Circadian Clock-controlled Gene Research Articles

Spatio-temporal coherence of circadian clocks and temporal control of differentiation in Anabaena filaments.

Circadian clock arrays in multicellular filaments of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 display remarkable spatio-temporal coherence under nitrogen-replete conditions. To shed light on the interplay between circadian clocks and the formation of developmental patterns, we followed the expression of a clock-controlled gene under nitrogen deprivation, at the level of individual cells. Our experiments showed that differentiation into heterocysts took place preferentially within a limited interval of the circadian clock cycle, that gene expression in different vegetative intervals along a developed filament was discoordinated, and that the circadian clock was active in individual heterocysts. Furthermore, Anabaena mutants lacking the kaiABC genes encoding the circadian clock core components produced heterocysts but failed in diazotrophy. Therefore, genes related to some aspect of nitrogen fixation, rather than early or mid-heterocyst differentiation genes, are likely affected by the absence of the clock. A bioinformatics analysis supports the notion that RpaA may play a role as master regulator of clock outputs in Anabaena, the temporal control of differentiation by the circadian clock and the involvement of the clock in proper diazotrophic growth. Together, these results suggest that under nitrogen-deficient conditions, the clock coherent unit in Anabaena is reduced from a full filament under nitrogen-rich conditions to the vegetative cell interval between heterocysts.IMPORTANCECircadian clocks, from unicellular organisms to animals, temporally align biological processes to day and night cycles. We study the dynamics of a circadian clock-controlled gene at the individual cell level in the multicellular filamentous cyanobacterium Anabaena, under nitrogen-stress conditions. Under these conditions, some cells along filaments differentiate to carry out atmospheric nitrogen fixation and lose their capability for oxygenic photosynthesis. We found that clock synchronization is limited to organismic units of contiguous photosynthetic cells, contrary to nitrogen-replete conditions in which clocks are synchronized over a whole filament. We provided evidence that the circadian clock regulates the process of differentiation, allowing it to occur preferentially within a limited time window during the circadian clock period. Lastly, we present evidence that the signal from the core clock to clock-regulated genes is conveyed in Anabaena as in unicellular cyanobacteria.

Cell-specific clock-controlled gene expression program regulates rhythmic fiber cell growth in cotton

BackgroundThe epidermis of cotton ovule produces fibers, the most important natural cellulose source for the global textile industry. However, the molecular mechanism of fiber cell growth is still poorly understood.ResultsHere, we develop an optimized protoplasting method, and integrate single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) to systematically characterize the cells of the outer integument of ovules from wild type and fuzzless/lintless (fl) cotton (Gossypium hirsutum). By jointly analyzing the scRNA-seq data from wildtype and fl, we identify five cell populations including the fiber cell type and construct the development trajectory for fiber lineage cells. Interestingly, by time-course diurnal transcriptomic analysis, we demonstrate that the primary growth of fiber cells is a highly regulated circadian rhythmic process. Moreover, we identify a small peptide GhRALF1 that circadian rhythmically controls fiber growth possibly through oscillating auxin signaling and proton pump activity in the plasma membrane. Combining with scATAC-seq, we further identify two cardinal cis-regulatory elements (CREs, TCP motif, and TCP-like motif) which are bound by the trans factors GhTCP14s to modulate the circadian rhythmic metabolism of mitochondria and protein translation through regulating approximately one third of genes that are highly expressed in fiber cells.ConclusionsWe uncover a fiber-specific circadian clock-controlled gene expression program in regulating fiber growth. This study unprecedentedly reveals a new route to improve fiber traits by engineering the circadian clock of fiber cells.

Disruption of day-to-night changes in circadian gene expression with chronic tendinopathy.

