InteractiveFly: GeneBrief

cycle: Biological Overview | References

Gene name - cycle

Synonyms -

Cytological map position-76C6-76C6

Function - transcription factor

Keywords - photoperiod response

Symbol - cyc

FlyBase ID: FBgn0023094

Genetic map position -3L: 19,806,892..19,809,038 [-]

Classification - Helix-loop-helix domain, PAS domain

Cellular location - nuclear

NCBI link: EntrezGene

cyc orthologs: Biolitmine
Recent literature
Qiao, B., Li, C., Allen, V. W., Shirasu-Hiza, M. and Syed, S. (2018). Automated analysis of long-term grooming behavior in Drosophila using a k-nearest neighbors classifier. Elife 7. PubMed ID: 29485401
Despite being pervasive, the control of programmed grooming is poorly understood. This study addressed this gap by developing a high-throughput platform that allows long-term detection of grooming in Drosophila melanogaster. In this method, a k-nearest neighbors algorithm automatically classifies fly behavior and finds grooming events with over 90% accuracy in diverse genotypes. The data show that flies spend ~13% of their waking time grooming, driven largely by two major internal programs. One of these programs regulates the timing of grooming and involves the core circadian clock components cycle, clock, and period. The second program regulates the duration of grooming and, while dependent on cycle and clock, appears to be independent of period. This emerging dual control model in which one program controls timing and another controls duration, resembles the two-process regulatory model of sleep. Together, this quantitative approach presents the opportunity for further dissection of mechanisms controlling long-term grooming in Drosophila.
Stone, R. A., McGlinn, A. M., Chakraborty, R., Lee, D. C., Yang, V., Elmasri, A., Landis, E., Shaffer, J., Iuvone, P. M., Zheng, X., Sehgal, A. and Pardue, M. T. (2019). Altered ocular parameters from circadian clock gene disruptions. PLoS One 14(6): e0217111. PubMed ID: 31211778
The pathophysiology of refractive errors is poorly understood. Myopia (nearsightedness) in particular both blurs vision and predisposes the eye to many blinding diseases during adulthood. Based on past findings of diurnal variations in the dimensions of the eyes of humans and other vertebrates, altered diurnal rhythms of these ocular dimensions with experimentally induced myopia, and evolving evidence that ambient light exposures influence refractive development, this study assessed whether disturbances in circadian signals might alter the refractive development of the eye. In mice, retinal-specific knockout of the clock gene Bmal1 induces myopia and elongates the vitreous chamber, the optical compartment separating the lens and the retina. These alterations simulate common ocular findings in clinical myopia. In Drosophila melanogaster, knockouts of the clock genes cycle or period lengthen the pseudocone, the optical component of the ommatidium that separates the facet lens from the photoreceptors. Disrupting circadian signaling thus alters optical development of the eye in widely separated species. It is proposed that mechanisms of myopia include circadian dysregulation, a frequent occurrence in modern societies where myopia also is both highly prevalent and increasing at alarming rates. Addressing circadian dysregulation may improve understanding of the pathogenesis of refractive errors and introduce novel therapeutic approaches to ameliorate myopia development in children.
Klemz, S., Wallach, T., Korge, S., Rosing, M., Klemz, R., Maier, B., Fiorenza, N. C., Kaymak, I., Fritzsche, A. K., Herzog, E. D., Stanewsky, R. and Kramer, A. (2021). Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity. Genes Dev 35(15-16): 1161-1174. PubMed ID: 34301769
In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, a systematic RNAi screen was performed in human cells and protein phosphatase 4 (PPP4) was identified with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila. Genetic depletion of PPP4 (Pp4-19C in Drosophila) shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 (Cycle in Drosophila) and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the "kamikaze" properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1Δ-mediated inhibition of CLOCK/BMAL1.
Lin, Z., Green, E. W., Webster, S. G., Hastings, M. H., Wilcockson, D. C., Kyriacou, C. P. (2023). The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach). PLoS Genet, 19(10):e1011011 PubMed ID: 37856540
Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 (see Drosophila Cryptochrome) and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal (~12.4 hour) or other lunar-mediated cycles (~15 day, ~29 day), widely expressed in coastal organisms, is unknown. Pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. This study shows that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between these two positive circadian regulators and circatidal behaviour. DsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2 was performed in animals taken from the wild. Epcry2 and Epbmal1 knockdown disrupted Eurydice's circadian phenotypes of chromatophore dispersion, tim mRNA cycling and the circadian modulation of circatidal swimming, as expected. However, circatidal behaviour was particularly sensitive to Epbmal1 knockdown with consistent effects on the power, amplitude and rhythmicity of the circatidal swimming cycle. Thus, three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM (from a previous study), do not appear to be required for the expression of robust circatidal behaviour, in contrast to the positive regulator EpBMAL1. A neurogenetic model is suggested whereby the positive circadian regulators EpBMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator.

A searched for novel Drosophila circadian rhythm genes was carried out by chemically mutagenizing flies and screening for altered or aberrant locomotor activity rhythms. A recessive arrhythmic mutant fly line was identified, which has been named cycle (cyc0). These flies show arrhythmic locomotor activity patterns when they carry two mutant third chromosomes. cyc0/cyc0 flies also manifest arrhythmic eclosion. Cyc is a potential dimerization partner for Clock. The homozygous mutant flies are always arrhythmic, regardless of the period genetic background. Unlike the semidominant Clk mutant, heterozygous cyc0/+ flies manifest robust rhythms with no hint of arrhythmicity. But they have altered periods, with rhythms 1 hr longer than their per genotype would otherwise dictate. This indicates that either cyc has a dominant phenotype or that the locus has dosage sensitive effects on period. The data suggest that cyc might identify a circadian clock component (Rutila, 1998).

Homozygous mutant cyc0/cyc0 flies also have difficulties under 12:12 light/dark (LD) conditions, since only 64% of them give discernable rhythms by chi2 analysis. Greater than 90% of arrhythmic per0 and tim0 flies are light responsive and therefore show robust 24 hr rhythms under these conditions. Since the cyc0/cyc0 flies show no visual difficulties in optomotor behavior, the data suggest that the flies are specifically impaired in circadian light perception. A similar phenotype is seen with Clk mutant flies (Rutila, 1998).

