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1.  Molecular Clocks in Pharmacology 
Circadian rhythms regulate a vast array of biological processes and play a fundamental role in mammalian physiology. As a result, considerable diurnal variation in the pharmacokinetics, efficacy, and side effect profiles of many therapeutics has been described. This variation has subsequently been tied to diurnal rhythms in absorption, distribution, metabolism, and excretion, as well as in pharmacodynamic variables, such as target expression. More recently, the molecular basis of circadian rhythmicity has been elucidated with the identification of clock genes, which oscillate in a circadian manner in most cells and tissues and regulate transcription of large sets of genes. Ongoing research efforts are beginning to reveal the critical role of circadian clock genes in the regulation of pharmacologic parameters, as well as the reciprocal impact of drugs on circadian clock function. This chapter will review the role of circadian clocks in the pharmacokinetics and pharmacodynamics of drug response, and provide several examples of the complex regulation of pharmacologic systems by components of the molecular circadian clock.
doi:10.1007/978-3-642-25950-0_10
PMCID: PMC3684693  PMID: 23604482
circadian clock; pharmacology; pharmacokinetics; pharmacodynamics; CLOCK; Bmal1
2.  Regulation of alternative splicing by the circadian clock and food related cues 
Genome Biology  2012;13(6):R54.
Background
The circadian clock orchestrates daily rhythms in metabolism, physiology and behaviour that allow organisms to anticipate regular changes in their environment, increasing their adaptation. Such circadian phenotypes are underpinned by daily rhythms in gene expression. Little is known, however, about the contribution of post-transcriptional processes, particularly alternative splicing.
Results
Using Affymetrix mouse exon-arrays, we identified exons with circadian alternative splicing in the liver. Validated circadian exons were regulated in a tissue-dependent manner and were present in genes with circadian transcript abundance. Furthermore, an analysis of circadian mutant Vipr2-/- mice revealed the existence of distinct physiological pathways controlling circadian alternative splicing and RNA binding protein expression, with contrasting dependence on Vipr2-mediated physiological signals. This view was corroborated by the analysis of the effect of fasting on circadian alternative splicing. Feeding is an important circadian stimulus, and we found that fasting both modulates hepatic circadian alternative splicing in an exon-dependent manner and changes the temporal relationship with transcript-level expression.
Conclusions
The circadian clock regulates alternative splicing in a manner that is both tissue-dependent and concurrent with circadian transcript abundance. This adds a novel temporal dimension to the regulation of mammalian alternative splicing. Moreover, our results demonstrate that circadian alternative splicing is regulated by the interaction between distinct physiological cues, and illustrates the capability of single genes to integrate circadian signals at different levels of regulation.
doi:10.1186/gb-2012-13-6-r54
PMCID: PMC3446320  PMID: 22721557
3.  Possible contribution of chronobiology to cardiovascular health 
The daily variations found in many aspects of physiology are collectively known as circadian rhythm (from “circa” meaning “about” and “dien” meaning “day”). Circadian oscillation in clock gene expression can generate quantitative or functional variations of the molecules directly involved in many physiological functions. This paper reviews the molecular mechanisms of the circadian clock, the transmission of circadian effects to cardiovascular functions, and the effects of circadian dysfunction on cardiovascular diseases. An evaluation of the operation of the internal clock is needed in clinical settings and will be an effective tool in the diagnosis of circadian rhythm disorders. Toward this end, we introduce a novel non-invasive method for assessing circadian time-regulation in human beings through the utilization of hair follicle cells.
doi:10.3389/fphys.2013.00409
PMCID: PMC3895809  PMID: 24478711
circadian; clock gene; non-invasive method; cardiovascular diseases; hair follicle
4.  Characterization of orderly spatiotemporal patterns of clock gene activation in mammalian suprachiasmatic nucleus 
The European journal of neuroscience  2011;33(10):1851-1865.
Because we can observe oscillation within individual cells and in the tissue as a whole, the suprachiasmatic nucleus (SCN) presents a unique system in the mammalian brain for the analysis of individual cells and the networks of which they are a part. While dispersed cells of the SCN sustain circadian oscillations in isolation, they are unstable oscillators that require network interactions for robust cycling. Using cluster analysis to assess bioluminescence in acute brain slices from PERIOD2∷Luciferase (PER2∷LUC) knockin mice, and immunochemistry of SCN from animals harvested at various circadian times, we assessed the spatiotemporal activation patterns of PER2 to explore the emergence of a coherent oscillation at the tissue level. The results indicate that circadian oscillation is characterized by a stable daily cycle of PER2 expression involving orderly serial activation of specific SCN subregions, followed by a silent interval, with substantial symmetry between the left and right side of the SCN. The biological significance of the clusters identified in living slices was confirmed by co-expression of LUC and PER2 in fixed, immunochemically stained brain sections, with the spatiotemporal pattern of LUC expression resembling that revealed in the cluster analysis of bioluminescent slices. We conclude that the precise timing of PER2 expression within individual neurons is dependent on their location within the nucleus, and that small groups of neurons within the SCN give rise to distinctive and identifiable subregions. We propose that serial activation of these subregions is the basis of robustness and resilience of the daily rhythm of the SCN.
doi:10.1111/j.1460-9568.2011.07682.x
PMCID: PMC3423955  PMID: 21488990
circadian rhythms; luciferase; networks; PER2; SCN; synchronization
5.  Harmonics of Circadian Gene Transcription in Mammals 
PLoS Genetics  2009;5(4):e1000442.
