Clock Regulation Research Articles

A circadian clock in Neurospora crassa functions during plant cell wall deconstruction

Circadian clocks are autonomous timers that are believed to confer organisms a selective advantage by enabling processes to occur at appropriate times of the day. In the model fungus Neurospora crassa, 20–40 % of its genes are reported to be under circadian regulation, as assayed in simple sugar media. Although it has been well-described that Neurospora efficiently deconstructs plant cell wall components, little is known regarding the status of the clock when Neurospora grows on cellulosic material, or whether such a clock has an impact on any of the genes involved in this process. Through luciferase-based reporters and fluorescent detection assays, we show that a clock is functioning when Neurospora grows on cellulose-containing wheat straw as the only carbon and nitrogen source. Additionally, we found that the major cellobiohydrolase encoding gene involved in plant cell wall deconstruction, cbh-1, is rhythmically regulated by the Neurospora clock, in a manner that depends on cellulose concentration and on the transcription factor CRE-1, known as a key player in carbon-catabolite repression in this fungus. Our findings are a step towards a more comprehensive understanding on how clock regulation modulates cellulose degradation, and thus Neurospora’s physiology.

Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176

G-protein-coupled receptors (GPCRs) are important drug targets with diverse therapeutic applications. However, there are still more than a hundred orphan GPCRs, whose protein functions and biochemical features remain unidentified. Gpr176 encodes a class-A orphan GPCR that has a role in circadian clock regulation in mouse hypothalamus and is also implicated in human breast cancer transcriptional response. Here we show that Gpr176 is N-glycosylated. Peptide-N-glycosidase treatment of mouse hypothalamus extracts revealed that endogenous Gpr176 undergoes N-glycosylation. Using a heterologous expression system, we show that N-glycosylation occurs at four conserved asparagine residues in the N-terminal region of Gpr176. Deficient N-glycosylation due to mutation of these residues reduced the protein expression of Gpr176. At the molecular function level, Gpr176 has constitutive, agonist-independent activity that leads to reduced cAMP synthesis. Although deficient N-glycosylation did not compromise this intrinsic activity, the resultant reduction in protein expression was accompanied by attenuation of cAMP-repressive activity in the cells. We also demonstrate that human GPR176 is N-glycosylated. Importantly, missense variations in the conserved N-glycosylation sites of human GPR176 (rs1473415441; rs761894953) affected N-glycosylation and thereby attenuated protein expression and cAMP-repressive activity in the cells. We show that N-glycosylation is a prerequisite for the efficient protein expression of functional Gpr176/GPR176.

Diurnal patterns of growth and transient reserves of sink and source tissues are affected by cold nights in barley.

Barley is described to mostly use sucrose for night carbon requirements. To understand how the transient carbon is accumulated and utilized in response to cold, barley plants were grown in a combination of cold days and/or nights. Both daytime and night cold reduced growth. Sucrose was the main carbohydrate supplying growth at night, representing 50-60% of the carbon consumed. Under warm days and nights, starch was the second contributor with 26% and malate the third with 15%. Under cold nights, the contribution of starch was severely reduced, due to an inhibition of its synthesis, including under warm days, and malate was the second contributor to C requirements with 24-28% of the total amount of carbon consumed. We propose that malate plays a critical role as an alternative carbon source to sucrose and starch in barley. Hexoses, malate, and sucrose mobilization and starch accumulation were affected in barley elf3 clock mutants, suggesting a clock regulation of their metabolism, without affecting growth and photosynthesis however. Altogether, our data suggest that the mobilization of sucrose and malate and/or barley growth machinery are sensitive to cold.

