western society.

The recovery of KaiA’s activity depends on its N-terminal domain and KaiB in the cyanobacterial circadian clock

Objective: A circadian rhythm in mammals controls the sleep-wake cycle, blood pressure, hormone secretion, metabolism and many other physiological processes. The circadian clock mechanism is regulated by four genes: Bmal1, Clock, Cry, and Per. Mutations in these regulatory genes are associated with sleep and mood disorders, obesity, and cancer. Several PER2 and CRY2 SNPs are associated with advanced sleep phase syndrome. It is, therefore, critical to understand the effect of clock genes’ SNPs on the circadian clock. In this study, we determined “pathogenic” BMAL1 and CLOCK SNPs in the Ensembl database for biochemical characterization. Materials and Methods: BMAL1 and CLOCK SNPs in the Ensemble database were filtered out for only missense mutations. Among the missense mutations, pathogenic ones were determined according to SIFT, PolyPhen, and CADD scores, REVEL, MetalR, Mutation Assessor, I-Mutant, PROVEAN, and FireDock programs. BMAL1 and CLOCK SNPs were visualized by using PyMol. Results: Thousands of BMAL1 and CLOCK missense SNP mutations were reported in the Ensembl database. After the classification of those SNPs according to their SIFT, PolyPhen, and CADD pathogenicity, twelve SNPs for each protein remained as pathogenic. A further analysis with all in silico tools revealed that BMAL1 SNPs causing Ala154Val, Arg166Gln, and Val440Gly mutations; and CLOCK SNPs causing Gly120Val, Asp119Val, Gly120Ser, Ala117Val, and Cys371Gly mutations were predicted as the most “pathogenic” ones. Conclusion: Overall, by using in silico tools, we provided a starting point for experimental studies for determining the effect of pathogenic BMAL1 and CLOCK SNPs on the circadian clock mechanism.

Mood disorders are multifactorial and heterogeneous diseases caused by the interplay of several genetic and environmental factors. In humans, mood disorders are often accompanied by abnormalities in the organization of the circadian system, which normally synchronizes activities and functions of cells and tissues. Studies on animal models suggest that the basic circadian clock mechanism, which runs in essentially all cells, is implicated in the modulation of biological phenomena regulating affective behaviors. In particular, recent findings highlight the importance of the circadian clock mechanisms in neurological pathways involved in mood, such as monoaminergic neurotransmission, hypothalamus-pituitary-adrenal axis regulation, suprachiasmatic nucleus and olfactory bulb activities, and neurogenesis. Defects at the level of both, the circadian clock mechanism and system, may contribute to the etiology of mood disorders. Modification of the circadian system using chronotherapy appears to be an effective treatment for mood disorders. Additionally, understanding the role of circadian clock mechanisms, which affect the regulation of different mood pathways, will open up the possibility for targeted pharmacological treatments.

Organisms from all the major kingdoms of life exhibit periodic rhythms of approximately 24 hours in vital cellular and physiological processes, including animal locomotor activity and photosynthesis in cyanobacteria and plants. These rhythms sustain most of their properties under constant environmental conditions, which is a hallmark of the intracellular pacemakers known as circadian clocks. The existence of the circadian clock in plants has been known for hundreds of years; however, only recently has work been focused on understanding the molecular makeup of the plant clockworks. Using the model plant Arabidopsis thaliana, significant progress has been made in identifying key genes in the oscillator (Salome & McClung, 2004). As anticipated from the more mature work on the Neurospora and Drosophila clocks, the minimal Arabidopsis clock is composed of a negative feedback loop. Even with the immense progress made in the past ten years, the molecular mechanism of the plant circadian clock remains unclear, especially in comparison to the detail available for fungal and animal clocks (Rosbash & Hall, 1989). Therefore, it is the task of the plant clock community to add color and depth to this relatively bare canvas by defining the function of the proteins now in hand, identifying the factors missing from the current picture, and connecting the function of each to generate a more comprehensive model of the plant oscillator. This chapter summarizes the work that has brought the plant clock field to this point, and goes on to illuminate avenues likely to be fruitful in the future. A current view of the plant oscillator is also presented based on the list of circadian genes identified so far.

