Clock Output Pathway Research Articles

Circadian Clock in Muscle Disease Etiology and Therapeutic Potential for Duchenne Muscular Dystrophy.

Circadian clock and clock-controlled output pathways exert temporal control in diverse aspects of skeletal muscle physiology, including the maintenance of muscle mass, structure, function, and metabolism. They have emerged as significant players in understanding muscle disease etiology and potential therapeutic avenues, particularly in Duchenne muscular dystrophy (DMD). This review examines the intricate interplay between circadian rhythms and muscle physiology, highlighting how disruptions of circadian regulation may contribute to muscle pathophysiology and the specific mechanisms linking circadian clock dysregulation with DMD. Moreover, we discuss recent advancements in chronobiological research that have shed light on the circadian control of muscle function and its relevance to DMD. Understanding clock output pathways involved in muscle mass and function offers novel insights into the pathogenesis of DMD and unveils promising avenues for therapeutic interventions. We further explore potential chronotherapeutic strategies targeting the circadian clock to ameliorate muscle degeneration which may inform drug development efforts for muscular dystrophy.

CRY2 isoform selectivity of a circadian clock modulator with antiglioblastoma efficacy

The mammalian cryptochrome isoforms, CRY1 and CRY2, are core circadian clock regulators that work redundantly. Recent studies revealed distinct roles of these closely related homologs in clock output pathways. Isoform-selective control of CRY1 and CRY2 is critical for further understanding their redundant and distinct roles. KL001 was the first identified small-molecule CRY modulator that activates both CRY1 and CRY2. SHP656 is an orally available KL001 derivative and has shown efficacy in blood glucose control and inhibition of glioblastoma stem cell (GSC) growth in animal models. However, CRY isoform selectivity of SHP656 was uncharacterized, limiting understanding of the roles of CRY1 and CRY2. Here, we report the elucidation of CRY2 selectivity of SHP656. SHP656 lengthened cellular circadian period in a CRY2-dependent manner and selectively interacted with CRY2. By determining the X-ray crystal structure of CRY2 in complex with SHP656 and performing molecular dynamics simulations, we elucidated compound interaction mechanisms. SHP656 binding was compatible with the intrinsic CRY2 gatekeeper W417 "in" orientation and also a close "further in" conformation. Perturbation of W417 interaction with the lid loop resulted in a reduced effect of SHP656 on CRY2, supporting an important role of gatekeeper orientation in isoform selectivity. We also identified the R form of SHP656 (called SHP1703) as the active isomer. Treatment with SHP1703 effectively reduced GSC viability. Our results suggest a direct role of CRY2 in glioblastoma antitumorigenesis and provide a rationale for the selective modulation of CRY isoforms in the therapeutic treatment of glioblastoma and other circadian clock-related diseases.

Daily Regulation of Key Metabolic Pathways in Two Seagrasses Under Natural Light Conditions

The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external environmental cycles. It produces rhythms of plant metabolism and physiology, and interacts with signaling pathways controlling daily and seasonal environmental responses through gene expression regulation. Downstream metabolic outputs, such as photosynthesis and sugar metabolism, besides being affected by the clock, can also contribute to the circadian timing itself. In marine plants, studies of circadian rhythms are still way behind in respect to terrestrial species, which strongly limits the understanding of how they coordinate their physiology and energetic metabolism with environmental signals at sea. Here, we provided a first description of daily timing of key core clock components and clock output pathways in two seagrass species, Cymodocea nodosa and Zostera marina (order Alismatales), co-occurring at the same geographic location, thus exposed to identical natural variations in photoperiod. Large differences were observed between species in the daily timing of accumulation of transcripts related to key metabolic pathways, such as photosynthesis and sucrose synthesis/transport, highlighting the importance of intrinsic biological, and likely ecological attributes of the species in determining the periodicity of functions. The two species exhibited a differential sensitivity to light-to-dark and dark-to-light transition times and could adopt different growth timing based on a differential strategy of resource allocation and mobilization throughout the day, possibly coordinated by the circadian clock. This behavior could potentially derive from divergent evolutionary adaptations of the species to their bio-geographical range of distributions.

MicroRNA-21 Controls Circadian Regulation of Apoptosis in Atherosclerotic Lesions.