Circadian analysis of the mouse retinal pigment epithelium transcriptome

The presence of a phagocytic peak of photoreceptor outer segments by the retinal pigment epithelium (RPE) one or 2 h after the onset of light has been reported for several diurnal and nocturnal species. This peak in phagocytic activity also persists under constant lighting conditions (i.e., constant light or dark) thus demonstrating that the timing of this peak is driven by a circadian clock. The aim of this study was to investigate the change in RPE whole transcriptome at two different circadian times (CT; 1 h before (CT23) and 1 h after (CT1) subjective light onset). C57BL/6J male mice were maintained in constant dark conditions for three days and euthanized under red light (<1 lux) at CT23 and CT1. RPE was isolated from whole eyes for RNA library preparation and sequencing on an Illumina HiSeq4000 platform. 14,083 mouse RPE transcripts were detected in common between CT23 and CT1. 12,005 were protein coding transcripts and 2078 were non-protein coding transcripts. 2421 protein coding transcripts were significantly upregulated whereas only 3 transcripts were significantly downregulated and 12 non-protein coding transcripts were significantly upregulated and 31 non-protein coding transcripts were significantly downregulated at CT1 when compared to CT23 (p < 0.05, fold change ≥ ±2.0). Of the protein coding transcripts, most of them were characterized as: enzymes, kinases, and transcriptional regulators with a large majority of activity in the cytoplasm, nucleus, and plasma membrane. Non-protein coding transcripts included biotypes such as long-non coding RNAs and pseudogenes. Gene ontology analysis and ingenuity pathway analysis revealed that differentially expressed transcripts were associated with integrin signaling, oxidative phosphorylation, protein phosphorylation, and actin cytoskeleton remodeling suggesting that these previously identified phagocytic pathways are under circadian control. Our analysis identified new pathways (e.g., increased mitochondrial respiration via increased oxidative phosphorylation) that may be involved in the circadian control of phagocytic activity. In addition, our dataset suggests a possible regulatory role for the identified non-protein coding transcripts in mediating the complex function of RPE phagocytosis. Finally, our results also indicate, as seen in other tissues, about 20% of the whole RPE transcriptome may be under circadian clock regulation.

Kalanchoë PPC1 Is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling Genes.

Unlike C3 plants, Crassulacean acid metabolism (CAM) plants fix CO2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines phosphoenolpyruvate with CO2 (as HCO3 -), forming oxaloacetate. The oxaloacetate is converted to malate, leading to malic acid accumulation in the vacuole, which peaks at dawn. During the light period, malate decarboxylation concentrates CO2 around Rubisco for secondary fixation. CAM mutants lacking PPC have not been described. Here, we employed RNA interference to silence the CAM isogene PPC1 in Kalanchoë laxiflora Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO2 fixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance and arrhythmia of the CAM CO2 fixation circadian rhythm under constant light and temperature free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1 Furthermore, a subset of gene transcripts within the central circadian oscillator was upregulated and oscillated robustly in this line. The regulation of guard cell genes involved in controlling stomatal movements was also perturbed in rPPC1-B These findings provide direct evidence that the regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM.

The dynamic nature and regulation of the root clock.

Plants explore the soil by continuously expanding their root system, a process that depends on the production of lateral roots (LRs). Sites where LRs can be produced are specified in the primary root axis through a pre-patterning mechanism, determined by a biological clock that is coordinated by temporal signals and positional cues. This 'root clock' generates an oscillatory signal that is translated into a developmental cue to specify a set of founder cells for LR formation. In this Review, we summarize recent findings that shed light on the mechanisms underlying the oscillatory signal and discuss how a periodic signal contributes to the conversion of founder cells into LR primordia. We also provide an overview of the phases of the root clock that may be influenced by endogenous factors, such as the plant hormone auxin, and by exogenous environmental cues. Finally, we discuss additional aspects of the root-branching process that act independently of the root clock.

Genome-Wide DNA Methylation Patterns in Persistent Attention-Deficit/Hyperactivity Disorder and in Association With Impulsive and Callous Traits.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that often persists into adulthood. ADHD and related personality traits, such as impulsivity and callousness, are caused by genetic and environmental factors and their interplay. Epigenetic modifications of DNA, including methylation, are thought to mediate between such factors and behavior and may behave as biomarkers for disorders. Here, we set out to study DNA methylation in persistent ADHD and related traits. We performed epigenome-wide association studies (EWASs) on peripheral whole blood from participants in the NeuroIMAGE study (age range 12–23 years). We compared participants with persistent ADHD (n = 35) with healthy controls (n = 19) and with participants with remittent ADHD (n = 19). Additionally, we performed EWASs of impulsive and callous traits derived from the Conners Parent Rating Scale and the Callous-Unemotional Inventory, respectively, across all participants. For every EWAS, the linear regression model analyzed included covariates for age, sex, smoking scores, and surrogate variables reflecting blood cell type composition and genetic background. We observed no epigenome-wide significant differences in single CpG site methylation between participants with persistent ADHD and healthy controls or participants with remittent ADHD. However, epigenome-wide analysis of differentially methylated regions provided significant findings showing that hypermethylated regions in the APOB and LPAR5 genes were associated with ADHD persistence compared to ADHD remittance (p = 1.68 * 10−24 and p = 9.06 * 10−7, respectively); both genes are involved in cholesterol signaling. Both findings appeared to be linked to genetic variation in cis. We found neither significant epigenome-wide single CpG sites nor regions associated with impulsive and callous traits; the top-hits from these analyses were annotated to genes involved in neurotransmitter release and the regulation of the biological clock. No link to genetic variation was observed for these findings, which thus might reflect environmental influences. In conclusion, in this pilot study with a small sample size, we observed several DNA-methylation–disorder/trait associations of potential significance for ADHD and the related behavioral traits. Although we do not wish to draw conclusions before replication in larger, independent samples, cholesterol signaling and metabolism may be of relevance for the onset and/or persistence of ADHD.

