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TIMELESS (TIM) was first identified as a molecular cog in the Drosophila circadian clock. Almost three decades of investigations have resulted in an insightful model describing the critical role of Drosophila TIM (dTIM) in circadian timekeeping in insects, including its function in mediating light entrainment and temperature compensation of the molecular clock. Furthermore, exciting discoveries on its sequence polymorphism and thermosensitive alternative RNA splicing have also established its role in regulating seasonal biology. Although mammalian TIM (mTIM), its mammalian paralog, was first identified as a potential circadian clock component in 1990s due to sequence similarity to dTIM, its role in clock regulation has been more controversial. Mammalian TIM has now been characterized as a DNA replication fork component and has been shown to promote fork progression and participate in cell cycle checkpoint signaling in response to DNA damage. Despite defective circadian rhythms displayed by mtim mutants, it remains controversial whether the regulation of circadian clocks by mTIM is direct, especially given the interconnection between the cell cycle and circadian clocks. In this review, we provide a historical perspective on the identification of animal tim genes, summarize the roles of TIM proteins in biological timing and genomic stability, and draw parallels between dTIM and mTIM despite apparent functional divergence.

We developed a method for single-cell resolution longitudinal bioluminescence imaging of PERIOD (PER) protein and TIMELESS (TIM) oscillations in cultured male adult Drosophila brains that captures circadian circuit-wide cycling under simulated day/night cycles. Light input analysis confirms that CRYPTOCHROME (CRY) is the primary circadian photoreceptor and mediates clock disruption by constant light (LL), and that eye light input is redundant to CRY; 3-h light phase delays (Friday) followed by 3-h light phase advances (Monday morning) simulate the common practice of staying up later at night on weekends, sleeping in later on weekend days then returning to standard schedule Monday morning [weekend light shift (WLS)]. PER and TIM oscillations are highly synchronous across all major circadian neuronal subgroups in unshifted light schedules for 11 d. In contrast, WLS significantly dampens PER oscillator synchrony and rhythmicity in most circadian neurons during and after exposure. Lateral ventral neuron (LNv) oscillations are the first to desynchronize in WLS and the last to resynchronize in WLS. Surprisingly, the dorsal neuron group-3 (DN3s) increase their within-group synchrony in response to WLS. In vivo, WLS induces transient defects in sleep stability, learning, and memory that temporally coincide with circuit desynchrony. Our findings suggest that WLS schedules disrupt circuit-wide circadian neuronal oscillator synchrony for much of the week, thus leading to observed behavioral defects in sleep, learning, and memory.

MicroRNAs are important coordinators of circadian regulation that mediate the fine-tuning of gene expression. Although many studies have shown the effects of individual miRNAs on the circadian clock, the global functional miRNA-mRNA interaction network involved in the circadian system remains poorly understood. Here, we used CLEAR (Covalent Ligation of Endogenous Argonaute-bound RNAs)-CLIP (Cross-Linking and Immuno-Precipitation) to explore the regulatory functions of miRNAs in the circadian system by comparing the miRNA-mRNA interactions between Drosophila wild-type strain W(1118) and a mutant of the key circadian transcriptional regulator Clock (Clk(jrk)). This experimental approach unambiguously identified tens of thousands of miRNA-mRNA interactions in both the head and body. The miRNA-mRNA interactome showed dramatic changes in the Clk(jrk) flies. Particularly, among ~300 miRNA-mRNA circadian relevant interactions, multiple interactions involving core clock genes pdp1, tim and vri displayed distinct changes as a result of the Clk mutation. Based on the CLEAR-CLIP analysis, we found a novel regulation of the circadian rhythm and sleep by the miR-375-timeless interaction. The results indicated that Clk disruption abolished normal rhythmic expression of miR-375 and the functional regulation occurred in the l-LNv neurons, where miR-375 modulated circadian rhythm and sleep via targeting timeless. This work provides the first global view of miRNA regulation in the circadian rhythm.

Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, upregulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents overproliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target., Disruption of different components of molecular circadian clocks has varying effects on health and lifespan of model organisms. Here the authors show that loss of period extends life in drosophila melanogaster.