To examine clock function more directly, the fluctuations of the clock proteins Period and Timeless were examined in wild-type, heterozygous, and homozygous cyc flies under LD conditions. Western analysis with an anti-Per antibody reveals very little protein in cyc0/cyc0 fly heads at any time of day. As predicted from the robust rhythms, cyc0/+ heterozygotes show normal Per cycling, with normal levels and a normal temporal phosphorylation program. For all genotypes, similar results were obtained for Tim. The low Per and Tim levels could be due to reduced protein stability or to reduced protein synthesis in the homozygous mutant strain. To distinguish between these possibilities, per and tim mRNA levels were measured. Low RNA levels and little or no cycling are found in the cyc0/cyc0 head extracts, suggesting reduced synthesis rather than reduced stability. As expected, RNA levels and cycling in cyc0/+ heterozygotes are indistinguishable from those in wild-type extracts (Rutila, 1998).

The cyc effect on per and tim RNA levels and cycling could be transcriptional or posttranscriptional. To directly measure transcription rates, nuclear run-on assays were performed in homozygous cyc flies. In this genotype, per and tim transcription rates show no evidence of cycling and are approximately equal to the very low trough levels of wild-type flies observed at ZT1. The result is essentially identical to that observed in homozygous Clk flies (Allada, 1998). It is interesting to note that the transcription rates in both genotypes are much lower than those observed in either per0 or tim0 head extracts, which also manifest little or no transcriptional oscillations. This suggests that cyc as well as Clk are epistatic to and upstream of per0 and tim0. Taken together, the data suggest that cyc, like Clk, affects the transcription of the clock genes per and tim (Rutila, 1998).

To identify specific sequence elements mediating the mutant effects on transcription, the effects of the cyc mutation were examined on a minimal per promoter element. This 69 bp enhancer contains a critical per-derived E box and drives rhythmic expression of a reporter gene (lacZ). To this end, the E box/lacZ construct was crossed into cyc0/cyc0 mutant flies and lacZ RNA levels were assayed for cycling by RNase protection. The results are dramatic and indicate that there is little or no cycling lacZ RNA transcription in the homozygous mutant flies, suggesting that Cyc affects the transcriptional activity of the per circadian transcriptional enhancer. These features of Cyc are similar to those of Clk, which probably binds to and activates transcription at the per E box (Rutila, 1998).

The mammalian protein BMAL1 (see Clock Evolutionary homologs section for information on the Cycle homolog BMAL1) was cloned as an "orphan" protein of the bHLH-PAS transcription factor family with no known biological function (Ikeda, 1997 and Hogenesch, 1997). However, there are recent biochemical experiments indicating that it may play a role in circadian rhythm-relevant transcription in mammals: it can act as a heterodimeric partner of mouse Clock (mClock) in DNA binding and transcriptional activation. The BMAL1:mClock heterodimer selects a DNA-binding sequence that resembles the critical E box sequence within the cycling element in the Drosophila per upstream region, and there are features of this enhancer in addition to the central CACGTG hexamer that provide specificity for the BMAL1:mCLOCK heterodimer. As transcripts from mouse per genes undergo circadian oscillations in level, these genes may contain a similar target cycling element to that of Drosophila per. The BMAL1:mCLOCK heterodimer could be the heterodimeric factor that binds to this cycling element and activates clock-relevant transcription (Hogenesch, 1998 and Rutila, 1998 and references).

By analogy, it is proposed that Cyc and Clk heterodimerize, bind to Drosophila clock gene E boxes, and function to drive the circadian-regulated transcription of these genes. This makes cyc and Clk the first Drosophila circadian rhythm genes with a known biochemical role and a defined place in the clock circuit; it also places them upstream of per and tim. RNA and transcription experiments in cyc0 and in Clk mutant flies (Allada, 1998) fully support such an assignment (Rutila, 1998).

Cyc is approximately half as big as Clk; the difference appears to be largely the extensive glutamine-rich C-terminal half of Clk (Allada, 1998). This may indicate that Clk brings the transcriptional activation domain to the complex. The dominant phenotype of the Clk mutant and the elimination of the Q-rich region by the mutation (Allada, 1998) are consistent with this notion. The mutant protein would then be able to dimerize with Cyc and bind DNA but would be unable to activate transcription. This would explain its recessive as well as its dominant features (Rutila, 1998).

Clock genes: Stress response genes protect against lethal effects of sleep deprivation in Drosophila

Sleep is controlled by two processes: a homeostatic drive that increases during waking and dissipates during sleep, and a circadian pacemaker that controls its timing. Although these two systems can operate independently, recent studies indicate a more intimate relationship. To study the interaction between homeostatic and circadian processes in Drosophila, homeostasis was examined in the canonical loss-of-function clock mutants period (per01), timeless (tim01), clock (Clkjrk) and cycle (cyc01). cyc01 mutants show a disproportionately large sleep rebound and die after 10 hours of sleep deprivation, although they are more resistant than other clock mutants to various stressors. Unlike other clock mutants, cyc01 flies show a reduced expression of heat-shock genes after sleep loss. However, activating heat-shock genes before sleep deprivation rescues cyc01 flies from its lethal effects. Consistent with the protective effect of heat-shock genes, is the observation that flies carrying a mutation for the heat-shock protein Hsp83 (Hsp8308445) show exaggerated homeostatic response and die after sleep deprivation. These data represent the first step in identifying the molecular mechanisms that constitute the sleep homeostat (Shaw, 2002).

A sleep-like state has been described in Drosophila on the basis of its similarities to mammalian sleep. This state is characterized by increased arousal thresholds and is regulated homeostatically. Like mammalian sleep, it is abundant in young flies, decreases in older animals and is modulated by stimulants and hypnotics. Perhaps the most important similarity between mammals and flies is homeostatic regulation: when flies are kept awake, they show a large compensatory increase in sleep the next day (Shaw, 2002).

In mammals, the circadian pacemaker alternately promotes and maintains both wakefulness and sleep. Although the circadian pacemaker and the sleep homeostat can interact, little is known about the underlying mechanisms. To evaluate this relationship, homeostasis was evaluated in clock mutants maintained in constant darkness (DD) and deprived of sleep for 3, 6, 9 and 12 h. Under these conditions, sleep is evenly distributed across the day. Upon release from sleep deprivation, wild-type Canton-S flies recover ~30%-40% of the sleep that they lost within 12 h. per01 and Clkjrk show a more prominent sleep rebound, reclaiming ~100% of lost sleep within 12 h. tim01 flies did not show a homeostatic response after 3–6 h of sleep deprivation but displayed a sleep rebound similar to that of per01 and Clkjrk flies after 7, 9 and 12 h of sleep deprivation (Shaw, 2002).