Overuse injury in tendon tissue (tendinopathy) is a frequent and costly musculoskeletal disorder and represents a major clinical problem with unsolved pathogenesis. Studies in mice have demonstrated that circadian clock-controlled genes are vital for protein homeostasis and important in the development of tendinopathy. We performed RNA sequencing, collagen content and ultrastructural analyses on human tendon biopsies obtained 12h apart in healthy individuals to establish whether human tendon is a peripheral clock tissue and we performed RNA sequencing on patients with chronic tendinopathy to examine the expression of circadian clock genes in tendinopathic tissues. We found time-dependent expression of 280 RNAs including 11 conserved circadian clock genes in healthy tendons and markedly fewer (23) differential RNAs with chronic tendinopathy. Further, the expression of COL1A1 and COL1A2 was reduced at night but was not circadian rhythmic in synchronised human tenocyte cultures. In conclusion, day-to-night changes in gene expression in healthy human patellar tendons indicate a conserved circadian clock as well as the existence of a night reduction in collagen I expression. KEY POINTS: Tendinopathy is a major clinical problem with unsolved pathogenesis. Previous work in mice has shown that a robust circadian rhythm is required for collagen homeostasis in tendons. The use of circadian medicine in the diagnosis and treatment of tendinopathy has been stifled by the lack of studies on human tissue. Here, we establish that the expression of circadian clock genes in human tendons is time dependent, and now we have data to corroborate that circadian output is reduced in diseased tendon tissues. We consider our findings to be of significance in advancing the use of the tendon circadian clock as a therapeutic target or preclinical biomarker for tendinopathy.

The circadian demethylation of a unique intronic deoxymethylCpG-rich island boosts the transcription of its cognate circadian clock output gene

We demonstrate that there is a tight functional relationship between two highly evolutionary conserved cell processes, i.e., the circadian clock(CC) and the circadian DNA demethylation-methylation of cognate deoxyCpG-rich islands. We have discovered that every circadian clock-controlled output gene (CCG), but not the core clock nor its immediate-output genes, contains a single cognate intronic deoxyCpG-rich island, the demethylation-methylation of which is controlled by the CC. During the transcriptional activation period, these intronic islands are demethylated and, upon dimerization of two YY1 protein binding sites located upstream to the transcriptional enhancer and downstream from the deoxyCpG-rich island, store activating components initially assembled on a cognate active enhancer (a RORE, a D-box or an E-box), in keeping with the generation of a transcriptionally active condensate that boosts the initiation of transcription of their cognate pre-mRNAs. We report how these single intronic deoxyCpG-rich islands are instrumental in such a circadian activation/repression transcriptional process.

Loss-of-function in GIGANTEA confers resistance to PPO-inhibiting herbicide tiafenacil through transcriptional activation of antioxidant genes in Arabidopsis

Herbicides play a crucial role in maintaining crop productivity by reducing competition between weeds and crops. Protoporphyrinogen oxidase (PPO)-inhibiting herbicides trigger the photooxidative damage that destroys cell membranes. Tiafenacil is a recently developed pyrimidinedione-type PPO-inhibiting herbicide that has low IC50 values in plants and is less toxic in humans compared to other PPO inhibitors. Previous reports confirmed that mutations in Arabidopsis circadian clock-controlled gene GIGANTEA (GI) were insensitive to phytooxidants, including chloroplast biogenesis inhibitors and herbicides. Here, we examined whether GI regulates the resistance to tiafenacil. Both gi mutant alleles, gi-1 and gi-2, were resistant to tiafenacil with survival rates of 97% and 83%, respectively, under 1 µM tiafenacil treatments, while 56% of wild-type and GI-overexpressing plants (GI-OX) survived. Both gi mutants were insensitive to tiafenacil-induced inhibition of photosystem efficiency and alleviated photooxidative damage. The gi mutants showed significant increases in transcriptional expressions and enzyme activities of antioxidants compared to wild-type and GI-OX. Moreover, loss-of-function in GI enhanced resistance to tiafenacil-containing commercial herbicide Terrad’or Plus®. Collectively, based on our results together with previous reports, mutations in GI confer resistance to herbicides with different MoAs and would be a crucial molecular target for non-target-site resistance strategies to develop herbicide-resistant crops.

Robust, coherent, and synchronized circadian clock-controlled oscillations along Anabaena filaments.