Background The incidence rate of non-small cell lung cancer (NSCLC) has been increasing worldwide, and the correlation of circadian rhythm disruption with a raised risk of cancer and worse prognosis has been shown by accumulating evidences recently. On the other hand, drug resistance and the impact of tumor heterogeneity have been inevitable in NSCLC therapy. These both lead to an urgent need to identify more useful prognostic and predictive markers for NSCLC diagnosis and treatment, especially on the aspect of circadian clock genes. Methods The expression of the main clock genes in cancer was probed with TIMER and Oncomine databases. The prognostic value of key clock genes was probed systematically with the Kaplan–Meier estimate and Cox regression on samples from TCGA database. RT-qPCR was performed on patient tissue samples to further validate the results from databases. The functional enrichment analysis was performed using the “ClusterProfiler” R package, and the correlation of key clock genes with tumor mutation burden, immune checkpoint, and immune infiltration levels were also assessed using multiple algorithms including TIDE, TIMER2.0, and XCELL. Results TIMELESS was significantly upregulated in lung tissue of clinical lung cancer patients as well as TCGA and Oncomine databases, while RORA was downregulated. Multivariate Cox regression analysis indicated that TIMELESS (P = 0.004, HR = 1.21 [1.06, 1.38]) and RORA (P = 0.047, HR = 0.868 [0.755, 0.998]) has a significant correlation with overall survival in NSCLC. Genes related to TIMELESS were enriched in the cell cycle and immune system, and the function of RORA was mainly focused on oncogenic signaling pathways or glycosylation and protein activation. Also, TIMELESS was positively correlated with tumor mutation burden while RORA was negatively correlated with it. TIMELESS and RORA were also significantly correlated with immune checkpoint and immune infiltration levels in NSCLC. Additionally, TIMELESS showed a significant positive relationship with lipid metabolism. Conclusions TIMELESS and RORA were identified as key clock genes in NSCLC, and were independent prognostic factors for overall survival in NSCLC. The function of them were assessed in many aspects, indicating the strong potential of the two genes to serve as biomarkers for NSCLC progression and prognosis.

Significance In animals, the control of daily sleep–wake rhythms is mediated by discrete circadian clock neurons via their rhythmic activity–dependent release of neuropeptides and neurotransmitters. Here, we describe a gene, Tango10, critical for daily behavioral rhythms. TANGO10 functions as an adaptor with its partner, the E3 ubiquitin ligase CULLIN 3, to regulate protein ubiquitination, neuronal excitability via voltage-gated potassium channels, and neuropeptide accumulation. These studies define a mechanism for control of neuronal excitability and daily rhythmicity., Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. Here we reveal a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, we identified mutants of Transport and Golgi organization 10 (Tango10) with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless. Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. We propose that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms.

Simple Summary One of the major characteristics of the circadian clock is temperature compensation, and previous studies suggested a single clock gene may determine the temperature compensation. In this study, we report the first full collection of clock gene knockout cell lines using CRISPR/Cas9 tools. Our full collections indicate that the temperature compensation is a complex gene regulation system instead of being regulated by any single gene. Besides, we systematically compared the proliferation rates and circadian periods using our full collections, and we found that the cell growth rate is not dependent on the circadian period. Therefore, complex interaction between clock genes and their protein products may underlie the mechanism of temperature compensation, which needs further investigations. Abstract Mammalian circadian genes are capable of producing a self-sustained, autonomous oscillation whose period is around 24 h. One of the major characteristics of the circadian clock is temperature compensation. However, the mechanism underlying temperature compensation remains elusive. Previous studies indicate that a single clock gene may determine the temperature compensation in several model organisms. In order to understand the influence of each individual clock gene on the temperature compensation, twenty-three well-known mammalian clock genes plus Timeless and Myc genes were knocked out individually, using a powerful gene-editing tool, CRISPR/Cas9. First, Bmal1, Cry1, and Cry2 were knocked out as examples to verify that deleting genes by CRISPR is effective and precise. Cell lines targeting twenty-two genes were successfully edited in mouse fibroblast NIH3T3 cells, and off-target analysis indicated these genes were correctly knocked out. Through measuring the luciferase reporters, the circadian periods of each cell line were recorded under two different temperatures, 32.5 °C and 37 °C. The temperature compensation coefficient Q10 was subsequently calculated for each cell line. Estimations of the Q10 of these cell lines showed that none of the individual cell lines can adversely affect the temperature compensation. Cells with a longer period at lower temperature tend to have a shorter period at higher temperature, while cells with a shorter period at lower temperature tend to be longer at higher temperature. Thus, the temperature compensation is a fundamental property to keep cellular homeostasis. We further conclude that the temperature compensation is a complex gene regulation system instead of being regulated by any single gene. We also estimated the proliferation rates of these cell lines. After systematically comparing the proliferation rates and circadian periods, we found that the cell growth rate is not dependent on the circadian period.

