scholarly journals Period2 3′-UTR and microRNA-24 regulate circadian rhythms by repressing PERIOD2 protein accumulation

2017 ◽  
Vol 114 (42) ◽  
pp. E8855-E8864 ◽  
Author(s):  
Seung-Hee Yoo ◽  
Shihoko Kojima ◽  
Kazuhiro Shimomura ◽  
Nobuya Koike ◽  
Ethan D. Buhr ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Light Pulse ◽  
Protein Level ◽  
Temperature Compensation ◽  
Clock Genes ◽  
Protein Translation ◽  
Protein Accumulation ◽  
Free Running ◽  
Protein Oscillation

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the Per2 3′-UTR region: Per2::Luc, which retains the endogenous Per2 3′-UTR and Per2::LucSV, where the endogenous Per2 3′-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of Per2 3′-UTR, we analyzed circadian rhythms of Per2::LucSV mice. Interestingly, Per2::LucSV mice displayed more than threefold stronger amplitude in bioluminescence rhythms than Per2::Luc mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Per2::Luc. Analysis of the Per2 3′-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in Per2::LucSV augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, Bmal1 mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in Per2::LucSV mice, suggesting rhythmic overexpression of PER2 enhances expression of Per2 and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of Per2 3′-UTR, miR-24, and PER2 in Per2 expression and core clock function.

scholarly journals Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora

2021 ◽  
pp. 074873042199994
Author(s):  
Rosa Eskandari ◽  
Lalanthi Ratnayake ◽  
Patricia L. Lakin-Thomas
Keyword(s):  
Circadian Rhythms ◽  
Clock Genes ◽  
Nutrient Sensing ◽  
Mass Spectrometry Analysis ◽  
Growth Responses ◽  
Tor Pathway ◽  
Spectrometry Analysis ◽  
Binding Partner ◽  
Binding Partners ◽  
Free Running

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes ( frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation ( vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δ gtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δ gtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δ gtr2 is defective in entrainment. In all of these assays, Δ gtr2 is similar to Δ vta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

scholarly journals Genetic variation and phenotypic plasticity in circadian rhythms in an armed beetle, Gnatocerus cornutus (Tenebrionidae)

2020 ◽  
Vol 130 (1) ◽  
pp. 34-40 ◽  
Author(s):  
Kentarou Matsumura ◽  
Masato S Abe ◽  
Manmohan D Sharma ◽  
David J Hosken ◽  
Taishi Yoshii ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Phenotypic Plasticity ◽  
Circadian Rhythms ◽  
Temperature Effects ◽  
Temperature Compensation ◽  
Biological Rhythms ◽  
Total Activity ◽  
Biological Clocks ◽  
Isofemale Lines ◽  
Free Running

Abstract Circadian rhythms, their free-running periods and the power of the rhythms are often used as indicators of biological clocks, and there is evidence that the free-running periods of circadian rhythms are not affected by environmental factors, such as temperature. However, there are few studies of environmental effects on the power of the rhythms, and it is not clear whether temperature compensation is universal. Additionally, genetic variation and phenotypic plasticity in biological clocks are important for understanding the evolution of biological rhythms, but genetic and plastic effects are rarely investigated. Here, we used 18 isofemale lines (genotypes) of Gnatocerus cornutus to assess rhythms of locomotor activity, while also testing for temperature effects. We found that total activity and the power of the circadian rhythm were affected by interactions between sex and genotype or between sex, genotype and temperature. The males tended to be more active and showed greater increases in activity, but this effect varied across both genotypes and temperatures. The period of activity varied only by genotype and was thus independent of temperature. The complicated genotype–sex–environment interactions we recorded stress the importance of investigating circadian activity in more integrated ways.

scholarly journals Machine Learning and In-Vivo Expression Analyses Reveal Circadian Clock Features Predictive of Anxiety in Humans

2021 ◽  
Author(s):  
Aziz Zafar ◽  
Rebeccah Overton ◽  
Ziad Attia ◽  
Ahmet Ay ◽  
Krista Ingram
Keyword(s):  
Machine Learning ◽  
Circadian Rhythms ◽  
Circadian Clock ◽  
Clock Gene ◽  
Molecular Mechanisms ◽  
Clock Genes ◽  
Anxiety Symptoms ◽  
Circadian Phase ◽  
Functional Studies