Circadian Clock, Time-Restricted Feeding and Reproduction.

The goal of this review was to seek a better understanding of the function and differential expression of circadian clock genes during the reproductive process. Through a discussion of how the circadian clock is involved in these steps, the identification of new clinical targets for sleep disorder-related diseases, such as reproductive failure, will be elucidated. Here, we focus on recent research findings regarding circadian clock regulation within the reproductive system, shedding new light on circadian rhythm-related problems in women. Discussions on the roles that circadian clock plays in these reproductive processes will help identify new clinical targets for such sleep disorder-related diseases.

Recent advances in understanding regulation of the Arabidopsis circadian clock by local cellular environment.

Circadian clocks have evolved to synchronise an organism’s physiology with the environmental rhythms driven by the Earth’s rotation on its axis. Over the past two decades, many of the genetic components of the Arabidopsis thaliana circadian oscillator have been identified. The interactions between these components have been formulized into mathematical models that describe the transcriptional translational feedback loops of the oscillator. More recently, focus has turned to the regulation and functions of the circadian clock. These studies have shown that the system dynamically responds to environmental signals and small molecules. We describe advances that have been made in discovering the cellular mechanisms by which signals regulate the circadian oscillator of Arabidopsis in the context of tissue-specific regulation.

12-h clock regulation of genetic information flow by XBP1s.

Our group recently characterized a cell-autonomous mammalian 12-h clock independent from the circadian clock, but its function and mechanism of regulation remain poorly understood. Here, we show that in mouse liver, transcriptional regulation significantly contributes to the establishment of 12-h rhythms of mRNA expression in a manner dependent on Spliced Form of X-box Binding Protein 1 (XBP1s). Mechanistically, the motif stringency of XBP1s promoter binding sites dictates XBP1s’s ability to drive 12-h rhythms of nascent mRNA transcription at dawn and dusk, which are enriched for basal transcription regulation, mRNA processing and export, ribosome biogenesis, translation initiation, and protein processing/sorting in the Endoplasmic Reticulum (ER)-Golgi in a temporal order consistent with the progressive molecular processing sequence described by the central dogma information flow (CEDIF). We further identified GA-binding proteins (GABPs) as putative novel transcriptional regulators driving 12-h rhythms of gene expression with more diverse phases. These 12-h rhythms of gene expression are cell autonomous and evolutionarily conserved in marine animals possessing a circatidal clock. Our results demonstrate an evolutionarily conserved, intricate network of transcriptional control of the mammalian 12-h clock that mediates diverse biological pathways. We speculate that the 12-h clock is coopted to accommodate elevated gene expression and processing in mammals at the two rush hours, with the particular genes processed at each rush hour regulated by the circadian and/or tissue-specific pathways.

Computer-Aided Design and Kinematic Simulation of Huygens’s Pendulum Clock

This article presents both the three-dimensional modelling of the isochronous pendulum clock and the simulation of its movement, as designed by the Dutch physicist, mathematician, and astronomer Christiaan Huygens, and published in 1673. This invention was chosen for this research not only due to the major technological advance that it represented as the first reliable meter of time, but also for its historical interest, since this timepiece embodied the theory of pendular movement enunciated by Huygens, which remains in force today. This 3D modelling is based on the information provided in the only plan of assembly found as an illustration in the book Horologium Oscillatorium, whereby each of its pieces has been sized and modelled, its final assembly has been carried out, and its operation has been correctly verified by means of CATIA V5 software. Likewise, the kinematic simulation of the pendulum has been carried out, following the approximation of the string by a simple chain of seven links as a composite pendulum. The results have demonstrated the exactitude of the clock.