Regulation of Monoamine Oxidase A by Circadian-Clock Components Implies Clock Influence on Mood

BackgroundAs a group of highly conserved small non-coding RNAs with a length of 21~23 nucleotides, microRNAs (miRNAs) regulate the gene expression post-transcriptionally by base pairing with the partial or full complementary sequences in target mRNAs, thus resulting in the repression of mRNA translation and the acceleration of mRNA degradation. Recent work has revealed that miRNAs are essential for the development and functioning of the skeletal muscles where they are. In particular, miR-206 has not only been identified as the only miRNA expressed in skeletal muscles, but also exhibited crucial roles in regulation of the muscle development. Although miRNAs are known to regulate various biological processes ranging from development to cancer, much less is known about their role in the dynamic regulation of the mammalian circadian clock.ResultsA detailed dynamic model of miR-206-mediated mammalian circadian clock system was developed presently by using Hill-type terms, Michaelis-Menten type and mass action kinetics. Based on a system-theoretic approach, the model accurately predicts both the periodicity and the entrainment of the circadian clock. It also explores the dynamics properties of the oscillations mediated by miR-206 by means of sensitivity analysis and alterations of parameters. Our results show that miR-206 is an important regulator of the circadian clock in skeletal muscle, and thus by study of miR-206 the main features of its mediation on the clock may be captured. Simulations of these processes display that the amplitude and frequency of the oscillation can be significantly altered through the miR-206-mediated control.ConclusionsMiR-206 has a profound effect on the dynamic mechanism of the mammalian circadian clock, both by control of the amplitude and control or alteration of the frequency to affect the level of the gene expression and to interfere with the temporal sequence of the gene production or delivery. This undoubtedly uncovers a new mechanism for regulation of the circadian clock at a post-transcriptional level and provides important insights into the normal development as well as the pathological conditions of skeletal muscles, such as the aging, chronic disease and cancer.

Virus-like particles in certain slow-growing strains of Neurospora crassa

The circadian clock exists in almost all life forms, and is an internal activity generated by organisms adapting to the daily periodic changes of the external environment. The circadian clock is regulated by the transcription-translation-negative feedback loop in the body, which can regulate the activities of tissues and organs. Its normal maintenance is important for the health, growth, and reproduction of organisms. In contrast, due to the season changes of the environment, organisms have also formed annual cycle physiological changes in their bodies, such as seasonal estrus, etc. The annual rhythm of living things is mainly affected by environmental factors such as photoperiod, and is related to gene expression, hormone content, morphological changes of cell and tissues in vivo. Melatonin is an important signal to recognize the changes of photoperiod, and the circadian clock plays an important role in the pituitary to interpret the signal of melatonin and regulate the changes of downstream signals, which plays an important guiding role in the recognition of annual changes in the environment and the generation of the body's annual rhythm. In this review, we summarize the progress of research on the mechanism of action of circadian clocks in influencing annual rhythms, by introducing the mechanisms of circadian and annual rhythms generation in insects and mammals, and in the context of annual rhythms in birds, with the aim of providing a broader range of ideas for future research on the mechanism of annual rhythms influence.

Natural Variation of the Circadian Clock in Neurospora.

The circadian clock plays an integral role in cellular functioning by temporally controlling gene expression, and there is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study we aimed to investigate circadian clock gene expression and oscillation patterns in cervical and oesophageal cancers, in order to determine whether disruptions in circadian clock functioning occur in these cancer types. Microarray gene expression analysis revealed the circadian clock gene, Per2, to be one of the most significantly downregulated genes in cervical cancer patient tissue compared to normal epithelium. In addition, Oncomine data-mining revealed significant downregulation of not only Per2, but multiple members of the circadian clock gene family (Clock, Bmal1, Per1, Per2, Cry1, Rev-erbα and RORα) in cervical cancer tissue compared to normal. Real-time RT-PCR analysis of circadian clock genes in oesophageal cancer patient tissue compared to matched normal epithelium revealed significant downregulation of Clock, Per2, Cry1 and RORα in oesophageal tumour tissue. Significant positive correlations were observed where circadian clock genes were simultaneously dysregulated in tumour tissue specimens. In cell line models, Clock, Bmal1, Per2, Cry1 and RORα circadian clock genes were significantly downregulated in transformed cells, compared to their untransformed counterparts, as well as in most cervical and oesophageal cancer cell lines compared to non-cancer epithelial cells. Patterns of protein expression did not accurately match mRNA expression, likely due to extensive post-translational processing, but Clock and Cry1 protein levels were considerably reduced in the cancer cell lines compared to normal. Overexpression of circadian clock genes in cancer cell lines negatively affected cell proliferation, highlighting the tumour suppressor properties of these genes in cervical and oesophageal cancer cells. Despite downreglated expression of circadian clock genes, cervical and oesophageal cancer cells maintain functional circadian oscillations, after synchronisation with Dexamethasone, as shown using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are still intact. Together, this study is a first to describe deregulated circadian clock machinery in cervical and oesophageal cancer cells, although the cells maintain a functional circadian rhythm. The study has relevance to the field of chronotherapy, where elucidating differences in circadian clock functioning in normal and cancer cells could yield better insights into the timing of administration of chemotherapy, ultimately ensuring a better patient response. Citation Format: Pauline J. van der Watt, Kate Davis, Virna Leaner. Investigating deregulated circadian clock machinery in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1729.