The necrotic core partly formed by ineffective efferocytosis increases the risk of an atherosclerotic plaque rupture. Microribonucleic acids contribute to necrotic core formation by regulating efferocytosis and macrophage apoptosis. Atherosclerotic plaque rupture occurs at increased frequency in the early morning, indicating diurnal changes in plaque vulnerability. Although circadian rhythms play a role in atherosclerosis, the molecular clock output pathways that control plaque composition and rupture susceptibility are unclear. Circadian gene expression, necrotic core size, apoptosis, and efferocytosis in aortic lesions were investigated at different times of the day in Apoe-/-Mir21+/+ mice and Apoe-/-Mir21-/- mice after consumption of a high-fat diet for 12 weeks. Genome-wide gene expression and lesion formation were analyzed in bone marrow-transplanted mice. Diurnal changes in apoptosis and clock gene expression were determined in human atherosclerotic lesions. The expression of molecular clock genes, lesional apoptosis, and necrotic core size were diurnally regulated in Apoe-/- mice. Efferocytosis did not match the diurnal increase in apoptosis at the beginning of the active phase. However, in parallel with apoptosis, expression levels of oscillating Mir21 strands decreased in the mouse atherosclerotic aorta. Mir21 knockout abolished circadian regulation of apoptosis and reduced necrotic core size but did not affect core clock gene expression. Further, Mir21 knockout upregulated expression of proapoptotic Xaf1 (XIAP-associated factor 1) in the atherosclerotic aorta, which abolished circadian expression of Xaf1. The antiapoptotic effect of Mir21 was mediated by noncanonical targeting of Xaf1 through both Mir21 strands. Mir21 knockout in bone marrow cells also reduced atherosclerosis and necrotic core size. Circadian regulation of clock gene expression was confirmed in human atherosclerotic lesions. Apoptosis oscillated diurnally in phase with XAF1 expression, demonstrating an early morning peak antiphase to that of the Mir21 strands. Our findings suggest that the molecular clock in atherosclerotic lesions induces a diurnal rhythm of apoptosis regulated by circadian Mir21 expression in macrophages that is not matched by efferocytosis, thus increasing the size of the necrotic core.

The Transcriptional Network in the Arabidopsis Circadian Clock System.

The circadian clock is the biological timekeeping system that governs the approximately 24-h rhythms of genetic, metabolic, physiological and behavioral processes in most organisms. This oscillation allows organisms to anticipate and adapt to day–night changes in the environment. Molecular studies have indicated that a transcription–translation feedback loop (TTFL), consisting of transcriptional repressors and activators, is essential for clock function in Arabidopsis thaliana (Arabidopsis). Omics studies using next-generation sequencers have further revealed that transcription factors in the TTFL directly regulate key genes implicated in clock-output pathways. In this review, the target genes of the Arabidopsis clock-associated transcription factors are summarized. The Arabidopsis clock transcriptional network is partly conserved among angiosperms. In addition, the clock-dependent transcriptional network structure is discussed in the context of plant behaviors for adapting to day–night cycles.

Size‐selective mortality induces evolutionary changes in group risk‐taking behaviour and the circadian system in a fish

Intensive and trait-selective mortality of fish and wildlife can cause evolutionary changes in a range of life-history and behavioural traits. These changes might in turn alter the circadian system due to co-evolutionary mechanisms or correlated selection responses both at behavioural and molecular levels, with knock-on effects on daily physiological processes and behavioural outputs. We examined the evolutionary impact of size-selective mortality on group risk-taking behaviour and the circadian system in a model fish species. We exposed zebrafish Danio rerio to either large or small size-selective harvesting relative to a control over five generations, followed by eight generations during which harvesting was halted to remove maternal effects. Size-selective mortality affected fine-scale timing of behaviours. In particular, small size-selective mortality, typical of specialized fisheries and gape-limited predators targeting smaller size classes, increased group risk-taking behaviuor during feeding and after simulated predator attacks. Moreover, small size-selective mortality increased early peaks of daily activity as well as extended self-feeding daily activity to the photophase compared to controls. By contrast large size-selective mortality, typical of most wild capture fisheries, only showed an almost significant effect of decreasing group risk-taking behaviour during the habituation phase and no clear changes in fine-scale timing of daily behavioural rhythms compared to controls. We also found changes in the molecular circadian core clockwork in response to both size-selective mortality treatments. These changes disappeared in the clock output pathway because both size-selected lines showed similar transcription profiles. This switch downstream to the molecular circadian core clockwork also resulted in similar overall behavioural rhythms (diurnal swimming and self-feeding in the last hours of darkness) independent of the underlying molecular clock. To conclude, our experimental harvest left an asymmetrical evolutionary legacy in group risk-taking behaviour and in fine-scale daily behavioural rhythms. Yet, the overall timing of activity showed evolutionary resistance probably maintained by a molecular switch. Our experimental findings suggest that size-selective mortality can have consequences for behaviour and physiological processes.