Surprisingly, cyc01 mutants showed an exaggerated response to 3 h of sleep deprivation, reclaiming ~3 min of sleep over baseline for each minute of sleep lost. Further increasing sleep debt produces a change in the regulation of sleep not seen in other clock mutants: cyc01 flies showed large increases in sleep that persisted for as long as the flies were recorded (up to 16 days). These periods of quiescence are associated with increased arousal thresholds, indicating that the deprivation has produced an increase in the amount of sleep and does not result in an injured fly. If sleep deprivation produces an increased need for sleep in cyc01 flies, they should show higher amounts of sleep when sleep deprived for a second time. Indeed, deprivations of an additional 6 h results in further increases in sleep (Shaw, 2002).

The extreme sensitivity of cyc01 flies is revealed when sleep deprivation extends past 10 h: the flies begin to die. This effect was not observed in wild-type flies, in mutant lines representing 45 other genetic loci, or in other clock mutants, indicating that the mutations do not in themselves increase vulnerability to sleep deprivation. Note that the most sensitive of the clock mutants (cyc01) is the only one that does not cycle (Shaw, 2002).

To determine whether death in the cyc01 flies was due to the stimuli used for sleep deprivation, flies were deprived of sleep for 30 min each hour for 24 h, ensuring that the flies received the same number of stimuli that accrued during 12 h of sleep deprivation but without producing 12 h of continuous wakefulness. No deaths were observed after this protocol, indicating that the deprivation stimulus was not responsible for the deaths. Further supporting this conclusion, stress-sensitive B (sesB1) flies, which are extremely sensitive to mechanical shock, survived 12 h of sleep deprivation and showed activity patterns during the deprivation that were similar to those of wild-type flies. cyc01 flies were also deprived of sleep by gentle handling. The proportion of flies that succumbed to sleep deprivation and the size of the homeostatic response in surviving flies were indistinguishable from the automated deprivation method. Similar results were obtained with a rotating deprivation apparatus (Shaw, 2002).

To determine whether death in cyc01 flies is due specifically to sleep deprivation or to hypersensitivity to any environmental challenge, per01, tim01, Clkjrk, cyc01 and Canton-S flies were subjected to several stressors including heat stress, oxidative stress, starvation, desiccation and physical stress. cyc01 flies were as sensitive, but no more so than other genotypes to desiccation and vortex-mixing and survived longer than per01, tim01 and Clkjrk flies when challenged with heat, oxidative stress and starvation. Canton-S flies, which have an intact clock, were more resistant to starvation and desiccation than tim01, Clkjrk and cyc01 flies. These data indicate that cyc01 mutants are vulnerable to prolonged wakefulness in itself and are not merely hypersensitive to non-related stressors (Shaw, 2002).

To confirm that this phenotype maps to the cyc locus, cyc01 homozygotes were crossed with flies carrying the appropriate deficiency Df(3L)kto2/TM6B, Tb1. The resulting cyc01/Df transheterozygote flies showed an exaggerated homeostatic response and deaths after 12 h of sleep deprivation. Furthermore, cyc01 heterozygotes with and without a functioning clock (cyc01/+ and per0w;cyc01/+) also showed exaggerated homeostasis (Shaw, 2002).

Prolonged sleep deprivation (2–4 weeks) is invariably fatal in normal rats. Is the rapid demise after a few hours of sleep deprivation the result of an anomalous reaction in cyc01 mutants, or is it an increased susceptibility to the lethal consequences of sleep loss? Individual Canton-S flies were kept awake for 70 h by tapping on their tubes when they stopped moving to ensure that lethality was not due to excessive handling; 2 of 12 Canton-S flies died after ~60 h of continuous wakefulness, whereas 2 more died by ~67–70 h. The behavior of the flies during the last hours of the sleep deprivation protocol resembled that seen in cyc01 flies, indicating that the deaths were due to sleep loss and not to the deprivation stimulus itself. To test this, an additional group of Canton-S flies was kept awake by using a different deprivation method and again it was found that flies began to die between 60 and 70 h. These data indicate that sleep does indeed serve a vital biological role in the fly and that specific mutations that increase susceptibility to death might help to clarify such a role (Shaw, 2002).

Given that cyc01 flies are equally well or better equipped than other clock mutants to tolerate chronic heat and other stressors, why do they die in response to sleep deprivation? There is much evidence that stress response genes can protect an organism during challenging conditions. The ability of heat and sleep deprivation to activate stress response genes was examined, by using real-time quantitative polymerase chain reaction (QPCR). All clock mutants respond to 3 h of heat with an induction of genes such as hsp70, Hsp83, droj1 and hsc70-3 coding for chaperone proteins. After 3 h of sleep deprivation, the levels of these genes were near baseline in Clkjrk flies (with the exception of hsp70) and were unchanged or mildly increased in per01, tim01 and Canton-S flies. Interestingly, levels of chaperone proteins are also elevated after sleep deprivation in rodents. However, sleep deprivation produces a decrease in the expression of these genes in cyc01 flies. Genes activated by qualitatively different stressors, including metabolic stress (SNF4a, Hif1), chemical stress (mpk2) and humoral stress (turandot), were reduced in all lines, indicating that sleep deprivation is not inherently stressful (Shaw, 2002).

To evaluate the relationship between heat-shock genes and sleep deprivation in cyc01 flies, heat-shock genes were induced before sleep deprivation. When 12 h of sleep deprivation was preceded by 3 h of heat exposure at 36°C, the mortality rate was reduced compared to unheated cyc01 flies (note that one would have predicted increased mortality because preheating results in a further 3 h of wakefulness). Moreover, heat exposure reduces homeostatic drive in cyc01 and Clkjrk flies). When cyc01 flies were pre-exposed to 37°C, homeostasis was reduced further (Shaw, 2002).