Circadian clocks display remarkable reliability despite significant stochasticity in biomolecular reactions. We study the dynamics of a circadian clock-controlled gene at the individual cell level in Anabaena sp. PCC 7120, a multicellular filamentous cyanobacterium. We found significant synchronization and spatial coherence along filaments, clock coupling due to cell-cell communication, and gating of the cell cycle. Furthermore, we observed low-amplitude circadian oscillatory transcription of kai genes encoding the post-transcriptional core oscillatory circuit and high-amplitude oscillations of rpaA coding for the master regulator transducing the core clock output. Transcriptional oscillations of rpaA suggest an additional level of regulation. A stochastic one-dimensional toy model of coupled clock cores and their phosphorylation states shows that demographic noise can seed stochastic oscillations outside the region where deterministic limit cycles with circadian periods occur. The model reproduces the observed spatio-temporal coherence along filaments and provides a robust description of coupled circadian clocks in a multicellular organism.

Thyrotroph embryonic factor polymorphism predicts faster progression of Parkinson's disease in a longitudinal study.

The thyrotroph embryonic factor gene is a circadian clock-controlled gene. The rs738499 polymorphism of this gene has been suggested to be associated with depression and sleep disturbance in Parkinson's disease in previous cross-sectional studies. We aimed to investigate whether this single nucleotide polymorphism is associated with the progression rates of various motor and non-motor symptoms in patients with Parkinson's disease. We recruited 186 patients with Parkinson's disease for a longitudinal study. Motor and non-motor symptoms were assessed at baseline and follow-up, and 170 Parkinson's disease patients completed the clinical evaluation twice with an average follow-up period of 3.3 ± 1.1 years. A stepwise linear regression model was used to validate factors associated with Parkinson's disease symptoms' annual progression rates. Faster annual worsening rates of sleep quality and Hoehn-Yahr stage were found in carriers with the homozygous dominant (TT). After adjustment for related clinical factors, the rs738499 polymorphism showed a contribution of 3.1% to the annual decline rate on the Parkinson's Disease Sleep Scale score and a contribution of 5.5% to the annual increase rate of the Hoehn-Yahr stage. Additionally, anxiety and axial symptoms predicted the progression of sleep disturbances and motor staging. The TT genotype of rs738499 might be a potential predictor of rapid deterioration in sleep quality and Hoehn-Yahr stage in patients with Parkinson's disease and may advance the understanding of the genetic contributions to Parkinson's disease.

Circadian clock-controlled gene expression in co-cultured, mat-forming cyanobacteria

Natural coastal microbial mat communities are multi-species assemblages that experience fluctuating environmental conditions and are shaped by resource competition as well as by cooperation. Laboratory studies rarely address the natural complexity of microbial communities but are usually limited to homogeneous mono-cultures of key species grown in liquid media. The mat-forming filamentous cyanobacteria Lyngbya aestuarii and Coleofasciculus chthonoplastes were cultured under different conditions to investigate the expression of circadian clock genes and genes that are under their control. The cyanobacteria were grown in liquid medium or on a solid substrate (glass beads) as mono- or as co-cultures under a light–dark regime and subsequently transferred to continuous light. TaqMan-probe based qPCR assays were used to quantify the expression of the circadian clock genes kaiA, kaiB, and kaiC, and of four genes that are under control of the circadian clock: psbA, nifH, ftsZ, and prx. Expression of kaiABC was influenced by co-culturing the cyanobacteria and whether grown in liquid media or on a solid substrate. Free-running (i.e. under continuous light) expression cycle of the circadian clock genes was observed in L. aestuarii but not in C. chthonoplastes. In the former organism, maximum expression of psbA and nifH occurred temporally separated and independent of the light regime, although the peak shifted in time when the culture was transferred to continuous illumination. Although functionally similar, both species of cyanobacteria displayed different 24-h transcriptional patterns in response to the experimental treatments, suggesting that their circadian clocks have adapted to different life strategies adopted by these mat-forming cyanobacteria.

Arabidopsis GIGANTEA negatively regulates chloroplast biogenesis and resistance to herbicide butafenacil.