Abstract Mood disorders, including anxiety, are associated with disruptions in circadian rhythms and are linked to polymorphisms in circadian clock genes. Molecular mechanisms underlying these connections may be direct—via transcriptional activity of clock genes on downstream mood pathways in the brain, or indirect—via clock gene influences on the phase and amplitude of circadian rhythms which, in turn, modulate physiological processes influencing mood. Employing machine learning combined with statistical approaches, we explored clock genotype combinations that predict risk for anxiety symptoms in a deeply phenotyped population. We identified multiple novel circadian genotypes predictive of anxiety, with the PER3B-AG/CRY1-CG genotype being the strongest predictor of anxiety risk in males. Molecular chronotyping, using clock gene expression oscillations, revealed that advanced circadian phase and robust circadian amplitudes are associated with high levels of anxiety symptoms. Further analyses revealed that individuals with advanced phases and pronounced circadian misalignment were at higher risk for severe anxiety symptoms. Our results support both direct and indirect influences of clock gene variants on mood: while sex-specific clock genotype combinations predictive of anxiety symptoms suggest direct effects on mood pathways, the mediation of PER3B effects on anxiety via diurnal preference measures and the association of circadian phase with anxiety symptoms provide evidence for indirect effects of the molecular clockwork on mood. Unraveling the complex molecular mechanisms underlying the links between circadian physiology and mood is essential to identifying the core clock genes to target in future functional studies, thereby advancing the development of non-invasive treatments for anxiety-related disorders.

scholarly journals Gpr19 is a circadian clock-controlled orphan GPCR with a role in modulating free-running period and light resetting capacity of the circadian clock

2021 ◽  
Author(s):  
Yoshiaki Yamaguchi ◽  
Iori Murai ◽  
Kaoru Goto ◽  
Shotaro Doi ◽  
Huihua Zhou ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Clock Genes ◽  
Dorsal Region ◽  
Circadian Expression ◽  
Delayed Initiation ◽  
Free Running ◽  
Responsive Element ◽  
Free Running Period ◽  
Orphan Gpcr

Background and Purpose: Gpr19 encodes an evolutionarily conserved orphan G-protein-coupled receptor (GPCR) with no established physiological function in vivo. The purpose of this study was to determine the role of Gpr19 in the circadian clock system. Experimental Approach: We examined whether and how the master circadian clock neurons in the suprachiasmatic nucleus (SCN) express Gpr19. By analysing Gpr19-deficient (Gpr19−/−) mice, we asked whether Gpr19 has a role in modulating free-running period and light resetting capacity of the circadian clock. Key Results: Compared with the known common core clock genes, Gpr19 was identified to show several distinct yet limited features related to the circadian clock. Gpr19 mRNA was mainly expressed in the middle-to-dorsal region of the SCN. A conserved cAMP-responsive element within the Gpr19 promoter drove the circadian expression of Gpr19. Gpr19−/− mice exhibited a prolonged circadian period and a delayed initiation of daily locomotor activity in a 12-h light/12-h dark cycle. Gpr19 deficiency caused the downregulation of several genes that normally peak during the night, including Bmal1 and Gpr176. Gpr19−/− mice had a reduced capacity for phase shift to early subjective night light. The defect was only observed for phase-delay, but not phase-advance, and accompanied by reduced response of c-Fos expression in the dorsal region of the SCN, while apparently normal in the ventral part of the SCN, in Gpr19−/− mice. Conclusion and Implications: Gpr19 is an SCN-enriched orphan GPCR with a distinct role in circadian regulation and thus may be a potential target for alleviating circadian clock disorders.

scholarly journals Cancer clocks in tumourigenesis: the p53 pathway and beyond

2021 ◽  
Vol 28 (4) ◽  
pp. R95-R110
Author(s):  
Ewan M Stephenson ◽  
Laura E J Usselmann ◽  
Vinay Tergaonkar ◽  
David M Virshup ◽  
Robert Dallmann
Keyword(s):  
Circadian Rhythms ◽  
Circadian Clock ◽  
Cancer Biology ◽  
Cell Cycle Progression ◽  
Clock Genes ◽  
Human Cancer ◽  
Epidemiologic Studies ◽  
Current Evidence