The importance of heat-shock genes in sleep deprivation was also shown by examining flies mutant for Hsp83 (Hsp8308445). After 12 h of sleep deprivation, Hsp8308445 mutants exhibit a mortality rate similar to that of cyc01 and show a homeostatic response corresponding to fivefold that of wild-type flies. The sensitivity to sleep deprivation in Hsp8308445 mutants is present even in heterozygous flies, which have only a modest reduction in gene product. Heterozygous Hsp8308445 flies display a sleep rebound that is not statistically different from either homozygous Hsp8308445 or heterozygous Hsp83e6A flies. However, both Hsp83 heterozygotes exhibit a sleep rebound that is significantly different from that of Hsp60RA75 heterozygotes, indicating that a limited set of chaperone proteins are involved in homeostasis. Finally, whereas preheating cyc01 flies prevents the lethal effects of sleep deprivation, this does not occur in Hsp8308445 flies. It should be noted that sleep homeostasis has been evaluated in mutant lines representing 45 other genetic loci; cyc01 and Hsp8308445 flies are the only mutants that show both an exaggerated homeostatic response and death after sleep deprivation. Although it is unlikely that these are the only two genes involved in the sleep homeostat, it is worth noting that their mammalian homologs have been shown to interact physically. Nevertheless, it is possible that the increased homeostatic response and lethality that is observed in cyc01 mutants is due to factors other than Hsp83 (Shaw, 2002).

Although it is believed that sleep is an essential biological process, its function remains a mystery. So far, death after chronic total sleep deprivation in the rat provides the best evidence in support of a vital role for sleep. The data reinforce these findings and indicate that the vital role of sleep extends beyond mammals; the data also indicate a connection between vulnerability to sleep loss and increased homeostatic drive. Most importantly, the observation that the induction of certain chaperone proteins protects against the lethal effects of sleep loss provides a first hint about the functional targets of sleep and its molecular mechanisms (Shaw, 2002).

Identification of genes associated with resilience/vulnerability to sleep deprivation and starvation in Drosophila

Flies mutant for the canonical clock protein cycle (cyc01) exhibit a sleep rebound that is approximately 10 times larger than wild-type flies and die after only 10 h of sleep deprivation. Surprisingly, when starved, cyc01 mutants can remain awake for 28 h without demonstrating negative outcomes. Thus, it was hypothesized that identifying transcripts that are differentially regulated between waking induced by sleep deprivation and waking induced by starvation would identify genes that underlie the deleterious effects of sleep deprivation and/or protect flies from the negative consequences of waking. Partial complementary DNA microarrays to identify transcripts that are differentially expressed between cyc01 mutants that had been sleep deprived or starved for 7 h. Genetics was used to determine whether disrupting genes involved in lipid metabolism would exhibit alterations in their response to sleep deprivation. 84 genes were identified with transcript levels that were differentially modulated by 7 h of sleep deprivation and starvation in cyc01 mutants and were confirmed in independent samples using quantitative polymerase chain reaction. Several of these genes were predicted to be lipid metabolism genes, including bubblegum, cueball, and CG4500, which based on data obtained in this study was renamed heimdall (hll). Using lipidomics it was confirmed that knockdown of hll using RNA interference significantly decreased lipid stores. Importantly, genetically modifying bubblegum, cueball, or hll resulted in sleep rebound alterations following sleep deprivation compared to genetic background controls. This study has identified a set of genes that may confer resilience/vulnerability to sleep deprivation and demonstrate that genes involved in lipid metabolism modulate sleep homeostasis (Thimgan, 2014).

Gender dimorphism in the role of cycle (BMAL1) in rest, rest regulation, and longevity in Drosophila melanogaster

The central clock is generally thought to provide timing information for rest/activity but not to otherwise participate in regulation of these states. To test the hypothesis that genes that are components of the molecular clock also regulate rest, the authors quantified the duration and intensity of consolidated rest and activity for the four viable Drosophila mutations of the central clock that led to arrhythmic locomotor behavior and for the pdf mutant that lacked pigment-dispersing factor, an output neuropeptide. Only the cycle (cyc01) and Clock (ClkJrk) mutants had abnormalities that mapped to the mutant locus, namely, decreased consolidated rest and grossly extended periods of activity. All mutants with the exception of the cyc01 fly exhibited a qualitatively normal compensatory rebound after rest deprivation. This abnormal response in cyc01 was sexually dimorphic, being reduced or absent in males and exaggerated in females. Finally, the cyc01 mutation shortened the life span of male flies. These data indicate that cycle regulates rest and life span in male Drosophila (Hendricks, 2003).

PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription

Transcriptional activation by Clock-Cycle (Clk-Cyc) heterodimers and repression by Period-Timeless (Per-Tim) heterodimers are essential for circadian oscillator function in Drosophila (see reviews by Hardin, The Circadian Timekeeping System of Drosophila and Vallone, Start the clock! Circadian rhythms and development). Per-Tim has been found to interact with Clk-Cyc to repress transcription, and this interaction is shown to inhibit binding of Clk-Cyc to E-box regulatory elements in vivo. Coincident with the interaction between Per-Tim and Clk-Cyc is the hyperphosphorylation of Clk. This hyperphosphorylation occurs in parallel with the Per-dependent entry of Double-time (Dbt) kinase into a complex with Clk-Cyc, where Dbt destabilizes both Clk and Per. Once Per and Clk are degraded, a novel hypophosphorylated form of Clk accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that Per-dependent rhythms in Clk phosphorylation control rhythms in E-box-dependent transcription and Clk stability, thus linking Per and Clk function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals (Yu, 2006).

ChIP studies demonstrate that Clk-Cyc is only bound to E-boxes when target genes are being actively transcribed (see Clock 'Targets of Activity' for Cycle transcriptional targets). Since Per-Dbt/Per-Tim-Dbt complexes interact with Clk-Cyc to inhibit transcription, these data imply that these Per-containing complexes inhibit transcription by removing Clk-Cyc from E-boxes. It is also possible that binding of these Per-containing complexes to Clk-Cyc effectively blocks Clk and Cyc antibody access, in which case Per complexes would inhibit transcription while Clk-Cyc is bound to E-boxes. Given that the polyclonal Clk and Cyc antibodies used in this study were raised against full-length proteins and have been used to immunoprecipitate Per-containing complexes (Lee, 1998), it is highly unlikely that all Clk and Cyc epitopes are fully blocked by Per complex binding. Thus, it is concluded that Clk-Cyc rhythmically binds E-boxes in concert with target gene activation (Yu, 2006).