The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior. Numerous investigations have addressed the contribution of circadian rhythmicity to cellular, organ, and organismal physiology. We recently developed a method to look at transcriptional oscillations with unprecedented precision and accuracy using high-density time sampling. Here, we report a comparison of oscillating transcription from mouse liver, NIH3T3, and U2OS cells. Several surprising observations resulted from this study, including a 100-fold difference in the number of cycling transcripts in autonomous cellular models of the oscillator versus tissues harvested from intact mice. Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells. Validation experiments show that 12-hour oscillatory transcripts occur in several other peripheral tissues as well including heart, kidney, and lungs. These harmonics are lost ex vivo, as well as under restricted feeding conditions. Taken in sum, these studies illustrate the importance of time sampling with respect to multiple testing, suggest caution in use of autonomous cellular models to study clock output, and demonstrate the existence of harmonics of circadian gene expression in the mouse.
Author Summary
Circadian rhythms confer adaptive advantages by allowing organisms to anticipate daily changes in their environment. Over the last few years, many groups have used microarray technology to systematically identify genes under circadian regulation. We have extended on these studies by profiling the circadian transcriptome from the mouse liver and two immortalized cell lines at an unprecedentedly high temporal resolution. We identified over 3,000 different transcripts in the mouse liver that cycle with a period length of approximately 24 hours. To our surprise, we also identified two classes of genes which cycle with period lengths of 12 and 8 hours; i.e., harmonics of the circadian clock. Importantly, we were able to identify harmonics in five other tissue types; however, these rhythms were undetectable in disassociated cells. Moreover, harmonics were lost in the liver when mice are subjected to restricted feeding, suggesting that at least one component of circadian harmonics is driven by feeding.
doi:10.1371/journal.pgen.1000442
PMCID: PMC2654964  PMID: 19343201
6.  Basis of Robustness and Resilience in the Suprachiasmatic Nucleus: Individual Neurons Form Nodes in Circuits that Cycle Daily 
Journal of biological rhythms  2009;24(5):340-352.
How the cellular elements of the SCN are synchronized to each other is not well understood. We explore circadian oscillations manifest at the level of the cell, the tissue, and the whole animal to better understand intra-SCN synchrony and master clock function of the nucleus. At each level of analysis, responses to variations in operating environment (robustness), and following damage to components of the system (resilience), provide insight into the mechanisms whereby the SCN orchestrates circadian timing. Tissue level rhythmicity reveals circuits associated with an orderly spatiotemporal daily pattern of activity that is not predictable from their cellular elements. Specifically, in stable state, some SCN regions express low amplitude or undetectable rhythms in clock gene expression while others produce high amplitude oscillations. Within the SCN, clock gene expression follows a spatially ordered, repeated pattern of activation and inactivation. This pattern of activation is plastic and subserves responses to changes in external and internal conditions. Just as daily rhythms at the cellular level depend on sequential expression and interaction of clock genes, so too do rhythms at the SCN tissue level depend on sequential activation of local nodes. We hypothesize that individual neurons are organized into nodes that are themselves sequentially activated across the volume of the SCN in a cycle that repeats on a daily basis. We further propose that robustness is expressed in the ability of the SCN to sustain rhythmicity over a wide range of internal and external conditions, and that this reflects plasticity of the underlying nodes and circuits. Resilience is expressed in the ability of SCN cells to oscillate and to sustain activity-related rhythms at the behavioral level. Importantly, other aspects of pacemaker function remain to be examined.
doi:10.1177/0748730409344800
PMCID: PMC3104278  PMID: 19755580
circadian; coupling; connectome; emergent properties; network plasticity; node
7.  Chronobiology of Melatonin beyond the Feedback to the Suprachiasmatic Nucleus—Consequences to Melatonin Dysfunction 
The mammalian circadian system is composed of numerous oscillators, which gradually differ with regard to their dependence on the pacemaker, the suprachiasmatic nucleus (SCN). Actions of melatonin on extra-SCN oscillators represent an emerging field. Melatonin receptors are widely expressed in numerous peripheral and central nervous tissues. Therefore, the circadian rhythm of circulating, pineal-derived melatonin can have profound consequences for the temporal organization of almost all organs, without necessarily involving the melatonin feedback to the suprachiasmatic nucleus. Experiments with melatonin-deficient mouse strains, pinealectomized animals and melatonin receptor knockouts, as well as phase-shifting experiments with explants, reveal a chronobiological role of melatonin in various tissues. In addition to directly steering melatonin-regulated gene expression, the pineal hormone is required for the rhythmic expression of circadian oscillator genes in peripheral organs and to enhance the coupling of parallel oscillators within the same tissue. It exerts additional effects by modulating the secretion of other hormones. The importance of melatonin for numerous organs is underlined by the association of various diseases with gene polymorphisms concerning melatonin receptors and the melatonin biosynthetic pathway. The possibilities and limits of melatonergic treatment are discussed with regard to reductions of melatonin during aging and in various diseases.
doi:10.3390/ijms14035817
PMCID: PMC3634486  PMID: 23481642
age-related diseases; aging; circadian; melatonergic agonists; melatonin; MT1, MT2, peripheral oscillators; polymorphisms
8.  Altered behavioral and metabolic circadian rhythms in mice with disrupted NAD+ oscillation 
Aging (Albany NY)  2011;3(8):794-802.