Intercellular Coupling Confers Robustness against Mutations in the SCN Circadian Clock Network

Nutrient and energy metabolism in organisms oscillates in a time-of the-day-dependent manner under the control of an endogenous timing mechanism called the circadian clock. This is a cell autonomous, self-sustained molecular mechanism, which is synchronized by a key environmental signals, notably light and food availability. There is a wealth of evidence showing a bidirectional interaction between food-regulated clocks and the rhythmic expression of metabolic genes in peripheral tissues, notably the liver. For example, genetic or environmental disruption of the circadian clock is linked with metabolic disease, such as obesity and type 2 diabetes. Furthermore, cycling changes in cellular redox potential impact on the expression of circadian clock genes and influence energy metabolism. Therefore, it is vital to understand how animals integrate input from lighting conditions and food availability to ultimately coordinate their daily metabolic rhythms. In this regard, one key issue is whether there are genetically distinct light and food regulated circadian clock mechanisms. The Foulkes group has used zebrafish and blind cavefish models to demonstrate that certain metabolic pathways cycle according to the light dark cycle and are unaffected by the timing of feeding activity, while other pathways are predominantly feeding time regulated. Based on these preliminary data, this thesis project used fish models and fish-derived cell lines to explore the genetic mechanisms linking metabolism with light and food regulated circadian clocks. The first part of this project aimed to explore at which stage during early zebrafish development a feeding-regulated clock first appears. Due to reduced feeding activity in constant darkness it was not feasible to examine the impact of feeding on clock gene expression. However, it was revealed that regular handling and disturbance of fish larvae, under otherwise constant environmental conditions results in the emergence of circadian clock rhythmicity. Several lines of evidence indicate that stress serves as a Zeitgeber and results in the emergence of rhythmicity in clock gene expression as well as clock outputs such as the cell cycle. The second part of this thesis explored whether genetically distinct light and food regulated clocks coexist in fish cells and which transcriptional control mechanisms link food-regulated circadian clocks with metabolism. It was demonstrated that during restricted feeding in zebrafish, rhythmic expression of core clock genes in the liver is regulated according to the timing of light-dark cycles, whereas the expression of genes involved in the control of metabolism are influenced by feeding time. However, this study was unable to confirm previous data obtained using NMR, where it was shown that circadian rhythmicity in the levels of essential amino acids is regulated by the light-dark cycle while rhythmic non-essential amino acid levels are influenced by feeding time. Instead, by UPLC-MS/MS analysis, daily changes in the concentration of both essential and non-essential amino acids were shown to be set by the phase of regular timed feeding and not by the light dark cycle. Furthermore, the NAD+ biosynthesis pathway and autophagy were affected by a clock which is set by feeding time and not by light-dark cycles. In addition, regular nocturnal feeding resulted in an increase in obesity. These findings point to the presence of at least two distinct clock mechanism in the zebrafish liver. In order to explore in more detail, the nature of the multiple clock mechanisms in zebrafish cells, the next part of this project employed multi-omics approaches and revealed infradian rhythmicity in amino acid concentrations in cultured fish cell lines. However, neither the expression of amino acid transporters nor autophagy exhibited infradian rhythmicity, instead showing circadian rhythmicity. In order to explore the involvement of the classical circadian clock mechanism in generating infradian rhythmicity, a cell line expressing a dominant negative form of clock1 gene was examined and shown to lack infradian rhythmicity in amino acid levels. Interestingly, the mRNA expression of Asparagine synthetase (asns) shows infradian rhythmicity, which are disrupted in Δclock1 cells. These data lead to the hypothesis that asns may be involved in the regulation of infradian rhythms in amino acid levels and point to a complex interplay between circadian and infradian rhythmicity.