Evolutionary Insight into the Clock-Associated PRR5 Transcriptional Network of Flowering Plants

Circadian clocks regulate the daily timing of metabolic, physiological, and behavioral activities to adapt organisms to day-night cycles. In the model plant Arabidopsis thaliana, transcript-translational feedback loops (TTFL) constitute the circadian clock, which is conserved among flowering plants. Arabidopsis TTFL directly regulates key genes in the clock-output pathways, whereas the pathways for clock-output control in other plants is largely unknown. Here, we propose that the transcriptional networks of clock-associated pseudo-response regulators (PRRs) are conserved among flowering plants. Most PRR genes from Arabidopsis, poplar, and rice encode potential transcriptional repressors. The PRR5-target-like gene group includes genes that encode key transcription factors for flowering time regulation, cell elongation, and chloroplast gene expression. The 5′-upstream regions of PRR5-target-like genes from poplar and rice tend to contain G-box-like elements that are potentially recognized by PRRs in vivo as has been shown in Arabidopsis. Expression of PRR5-target-like genes from poplar and rice tends to decrease when PRRs are expressed, possibly suggesting that the transcriptional network of PRRs is evolutionarily conserved in these plants.

Molecular evidence for an intrinsic circadian pacemaker in the cardiac ganglion of the American lobster, Homarus americanus - Is diel cycling of heartbeat frequency controlled by a peripheral clock system?

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.

Circadian signaling in Homarus americanus: Region-specific de novo assembled transcriptomes show that both the brain and eyestalk ganglia possess the molecular components of a putative clock system

Essentially all organisms exhibit recurring patterns of physiology/behavior that oscillate with a period of ~24-h and are synchronized to the solar day. Crustaceans are no exception, with robust circadian rhythms having been documented in many members of this arthropod subphylum. However, little is known about the molecular underpinnings of their circadian rhythmicity. Moreover, the location of the crustacean central clock has not been firmly established, although both the brain and eyestalk ganglia have been hypothesized as loci. The American lobster, Homarus americanus, is known to exhibit multiple circadian rhythms, and immunodetection data suggest that its central clock is located within the eyestalk ganglia rather than in the brain. Here, brain- and eyestalk ganglia-specific transcriptomes were generated and used to assess the presence/absence of transcripts encoding the commonly recognized protein components of arthropod circadian signaling systems in these two regions of the lobster central nervous system. Transcripts encoding putative homologs of the core clock proteins clock, cryptochrome 2, cycle, period and timeless were found in both the brain and eyestalk ganglia assemblies, as were transcripts encoding similar complements of putative clock-associated, clock input pathway and clock output pathway proteins. The presence and identity of transcripts encoding core clock proteins in both regions were confirmed using PCR. These findings suggest that both the brain and eyestalk ganglia possess all of the molecular components needed for the establishment of a circadian signaling system. Whether the brain and eyestalk clocks are independent of one another or represent a single timekeeping system remains to be determined. Interestingly, while most of the proteins deduced from the identified transcripts are shared by both the brain and eyestalk ganglia, assembly-specific isoforms were also identified, e.g., several period variants, suggesting the possibility of region-specific variation in clock function, especially if the brain and eyestalk clocks represent independent oscillators.

Atomic force microscopy analysis of SasA-KaiC complex formation involved in information transfer from the KaiABC clock machinery to the output pathway in cyanobacteria.