The Intracellular levels of nicotinamide adenine dinucleotide (NAD+) are rhythmic and controlled by the circadian clock. However, whether NAD+ oscillation in turn contributes to circadian physiology is not fully understood. To address this question we analyzed mice mutated for the NAD+ hydrolase CD38. We found that rhythmicity of NAD+ was altered in the CD38-deficient mice. The high, chronic levels of NAD+ results in several anomalies in circadian behavior and metabolism. CD38-null mice display a shortened period length of locomotor activity and alteration in the rest-activity rhythm. Several clock genes and, interestingly, genes involved in amino acid metabolism were deregulated in CD38-null livers. Metabolomic analysis identified alterations in the circadian levels of several amino acids, specifically tryptophan levels were reduced in the CD38-null mice at a circadian time paralleling with elevated NAD+ levels. Thus, CD38 contributes to behavioral and metabolic circadian rhythms and altered NAD+ levels influence the circadian clock.
PMCID: PMC3184980  PMID: 21937766
Circadian Rhythm; Clock; NAD+; CD38; Amino acid metabolism
9.  In-Vivo Quantitative Proteomics Reveals a Key Contribution of Post-Transcriptional Mechanisms to the Circadian Regulation of Liver Metabolism 
PLoS Genetics  2014;10(1):e1004047.
Circadian clocks are endogenous oscillators that drive the rhythmic expression of a broad array of genes, orchestrating metabolism and physiology. Recent evidence indicates that post-transcriptional and post-translational mechanisms play essential roles in modulating temporal gene expression for proper circadian function, particularly for the molecular mechanism of the clock. Due to technical limitations in large-scale, quantitative protein measurements, it remains unresolved to what extent the circadian clock regulates metabolism by driving rhythms of protein abundance. Therefore, we aimed to identify global circadian oscillations of the proteome in the mouse liver by applying in vivo SILAC mouse technology in combination with state of the art mass spectrometry. Among the 3000 proteins accurately quantified across two consecutive cycles, 6% showed circadian oscillations with a defined phase of expression. Interestingly, daily rhythms of one fifth of the liver proteins were not accompanied by changes at the transcript level. The oscillations of almost half of the cycling proteome were delayed by more than six hours with respect to the corresponding, rhythmic mRNA. Strikingly we observed that the length of the time lag between mRNA and protein cycles varies across the day. Our analysis revealed a high temporal coordination in the abundance of proteins involved in the same metabolic process, such as xenobiotic detoxification. Apart from liver specific metabolic pathways, we identified many other essential cellular processes in which protein levels are under circadian control, for instance vesicle trafficking and protein folding. Our large-scale proteomic analysis reveals thus that circadian post-transcriptional and post-translational mechanisms play a key role in the temporal orchestration of liver metabolism and physiology.
Author Summary
The circadian clock is an evolutionary system that allows organisms to anticipate and thus adapt to daily changes in the environment. In mammals, the circadian clock is found in virtually every tissue regulating rhythms of metabolism and physiology. While a lot of studies have focused in how circadian clocks regulate gene expression little is known about daily control of protein abundance. Here we applied state of the art mass spectrometry in combination with quantitative proteomics to investigate global circadian oscillations of the proteome in the mouse liver. We found that approximately 6% of the liver proteins are cycling daily and interestingly the majority of these oscillations diverge from the behavior of their transcripts. Our data indicates that post-transcriptional mechanisms play an essential role in shaping the phase of rhythmic proteins downstream of transcription regulation to ultimately drive rhythms of metabolism. Moreover, the contribution of post-transcriptional regulation seems to differ among distinct metabolic pathways. Overall we not only found circadian oscillations in the abundance of proteins involved in liver specific metabolic pathways but also in essential cellular processes.
doi:10.1371/journal.pgen.1004047
PMCID: PMC3879213  PMID: 24391516
10.  Scheduled Exercise Phase Shifts the Circadian Clock in Skeletal Muscle 
Purpose
It has been well established in mammals that circadian behavior as well as the molecular clockwork can be synchronized to the light-dark (LD) cycle via the suprachiasmatic nucleus of the hypothalamus (SCN). In addition to light, it has been demonstrated that non-photic time cues, such as restricting the time of food availability, can alter circadian behavior and clock gene expression in selected peripheral tissues such as liver. Studies have also suggested that scheduled physical activity (exercise) can alter circadian rhythms in behavior and clock gene expression, however currently the effects of exercise alone are largely unknown and have not been explored in skeletal muscle.
Methods
Period2∷Luciferase (Per2∷Luc) mice were maintained under 12 hours of light followed by 12 hours of darkness (12L:12D) then exposed to 2 hours of voluntary or involuntary exercise during the light phase for 4 weeks. Control mice were left in home cages or moved to the exercise environment (sham). A second group of mice had restricted access to food (4 hours per day for 2 weeks) in order to compare the effects of two non-photic cues on PER2∷LUC bioluminescence. Skeletal muscles, lung and SCN tissue explants were cultured for 5-6 days to study molecular rhythms.
Results
In the exercised mice, the phase of peak PER2∷LUC bioluminescence was shifted in the skeletal muscle and lung explants but not the SCN suggesting a specific synchronizing effect of exercise on the molecular clockwork in peripheral tissues.