A New Set of Stability Criteria Extending Lyapunov's Direct Method

A dynamical system is a mathematical model described by a high dimensional ordinary differential equation for a wide variety of real world phenomena, which can be as simple as a clock pendulum or as complex as a chaotic Lorenz system. Stability is an important topic in the studies of the dynamical system. A major challenge is that the analytical solution of a time-varying nonlinear dynamical system is in general not known. Lyapunov's direct method is a classical approach used for many decades to study stability without explicitly solving the dynamical system, and has been successfully employed in numerous applications ranging from aerospace guidance systems, chaos theory, to traffic assignment. Roughly speaking, an equilibrium is stable if an energy function monotonically decreases along the trajectory of the dynamical system. This paper extends Lyapunov's direct method by allowing the energy function to follow a rich set of dynamics. More precisely, the paper proves two theorems, one on globally uniformly asymptotic stability and the other on stability in the sense of Lyapunov, where stability is guaranteed provided that the evolution of the energy function satisfies an inequality of a non-negative Hurwitz polynomial differential operator, which uses not only the first-order but also high-order time derivatives of the energy function. The classical Lyapunov theorems are special cases of the extended theorems. the paper provides an example in which the new theorem successfully determines stability while the classical Lyapunov's direct method fails.

Clock regulation of protein secretion.

The molecular clock regulates the rhythmic transcription of myriad genes, leading to a circadian pattern of expression of the encoded proteins. A study demonstrates circadian regulation of expression of components of the protein secretory pathway, providing a mechanism to generate circadian patterns of secreted protein expression.

Circadian Clock Regulation on Lipid Metabolism and Metabolic Diseases.

The basic helix-loop-helix-PAS transcription factor (CLOCK, Circadian locomotor output cycles protein kaput) was discovered in 1994 as a circadian clock. Soon after its discovery, the circadian clock, Aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL, also call BMAL1), was shown to regulate adiposity and body weight by controlling on the brain hypothalamic suprachiasmatic nucleus (SCN). Farther, circadian clock genes were determined to exert several of lipid metabolic and diabetes effects, overall indicating that CLOCK and BMAL1 act as a central master circadian clock. A master circadian clock acts through the neurons and hormones, with expression in the intestine, liver, kidney, lung, heart, SCN of brain, and other various cell types of the organization. Among circadian clock genes, numerous metabolic syndromes are the most important in the regulation of food intake (via regulation of circadian clock genes or clock-controlled genes in peripheral tissue), which lead to a variation in plasma phospholipids and tissue phospholipids. Circadian clock genes affect the regulation of transporters and proteins included in the regulation of phospholipid metabolism. These genes have recently received increasing recognition because a pharmacological target of circadian clock genes may be of therapeutic worth to make better resistance against insulin, diabetes, obesity, metabolism syndrome, atherosclerosis, and brain diseases. In this book chapter, we focus on the regulation of circadian clock and summarize its phospholipid effect as well as discuss the chemical, physiology, and molecular value of circadian clock pathway regulation for the treatment of plasma lipids and atherosclerosis.

Study of the modulatory mechanism of the miR-182-Clock axis in circadian rhythm disturbance after hypoxic–ischemic brain damage

Hypoxic–ischemic encephalopathy (HIE) in neonates can lead to severe chronic neurological deficit, including mental retardation, epilepsy, and sleep–wake cycle (SWC) disorder. Among these defects, little is known about the molecular mechanism of circadian rhythm disorder after HIE. Therefore, further study of sleep problems and its mechanism in HIE children will provide new ideas for clinical treatment of HIE children. For pediatric patients with cerebral ischemia, somnipathy often occurs due to visual and airway abnormalities. From May 2010 to August 2013, 128 newborns with history of HIE were followed up. Meanwhile, 88 normal full-term newborns in the same period were taken as the control group. The clinical data of the patients were collected and the sleep status was assessed by questionnaire. To establish the hypoxic–ischemic brain injury model of neonatal rats and analyze the mechanism of mir-182 in the circadian rhythm disorder caused by pineal function injury. The core clock genes during the regulation of the circadian clock were explored by bioinformatics methods. Patients’ sleep quality was affected by the circadian rhythm and respiratory problems; the pineal gland can regulate the core clock genes in the circadian clock during regulation. miR-182 was highly expressed in the pineal gland after hypoxic–ischemic brain damage (HIBD). Children with mild and moderate HIE showed significant sleep disorders in varying degrees, which provided a clinical basis for improving the long-term prognosis of children with HIE through targeted treatment of sleep disorders. MiR-182 is highly expressed in the pineal gland and is related to the expression of CLOCK protein. CLOCK gene is the target gene of miR-182, which provides a new target for the treatment of rhythm disorder related to the damage of pineal function caused by HIBD.