Arabidopsis GI negatively regulates chloroplast biogenesis and resistance to the herbicide butafenacil by enhanced activity and transcriptional levels of antioxidant enzymes Chloroplast biogenesis is blocked by retrograde signaling triggered by diverse internal and external cues, including sugar, reactive oxygen species (ROS), phytohormones, and abiotic stress. Efficient chloroplast biogenesis is essential for crop productivity due to its effect on photosynthetic efficiency, and is associated with agronomic traits such as insect/disease resistance, herbicide resistance, and abiotic stress tolerance. Here, we show that the circadian clock-controlled gene GIGANTEA (GI) regulates chloroplast biogenesis in Arabidopsis thaliana. The gi-2 mutant showed reduced sensitivity to the chloroplast biogenesis inhibitor lincomycin, maintaining high levels of photosynthetic proteins. By contrast, wild-type and GI-overexpressing plants were sensitive to lincomycin, with variegated leaves and reduced photosynthetic protein levels. GI is degraded by lincomycin, suggesting that GI is genetically linked to chloroplast biogenesis. The GI mutant alleles gi-1 and gi-2 were resistant to the herbicide butafenacil, which inhibits protoporphyrinogen IX oxidase activity and triggers ROS-mediated cell death via the accumulation of chlorophyll precursors. Butafenacil-mediated accumulation of superoxide anions and H2O2 was not detected in gi-1 or gi-2, as revealed by histochemical staining. The activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase were 1.2-1.4-fold higher in both gi mutants compared to the wild type. Finally, the expression levels of antioxidant enzyme genes were 1.5-2-fold higher in the mutants than in the wild type. These results suggest that GI negatively regulates chloroplast biogenesis and resistance to the herbicide butafenacil, providing evidence for a genetic link between GI and chloroplast biogenesis, which could facilitate the development of herbicide-resistant crops.

Impact of Short-Term Fasting on The Rhythmic Expression of the Core Circadian Clock and Clock-Controlled Genes in Skeletal Muscle of Crucian Carp (Carassius auratus).

The peripheral tissue pacemaker is responsive to light and other zeitgebers, especially food availability. Generally, the pacemaker can be reset and entrained independently of the central circadian structures. Studies involving clock-gene expressional patterns in fish peripheral tissues have attracted considerable attention. However, the rhythmic expression of clock genes in skeletal muscle has only scarcely been investigated. The present study was designed to investigate the core clock and functional gene expression rhythms in crucian carp. Meanwhile, the synchronized effect of food restrictions (short-term fasting) on these rhythms in skeletal muscle was carefully examined. In fed crucian carp, three core clock genes (Clock, Bmal1a, and Per1) and five functional genes (Epo, Fas, IGF1R2, Jnk1, and MyoG) showed circadian rhythms. By comparison, four core clock genes (Clock, Bmal1a, Cry3, and Per2) and six functional genes (Epo, GH, IGF2, Mstn, Pnp5a, and Ucp1) showed circadian rhythms in crucian carp muscle after 7-day fasting. In addition, three core clock genes (Clock, Per1, and Per3) and six functional genes (Ampk1a, Lpl, MyoG, Pnp5a, PPARα, and Ucp1) showed circadian rhythms in crucian carp muscle after 15-day fasting. However, all gene rhythmic expression patterns differed from each other. Furthermore, it was found that the circadian genes could be altered by feed deprivation in crucian carp muscle through the rhythms correlation analysis of the circadian genes and functional genes. Hence, food-anticipatory activity of fish could be adjusted through the food delivery restriction under a light–dark cycle. These results provide a potential application in promoting fish growth by adjusting feeding conditions and nutritional state.

Atorvastatin alters the expression of genes related to bile acid metabolism and circadian clock in livers of mice

AimAtorvastatin is a HMG-CoA reductase inhibitor used for hyperlipidemia. Atorvastatin is generally safe but may induce cholestasis. The present study aimed to examine the effects of atorvastatin on hepatic gene expression related to bile acid metabolism and homeostasis, as well as the expression of circadian clock genes in livers of mice.MethodsAdult male mice were given atorvastatin (10, 30, and 100 mg/kg, po) daily for 30 days, and blood biochemistry, histopathology, and gene expression were examined.ResultsRepeated administration of atorvastatin did not affect animal body weight gain or liver weights. Serum enzyme activities were in the normal range. Histologically, the high dose of atorvastatin produced scattered swollen hepatocytes, foci of feathery-like degeneration, together with increased expression of Egr-1 and metallothionein-1. Atorvastatin increased the expression of Cyp7a1 in the liver, along with FXR and SHP. In contract, atorvastatin decreased the expression of bile acid transporters Ntcp, Bsep, Ostα, and Ostβ. The most dramatic change was the 30-fold induction of Cyp7a1. Because Cyp7a1 is a circadian clock-controlled gene, we further examined the effect of atorvastatin on clock gene expression. Atorvastatin increased the expression of clock core master genes Bmal1 and Npas2, decreased the expression of clock feedback genes Per2, Per3, and the clock targeted genes Dbp and Tef, whereas it had no effect on Cry1 and Nr1d1 expression.ConclusionRepeated administration of atorvastatin affects bile acid metabolism and markedly increases the expression of the bile acid synthesis rate-limiting enzyme gene Cyp7a1, together with alterations in the expression of circadian clock genes.