Conclusions
These data provide evidence that the molecular circadian clock in peripheral tissues can respond to the time of exercise suggesting that physical activity contributes important timing information for synchronization of circadian clocks throughout the body.
doi:10.1249/MSS.0b013e318255cf4c
PMCID: PMC3414645  PMID: 22460470
PERIOD2∷LUCIFERASE; CIRCADIAN; NON-PHOTIC; TIME CUE
11.  Exploring the transcriptional landscape of plant circadian rhythms using genome tiling arrays 
Genome Biology  2009;10(2):R17.
Whole genome tiling array analysis reveals the extent of transcriptional oscillation for both coding and non-coding genes in regulating Arabidopsis thaliana circadian rhythms
Background
Organisms are able to anticipate changes in the daily environment with an internal oscillator know as the circadian clock. Transcription is an important mechanism in maintaining these oscillations. Here we explore, using whole genome tiling arrays, the extent of rhythmic expression patterns genome-wide, with an unbiased analysis of coding and noncoding regions of the Arabidopsis genome.
Results
As in previous studies, we detected a circadian rhythm for approximately 25% of the protein coding genes in the genome. With an unbiased interrogation of the genome, extensive rhythmic introns were detected predominantly in phase with adjacent rhythmic exons, creating a transcript that, if translated, would be expected to produce a truncated protein. In some cases, such as the MYB transcription factor AT2G20400, an intron was found to exhibit a circadian rhythm while the remainder of the transcript was otherwise arrhythmic. In addition to several known noncoding transcripts, including microRNA, trans-acting short interfering RNA, and small nucleolar RNA, greater than one thousand intergenic regions were detected as circadian clock regulated, many of which have no predicted function, either coding or noncoding. Nearly 7% of the protein coding genes produced rhythmic antisense transcripts, often for genes whose sense strand was not similarly rhythmic.
Conclusions
This study revealed widespread circadian clock regulation of the Arabidopsis genome extending well beyond the protein coding transcripts measured to date. This suggests a greater level of structural and temporal dynamics than previously known.
doi:10.1186/gb-2009-10-2-r17
PMCID: PMC2688271  PMID: 19210792
12.  Expression Levels of Estrogen Receptor β Are Modulated by Components of the Molecular Clock▿  
Molecular and Cellular Biology  2007;28(2):784-793.
Circadian regulation of gene expression plays a major role in health and disease. The precise role of the circadian system remains to be clarified, but it is known that circadian proteins generate physiological rhythms in organisms by regulating clock-controlled target genes. The estrogen receptor beta (ERβ) is, together with ERα, a member of the nuclear receptor superfamily and a key mediator of estrogen action. Interestingly, recent studies show that disturbed circadian rhythmicity in humans can increase the risk of reproductive malfunctions, suggesting a link between the circadian system and ER-mediated transcription pathways. Here, we identify a novel level of regulation of estrogen signaling where ERβ, but not ERα, is controlled by circadian clock proteins. We show that ERβ mRNA levels fluctuate in different peripheral tissues following a robust circadian pattern, with a peak at the light-dark transition, which is maintained under free-running conditions. Interestingly, this oscillation is abolished in clock-deficient BMAL1 knockout mice. Circadian control of ERβ expression is exerted through a conserved E-box element in the ERβ promoter region that recruits circadian regulatory factors. Furthermore, using small interfering RNA-mediated knockdown assays, we show that the expression levels of the circadian regulatory factors directly influence estrogen signaling by regulating the intracellular levels of endogenous ERβ.
doi:10.1128/MCB.00233-07
PMCID: PMC2223432  PMID: 18039858
13.  Differential Rescue of Light- and Food-Entrainable Circadian Rhythms 
Science (New York, N.Y.)  2008;320(5879):1074-1077.
When food is plentiful, circadian rhythms of animals are powerfully entrained by the light-dark cycle. However, if animals have access to food only during their normal sleep cycle, they will shift most of their circadian rhythms to match the food availability. We studied the basis for entrainment of circadian rhythms by food and light in mice with targeted disruption of the clock gene Bmal1, which lack circadian rhythmicity. Injection of a viral vector containing the Bmal1 gene into the suprachiasmatic nuclei of the hypothalamus restored light-entrainable, but not food-entrainable, circadian rhythms. In contrast, restoration of the Bmal1 gene only in the dorsomedial hypothalamic nucleus restored the ability of animals to entrain to food but not to light. These results demonstrate that the dorsomedial hypothalamus contains a Bmal1-based oscillator that can drive food entrainment of circadian rhythms.
doi:10.1126/science.1153277
PMCID: PMC3489954  PMID: 18497298
14.  Circadian modulation of melanopsin-driven light response in rat ganglion-cell photoreceptors 
Journal of biological rhythms  2009;24(5):391-402.