Per complex binding could remove Clk-Cyc from E-boxes by directly altering their conformation or by promoting Clk phosphorylation. The region of Per that inhibits Clk-Cyc transcription, called the Clk-Cyc inhibitory domain or CCID, is near the C terminus. The CCID can act independently of the N terminus of Per, where the Dbt-binding domain resides. This observation argues that Per does not inhibit Clk-Cyc binding to E-boxes by promoting Dbt-dependent Clk phosphorylation. Dbt- and CK2-dependent phosphorylation nevertheless enhances transcriptional repression in S2 cells by potentiating Per inhibition or by inhibiting Clk activity directly. Unfortunately, these disparate results from S2 cells do not allow distinguishing between the different effects of Per complex binding to inhibit transcription outlined above (Yu, 2006).

In mammals, mCry complexes bind to CLOCK-BMAL1 and repress transcription without removing CLOCK-BMAL1 from E-boxes. This contrasts with the situation in flies, where Per complexes inhibit transcription by inhibiting Clk-Cyc E-box binding, and suggests that these Per and mCry complexes repress transcription via different mechanisms. Although mCry complexes do not remove CLOCK-BMAL1 from E-boxes, they repress transcription by inhibiting the CLOCK-BMAL1-induced acetylation of histones by blocking p300 histone acetyl transferase function or introducing a histone deacetylase. Even though Per complexes repress transcription by inhibiting Clk-Cyc binding to E-boxes, this does not exclude the possibility that rhythms in histone acetylation are also involved in regulating rhythmic transcription in flies. Since chromatin remodeling is generally accepted as a prerequisite for transcription initiation, it would be surprising if rhythms in transcription were not accompanied by rhythms in histone acetylation or some other form of chromatin remodeling (Yu, 2006).

A rhythm in Clk phosphorylation has been defined in which hyperphosphorylated Clk predominates during times of transcriptional repression and hypophosphorylated Clk predominates during times of transcriptional activation. This rhythm occurs in parallel to the rhythm in Per phosphorylation; hyperphosphorylated Per and Clk accumulate in nuclei during the late night and early morning, then these forms are degraded and hypophosphorylated forms of Per and Clk accumulate in the cytoplasm and nucleus, respectively, during the late day and early evening. The rhythm in Clk and Per phosphorylation are not merely coincidental; the accumulation of hyperphosphorylated Clk is Per dependent. Although Per is not itself a kinase, it is bound by Dbt kinase. Per brings Dbt into the nucleus, where Per-Dbt or Per-Tim-Dbt complexes bind Clk-Cyc to inhibit transcription (Yu, 2006 and references therein).

Since Dbt enters a complex containing Clk-Cyc at times when Clk becomes hyperphosphorylated, Dbt may also act to phosphorylate Clk. However, an in vitro assay for Dbt phosphorylation is not available, thus it iw not known whether Dbt directly phosphorylates Clk. Dbt acts to reduce Clk levels in S2 cells even though Per levels are very low. It is therefore possible that Dbt can act to destabilize Clk in a Per-independent manner, although it is believed this is unlikely to be the case since Clk hyperphosphorylation and complex formation with Dbt are both Per dependent (Yu, 2006).

Clk is phosphorylated to some extent in the absence of Per and is hyperphosphorylated in the absence of functional Dbt, indicating that other kinases act to phosphorylate Clk. The accumulation of hyperphosphorylated Clk in dbtAR/dbtP flies suggests that Dbt triggers Clk degradation subsequent to Clk hyperphosphorylation. A similar situation is seen for Per, where hyperphosphorylated Per accumulates in the absence of functional Dbt, and phosphorylation by CK2 precedes Dbt-dependent phosphorylation and Per destabilization. In addition, rhythmically expressed phosphatases may also contribute to Clk phosphorylation rhythms (Yu, 2006 and references therein).

Rhythms in Clk phosphorylation may function to modulate Clk stability, subcellular localization, and/or activity. Clk levels do not change appreciably throughout the daily cycle despite approximately fivefold higher levels of Clk mRNA at dawn than at dusk. If a less stable hyperphosphorylated form of Clk accumulates when Clk mRNA is high and a more stable hypophosphorylated form of Clk accumulates when Clk mRNA is low, they would tend to equalize total Clk levels over the daily cycle. This possibility is supported by results in ARK flies, which express Clk mRNA in the opposite circadian phase (i.e., Clk mRNA peak at dusk rather than dawn). The overall level of Clk cycles in ARK flies with a peak in (hypophosphorylated) Clk around dusk, consistent with hypophosphorylated Clk being more stable than hyperphosphorylated Clk. This possibility is also supported by Dbt-dependent destabilization of Clk in S2 cells since Dbt associates with Clk as hyperphosphorylated Clk accumulates in wild-type flies. If hypophosphorylated Clk is relatively stable, higher levels of Clk might be expected to accumulate in per01 flies. However, constant low levels of Clk mRNA likely limit Clk accumulation in per01 flies. Clock phosphorylation is coupled to its degradation in cultured mammalian cells, yet degradation of phosphorylated Clock does not lead to a rhythm in Clock abundance even though Clock mRNA levels are constant (Yu, 2006).

Studies in cultured mammalian cells also demonstrate that Clock phosphorylation promotes Clock-BMAL1 nuclear localization, although the significance of this nuclear localization is not clear given that Clock-BMAL1 binding to E-boxes is either constant or more robust during transcriptional repression in vivo. In contrast, Clk is nuclear throughout the daily cycle in flies (Yu, 2006).