Aphids are paradigmatic photoperiodic animals often used to study the role of the circadian clock in the seasonal response. Previously, we described some elements of the circadian clock core (genes period and timeless) and output (melatonin, AANATs and PTTH) that could have a role in the regulation of the aphid seasonal response. More recently we identified two opsins (C-ops and SWO4) as candidate input photoperiodic receptors. In the present report, we focus on the study of cryptochromes (cry) as photoreceptors of the circadian clock and discuss their involvement in the seasonal response. We analyze the expression of cry1 and cry2 genes in a circadian and seasonal context, and map their expression sites in the brain. We observe a robust rhythmic expression of cry2 peaking at dusk in phase with core clock genes period and timeless, while cry1 shows a weaker rhythm. Changes in cry1 and cry2 expression correlate with activation of the seasonal response, suggesting a possible link. Finally, we map the expression of cry1 and cry2 genes to clock neurons in the pars lateralis, a region essential for the photoperiodic response. Our results support a role for cryptochromes as elements of the aphid circadian clock and suggest a role in photoreception for cry1 and in clock repression for cry2. This article is protected by copyright. All rights reserved.

We established a knockout strain of a clock gene, period (per), by using TALEN in a bivoltine strain (Kosetsu) of Bombyx mori (Insecta, Lepidoptera), and examined the effect of per knockout on the circadian rhythm and photoperiodism. The generated per knockout allele was considered to be null, because a new stop codon was present in the insertion allele. The wild type (Kosetsu) showed clear circadian rhythms in eclosion and hatching, whereas the per knockout strain showed arrhythmic eclosion and hatching under constant darkness. In this strain, moreover, temporal expression changes of clock genes per and timeless were disrupted. The wild type showed a clear long-day response for induction of embryonic diapause: when larvae were reared under long-day and short-day conditions at 25°C, adults produced nondiapause and diapause eggs, respectively. However, the per knockout strain lost the sensitivity to photoperiod and laid nondiapause eggs under both conditions. We conclude that per plays an important role both in circadian rhythms and in photoperiodism of B. mori, indicating the involvement of the circadian clock consisting of per in the photoperiodism.

Background The genetic interactions in the circadian rhythm biological system are promising as a source of pathophysiology in mood disorder. We examined the role of the gene–gene interactions of clock genes in mood disorder. Methods We included 413 patients with mood disorder and 1294 controls. The clock genes investigated were BHLHB2, CLOCK, CSNK1E, NR1D1, PER2, PER3, and TIMELESS. Allele, genotype, and haplotype associations were tested. Gene–-gene interactions were analyzed using the non-parametric model-free multifactor-dimensionality reduction (MDR) method. Results TIMELESS rs4630333 and CSNK1E rs135745 were significantly associated with both major depressive disorder and bipolar disorder. The CLOCK haplotype was also strongly associated. The genetic roles of these SNPs were consistent from the allele and genotypic associations to the MDR interaction results. In MDR analysis, the combination of TIMELESS rs4630333 and CSNK1E rs135745 exhibited the most significant association with mood disorders in the two-locus model. BHLHB2 rs2137947 for major depressive disorder and CLOCK rs12649507 for bipolar disorder were the most significant third loci in the three-locus combination model. The four-locus SNP combination model showed the best balanced accuracy (BA), but its cross-validation consistency (CVC) was unsatisfactory. Limitations We included only 17 SNPs for seven circadian genes due to our limited resources; all subjects were ethnically Korean. Conclusions Our results suggest significant single-gene associations and gene–gene interactions of circadian genes with mood disorder. Gene–gene interactions play a crucial role in mood disorder, even when individual clock genes do not have significant roles.

Mutations in CHMP2B, an ESCRT‐III (endosomal sorting complexes required for transport) component, are associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Neurodegenerative disorders including FTD are also associated with a disruption in circadian rhythms, but the mechanism underlying this defect is not well understood. Here, we ectopically expressed the human CHMP2B variant associated with FTD (CHMP2BIntron5) in flies using the GMR‐GAL4 driver (GMR>CHMP2BIntron5) and analyzed their circadian rhythms at behavioral, cellular, and biochemical level. In GMR>CHMP2BIntron5 flies, we observed disrupted eclosion rhythms, shortened free‐running circadian locomotor period, and reduced levels of timeless (tim) mRNA—a circadian pacemaker gene. We also observed that the GMR‐GAL4 driver, primarily known for its expression in the retina, drives expression in a subset of tim expressing neurons in the optic lobe of the brain. The patterning of these GMR‐ and tim‐positive neurons in the optic lobe, which appears distinct from the putative clusters of circadian pacemaker neurons in the fly brain, was disrupted in GMR>CHMP2BIntron5 flies. These results demonstrate that CHMP2BIntron5 can disrupt the normal function of the circadian clock in Drosophila.