Intrinsically photosensitive retinal ganglion cells (ipRGCs) project to the suprachiasmatic nucleus (SCN), and are essential for normal photic entrainment of global circadian rhythms in physiology and behavior. The effect of light on the central clock is dependent on circadian phase, and the retina itself contains intrinsic circadian oscillators that can alter its sensitivity to light. This raises the possibility that the ipRGCs, and hence the photoentraining signals in the retinohypothalamic tract, are subject to circadian modulation. Although the ipRGC photopigment, melanopsin, reportedly exhibits circadian variations in expression, there has been no direct test of the hypothesis that ipRGC sensitivity is under circadian control. Here, we provide such a test by measuring the sensitivity of intrinsic photoresponses of rat ipRGCs at four circadian times (CTs) using multielectrode array (MEA) recording. We observe little if any circadian modulation in the threshold of intrinsic ipRGC photoresponses. We do observe, however, that very bright light evoked significantly more spiking early in the subjective night (CT12-13) than at other circadian phases. Thus, the gain of the melanopsin-driven response is slightly increased in the early night, at roughly the circadian phase when melanopsin synthesis is thought to be elevated. However, this gain change is probably too modest to contribute much to shape the phase response curve for behavioral photoentrainment.
doi:10.1177/0748730409343767
PMCID: PMC2893738  PMID: 19755584
ipRGC; melanopsin; entrainment; sensitivity; gain; retina
15.  Clock Gene Expression in the Rat Retina: Effects of Lighting Conditions and Photoreceptor Degeneration 
Brain research  2007;1159:134-140.
Previous studies have shown that, in the Royal College of Surgeon rat, circadian rhythms in the retinal dopaminergic and melatonergic systems are still present after the photoreceptors have degenerated, thus demonstrating that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors. The aim of the present study was to investigate the pattern of expression of the clock genes in the retina of the Royal College of Surgeons rat under different lighting conditions. Expression of clock genes was investigated in the retina of normal and dystrophic Royal College of Surgeons rats under 12 hours of light/12 hours of dark (LD), constant darkness (DD) and constant light (LL) using Real Time Quantitative RT-PCR. Our data indicate that, in control animals, Period1, Period2, Cryptochrome1, Cryptochrome2, Clock, Rora, Rev-Erbα and Npas2 mRNA levels showed a significant variation over the sampling period in LD cycles and in DD, whereas Bmal1 mRNA did not show any significant variation. In LL, the transcripts for Per1, Per2, Clock and Rev-Erbα showed significant the temporal variations. In the dystrophic retina, only Per1 and Per2 mRNA levels showed a temporal variation over the 20-hours period. Our work indicates that degeneration of the photoreceptor cells dramatically affected the expression levels and patterns of many clock genes. Finally, the present study suggests that investigating the expression pattern of clock genes using the whole retina or animals with photoreceptor degeneration may not provide any definitive answers about the working of the retinal circadian clock system.
doi:10.1016/j.brainres.2007.05.023
PMCID: PMC1994112  PMID: 17560558
Retina; Circadian rhythm; Photoreceptor degeneration, Royal College Surgeon rat, Clock genes
16.  Circadian Rhythms of Fetal Liver Transcription Persist in the Absence of Canonical Circadian Clock Gene Expression Rhythms In Vivo 
PLoS ONE  2012;7(2):e30781.
The cellular circadian clock and systemic cues drive rhythmicity in the transcriptome of adult peripheral tissues. However, the oscillating status of the circadian clocks in fetal tissues, and their response to maternal cues, are less clear. Most clock genes do not cycle in fetal livers from mice and rats, although tissue level rhythms rapidly emerge when fetal mouse liver explants are cultured in vitro. Thus, in the fetal mouse liver, the circadian clock does not oscillate at the cellular level (but is induced to oscillate in culture). To gain a comprehensive overview of the clock status in the fetal liver during late gestation, we performed microarray analyses on fetal liver tissues. In the fetal liver we did not observe circadian rhythms of clock gene expression or many other transcripts known to be rhythmically expressed in the adult liver. Nevertheless, JTK_CYCLE analysis identified some transcripts in the fetal liver that were rhythmically expressed, albeit at low amplitudes. Upon data filtering by coefficient of variation, the expression levels for transcripts related to pancreatic exocrine enzymes and zymogen secretion were found to undergo synchronized daily fluctuations at high amplitudes. These results suggest that maternal cues influence the fetal liver, despite the fact that we did not detect circadian rhythms of canonical clock gene expression in the fetal liver. These results raise important questions on the role of the circadian clock, or lack thereof, during ontogeny.
doi:10.1371/journal.pone.0030781
PMCID: PMC3285613  PMID: 22383974
17.  When Clocks Go Bad: Neurobehavioural Consequences of Disrupted Circadian Timing 
PLoS Genetics  2008;4(5):e1000040.
Progress in unravelling the cellular and molecular basis of mammalian circadian regulation over the past decade has provided us with new avenues through which we can explore central nervous system disease. Deteriorations in measurable circadian output parameters, such as sleep/wake deficits and dysregulation of circulating hormone levels, are common features of most central nervous system disorders. At the core of the mammalian circadian system is a complex of molecular oscillations within the hypothalamic suprachiasmatic nucleus. These oscillations are modifiable by afferent signals from the environment, and integrated signals are subsequently conveyed to remote central neural circuits where specific output rhythms are regulated. Mutations in circadian genes in mice can disturb both molecular oscillations and measurable output rhythms. Moreover, systematic analysis of these mutants indicates that they can express an array of abnormal behavioural phenotypes that are intermediate signatures of central nervous system disorders. Furthermore, the response of these mutants to psychoactive drugs suggests that clock genes can modify a number of the brain’s critical neurotransmitter systems. This evidence has led to promising investigations into clock gene polymorphisms in psychiatric disease. Preliminary indications favour the systematic investigation of the contribution of circadian genes to central nervous system disease.