The coincidence between Clock phosphorylation and transcriptional repression in mice supports the possibility that phosphorylation inhibits Clock-BMAL1 activity, perhaps by promoting HDAC binding or inhibiting HAT binding. Likewise, hypophosphorylated and hyperphosphorylated Clk accumulate in parallel with target gene activation and repression, respectively, in flies. This relationship suggests that the state of Clk phosphorylation may alter its ability to activate target genes. Given that target gene activation occurs when Clk-Cyc is bound to E-boxes and that E-box binding coincides with the accumulation of hypophosphorylated Clk, it is possible that Clk hyperphosphorylation compromises Clk-Cyc binding to E-boxes and, consequently, target gene transcription is repressed. Precedent for such a regulatory mechanism is seen in the Neurospora clock, where limiting levels of FREQUENCY (FRQ) promote phosphorylation of WHITE COLLAR 1 (WC1) and WHITE COLLAR 2 (WC2), thereby inhibiting WC1-WC2 binding to C-box regulatory elements and repressing transcription. In contrast to FRQ in Neurospora, Per is considerably more abundant than Clk in Drosophila and forms stable complexes with Clk-Cyc. In addition, Per/Per-Tim can release Clk-Cyc from E-boxes in vitro, thus demonstrating that Per/Per-Tim binding is sufficient to release Clk-Cyc from E-boxes independent of Clk phosphorylation. Taken together with the in vitro E-box binding results, the high levels of Per relative to Clk and the formation of stable Per-Tim-Clk-Cyc complexes in flies argue that Per/Per-Tim binding may also be sufficient to inhibit E-box binding by Clk-Cyc in vivo, although they do not rule out a role for Clk hyperphosphorylation in inhibiting Clk-Cyc E-box binding. For instance, Per/Per-Tim binding could function to initially remove Clk-Cyc from E-boxes, and subsequent Clk phosphorylation could maintain Clk-Cyc in a form that is incapable of binding E-boxes (Yu, 2006).

The constant levels and rhythmic phosphorylation of Clk defined in this study are similar to those previously characterized for mammalian Clock. This similarity extends beyond metazoans to fungi, where positive elements of the Neurospora circadian feedback loop; i.e., WC1 and WC2, are also rhythmically phosphorylated. In each of these organisms, phosphorylation of positive factors increases when they interact with their respective negative feedback regulators, and decreases when they activate target gene transcription in the absence of these feedback inhibitors. This remarkable similarity suggests that phosphorylation controls one or more critical aspects of positive element function, and consequently, the rhythm in the positive element phosphorylation has become a conserved feature of circadian feedback loops in eukaryotes (Yu, 2006 and references therein).

Per-dependent regulation of Clk-Cyc binding to E-boxes, Per-dependent formation of Per-Dbt and/or Per-Dbt-Tim complexes with Clk-Cyc, and Per-dependent rhythms in Clk phosphorylation suggest a model for the regulation of rhythmic transcription. During the late day and early evening, hypophosphorylated Clk-Cyc binds E-boxes to activate transcription of per, tim, and other genes within and downstream of the transcriptional feedback loop. Accumulating levels of per mRNA peak during the early evening, but Per accumulation is delayed due to Dbt-dependent (and possibly CK2-dependent) phosphorylation, which destabilizes Per. Per is subsequently stabilized via Tim binding, which inhibits further phosphorylation of Per by Dbt. Phosphorylation of Tim by SGG then promotes the translocation of Tim-Per-Dbt complexes into the nucleus, where they bind (hypophosphorylated) Clk-Cyc and repress transcription by inhibiting E-box binding and promoting Clk hyperphosphorylation and degradation. These transcriptional repression mechanisms are not mutually exclusive; Clk hyperphosphorylation may inhibit E-box binding as well as promote Clk degradation. Dbt is able to enter the nucleus in per01 flies, but does not associate with Clk in the absence of Per. This suggests that Per is required to either bring Dbt into a complex with Clk-Cyc, enable phosphorylation of Clk after Dbt enters the complex, or both. Once the Tim-Per-Dbt-Clk-Cyc complex has formed, hyperphosphorylated Per and Clk levels decline in a coordinated fashion by mid-day. Tim is eliminated prior to hyperphosphorylated Per and Clk via separate light-dependent and light-independent mechanisms. As hyperphosphorylated Clk and Clk mRNA decline during the day, hypophosphorylated Clk accumulates. This hypophosphorylated Clk forms complexes with Cyc and binds E-boxes in the absence of nuclear Tim-Per-Dbt complexes, thus initiating the next cycle of transcription (Yu, 2006 and references therein).

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression

Clock (Clk) is a master transcriptional regulator of the circadian clock in Drosophila. To identify Clk direct target genes and address circadian transcriptional regulation in Drosophila, chromatin immunoprecipitation (ChIP) tiling array assays (ChIP-chip) were performed with a number of circadian proteins. Clk binding cycles on at least 800 sites with maximal binding in the early night. The Clk partner protein Cycle (Cyc) is on most of these sites. The Clk/Cyc heterodimer is joined 4-6 h later by the transcriptional repressor Period (Per), indicating that the majority of Clk targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. Clk has specific targets in different tissues, implying that important Clk partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation (Abruzzi, 2011).

Previous circadian models in Drosophila suggested a transcriptional cascade in which Clk directly controls a limited number of genes, including core clock genes, which then drive the oscillating expression of many different output genes. The results of this study indicate that Clk directly regulates not only the five core clock genes (i.e., pdp1, vri, tim, per, and cwo), but also many output genes, including ~60 additional transcription factors. Some of these are tissue-specific; e.g., lim1 and crp. In addition, Clk direct target gene regulation may impact timekeeping in yet unforeseen ways. For example, Clk, Per, and Cyc bind to three of the four known circadian kinases; i.e., dbt, nmo, and sgg. Although none of these mRNAs have been previously reported to cycle, both dbt and sgg have cycling Pol II, and dbt shows weak oscillations via qRT-PCR. nmo expression is enriched in circadian neurons and has been shown to cycle in l-LNvs. The data, taken together, indicate that this simple ChIP-chip strategy has uncovered important relationships and suggest that the transcriptional regulation of some of these new target genes warrants further investigation (Abruzzi, 2011).

Most of the 1500 Clk direct target genes are also bound by two other circadian transcription factors: Cyc and Per. Because a previous study showed that there is a progressive, ordered recruitment of Clk, Pol II, and Per on per and tim (Menet, 2010), the same basic mechanism is conserved on most Clk direct targets. Clk binding increases in late morning and gives rise to an increase in Pol II signal where detectable (ZT6-ZT10). Clk binding is maximal in the early night (ZT14), and both Clk binding and Pol II occupancy decrease rapidly after the repressor Per is bound to chromatin 4-6 h later, at ZT18. Interestingly, Per binds to nearly all Clk direct targets at the identical Clk/Cyc locations, suggesting Per recruitment via protein-protein interactions (Abruzzi, 2011).