Organisms use endogenous clocks to adapt to the rhythmicity of the environment and to synchronize social activities. Although the circadian cycle is implicated in aging, it is unknown whether natural variation in its function contributes to differences in lifespan between populations and whether the circadian clock of specific tissues is key for longevity. We have sequenced the genomes of Drosophila melanogaster strains with exceptional longevity that were obtained via multiple rounds of selection from a parental strain. Comparison of genomic, transcriptomic, and proteomic data revealed that changes in gene expression due to intergenic polymorphisms are associated with longevity and preservation of skeletal muscle function with aging in these strains. Analysis of transcription factors differentially modulated in long-lived versus parental strains indicates a possible role of circadian clock core components. Specifically, there is higher period and timeless and lower cycle expression in the muscle of strains with delayed aging compared to the parental strain. These changes in the levels of circadian clock transcription factors lead to changes in the muscle circadian transcriptome, which includes genes involved in metabolism, proteolysis, and xenobiotic detoxification. Moreover, a skeletal muscle-specific increase in timeless expression extends lifespan and recapitulates some of the transcriptional and circadian changes that differentiate the long-lived from the parental strains. Altogether, these findings indicate that the muscle circadian clock is important for longevity and that circadian gene variants contribute to the evolutionary divergence in longevity across populations.

Day length (photoperiod) and temperature oscillate daily and seasonally and are important cues for season-dependent behavior. Larval diapause of the parasitoid Nasonia vitripennis is maternally induced following a certain number of days (switch point) of a given critical photoperiod (CPP). Both the switch point and the CPP follow a latitudinal cline in European N. vitripennis populations. We previously showed that allelic frequencies of the clock gene period correlate with this diapause induction cline. Here we report that circadian expression of four clock genes-period (per), cryptochrome-2 (cry-2), clock (clk), and cycle (cyc)-oscillates as a function of photoperiod and latitude of origin in wasps from populations from the extremes of the cline. Expression amplitudes are lower in northern wasps, indicating a weaker, more plastic clock. Northern wasps also have a later onset of activity and longer free-running rhythms under constant conditions. RNA interference of per caused speeding up of the circadian clock, changed the expression of other clock genes, and delayed diapause in both southern and northern wasps. These results point toward adaptive latitudinal clock gene expression differences and to a key role of per in the timing of photoperiodic diapause induction of N. vitripennis.

The rice stem borer, Chilo suppressalis Walker, is one of the most important global agricultural pests. C. suppressalis has distinct rice and water-oat host populations. Asynchrony in sexual activity is thought to be the main factor maintaining reproductive segregation between these populations, particularly the obvious difference in the circadian rhythm of female calling activity between populations. However, the mechanism responsible for this difference in the timing of female calling is poorly understood. The circadian clock is an essential regulator of daily behavioral rhythms in insects, including female calling. We investigated the variation in circadian clock genes of the rice and water-oat populations of C. suppressalis. We did this by comparing deduced amino acid sequences and the expression patterns of seven circadian clock genes (clock, cycle, period, timeless, timeout, cryptochrome1, and cryptochrome2) between females from each population. We found that the two populations had different variants of the timeout and cryptochrome1 genes and differed in the expression of period, timeless and timeout. This suggests that population-related variation in the circadian clock genes period, timeless, timeout and cryptochrome1 could be responsible for the different circadian rhythms of female calling in these host population of C. suppressalis. These results provide new insights into the molecular mechanisms underlying asynchronous sexual activity in insect populations and suggest new topics for future research on the origins and maintenance of population differentiation in insects.

Significance Circadian oscillators rely on transcriptional negative feedback loops. In Drosophila , the key transcriptional repressor PERIOD (PER) slowly accumulates during the night under the control of its partner TIMELESS (TIM). A large number of posttranslational mechanisms regulate PER and TIM stability, but no mechanisms affecting the stability of their transcripts have been described. mRNA stability depends on the length of the poly(A) tail. We show that a deadenylase, POP2, shortens tim mRNA poly(A) tail, thus decreasing tim mRNA and TIM protein levels. Moreover, POP2 activity on tim mRNA appears to be inhibited by PER itself. These results reveal polyadenylation control of a core clock gene transcript and suggest that the repressor of the feedback loop also acts as a posttranscriptional regulator.