doi:10.1371/journal.pgen.1000040
PMCID: PMC2295261  PMID: 18516223
18.  Circadian Epigenomic Remodeling and Hepatic Lipogenesis: Lessons from HDAC3 
Circadian rhythms have evolved to anticipate metabolic needs across the 24-hour light/dark cycle. This is accomplished by circadian expression of metabolic genes orchestrated by transcription factors through chromatin remodeling and histone modifications. Our recent genome-wide study on histone deacetylase 3 (HDAC3) in mouse liver provides novel insights into the molecular link between circadian rhythm and hepatic de novo lipogenesis. We found that liver-specific knockout of HDAC3 in adult mouse display severe hepatic steatosis associated with enhanced de novo lipogenesis and increased expression of lipogenic genes. Genome-wide analysis (ChIP-seq) revealed a pronounced circadian pattern of HDAC3 occupancy on genes involved in lipid metabolism, which is inversely related to histone acetylation and RNA polymerase II recruitment at these sites. The cistromes of HDAC3 and its binding partner, nuclear receptor co-repressor (NCoR), significantly overlap with that of Rev-erbα, a nuclear receptor directly involved in the core circadian machinery. Knockout of Rev-erbα in mouse also leads to hepatic steatosis and enhanced de novo lipogenesis. Collectively, these data suggest that the circadian epigenomic remodeling controlled by HDAC3, and largely directed by Rev-erbα, is essential for homeostasis of the lipogenic process in liver.
doi:10.1101/sqb.2011.76.011494
PMCID: PMC3755609  PMID: 21900149
19.  Microarray Analysis of Natural Socially-Regulated Plasticity in Circadian Rhythms of Honey Bees 
Journal of Biological Rhythms  2012;27(1):12-24.
Honey bee workers care for ("nurse") the brood around the clock without circadian rhythmicity, but then they forage outside with strong circadian rhythms and a consolidated nightly rest. This chronobiological plasticity is associated with variation in the expression of the canonical “clock genes” that regulate the circadian clock: nurse bees show no brain rhythms of expression, while foragers do. These results suggest that the circadian system is organized differently in nurses and foragers. Nurses switch to activity with circadian rhythms shortly after removed from the hive suggesting that at least some clock cells in their brain continue to measure time while in the hive. We performed a microarray genome-wide survey to determine general patterns of brain gene expression in nurses and foragers sampled around the clock. We found 160 and 541 transcripts that exhibited significant sinusoidal oscillations in nurses and foragers, respectively, with peaks of expression distributed throughout the day in both task groups. Consistent with earlier studies, transcripts of genes involved in circadian rhythms, including Clockwork Orange that has not been studied before in bees, oscillated in foragers but not in nurses. The oscillating transcripts also were enriched for genes involved in the visual system, “development” and “response to stimuli” (foragers), “muscle contraction” and “microfilament motor gene expression” (nurses), and “generation of precursor metabolites” and “energy” (both). Transcripts of genes encoding P450 enzymes oscillated in both nurses and foragers but with a different phase. This study identified new putative clock-controlled genes in the honey bee and suggests that some brain functions show circadian rhythmicity even in nurse bees that are active around the clock.
doi:10.1177/0748730411431404
PMCID: PMC3313495  PMID: 22306970
transcriptome; social behavior; gene expression; clock genes; honey bee; P450
20.  Ischemic stroke destabilizes circadian rhythms 
Background
The central circadian pacemaker is a remarkably robust regulator of daily rhythmic variations of cardiovascular, endocrine, and neural physiology. Environmental lighting conditions are powerful modulators of circadian rhythms, but regulation of circadian rhythms by disease states is less clear. Here, we examine the effect of ischemic stroke on circadian rhythms in rats using high-resolution pineal microdialysis.
Methods
Rats were housed in LD 12:12 h conditions and monitored by pineal microdialysis to determine baseline melatonin timing profiles. After demonstration that the circadian expression of melatonin was at steady state, rats were subjected to experimental stroke using two-hour intralumenal filament occlusion of the middle cerebral artery. The animals were returned to their cages, and melatonin monitoring was resumed. The timing of onset, offset, and duration of melatonin secretion were calculated before and after stroke to determine changes in circadian rhythms of melatonin secretion. At the end of the monitoring period, brains were analyzed to determine infarct volume.
Results
Rats demonstrated immediate shifts in melatonin timing after stroke. We observed a broad range of perturbations in melatonin timing in subsequent days, with rats exhibiting onset/offset patterns which included: advance/advance, advance/delay, delay/advance, and delay/delay. Melatonin rhythms displayed prolonged instability several days after stroke, with a majority of rats showing a day-to-day alternation between advance and delay in melatonin onset and duration. Duration of melatonin secretion changed in response to stroke, and this change was strongly determined by the shift in melatonin onset time. There was no correlation between infarct size and the direction or amplitude of melatonin phase shifting.
Conclusion
This is the first demonstration that stroke induces immediate changes in the timing of pineal melatonin secretion, indicating that cortical and basal ganglia infarction impacts the timing of melatonin rhythms. The heterogeneous direction and amplitude of melatonin shifts suggests that the upstream regulation of hypothalamic timekeeping is likely anatomically diffuse and mechanistically complex. Finally, our study exemplifies the use of pineal microdialysis to evaluate the effect of neurological diseases on circadian function.
doi:10.1186/1740-3391-6-9
PMCID: PMC2584098  PMID: 18922153
21.  Reduced Anxiety and Depression-Like Behaviours in the Circadian Period Mutant Mouse Afterhours 
PLoS ONE  2012;7(6):e38263.