Achilles is a circadian clock-controlled gene that regulates immune function in Drosophila

The circadian clock is a transcriptional/translational feedback loop that drives the rhythmic expression of downstream mRNAs. Termed “clock-controlled genes,” these molecular outputs of the circadian clock orchestrate cellular, metabolic, and behavioral rhythms. As part of our on-going work to characterize key upstream regulators of circadian mRNA expression, we have identified a novel clock-controlled gene in Drosophila melanogaster, Achilles (Achl), which is rhythmic at the mRNA level in the brain and which represses expression of antimicrobial peptides in the immune system. Achilles knock-down in neurons dramatically elevates expression of crucial immune response genes, including IM1 (Immune induced molecule 1), Mtk (Metchnikowin), and Drs (Drosomysin). As a result, flies with knocked-down Achilles expression are resistant to bacterial challenges. Meanwhile, no significant change in core clock gene expression and locomotor activity is observed, suggesting that Achilles influences rhythmic mRNA outputs rather than directly regulating the core timekeeping mechanism. Notably, Achilles knock-down in the absence of immune challenge significantly diminishes the fly’s overall lifespan, indicating a behavioral or metabolic cost of constitutively activating this pathway. Together, our data demonstrate that (1) Achilles is a novel clock-controlled gene that (2) regulates the immune system, and (3) participates in signaling from neurons to immunological tissues.

Identification of intellectual disability genes showing circadian clock-dependent expression in the mouse hippocampus

Sleep is strongly implicated in learning, especially in the reprocessing of recently acquired memory. Children with intellectual disability (ID) tend to have sleep-wake disturbances, which may contribute to the pathophysiology of the disease. Given that sleep is partly controlled by the circadian clock, we decided to study the rhythmic expression of genes in the hippocampus, a brain structure which plays a key role in memory in humans and rodents.By investigating the hippocampal transcriptome of adult mice, we identified 663 circadian clock controlled (CCC) genes, which we divided into four categories based on their temporal pattern of expression. In addition to the standard core clock genes, enrichment analysis identified several transcription factors among these hippocampal CCC genes, and our findings suggest that genes from one cluster regulate the expression of those in another. Interestingly, these hippocampal CCC genes were highly enriched in sleep/wakefulness-related genes. We show here that several genes in the glucocorticoid signaling pathway, which is involved in memory, show a CCC pattern of expression. However, ID genes were not enriched among these CCC genes, suggesting that sleep or learning and memory disturbances observed in patients with ID are probably not related to the circadian clock in the hippocampus.

A day and night difference in the response of the hepatic transcriptome to cyclophosphamide treatment.

Application of omics-based technologies is a widely used approach in research aiming to improve testing strategies for human health risk assessment. In most of these studies, however, temporal variations in gene expression caused by the circadian clock are a commonly neglected pitfall. In the present study, we investigated the impact of the circadian clock on the response of the hepatic transcriptome after exposure of mice to the chemotherapeutic agent cyclophosphamide (CP). Analysis of the data without considering clock progression revealed common responses in terms of regulated pathways between light and dark phase exposure, including DNA damage, oxidative stress, and a general immune response. The overall response, however, was stronger in mice exposed during the day. Use of time-matched controls, thereby eliminating non-CP-responsive circadian clock-controlled genes, showed that this difference in response was actually even more pronounced: CP-related responses were only identified in mice exposed during the day. Only minor differences were found in acute toxicity pathways, namely lymphocyte counts and kidney weights, indicating that gene expression is subject to time of day effects. This study is the first to highlight the impact of the circadian clock on the identification of toxic responses by omics approaches.