Circadian clocks regulate ∼24 hour oscillations in gene expression, behavior, and physiology. While the molecular and neural mechanisms of circadian rhythms are well characterized, how cellular organization of clock components controls circadian clock regulation remains poorly understood. Here, we elucidate how clock proteins regulate circadian rhythms by controlling the spatiotemporal organization of clock genes. Using high-resolution live imaging techniques we demonstrate that Drosophila clock proteins are concentrated in a few discrete foci and are organized at the nuclear envelope; these results are in contrast to longstanding expectations that clock proteins are diffusely distributed in the nucleus. We also show that clock protein foci are highly dynamic and change in number, size, and localization over the circadian cycle. Further, we demonstrate that clock genes are positioned at the nuclear periphery by the clock proteins precisely during the circadian repression phase, suggesting that subnuclear localization of clock genes plays an important role in the control of rhythmic gene expression. Finally, we show that Lamin B receptor, a nuclear envelope protein, is required for peripheral localization of clock protein foci and clock genes and for normal circadian rhythms. These results reveal that clock proteins form dynamic nuclear foci and play a hitherto unexpected role in the subnuclear reorganization of clock genes to control circadian rhythms, identifying a novel mechanism of circadian regulation. Our results further suggest a new role for clock protein foci in the clustering of clock-regulated genes during the repression phase to control gene co-regulation and circadian rhythms.SIGNIFICANCEAlmost all living organisms have evolved circadian clocks to tell time. Circadian clocks regulate ∼24-hour oscillations in gene expression, behavior and physiology. Here, we reveal the surprisingly sophisticated spatiotemporal organization of clock proteins and clock genes and its critical role in circadian clock function. We show, in contrast to current expectations, that clock proteins are concentrated in a few discrete, dynamic nuclear foci at the nuclear envelope during the repression phase. Further, we uncovered several unexpected features of clock protein foci, including their role in positioning the clock genes at the nuclear envelope precisely during the repression phase to enable circadian rhythms. These studies provide fundamental new insights into the cellular mechanisms of circadian rhythms and establish direct links between nuclear organization and circadian clocks.

People are constantly exposed to phthalates, due to their common use in the production of plastics, pharmaceuticals, cosmetics and skin care products. The ability of phthalates to disrupt endocrine signaling, leading to developmental, reproductive and metabolic defects, has been studied, yet how phthalates interfere with these biological functions is still unclear. To uncover DBP interacting molecular pathways, we raised Drosophila melanogaster on food containing dibutyl phthalate (DBP) at various concentrations. Whole transcriptome analysis of adult Drosophila reveals that DBP exposure throughout development disrupts the expression of genes central to circadian rhythm regulation, including increased expression of vrille (vri, human NFIL3), timeless (tim, human TIMELESS) and period (per, human PER3), with decreased expression of Pigment-dispersing factor (Pdf). DBP exposure also alters the expression of the evolutionarily conserved nuclear receptor Hormone receptor-like in 38 (Hr38, human NR4A2), which is known to regulate Pdf expression. Furthermore, behavioral assays determined that exposing Drosophila to DBP throughout development modifies the circadian rhythm of adults. Although DBP inhibits the expression of signaling systems regulating vision, including Rh5 and Rh6, two light-sensing G-protein coupled receptors involved in the daily resetting of circadian rhythm, it does not influence eye development. Circadian rhythm genes are well conserved from flies to humans; therefore, we tested the effect of DBP exposure on human breast cells (MCF10A) and demonstrate that, similar to the fruit fly model, this exposure disrupts circadian rhythm (BMAL1 expression) at doses that promote the proliferation and migration ability of MCF10A cells. Our results are the first to provide comprehensive evidence that DBP interferes with circadian rhythm in both adult Drosophila and human cells, which may help to explain the broad physiological action of phthalates. De två första författarna delar förstaförfattarskapet