Circadian rhythms regulate a vast array of physiological and cellular processes, as well as the hormonal milieu, to keep our cells synchronised to the light–darkness cycle. Epidemiologic studies have implicated circadian disruption in the development of breast and other cancers, and numerous clock genes are dysregulated in human tumours. Here we review the evidence that circadian rhythms, when altered at the molecular level, influence cancer growth. We also note some common pitfalls in circadian-cancer research and how they might be avoided to maximise comparable results and minimise misleading data. Studies of circadian gene mutant mice, and human cancer models in vitro and in vivo, demonstrate that clock genes can impact tumourigenesis. Clock genes influence important cancer-related pathways, ranging from p53-mediated apoptosis to cell cycle progression. Confusingly, clock dysfunction can be both pro- or anti-tumourigenic in a model and cell type-specific manner. Due to this duality, there is no canonical mechanism for clock interaction with tumourigenic pathways. To understand the role of the circadian clock in patients’ tumours requires analysis of the molecular clock status compared to healthy tissue. Novel mathematical approaches are under development, but this remains largely aspirational, and is hampered by a lack of temporal information in publicly available datasets. Current evidence broadly supports the notion that the circadian clock is important for cancer biology. More work is necessary to develop an overarching model of this connection. Future studies would do well to analyse the clock network in addition to alterations in single clock genes.

scholarly journals Dissociation of Per1 and Bmal1 circadian rhythms in the suprachiasmatic nucleus in parallel with behavioral outputs

2017 ◽  
Vol 114 (18) ◽  
pp. E3699-E3708 ◽  
Author(s):  
Daisuke Ono ◽  
Sato Honma ◽  
Yoshihiro Nakajima ◽  
Shigeru Kuroda ◽  
Ryosuke Enoki ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Suprachiasmatic Nucleus ◽  
Clock Genes ◽  
Behavioral Rhythm ◽  
Calcium Indicator ◽  
Intracellular Ca ◽  
Regional Specificity ◽  
Free Running ◽  
Activity Onset ◽  
And Behavior

The temporal order of physiology and behavior in mammals is primarily regulated by the circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Taking advantage of bioluminescence reporters, we monitored the circadian rhythms of the expression of clock genes Per1 and Bmal1 in the SCN of freely moving mice and found that the rate of phase shifts induced by a single light pulse was different in the two rhythms. The Per1-luc rhythm was phase-delayed instantaneously by the light presented at the subjective evening in parallel with the activity onset of behavioral rhythm, whereas the Bmal1-ELuc rhythm was phase-delayed gradually, similar to the activity offset. The dissociation was confirmed in cultured SCN slices of mice carrying both Per1-luc and Bmal1-ELuc reporters. The two rhythms in a single SCN slice showed significantly different periods in a long-term (3 wk) culture and were internally desynchronized. Regional specificity in the SCN was not detected for the period of Per1-luc and Bmal1-ELuc rhythms. Furthermore, neither is synchronized with circadian intracellular Ca2+ rhythms monitored by a calcium indicator, GCaMP6s, or with firing rhythms monitored on a multielectrode array dish, although the coupling between the circadian firing and Ca2+ rhythms persisted during culture. These findings indicate that the expressions of two key clock genes, Per1 and Bmal1, in the SCN are regulated in such a way that they may adopt different phases and free-running periods relative to each other and are respectively associated with the expression of activity onset and offset.

THE STUDY OF CIRCADIAN RHYTHMS IN OVARIAN CANCER RAT MODEL: IS THERE CONNECTION BETWEEN TUMOR DEVELOPMENT AND RHYTHMICITY DISRUPTION IN INTACT TISSUES?

2019 ◽  
Vol 65 (6) ◽  
pp. 889-897
Author(s):  
Galina Kireeva ◽  
Yekaterina Gubareva ◽  
Natalya Mityushkina ◽  
Andrey Panchenko ◽  
Mikhail Maydin ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Circadian Rhythms ◽  
Tumor Cells ◽  
Clock Genes ◽  
Tumor Development ◽  
Tumor Model ◽  
Circadian Rhythmicity ◽  
Normal Tissues ◽  
Tumor Bearing

Considering emerging yet contradictory data on circadian rhythms disruption and its effects on tumor initiation and progression, we performed in vivo study to evaluate changes (if any) in clock genes expression in tumor compared to intact tissues as well as to see if tumor development affects normal tissues in tumor-bearing animals. The study was performed in 75 female Wistar rats, intraperitoneally transplanted ovarian cancer was used as a tumor model. Tumor-bearing rats had fragmented circadian rhythmicity of their locomotor activity compared to intact animals. No circadian rhythmicity in proliferation of tumor cells was detected. Precise proliferative rhythmicity was found in normal cells (intestinal epithelium) of intact rats, while significant disruption in such rhythmicity was observed in the same cells of tumor-bearing rats. Average clock genes (BMAL1, CLOCK, CRY1, PER2) expression rate was significantly reduced in tumor cells compared to intact tissues. The data from these experiments let us choosing 2 time points to perform chemotherapy in the following study where effects of chronochemotherapy will be evaluated.