The identical binding sites for Clk, Cyc, and Per suggest that binding is not background binding or 'sterile' binding with no functional consequence. This is because three components of the circadian transcription machinery are present with proper temporal regulation. Pol II cycling on ~30% of cycling Clk targets further supports this interpretation. The Pol II signal is maximal from mid- to late morning (ZT6-ZT10), which slightly anticipates the maximal transcription times of core circadian genes like per and tim. Most Pol II signals are promoter-proximal and may reflect poised Pol II complexes often found on genes that respond quickly to environmental stimuli (Abruzzi, 2011).

To address RNA cycling, ten direct target genes with Pol II cycling were examined. Eight of these genes show oscillating mRNA with >1.5-fold amplitude, suggesting that oscillating Pol II indeed reflects cycling transcription. Because this assay may underestimate cycling transcription due to tissue heterogeneity (i.e., masking by noncycling gene expression elsewhere in the head), ~30% is a minimal estimate of Clk direct targets with cyclical mRNA (Abruzzi, 2011).

Interestingly, previous microarray studies did not detect many of these genes. One possibility is that the alternative start sites that characterize 55% of Clk direct targets are not detectable on microarrays; e.g., moe and mnt. However, several mRNAs that cycle robustly by qRT-PCR are not isoform-specific and are also not consistently identified in microarray studies. A second possibility is that the relatively low cycling amplitude of many target genes -- twofold or less, compared with the much greater amplitudes of core clock genes such as tim, per, and pdp1, assayed in parallel -- may be more difficult to detect on microarrays (Abruzzi, 2011).

Low-amplitude cycling may result from relatively stable mRNA, which will dampen the amplitude of cycling transcription. Alternatively, or in addition, low-amplitude cycling may reflect cycling in one head location and noncycling elsewhere within the head, which will dampen head RNA cycling amplitude. This is likely for many eye-specific Clk targets, which appear expressed elsewhere in the head via a Clk-independent mechanism (Abruzzi, 2011).

A third and arguably more interesting explanation for low-amplitude cycling is that Clk binds on promoters with other transcription factors within single tissues. These could include chromatin modifiers and would function together with Clk in a gene- and tissue-specific fashion. For example, a gene could be constitutively expressed at a basal level by one transcription factor, with temporal Clk binding causing a modest boost to transcription. For example, gol is a Clk target exclusively in the eye, and gol mRNA cycles with a fourfold amplitude. Rather than cycling from 'OFF' (no or very low mRNA levels) to 'ON,' however, gol mRNA levels are quite high even at the trough or lowest time points. This suggests that gol cycles from a substantial basal level in the late night and daytime to an even higher level of expression in the evening and early night. Since mRNA levels decrease by >10-fold in GMR-hid flies, trough transcription levels are not likely from other tissues. Therefore, Clk probably acts on gol and other targets not as an 'ON/OFF switch,' but rather in concert with other factors to boost a basal level of gene expression at a particular time of day and cause low-amplitude cycling within a single tissue (Abruzzi, 2011).

The large number of Clk target genes in fly heads is explained in part by tissue-specific Clk binding. Transcription assays that measure the cycling of mRNA and Pol II binding in one head tissue can be masked by noncycling expression in another. The ChIP assays, in contrast, are not plagued with the same problem. They can identify a gene bound by the cycling circadian transcription machinery even if the same gene is constitutively expressed elsewhere in the head. Surprisingly 44% of Clk direct targets were no longer detected when eyes were ablated with GMR-hid. Because many of these mRNAs are not particularly eye-enriched, it is inferred that their genes are constitutively expressed under the control of other transcription factors elsewhere in the head (Abruzzi, 2011).

The large number of target genes is also explained by the efficiency and sensitivity of the ChIP assay. It is inferred that it can detect Clk binding from a relatively low number of cells within the fly head. Lim1 is one example and is expressed predominantly in a subset of circadian neurons (l-LNvs; enriched more than four times relative to head). Preliminary cell-specific Clk ChIP-chip experiments from LNvs confirm that lim1 is an enriched Clk direct target in these cells, suggesting that this is the source of a large fraction of the binding signal in the head ChIP-chip experiments. Experiments are under way to more clearly define circadian neuron-specific Clk-binding patterns (Abruzzi, 2011).

This tissue specificity also suggests the existence of factors and/or chromatin modifications that help regulate Clk-mediated gene expression. They could enable Clk binding to specific genes in one tissue or inhibit binding in another tissue. These tissue-specific factors are strongly indicated by the pdp1 and lk6 Clk-binding patterns, which change so strikingly and specifically in GMR-hid. Although not unprecedented, tissue-specific factors that enable or inhibit specific DNA-binding locations are intriguing and warrant further investigation and identification (Abruzzi, 2011).

cis-regulatory requirements for tissue-specific programs of the circadian clock

Broadly expressed transcriptions factors (TFs) control tissue-specific programs of gene expression through interactions with local TF networks. A prime example is the circadian clock: although the conserved TFs Clock (Clk) and Cycle (Cyc) control a transcriptional circuit throughout animal bodies, rhythms in behavior and physiology are generated tissue specifically. Yet, how Clk and Cyc determine tissue-specific clock programs has remained unclear. This study used a functional genomics approach to determine the cis-regulatory requirements for clock specificity. First Clk and Cyc genome-wide binding targets in heads and bodies were determined by ChIP-seq, and they were shown to have distinct DNA targets in the two tissue contexts. Computational dissection of Clk/Cyc context-specific binding sites reveals sequence motifs for putative partner factors, which are predictive for individual binding sites. Among them, it was shown that the opa and GATA motifs, differentially enriched in head and body binding sites respectively, can be bound by Opa and Serpent (Srp). They act synergistically with Clk/Cyc in the Drosophila feedback loop, suggesting that they help to determine their direct targets and therefore orchestrate tissue-specific clock outputs. In addition, using in vivo transgenic assays, it was validated that GATA motifs are required for proper tissue-specific gene expression in the adult fat body, midgut, and Malpighian tubules, revealing a cis-regulatory signature for enhancers of the peripheral circadian clock. These results reveal how universal clock circuits can regulate tissue-specific rhythms and, more generally, provide insights into the mechanism by which universal TFs can be modulated to drive tissue-specific programs of gene expression (Meireles-Filho, 2013).