The cyanobacterial clock oscillator is composed of three clock proteins: KaiA, KaiB and KaiC. SasA, a KaiC-binding EnvZ-like orthodox histidine kinase involved in the main clock output pathway, exists mainly as a trimer (SasA3mer ) and occasionally as a hexamer (SasA6mer ) in vitro. Previously, the molecular mass of the SasA-KaiCDD complex, where KaiCDD is a mutant KaiC with two Asp substitutions at the two phosphorylation sites, has been estimated by gel-filtration chromatography to be larger than 670kDa. This value disagrees with the theoretical estimation of 480kDa for a SasA3mer -KaiC hexamer (KaiC6mer ) complex with a 1:1 molecular ratio. To clarify the structure of the SasA-KaiC complex, we analyzed KaiCDD with 0.1mmol/L ATP and 5mmol/L MgCl2 (Mg-ATP), SasA and a mixture containing SasA and KaiCDD6mer with Mg-ATP by atomic force microscopy (AFM). KaiCDD images were classified into two types with height distribution corresponding to KaiCDD monomer (KaiCDD1mer ) and KaiCDD6mer , respectively. SasA images were classified into two types with height corresponding to SasA3mer and SasA6mer , respectively. The AFM images of the SasA-KaiCDD mixture indicated not only KaiCDD1mer , KaiCDD6mer , SasA3mer and SasA6mer , but also wider area "islands," suggesting the presence of a polymerized form of the SasA-KaiCDD complex.

A HAD family phosphatase CSP-6 regulates the circadian output pathway in Neurospora crassa.

Circadian clocks are ubiquitous in eukaryotic organisms where they are used to anticipate regularly occurring diurnal and seasonal environmental changes. Nevertheless, little is known regarding pathways connecting the core clock to its output pathways. Here, we report that the HAD family phosphatase CSP-6 is required for overt circadian clock output but not for the core oscillation. The loss of function Δcsp-6 deletion mutant is overtly arrhythmic on race tubes under free running conditions; however, reporter assays confirm that the FREQUENCY-WHITE COLLAR COMPLEX core circadian oscillator is functional, indicating a discrete block between oscillator and output. CSP-6 physically interacts with WHI-2, Δwhi-2 mutant phenotypes resemble Δcsp-6, and the CSP-6/WHI-2 complex physically interacts with WC-1, all suggesting that WC-1 is a direct target for CSP-6/WHI-2-mediated dephosphorylation and consistent with observed WC-1 hyperphosphorylation in Δcsp-6. To identify the source of the block to output, known clock-controlled transcription factors were screened for rhythmicity in Δcsp-6, identifying loss of circadian control of ADV-1, a direct target of WC-1, as responsible for the loss of overt rhythmicity. The CSP-6/WHI-2 complex thus participates in the clock output pathway by regulating WC-1 phosphorylation to promote proper transcriptional/translational activation of adv-1/ADV-1; these data establish an unexpected essential role for post-translational modification parallel to circadian transcriptional regulation in the early steps of circadian output.

The Circadian Clock Regulates Adipogenesis by a Per3 Crosstalk Pathway to Klf15

The generation of new adipocytes from precursor cells (adipogenesis) has implications for systemic metabolism and is a commonly used model for studying the process of cell differentiation invitro. Previous studies from us and others suggested that the peripheral circadian clock can influence adipogenesis invitro, but the mechanisms driving this activity and the relevance for adipogenesis invivo are unknown. Here we reveal that mouse adipocyte precursor cells (APCs) contain a circadian clock that oscillates invivo. We expose context-specific features of the clock in APCs: expression of the canonical core clock component Per1 does not significantly oscillate, whereas the lesser-understood paralog Per3 has a prominent rhythm. We discovered that deletion of Per3 promotes adipogenesis invivo by a clock output pathway in which PER3 and BMAL1 directly regulate Klf15 expression. These findings demonstrate that Per3 has a major role in the APC clock and regulates adipogenesis invivo.

LATE ELONGATED HYPOCOTYL regulates photoperiodic flowering via the circadian clock in Arabidopsis.