Background
Disruption of the circadian rhythm is a key feature of bipolar disorder. Variation in genes encoding components of the molecular circadian clock has been associated with increased risk of the disorder in clinical populations. Similarly in animal models, disruption of the circadian clock can result in altered mood and anxiety which resemble features of human mania; including hyperactivity, reduced anxiety and reduced depression-like behaviour. One such mutant, after hours (Afh), an ENU-derived mutant with a mutation in a recently identified circadian clock gene Fbxl3, results in a disturbed (long) circadian rhythm of approximately 27 hours.
Methodology
Anxiety, exploratory and depression-like behaviours were evaluated in Afh mice using the open-field, elevated plus maze, light-dark box, holeboard and forced swim test. To further validate findings for human mania, polymorphisms in the human homologue of FBXL3, genotyped by three genome wide case control studies, were tested for association with bipolar disorder.
Principal Findings
Afh mice showed reduced anxiety- and depression-like behaviour in all of the behavioural tests employed, and some evidence of increased locomotor activity in some tests. An analysis of three separate human data sets revealed a gene wide association between variation in FBXL3 and bipolar disorder (P = 0.009).
Conclusions
Our results are consistent with previous studies of mutants with extended circadian periods and suggest that disruption of FBXL3 is associated with mania-like behaviours in both mice and humans.
doi:10.1371/journal.pone.0038263
PMCID: PMC3376117  PMID: 22719873
22.  Circadian Genes Are Expressed during Early Development in Xenopus laevis 
PLoS ONE  2008;3(7):e2749.
Background
Circadian oscillators are endogenous time-keeping mechanisms that drive twenty four hour rhythmic changes in gene expression, metabolism, hormone levels, and physical activity. We have examined the developmental expression of genes known to regulate circadian rhythms in order to better understand the ontogeny of the circadian clock in a vertebrate.
Methodology/Principal Findings
In this study, genes known to function together in part of the core circadian oscillator mechanism (xPeriod1, xPeriod2, and xBmal1) as well as a rhythmic, clock-controlled gene (xNocturnin) were analyzed using in situ hybridization in embryos from neurula to late tailbud stages. Each transcript was present in the developing nervous system in the brain, eye, olfactory pit, otic vesicle and at lower levels in the spinal cord. These genes were also expressed in the developing somites and heart, but at different developmental times in peripheral tissues (pronephros, cement gland, and posterior mesoderm). No difference was observed in transcript levels or localization when similarly staged embryos maintained in cyclic light were compared at two times of day (dawn and dusk) by in situ hybridization. Quantitation of xBmal1 expression in embryonic eyes was also performed using qRT-PCR. Eyes were isolated at dawn, midday, dusk, and midnight (cylic light). No difference in expression level between time-points was found in stage 31 eyes (p = 0.176) but stage 40 eyes showed significantly increased levels of xBmal1 expression at midnight (RQ = 1.98+/−0.094) when compared to dawn (RQ = 1+/−0.133; p = 0.0004).
Conclusions/Significance
We hypothesize that when circadian genes are not co-expressed in the same tissue during development that it may indicate pleiotropic functions of these genes that are separate from the timing of circadian rhythm. Our results show that all circadian genes analyzed thus far are present during early brain and eye development, but rhythmic gene expression in the eye is not observed until after stage 31 of development.
doi:10.1371/journal.pone.0002749
PMCID: PMC2518526  PMID: 18716681
23.  Genetics and Neurobiology of Circadian Clocks in Mammals 
In animals circadian behavior can be analyzed as an integrated system - beginning with genes leading ultimately to behavioral outputs. In the last decade, the molecular mechanism of circadian clocks has been unraveled primarily by the use of phenotype-driven (forward) genetic analysis in a number of model systems. Circadian oscillations are generated by a set of genes forming a transcriptional autoregulatory feedback loop. In mammals, there is a “core” set of circadian genes that form the primary negative feedback loop of the clock mechanism (Clock/Npas2, Bmal1, Per1, Per2, Cry1, Cry2 and CK1ε). Another dozen candidate genes have been identified and play additional roles in the circadian gene network such as the feedback loop involving Rev-erbα. Despite this remarkable progress, it is clear that a significant number of genes that strongly influence and regulate circadian rhythms in mammals remain to be discovered and identified. As part of a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen using a wide range of nervous system and behavioral phenotypes, we have identified a number of new circadian mutants in mice. Here we describe a new short period circadian mutant, part-time (prtm), which is caused by a loss-of-function mutation in the Cryptochrome1 gene. We also describe a long period circadian mutant named Overtime (Ovtm). Positional cloning and genetic complementation reveal that Ovtm is encoded by the F-box protein FBXL3 a component of the SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligase complex. The Ovtm mutation causes an isoleucine to threonine (I364T) substitution leading to a loss-of-function in FBXL3 which interacts specifically with the CRYPTOCHROME (CRY) proteins. In Ovtm mice, expression of the PERIOD proteins PER1 and PER2 is reduced; however, the CRY proteins CRY1 and CRY2 are unchanged. The loss of FBXL3 function leads to a stabilization of the CRY proteins, which in turn leads to a global transcriptional repression of the Per and Cry genes. Thus, Fbxl3Ovtm defines a molecular link between CRY turnover and CLOCK/BMAL1-dependent circadian transcription to modulate circadian period.
doi:10.1101/sqb.2007.72.052
PMCID: PMC3749845  PMID: 18419282
24.  Nascent-Seq reveals novel features of mouse circadian transcriptional regulation 
eLife  2012;1:e00011.