Objectives Circadian rhythm controls complicated physiological activities in organisms. Circadian clock genes have been related to tumour progression, but its role in glioma is unknown. Therefore, we explored the relationship between dysregulated circadian clock genes and glioma progression. Materials and Methods Samples were divided into different groups based on circadian clock gene expression in training dataset (n = 672) and we verified the results in other four validating datasets (n = 1570). The GO and GSEA enrichment analysis were conducted to explore potential mechanism of how circadian clock genes affected glioma progression. The single‐cell RNA‐Seq analysis was conducted to verified previous results. The immune landscape was evaluated by the ssGSEA and CIBERSORT algorithm. Cell proliferation and viability were confirmed by the CCK8 assay, colony‐forming assay and flow cytometry. Results The cluster and risk model based on circadian clock gene expression can predict survival outcome. Samples were scoring by the least absolute shrinkage and selection operator regression analysis, and high scoring tumour was associated with worse survival outcome. Samples in high‐risk group manifested higher activation of immune pathway and cell cycle. Tumour immune landscape suggested high‐risk tumour infiltrated more immunocytes and more sensitivity to immunotherapy. Interfering TIMELESS expression affected circadian clock gene expression, inhibited tumour cell proliferation and arrested cell cycle at the G0/G1 phase. Conclusions Dysregulated circadian clock gene expression can affect glioma progression by affecting tumour immune landscape and cell cycle. The risk model can predict glioma survival outcome, and this model can also be applied to pan‐cancer., Dysregulated circadian clock genes were associated with glioma grades and the IDH status. Prognostic model suggests circadian clock genes affect glioma progression. The GO and GSEA enrichment analysis suggested dysregulated circadian clock genes can affect glioma through interfering cell cycle and influencing immunocytes infltration.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related mortality worldwide due to its asymptomatic onset and poor survival rate. This highlights the urgent need for developing novel diagnostic markers for early HCC detection. The circadian clock is important for maintaining cellular homeostasis and is tightly associated with key tumorigenesis-associated molecular events, suggesting the so-called chronotherapy. An analysis of these core circadian genes may lead to the discovery of biological markers signaling the onset of the disease. In this study, the possible functions of 13 core circadian clock genes (CCGs) in HCC were systematically analyzed with the aim of identifying ideal biomarkers and therapeutic targets. Profiles of HCC patients with clinical and gene expression data were downloaded from The Cancer Genome Atlas and International Cancer Genome Consortium. Various bioinformatics methods were used to investigate the roles of circadian clock genes in HCC tumorigenesis. We found that patients with high TIMELESS expression or low CRY2, PER1, and RORA expressions have poor survival. Besides, a prediction model consisting of these four CCGs, the tumor-node-metastasis (TNM) stage, and sex was constructed, demonstrating higher predictive accuracy than the traditional TNM-based model. In addition, pathway analysis showed that these four CCGs are involved in the cell cycle, PI3K/AKT pathway, and fatty acid metabolism. Furthermore, the network of these four CCGs-related coexpressed genes and immune infiltration was analyzed, which revealed the close association with B cells and nTreg cells. Notably, TIMELESS exhibited contrasting effects against CRY2, PER1, and RORA in most situations. In sum, our works revealed that these circadian clock genes TIMELESS, CRY2, PER1, and RORA can serve as potential diagnostic and prognostic biomarkers, as well as therapeutic targets, for HCC patients, which may promote HCC chronotherapy by rhythmically regulating drug sensitivity and key cellular signaling pathways.

Mosquitoes exhibit activity rhythms, crucial for the transmission of pathogens, under the control of a circadian clock.Aedes aegyptiis one of the world’s leading vectors. For decades, several studies have linked the rise in ambient temperature with the increase in their activity. Here, we identify candidate genes whose expression is influenced by temperature cycles and may affectAedeslocomotor activity. We observed thattimelesscompletely lost its rhythmic expression in light/dark, with out-of-phase temperature cycles, and by RNAi mediated knockdown ofnocte, an important gene forDrosophilacircadian synchronization by temperature cycles. Thus,timelessandnocteare important genes for synchronization by temperature cycles inAedes aegypti. To reinforce our findings, we simulated in the laboratory the gradual temperature fluctuations that were as close as possible to daily temperature variations in Brazil. We observed that the activity and the expression of the molecular circadian clock ofAe. aegyptidiffers significantly from that of mosquitoes subjected to constant or rectangular abrupt changes in temperature. We suggest that for understanding the circadian behavior ofAedeswith possible implications for intervention strategies, the seminatural paradigm needs to replace the traditional laboratory study.