BackgroundPlants constantly monitor changes in photoperiod or day length to trigger the flowering cycle at the most appropriate time of the year. It is well established that photoperiodic flowering is intimately associated with the circadian clock in Arabidopsis. In support of this notion, many clock-defective mutants exhibit altered photoperiodic sensitivity in inducing flowering. LATE ELONGATED HYPOCOTYL (LHY) and its functional paralogue CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) constitute the core of the circadian clock together with TIMING OF CAB EXPRSSION 1 (TOC1). While it is known that TOC1 contributes to the timing of flowering entirely by modulating the clock function, molecular mechanisms by which LHY and CCA1 regulate flowering time have not been explored.ResultsWe investigated how LHY and CCA1 regulate photoperiodic flowering through molecular genetic and biochemical studies. It was found that LHY-defective mutants (lhy-7 and lhy-20) exhibit accelerated flowering under both long days (LDs) and short days (SDs). Consistent with the accelerated flowering phenotypes, gene expression analysis revealed that expression of the floral integrator FLOWERING LOCUS T (FT) is up-regulated in the lhy mutants. In addition, the expression peaks of GIGANTEA (GI) and FLAVIN-BINDING, KELCH REPEAT, F-BOX PROTEIN 1 (FKF1) genes, which constitute the clock output pathway that is linked with photoperiodic flowering, were advanced by approximately 4 h in the mutants. Furthermore, the up-regulation of FT disappeared when the endogenous circadian period is matched to the external light/dark cycles in the lhy-7 mutant. Notably, whereas CCA1 binds strongly to FT gene promoter, LHY does not show such DNA-binding activity.ConclusionsOur data indicate that the advanced expression phases of photoperiodic flowering genes are associated with the clock defects in the lhy mutants and responsible for the reduced photoperiodic sensitivity of the mutant flowering, demonstrating that LHY regulates photoperiodic flowering via the circadian clock, similar to what has been shown with TOC1. It is notable that while LHY regulates photoperiodic flowering in a similar manner as with TOC1, the underlying molecular mechanism would be somewhat distinct from that exerted by CCA1 in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0810-8) contains supplementary material, which is available to authorized users.

Genome-wide identification of CCA1 targets uncovers an expanded clock network in Arabidopsis

The circadian clock in Arabidopsis exerts a critical role in timing multiple biological processes and stress responses through the regulation of up to 80% of the transcriptome. As a key component of the clock, the Myb-like transcription factor CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is able to initiate and set the phase of clock-controlled rhythms and has been shown to regulate gene expression by binding directly to the evening element (EE) motif found in target gene promoters. However, the precise molecular mechanisms underlying clock regulation of the rhythmic transcriptome, specifically how clock components connect to clock output pathways, is poorly understood. In this study, using ChIP followed by deep sequencing of CCA1 in constant light (LL) and diel (LD) conditions, more than 1,000 genomic regions occupied by CCA1 were identified. CCA1 targets are enriched for a myriad of biological processes and stress responses, providing direct links to clock-controlled pathways and suggesting that CCA1 plays an important role in regulating a large subset of the rhythmic transcriptome. Although many of these target genes are evening expressed and contain the EE motif, a significant subset is morning phased and enriched for previously unrecognized motifs associated with CCA1 function. Furthermore, this work revealed several CCA1 targets that do not cycle in either LL or LD conditions. Together, our results emphasize an expanded role for the clock in regulating a diverse category of genes and key pathways in Arabidopsis and provide a comprehensive resource for future functional studies.

Linked circadian outputs control elongation growth and flowering in response to photoperiod and temperature

Clock-regulated pathways coordinate the response of many developmental processes to changes in photoperiod and temperature. We model two of the best-understood clock output pathways in Arabidopsis, which control key regulators of flowering and elongation growth. In flowering, the model predicted regulatory links from the clock to CYCLING DOF FACTOR 1 (CDF1) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) transcription. Physical interaction data support these links, which create threefold feed-forward motifs from two clock components to the floral regulator FT. In hypocotyl growth, the model described clock-regulated transcription of PHYTOCHROME-INTERACTING FACTOR 4 and 5 (PIF4, PIF5), interacting with post-translational regulation of PIF proteins by phytochrome B (phyB) and other light-activated pathways. The model predicted bimodal and end-of-day PIF activity profiles that are observed across hundreds of PIF-regulated target genes. In the response to temperature, warmth-enhanced PIF4 activity explained the observed hypocotyl growth dynamics but additional, temperature-dependent regulators were implicated in the flowering response. Integrating these two pathways with the clock model highlights the molecular mechanisms that coordinate plant development across changing conditions.