Molecular markers for the E2 and E3 genes controlling flowering and maturity in soybean

Natural variation in flowering time may play a role in the adaptation of plants to various environments, and understanding the genetic basis of flowering and maturity would facilitate the development of early maturing cultivars. Molecular markers for the E2 and E3 loci, which control the time of flowering and maturity in soybean (Glycinemax), were developed in this study. Single nucleotide-amplified polymorphism (SNAP) markers were developed from the nonsense mutation in E2 (GmGIa), which is a circadian clock-controlled gene. The E2- and e2-specific SNAP markers were validated using six E2 isolines. The soybean E3 gene is a photoreceptor phytochrome A (GmPhyA3) gene, and a co-dominant marker was designed based on sequence deletions within the E3 allele. A multiplex PCR assay using three primers for the E3 gene allowed allelic discrimination based on the sizes of PCR products. Furthermore, this E3 marker successfully detected two alleles in a single reaction when two types of DNA were pooled. These markers determined the genotypes of our mapping population previously reported to detect flowering quantitative trait loci close to the E2 and E3 loci, confirming that the mutations are responsible for the early flowering phenotype. The use of SNAP markers for E2 and a co-dominant marker for E3 is a simple, fast, and reproducible method, requiring only PCR and agarose gel electrophoresis. The molecular resources developed in this study could accelerate marker-assisted selection and cultivar development for short-season areas in a soybean breeding program.

Tef polymorphism is associated with sleep disturbances in patients with Parkinson’s disease

Circadian mechanisms play an important role in the regulation of sleep. A circadian clock-controlled gene, Tef, has been suggested to be associated with depressive symptoms, restless legs syndrome, and slow wave sleep in patients with sleep disorders. The present study sought to explore the association between Tef and sleep disturbances in patients with Parkinson's disease (PD). Three hundred and ninety-two unrelated patients with PD were recruited for this study. All of them completed the PD Sleep Scale (PDSS) and other clinical and demographic assessments. rs738499, a single nucleotide polymorphism of the Tef gene, was genotyped by polymerase chain reaction-restriction fragment length polymorphism. Mean total PDSS scores were 111.5 (standard deviation [SD] 23.0) in the TT genotype and 122.2 (SD 18.2) in the TG+GG genotypes (P<0.01). Significant differences were found between genotypes (TT vs TG+GG) for 14 item scores (all P<0.05). Total and item scores displayed negative associations with the TT genotype (all P<0.05) except Item 2 (P=0.178). Linear regression adjusted for gender, duration, depression and disease severity showed that the polymorphism could explain 0.9% of the variance in PDSS scores. These preliminary results suggest that the TT genotype in Tef rs738499 is associated with sleep disturbances in PD. Depression and disease severity are the main contributors to these findings, but rs738499 itself is an independent risk factor.

Abnormal expressions of circadian-clock and circadian clock-controlled genes in the livers and kidneys of long-term, high-fat-diet-treated mice

Physiological and behavioral circadian rhythmicities are exhibited by all mammals and are generated by intracellular levels of circadian oscillators, which are composed of transcriptional/translational feedback loops involving a set of circadian-clock genes, such as Clock, Per1-3, Cry1-2, Bmal1, Dbp, E4BP4 and CK1varepsilon. These circadian-clock genes play important roles in regulating circadian rhythms and also energy homeostasis and metabolism. Determining whether obesity induced by high-fat diet affected the expressions of circadian-clock genes and their related genes in peripheral tissues, was the main focus of this study. To address this issue, we fed male C57BL/6 mice a high-fat diet for 11 months to induce obesity, hyperglycemic, hypercholesterolemic and hyperinsulinemic symptoms, and used quantitative real-time reverse transcription-PCR to measure gene expression levels. We found that the expressions of circadian-clock genes and circadian clock-controlled genes, including Per1-3, Cry1-2, Bmal1, Dbp, E4BP4, CK1varepsilon, PEPCK, PDK4 and NHE3, were altered in the livers and/or kidneys. These results indicate that obesity induced by high-fat diet alters the circadian-clock system, and obesity and metabolic syndrome are highly correlated with the expressions of circadian-clock genes and their downstream, circadian clock-controlled genes.