Chronotype is an important moderator of psychiatric illnesses, which seems to be controlled in some part by genetic factors. Clock genes are the most relevant genes for chronotype. In addition to the roles of individual genes, gene-gene interactions of clock genes substantially contribute to chronotype. We investigated genetic associations and gene-gene interactions of the clock genes BHLHB2, CLOCK, CSNK1E, NR1D1, PER1, PER2, PER3, and TIMELESS for chronotype in 1,293 healthy Korean individuals. Regression analysis was conducted to find associations between single nucleotide polymorphism (SNP) and chronotype. For gene-gene interaction analyses, the quantitative multifactor dimensionality reduction (QMDR) method, a nonparametric model-free method for quantitative phenotypes, were performed. No individual SNP or haplotype showed a significant association with chronotype by both regression analysis and single-locus model of QMDR. QMDR analysis identified NR1D1 rs2314339 and TIMELESS rs4630333 as the best SNP pairs among two-locus interaction models associated with chronotype (cross-validation consistency [CVC] = 8/10, p = 0.041). For the three-locus interaction model, the SNP combination of NR1D1 rs2314339, TIMELESS rs4630333, and PER3 rs228669 showed the best results (CVC = 4/10, p < 0.001). However, because the mean differences between genotype combinations were minor, the clinical roles of clock gene interactions are unlikely to be critical.

Many insects show daily and circadian changes in morphology and physiology in their compound eye. In this study, we investigated whether the compound eye had an intrinsic circadian rhythm in the cricket Gryllus bimaculatus. We found that clock genes period (per), timeless (tim), cryptochrome 2 (cry2), and cycle (cyc) were rhythmically expressed in the compound eye under 12-h light/12-h dark cycles (LD 12:12) and constant darkness (DD) at a constant temperature. After the optic nerves were severed (ONX), a weak but significant rhythmic expression persisted for per and tim under LD 12:12, while under DD, tim and cyc showed rhythmic expression. We also found that more than half of the ONX compound eyes exhibited weak but significant circadian electroretinographic rhythms. These results clearly demonstrate that the cricket compound eye possesses an intrinsic circadian oscillator which can drive the circadian light sensitivity rhythm in the eye, and that the circadian clock in the optic lobe exerts its influence on the oscillator in the eye.

Circadian rhythms, which are ubiquitous and adaptive, occur across all species, from microbes to humans, in which they organize and modify behavior and physiology. timeless (tim) is a canonical clock gene. The core composition of the Drosophila melanogaster endogenous circadian clock has been extensively investigated; however, in lepidopteran insects, including Bombyx mori, the mechanism is complicated and little is known regarding the participation of tim in the negative feedback loop responsible for behavioral activities. To arrive at a comprehensive understanding of the role of tim in the B. mori endogenous circadian clock, we exploited the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 gene editing system. We attempted to elucidate the functions of tim in the circadian clock of B. mori using Bmtim mutants. The knockouts affected two circadian behavioral activities: adult emergence and embryo hatching rhythms. Quantitative real-time polymerase chain reaction results confirmed that tim-knockouts induced relative reductions in the expression levels, and thereby the oscillation amplitudes, of Bmper and Bmclk messenger RNAs during both the photophase and scotophase. Additionally, the daily rhythmic expression of Bmdbt was upregulated in the photophase and downregulated in the scotophase in a tim-knockout. Our study reveals that tim is integral to the B. mori circadian clock and may be involved in regulating eclosion and hatching rhythms.

We have used the Cambridge Protein Trap resource (CPTI) to screen for flies whose locomotor rhythms are rhythmic in constant light (LL) as a means of identifying circadian photoreception genes. From the screen of ∼150 CPTI lines, we obtained seven hits, two of which targeted the glutamate pathway, Got1 (Glutamate oxaloacetate transaminase 1) and Gs2 (Glutamine synthetase 2). We focused on these by employing available mutants and observed that variants of these genes also showed high levels of LL rhythmicity compared with controls. It was also clear that the genetic background was important with a strong interaction observed with the common and naturally occurring timeless (tim) polymorphisms, ls-tim and s-tim. The less circadian photosensitive ls-tim allele generated high levels of LL rhythmicity in combination with Got1 or Gs2, even though ls-tim and s-tim alleles do not, by themselves, generate the LL phenotype. The use of dsRNAi for both genes as well as for Gad (Glutamic acid decarboxylase) and the metabotropic glutamate receptor DmGluRA driven by clock gene promoters also revealed high levels of LL rhythmicity compared to controls. It is clear that the glutamate pathway is heavily implicated in circadian photoreception. TIM levels in Got1 and Gs2 mutants cycled and were more abundant than in controls under LL. Got1 but not Gs2 mutants showed diminished phase shifts to 10 min light pulses. Neurogenetic dissection of the LL rhythmic phenotype using the gal4/gal80 UAS bipartite system suggested that the more dorsal CRY-negative clock neurons, DNs and LNds were responsible for the LL phenotype. Immunocytochemistry using the CPTI YFP tagged insertions for the two genes revealed that the DN1s but not the DN2 and DN3s expressed Got1 and Gs2, but expression was also observed in the lateral neurons, the LNds and s-LNvs. Expression of both genes was also found in neuroglia. However, downregulation of glial Gs2 and Got1 using repo-gal4 did not generate high levels of LL rhythmicity, so it is unlikely that this phenotype is mediated by glial expression. Our results suggest a model whereby the DN1s and possibly CRY-negative LNds use glutamate signaling to supress the pacemaker s-LNvs in LL.