A substantial fraction of the metazoan transcriptome undergoes circadian oscillations in many cells and tissues. Based on the transcription feedback loops important for circadian timekeeping, it is commonly assumed that this mRNA cycling reflects widespread transcriptional regulation. To address this issue, we directly measured the circadian dynamics of mouse liver transcription using Nascent-Seq (genome-wide sequencing of nascent RNA). Although many genes are rhythmically transcribed, many rhythmic mRNAs manifest poor transcriptional rhythms, indicating a prominent contribution of post-transcriptional regulation to circadian mRNA expression. This analysis of rhythmic transcription also showed that the rhythmic DNA binding profile of the transcription factors CLOCK and BMAL1 does not determine the transcriptional phase of most target genes. This likely reflects gene-specific collaborations of CLK:BMAL1 with other transcription factors. These insights from Nascent-Seq indicate that it should have broad applicability to many other gene expression regulatory issues.
DOI: http://dx.doi.org/10.7554/eLife.00011.001
eLife digest
Many biological processes oscillate with a period of roughly 24 hr, and the ability of organisms as diverse as bacteria and humans to maintain such circadian rhythms, even under conditions of continuous darkness, influences a range of phenomena, including sleep, migration and reproduction. One characteristic of circadian rhythms is that they can adjust to local time (with humans suffering from jet lag as they wait for this to happen).
Experiments have shown that the circadian system in mammals relies on feedback loops that operate at the level of individual cells. These loops are controlled by two particular proteins, which comprise the transcription factor complex called BMAL1:CLK. Transcription factors cause particular sequences of bases in the DNA of cells to be transcribed into messenger RNA, thus starting the process by which target genes are expressed as proteins. In the case of BMAL1:CLK, these proteins are then modified, which inhibits any further transcription of the target genes. A reversal of these modifications is then followed by the synthesis of new proteins, which allows a new cycle of the transcription process to begin.
The amounts of many messenger RNAs (mRNAs) in a cell also increases and decreases with a period of 24 hr, and it was generally assumed that this was due to the changes in the level of transcription. More recently, however, it was suggested that other processes, such as splicing and translation, might also contribute to rhythmic changes in the amount of mRNA associated with particular genes. Such post-transcriptional processes are known to have a role in other areas of cell biology, including aspects of the circadian system, but until very recently this had not been studied in detail for all genes.
Now Menet et al. have directly assayed rhythmic transcription by measuring the amount of nascent mRNA being produced at a given time, six times a day, across all the genes in mouse liver cells using a high-throughput sequencing approach called Nascent-Seq. They compared this with the amount of liver mRNA expressed at six time points of the day. Although the authors found that many genes exhibit rhythmic mRNA expression in the mouse liver, about 70% of them did not show comparable transcriptional rhythms. Post-transcriptional regulation must, therefore, have a major role in the circadian system of mice and, presumably, other mammals.
Menet et al. also found that the influence of CLK:BMAL1 differed from what was expected, which suggests that it collaborates with a number of other transcription factors to effect transcription of most target genes. In addition to showing that circadian systems of mammals are more complex than previously believed, the results also illustrate the potential of Nascent-Seq as a genome-wide assay technique for exploring a range of questions related to gene expression and gene regulation.
DOI: http://dx.doi.org/10.7554/eLife.00011.002
doi:10.7554/eLife.00011
PMCID: PMC3492862  PMID: 23150795
Circadian rhythms; transcription; nascent RNA; high-throughput sequencing; RNA processing; post-transcriptional regulation; Mouse
25.  The Interplay of cis-Regulatory Elements Rules Circadian Rhythms in Mouse Liver 
PLoS ONE  2012;7(11):e46835.
The mammalian circadian clock is driven by cell-autonomous transcriptional feedback loops that involve E-boxes, D-boxes, and ROR-elements. In peripheral organs, circadian rhythms are additionally affected by systemic factors. We show that intrinsic combinatorial gene regulation governs the liver clock. With a temporal resolution of 2 h, we measured the expression of 21 clock genes in mouse liver under constant darkness and equinoctial light-dark cycles. Based on these data and known transcription factor binding sites, we develop a six-variable gene regulatory network. The transcriptional feedback loops are represented by equations with time-delayed variables, which substantially simplifies modelling of intermediate protein dynamics. Our model accurately reproduces measured phases, amplitudes, and waveforms of clock genes. Analysis of the network reveals properties of the clock: overcritical delays generate oscillations; synergy of inhibition and activation enhances amplitudes; and combinatorial modulation of transcription controls the phases. The agreement of measurements and simulations suggests that the intrinsic gene regulatory network primarily determines the circadian clock in liver, whereas systemic cues such as light-dark cycles serve to fine-tune the rhythms.
doi:10.1371/journal.pone.0046835
PMCID: PMC3489864  PMID: 23144788

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