Circadian control of the secretory pathway maintains collagen homeostasis.

Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function.

Rev-erbα Negatively Regulates Osteoclast and Osteoblast Differentiation through p38 MAPK Signaling Pathway.

The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Rev-erbs, comprising Rev-erbα and Rev-erbβ, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erbα in osteoclast precursor cells enhanced receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erbα leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erbα in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erbα interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Rev-erbα in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erbα negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo. Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.

Habitat-Specific Clock Variation and Its Consequence on Reproductive Fitness.

The circadian clock controls daily activities at the cellular and organismic level, allowing an organism to anticipate incoming stresses and to use resources accordingly. The circadian clock has therefore been considered a fitness trait in multiple organisms. However, the mechanism of how circadian clock variation influences organismal reproductive fitness is still not well understood. Here we describe habitat-specific clock variation (HSCV) of asexual reproduction in Neurospora discreta, a species that is adapted to 2 different habitats, under or above tree bark. African (AF) N. discreta strains, whose habitat is above the tree bark in light-dark (LD) conditions, display a higher rhythmicity index compared with North American (NA) strains, whose habitat is under the tree bark in constant dark (DD). Although AF-type strains demonstrated an overall fitness advantage under LD and DD conditions, NA-type strains exhibit a habitat-specific fitness advantage in DD over the LD condition. In addition, we show that allelic variation of the clock-controlled gene, Ubiquinol cytochrome c oxidoreductase (NEUDI_158280), plays a role in HSCV by modulating cellular reactive oxygen species levels. Our results demonstrate a mechanism by which local adaptation involving circadian clock regulation influences reproductive fitness.

Molecular Cloning and Expression of a Cryptochrome Gene CiCRY-DASH1 from the Antarctic microalga Chlamydomonas sp. ICE-L.

Cryptochromes (CRYs) are flavin-binding proteins that sense blue and near-ultraviolet light and participate in the photoreactions of organisms and the regulation of biological clocks. In this study, the complete open reading frame (ORF) of CiCRY-DASH1 (GenBank ID MK392361), encoding one kind of cryptochrome, was cloned from the Antarctic microalga Chlamydomonas sp. ICE-L. The quantitative real-time PCR study showed that the CiCRY-DASH1 had the highest expression at 5°C and salinity of 32‰. The CiCRY-DASH1 was positively regulated by blue, yellow, or red light. Moreover, the CiCRY-DASH1 can positively respond to extreme polar day and night treatment and exhibit a certain circadian rhythm, which indicated that CiCRY-DASH1 participated in the circadian clock and its expression was regulated by circadian rhythms. And the CiCRY-DASH1 was more noticeably affected by ultraviolet-B radiation than ultraviolet-A radiation, indicating ultraviolet-B light does obvious damage to Antarctic microalgae.

The long term impact of Daylight Saving Time regulations in daily life at several circles of latitude

We analyze large scale (N ~ 10000) time use surveys in United States, Spain, Italy, France and Great Britain to ascertain seasonal variations in the sleep/wake cycle and the labor cycle after daylight saving time regulations have stood up for at least forty years. That is, not the usual search for the impact of the biannual transitions, but a search for how industrialized societies have answered to DST regulations at different circles of latitude. Results show that the labor cycle is equally distributed through seasons if measured in local time. It is an everyday experience which is a major outcome of DST. The sleep/wake cycle displays disturbances punctuated by solar events: sunrise, sunset and noon. In week-ends, under free preferences, sleep onset delays in summer, opposing to the regulation and following the delay in sunset time, while sleep offset advances, despite clock time already advanced in the spring transition. This advance still follows the advance in sunrise times. The best explanation for these findings is that human cycles are not misaligned by the size and direction of DST regulations, which explains the success of that practice. The sleep/wake cycle in Great Britain and France exhibit fewer statistically significant excursions than the sleep/wake cycle in Spain, Italy and United States, despite light and dark seasonal deviations are larger. That could be indicating that the preference for a seasonal regulation of time decreases with increasing latitude above 47°. The preferences for a seasonal regulation of clocks and for the choice of permanent summer time or permanent winter time are sketched from a previous report on human activity.