Regulatory properties of ADP glucose pyrophosphorylase are required for adjustment of leaf starch synthesis in different photoperiods.

Arabidopsis (Arabidopsis thaliana) leaves synthesize starch faster in short days than in long days, but the mechanism that adjusts the rate of starch synthesis to daylength is unknown. To understand this mechanism, we first investigated whether adjustment occurs in mutants lacking components of the circadian clock or clock output pathways. Most mutants adjusted starch synthesis to daylength, but adjustment was compromised in plants lacking the GIGANTEA or FLAVIN-BINDING, KELCH REPEAT, F BOX1 components of the photoperiod-signaling pathway involved in flowering. We then examined whether the properties of the starch synthesis enzyme adenosine 5'-diphosphate-glucose pyrophosphorylase (AGPase) are important for adjustment of starch synthesis to daylength. Modulation of AGPase activity is known to bring about short-term adjustments of photosynthate partitioning between starch and sucrose (Suc) synthesis. We found that adjustment of starch synthesis to daylength was compromised in plants expressing a deregulated bacterial AGPase in place of the endogenous AGPase and in plants containing mutant forms of the endogenous AGPase with altered allosteric regulatory properties. We suggest that the rate of starch synthesis is in part determined by growth rate at the end of the preceding night. If growth at night is low, as in short days, there is a delay before growth recovers during the next day, leading to accumulation of Suc and stimulation of starch synthesis via activation of AGPase. If growth at night is fast, photosynthate is used for growth at the start of the day, Suc does not accumulate, and starch synthesis is not up-regulated.

The ion transport peptide is a new functional clock neuropeptide in the fruit fly Drosophila melanogaster.

The clock network of Drosophila melanogaster expresses various neuropeptides, but a function in clock-mediated behavioral control was so far only found for the neuropeptide pigment dispersing factor (PDF). Here, we propose a role in the control of behavioral rhythms for the ion transport peptide (ITP), which is expressed in the fifth small ventral lateral neuron, one dorsal lateral neuron, and in only a few nonclock cells in the brain. Immunocytochemical analyses revealed that ITP, like PDF, is most probably released in a rhythmic manner at projection terminals in the dorsal protocerebrum. This rhythm continues under constant dark conditions, indicating that ITP release is clock controlled. ITP expression is reduced in the hypomorph mutant Clk(AR), suggesting that ITP expression is regulated by CLOCK. Using a genetically encoded RNAi construct, we knocked down ITP in the two clock cells and found that these flies show reduced evening activity and increased nocturnal activity. Overexpression of ITP with two independent timeless-GAL4 lines completely disrupted behavioral rhythms, but only slightly dampened PER cycling in important pacemaker neurons, suggesting a role for ITP in clock output pathways rather than in the communication within the clock network. Simultaneous knockdown (KD) of ITP and PDF made the flies hyperactive and almost completely arrhythmic under constant conditions. Under light-dark conditions, the double-KD combined the behavioral characteristics of the single-KD flies. In addition, it reduced the flies' sleep. We conclude that ITP and PDF are the clock's main output signals that cooperate in controlling the flies' activity rhythms.

Ablation of the Id2 Gene Results in Altered Circadian Feeding Behavior, and Sex-Specific Enhancement of Insulin Sensitivity and Elevated Glucose Uptake in Skeletal Muscle and Brown Adipose Tissue