scholarly journals Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms

2021 ◽  
Vol 7 (18) ◽  
pp. eabe8132
Author(s):  
Naohiro Kon ◽  
Hsin-tzu Wang ◽  
Yoshiaki S. Kato ◽  
Kyouhei Uemoto ◽  
Naohiro Kawamoto ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Mammalian Cells ◽  
Temperature Compensation ◽  
Clock Genes ◽  
Common Mechanism ◽  
Behavioral Rhythms ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Biochemical Oscillations ◽  
Response To Temperature

Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. Here, we show that Na+/Ca2+ exchanger (NCX) mediates cold-responsive Ca2+ signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca2+ signaling is conserved among mice, Drosophila, and Arabidopsis. The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. Our results suggest that NCX-mediated Ca2+ signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.

Spontaneous Recovery of Circadian Organization in Mice Lacking a Core Component of the Molecular Clockwork

2021 ◽  
pp. 074873042110608
Author(s):  
Jonathan P. Riggle ◽  
Kenneth G. Onishi ◽  
Jharnae A. Love ◽  
Dana E. Beach ◽  
Irving Zucker ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Spontaneous Recovery ◽  
Clock Genes ◽  
Feedback Loops ◽  
Constant Darkness ◽  
Core Component ◽  
Period 2 ◽  
Circadian Organization ◽  
Circadian Clock Genes ◽  
Free Running

Circadian rhythms are generated by interlocked transcriptional-translational feedback loops of circadian clock genes and their protein products. Mice homozygous for a functional deletion in the Period-2 gene ( Per2m/m mice) exhibit short free-running circadian periods and eventually lose behavioral circadian rhythmicity in constant darkness (DD). We investigated Per2m/m mice in DD for several months and identified a categorical sex difference in the dependence on Per2 for maintenance of circadian rhythms. Nearly all female Per2m/m mice became circadian arrhythmic in DD, whereas free-running rhythms persisted in 37% of males. Remarkably, with extended testing, Per2m/m mice did not remain arrhythmic in DD, but after varying intervals spontaneously recovered robust, free-running circadian rhythms, with periods shorter than those expressed prior to arrhythmia. Spontaneous recovery was strikingly sex-biased, occurring in 95% of females and 33% of males. Castration in adulthood resulted in male Per2m/m mice exhibiting female-like levels of arrhythmia in DD, but did not affect spontaneous recovery. The circadian pacemaker of many gonad-intact males, but not females, can persist in DD for long intervals without a functional PER2 protein; their circadian clocks may be in an unstable equilibrium, incapable of sustaining persistent coherent circadian organization, resulting in transient cycles of circadian organization and arrhythmia.

scholarly journals Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines

2019 ◽  
Author(s):  
Eric Poliner ◽  
Cameron Cummings ◽  
Linsey Newton ◽  
Eva M. Farré
Keyword(s):  
Cell Division ◽  
Circadian Rhythms ◽  
Molecular Mechanisms ◽  
Environmental Changes ◽  
Firefly Luciferase ◽  
Constant Light ◽  
Future Studies ◽  
Circadian Oscillators ◽  
Free Running

SummaryCircadian clocks allow organisms to predict environmental changes caused by the rotation of the Earth. Although circadian rhythms are widespread among different taxa, the core components of circadian oscillators are not conserved and differ between bacteria, plants, animals and fungi. Stramenopiles are a large group of organisms in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have investigated cell division and molecular rhythms in Nannochloropsis species. In the four strains tested, cell division occurred principally during the night period under diel conditions, however, rhythms dampened within 2-3 days after transfer to constant light. We developed firefly luciferase reporters for long-term monitoring of in vivo transcriptional rhythms in two Nannochlropsis species, N. oceanica CCMP1779 and N. salina CCMP537. The reporter lines express free-running bioluminescence rhythms with periods of ~21-31 h that dampen within ~3-4 days under constant light. Using different entrainment regimes, we demonstrate that these rhythms are regulated by a circadian-type oscillator. In addition, the phase of free-running luminescence rhythms can be modulated pharmacologically using a CK1 ε/δ inhibitor, suggesting a role of this kinase in the Nannochloropsis clock. Together with the molecular and genomic tools available for Nannochloropsis species, these reporter lines represent an excellent system for future studies on the molecular mechanisms of stramenopile circadian oscillators.Significance statementStramenopiles are a large and diverse line of eukaryotes in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have developed bioluminescence reporter lines in Nannochloropsis species and provide evidence for the presence of a circadian oscillator in stramenopiles; these lines will serve as tools for future studies to uncover the molecular mechanisms of circadian oscillations in these species.

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