Implication of the F-Box Protein FBXL21 in Circadian Pacemaker Function in Mammals

In mammals, the circadian clock relies on interlocked feedback loops involving clock genes and their protein products. Post-translational modifications control intracellular trafficking, functionality and degradation of clock proteins and are keys to the functioning of the clock as recently exemplified for the F-Box protein Fbxl3. The SCFFbxl3 complex directs degradation of CRY1/2 proteins and Fbxl3 murine mutants have a slower clock. To assess whether the role of Fbxl3 is phylogenetically conserved, we investigated its function in the sheep, a diurnal ungulate. Our data show that Fbxl3 function is conserved and further reveal that its closest homologue, the F-Box protein Fbxl21, also binds to CRY1 which impairs its repressive action towards the transcriptional activators CLOCK/BMAL1. However, while Fbxl3 appears to be ubiquitously expressed, Fbxl21 expression is tissue-specific. Furthermore, and in sharp contrast with Fbxl3, Fbxl21 is highly expressed within the suprachiasmatic nuclei, site of the master clock, where it displays marked circadian oscillations apparently driven by members of the PAR-bZIP family. Finally, for both Fbxl3 and Fbxl21 we identified and functionally characterized novel splice-variants, which might reduce CRY1 proteasomal degradation dependent on cell context. Altogether, these data establish Fbxl21 as a novel circadian clock-controlled gene that plays a specific role within the mammalian circadian pacemaker.

Circadian dynamics of vasopressin in mouse selection lines: Translation and release in the SCN

Arg 8-vasopressin (AVP), a circadian clock-controlled gene product, is released from the hypothalamic suprachiasmatic nuclei (SCN) in mice in a circadian fashion. Previously reported differences in two mouse lines, initially selected for thermoregulatory nest-building behavior (building small nests (S-mice) or big nests (B-mice)) with different circadian organization of behavior and in number of SCN-AVP immunoreactive neurons, were further investigated. We confirmed and expanded the finding that S-mice exhibited constant high levels of SCN-AVP content with no apparent circadian rhythmicity, whereas B-mice had lower numbers of AVP positive cells which varied with time of day. We found that AVP mRNA expression levels at midnight and midday were similar in both lines, as established by in situ hybridization. When AVP transport and release were blocked by colchicine, SCN-AVP immunoreactivity was similar in both lines. This suggests that differences in SCN-AVP content depend on transport or release. Organotypic SCN cultures of B-mice showed more AVP release per neuron than cultures of S-mice. These results reveal that on a mechanistic level the mouse lines differed in transport and/or release of AVP in the SCN, rather than differential regulation of AVP gene transcription or number of AVP immunoreactive neurons.

Light regulation of circadian clock-controlled gene expression in rice.

Using transgenic rice seedlings expressing a firefly luciferase (luc) gene under the control of a rice CAB (chlorophyll a/b binding protein) promoter, we demonstrated how light affects circadian clocks in the transcription of the CAB gene. Rhythmic luc expression was observed for more than 5 d under constant light and dark (DD) conditions after light/dark entrainment. After a light pulse was applied at different time points in DD various temporal patterns of CAB gene expression were individually observed. We first examined two distinct properties related to the entrainment mechanism of the circadian clock: fluence-rate dependence of free-running periods (FRPs) and phase resetting by a light pulse. Although fluence-rate dependent shortening of FRP was demonstrated, the FRP in DD was almost equal to that in constant light of a middle fluence-rate, indicating that this fluence-rate dependence may not fully describe the entrainment of the circadian clock in rice. Typical phase responses of the circadian clock by a single light pulse were also observed at the transcriptional level in rice seedlings. Thus, the phase resettings upon the light/dark transitions of daily cycles may be sufficient to explain the entrainment mechanisms of rice. We have further demonstrated that, in addition to having a gating effect to acute response, a light pulse can activate the circadian clock-controlled CAB1R gene expression at the first circadian peak in a phase-dependent manner. This suggests that light activates circadian clock activity in the diurnal CAB gene expression under daily light/dark cycles.