Inhibitor of DNA binding 2 (ID2) is a helix-loop-helix transcriptional repressor rhythmically expressed in many adult tissues. Our earlier studies have demonstrated a role for ID2 in the input pathway, core clock function and output pathways of the mouse circadian system. We have also reported that Id2 null (Id2−/−) mice are lean with low gonadal white adipose tissue deposits and lower lipid content in the liver. These results coincided with altered or disrupted circadian expression profiles of liver genes including those involved in lipid metabolism. In the present phenotypic study we intended to decipher, on a sex-specific basis, the role of ID2 in glucose metabolism and in the circadian regulation of activity, important components of energy balance. We find that Id2−/− mice exhibited altered daily and circadian rhythms of feeding and locomotor activity; activity profiles extended further into the late night/dark phase of the 24-hr cycle, despite mice showing reduced total locomotor activity. Also, male Id2−/− mice consumed a greater amount of food relative to body mass, and displayed less weight gain. Id2−/− females had smaller adipocytes, suggesting sexual-dimorphic programing of adipogenesis. We observed increased glucose tolerance and insulin sensitivity in male Id2−/− mice, which was exacerbated in older animals. FDG-PET analysis revealed increased glucose uptake by skeletal muscle and brown adipose tissue of male Id2−/− mice, suggesting increased glucose metabolism and thermogenesis in these tissues. Reductions in intramuscular triacylglycerol and diacylglycerol were detected in male Id2−/− mice, highlighting its possible mechanistic role in enhanced insulin sensitivity in these mice. Our findings indicate a role for ID2 as a regulator of glucose and lipid metabolism, and in the circadian control of feeding/locomotor behavior; and contribute to the understanding of the development of obesity and diabetes, particularly in shift work personnel among whom incidence of such metabolic disorders is elevated.

Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator

In the cyanobacteria Synechococcus elongatus and Thermosynechococcus elongatus, the KaiA, KaiB and KaiC proteins in the presence of ATP generate a post-translational oscillator (PTO) that can be reconstituted in vitro. KaiC is the result of a gene duplication and resembles a double doughnut with N-terminal CI and C-terminal CII hexameric rings. Six ATPs are bound between subunits in both the CI and CII ring. CI harbors ATPase activity, and CII catalyzes phosphorylation and dephosphorylation at T432 and S431 with a ca. 24-h period. KaiA stimulates KaiC phosphorylation, and KaiB promotes KaiC subunit exchange and sequesters KaiA on the KaiB-KaiC interface in the final stage of the clock cycle. Studies of the PTO protein-protein interactions are convergent in terms of KaiA binding to CII but have led to two opposing models of the KaiB-KaiC interaction. Electron microscopy (EM) and small angle X-ray scattering (SAXS), together with native PAGE using full-length proteins and separate CI and CII rings, are consistent with binding of KaiB to CII. Conversely, NMR together with gel filtration chromatography and denatured PAGE using monomeric CI and CII domains support KaiB binding to CI. To resolve the existing controversy, we studied complexes between KaiB and gold-labeled, full-length KaiC with negative stain EM. The EM data clearly demonstrate that KaiB contacts the CII ring. Together with the outcomes of previous analyses, our work establishes that only CII participates in interactions with KaiA and KaiB as well as with the His kinase SasA involved in the clock output pathway.

Transcriptional repressor PRR5 directly regulates clock-output pathways

The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external daily cycles. The clock coordinates biological activities with these cycles, mainly through genome-wide gene expression. However, the exact mechanism underlying regulation of circadian gene expression is poorly understood. Here we demonstrated that an Arabidopsis PSEUDO-RESPONSE REGULATOR 5 (PRR5), which acts in the clock genetic circuit, directly regulates expression timing of key transcription factors involved in clock-output pathways. A transient expression assay and ChIP-quantitative PCR assay using mutated PRR5 indicated that PRR5 associates with target DNA through binding at the CCT motif in vivo. ChIP followed by deep sequencing coupled with genome-wide expression profiling revealed the direct-target genes of PRR5. PRR5 direct-targets include genes encoding transcription factors involved in flowering-time regulation, hypocotyl elongation, and cold-stress responses. PRR5-target gene expression followed a circadian rhythm pattern with low, basal expression from noon until midnight, when PRR9, PRR7, and PRR5 were expressed. ChIP-quantitative PCR assays indicated that PRR7 and PRR9 bind to the direct-targets of PRR5. Genome-wide expression profiling using a prr9 prr7 prr5 triple mutant suggests that PRR5, PRR7, and PRR9 repress these targets. Taken together, our results illustrate a genetic network in which PRR5, PRR7, and PRR9 directly regulate expression timing of key transcription factors to coordinate physiological processes with daily cycles.