The animal circadian timing system interprets environmental time cues and internal metabolic status to orchestrate circadian rhythms of physiology, allowing animals to perform necessary tasks in a time-of-day-dependent manner. Normal progression of circadian rhythms is dependent on the daily cycling of core transcriptional factors that make up cell-autonomous molecular oscillators. InDrosophila, PERIOD (PER), TIMELESS (TIM), CLOCK (CLK), and CYCLE (CYC) are core clock proteins that function in a transcriptional–translational feedback mechanism to regulate the circadian transcriptome. Posttranslational modifications of core clock proteins provide precise temporal control over when they are active as regulators of clock-controlled genes. In particular, phosphorylation is a key regulatory mechanism that dictates the subcellular localization, stability, and transcriptional activity of clock proteins. Previously, casein kinase 1α (CK1α) has been identified as a kinase that phosphorylates mammalian PER1 and modulates its stability, but the mechanisms by which it modulates PER protein stability is still unclear. UsingDrosophilaas a model, we show that CK1α has an overall function of speeding up PER metabolism and is required to maintain the 24 h period of circadian rhythms. Our results indicate that CK1α collaborates with the key clock kinase DOUBLETIME (DBT) in both the cytoplasm and the nucleus to regulate the timing of PER-dependent repression of the circadian transcriptome. Specifically, we observe that CK1α promotes PER nuclear localization by antagonizing the activity of DBT to inhibit PER nuclear translocation. Furthermore, CK1α enhances DBT-dependent PER phosphorylation and degradation once PER moves into the nucleus.SIGNIFICANCE STATEMENTCircadian clocks are endogenous timers that integrate environmental signals to impose temporal control over organismal physiology over the 24 h day/night cycle. To maintain the 24 h period length of circadian clocks and to ensure that circadian rhythms are in synchrony with the external environment, key proteins that make up the molecular oscillator are extensively regulated by phosphorylation to ensure that they perform proper time-of-day-specific functions. Casein kinase 1α (CK1α) has previously been identified as a kinase that phosphorylates mammalian PERIOD (PER) proteins to promote their degradation, but the mechanism by which it modulates PER stability is unclear. In this study, we characterize the mechanisms by which CK1α interacts with DOUBLETIME (DBT) to achieve the overall function of speeding up PER metabolism and to ensure proper time-keeping.

Whether cardiac output in decapod crustaceans is under circadian control has long been debated, with mixed evidence for and against the hypothesis. Moreover, the locus of the clock system controlling cardiac activity, if it is under circadian control, is unknown. However, a report that the crayfish heart in organ culture maintains a circadian oscillation in heartbeat frequency suggests the presence of a peripheral pacemaker within the cardiac neuromuscular system itself. Because the decapod heart is neurogenic, with contractions controlled by the five motor and four premotor neurons that make up the cardiac ganglion (CG), a likely locus for a circadian clock is the CG itself. Here, a CG-specific transcriptome was generated for the lobster, Homarus americanus, and was used to assess the presence/absence of transcripts encoding putative clock-related proteins in the ganglion. Using known Homarus brain/eyestalk ganglia clock-related proteins as queries, BLAST searches of the CG transcriptome were conducted for the five proteins that form the core clock, i.e., clock, cryptochrome 2, cycle, period and timeless, as well as for a variety of clock-associated, clock input pathway and clock output pathway proteins. With the exception of pigment dispersing hormone receptor [PDHR], a putative clock output pathway protein, one or more transcripts encoding each of the proteins searched for were identified from the CG assembly; no PDHR-encoding transcripts were found. RT-PCR confirmed the expression of all core clock transcripts in multiple independent CG cDNAs; RNA-Seq data suggest that both the motor and premotor neurons could contribute to the cellular locus of a pacemaker. These data provide support for the possible existence of an intrinsic circadian clock in the H. americanus CG, and form a foundation for guiding future anatomical, molecular and physiological investigations of circadian signaling in the lobster cardiac neuromuscular system.