Although frequently not restricted to single cell types, individual TFs can control tissue-specific programs of gene expression through interactions with local TF networks. But despite substantial progress in identifying differential cell-specific circadian expression programs, how Clk and Cyc interact with local TF networks to generate output rhythms tissue specifically is still elusive (Meireles-Filho, 2013).

This study used an integrative genomics approach to shed light on how the circadian clock drives tissue-specific gene expression. While shared Clk/Cyc binding sites could not be explained by combinations of head- and body-specific motifs, yet were slightly more enriched in E box motifs and -- similar to highly occupied target [HOT] regions -- in Trithorax-like motifs [Trl/GAGA; 2-fold]), a substantial number of Clk and Cyc binding sites were specific to either heads or bodies and next to genes with different functional GO categories. These binding sites differed substantially in their motif content, and this motif signature was predictive of context-specific Clk/Cyc binding, suggesting that tissue-specific clock targets are determined by the binding site sequences (Meireles-Filho, 2013).

GATA motifs were enriched in Clk/Cyc binding sites in bodies and required for enhancer activity in the fat body, midgut, and Malpighian tubules. This suggests that GATA factors might play a key role for Clk/Cyc-bound enhancers in bodies, potentially by helping to establish the chromatin landscape in tissues where they are specifically expressed (e.g., srp in the fat body and GATAe in the gut). Interestingly, GATA motifs are also overrepresented in promoter regions of circadian genes in rodents, suggesting a conserved role for GATA factors in the circadian clock (Meireles-Filho, 2013).

This study found that the GATA factor Srp could act synergistically with Clk, suggesting that it is an important determinant of clock function in peripheral tissues. Srp has multiple functions in Drosophila, including the control of endodermal development and hematopoiesis in the embryo and the induction of immune response in the larval fat body. Interestingly, srp is coexpressed with Clk and Cyc in the fat body, a tissue with roles in metabolic activity, innate immunity response, and detoxification - all known to be controlled in a circadian manner. Clk body-specific peaks were 4.17-fold enriched close to cycling fat body genes, suggesting that srp might help determine the physiological outputs controlled by the fat body pacemaker. Interestingly, srp is also required for hormone-induced expression of the Fbp1 TF during fat body development, supporting the idea that it might be important for temporal or inducible regulation more generally (Meireles-Filho, 2013).

Similarly, Opa, which belongs to the Zic family of mammalian TFs with conserved roles in head formation in flies and mammals, is coexpressed with Clk and cyc in the adult brain. In addition, an enhancer of Slob, an output gene of the clock pacemaker involved in the generation of locomotor activity rhythms, responded to Clk and Cyc in an Opa-dependent manner, suggesting that Opa might be involved in the recruitment of Clk/Cyc to regulate genes controlling fly behavior. Further studies on Opa and additional predicted partner TFs might provide new insights into the Drosophila clock in the head (Meireles-Filho, 2013).

It is likely that different cofactors with functions equivalent to srp or opa exist in different cell types, which redirect Clk/ Cyc to tissue-specific binding sites and allow tissue-specific gene regulation. Indeed, this study has identified several other motifs that are tissue-specifically enriched. This is reminiscent of studies showing that TFs downstream of signaling pathways are redirected in a tissue-specific manner by cell-specific master regulators. The results might thus constitute an important example of how partner TFs adapt broadly active transcriptional regulators to achieve tissue-specific gene expression and function, contributing to a better understanding of gene regulatory networks more generally (Meireles-Filho, 2013).

These data on Clk/Cyc binding in different contexts not only provide novel insights into clock regulatory networks and enhancer structure but also exemplify a new strategy to uncover cofactors of the circadian clock via their cis-regulatory motifs. This approach is complementary to forward and reverse genetics or biochemistry, which have traditionally been used to reveal clock factors. It can also be applied more generally to identify factors that recruit broadly expressed TFs in different cell types or tissues. In addition, the tagging of endogenous loci allows the study of TFs under physiological conditions in their endogenous expression domains, which is crucial especially for TFs that have large and complex regulatory regions and/or for which physiological expression levels are of fundamental importance. In summary, the results in the Drosophila circadian clock reveal how universal TF circuits can be modulated to generate transcriptional tissue-specific outputs and demonstrate a novel approach to determine regulatory partners more generally (Meireles-Filho, 2013).

Circadian gene Bmal1 regulates diurnal oscillations of Ly6Chi) inflammatory monocytes

Circadian clocks have evolved to regulate physiologic and behavioral rhythms in anticipation of changes in the environment. Although the molecular clock is present in innate immune cells, its role in monocyte homeostasis remains unknown. This study report that Ly6Chi inflammatory monocytes exhibit diurnal variation, which controls their trafficking to sites of inflammation. This cyclic pattern of trafficking confers protection against Listeria monocytogenes and is regulated by the repressive activity of the circadian gene Bmal1. Accordingly, myeloid cell-specific deletion of Bmal1 induces expression of monocyte-attracting chemokines and disrupts rhythmic cycling of Ly6Chi monocytes, predisposing mice to development of pathologies associated with acute and chronic inflammation. These findings have unveiled a critical role for BMAL1 in controlling the diurnal rhythms in Ly6C(hi) monocyte numbers (Nguyen, 2013)

The circadian molecular clock regulates adult hippocampal neurogenesis by controlling the timing of cell-cycle entry and exit

The subgranular zone (SGZ) of the adult hippocampus contains a pool of quiescent neural progenitor cells (QNPs) that are capable of entering the cell cycle and producing newborn neurons. The mechanisms that control the timing and extent of adult neurogenesis are not well understood. This study shows that QNPs of the adult SGZ express molecular-clock components and proliferate in a rhythmic fashion. The clock proteins PERIOD2 and BMAL1 are critical for proper control of neurogenesis. The absence of PERIOD2 abolishes the gating of cell-cycle entrance of QNPs, whereas genetic ablation of bmal1 results in constitutively high levels of proliferation and delayed cell-cycle exit. Mathematical model simulations were used to show that these observations may arise from clock-driven expression of a cell-cycle inhibitor that targets the cyclin D/Cdk4-6 complex. These findings may have broad implications for the circadian clock in timing cell-cycle events of other stem cell populations throughout the body (Bouchard-Cannon, 2013).

Reprogramming of the circadian clock by nutritional challenge

Circadian rhythms and cellular metabolism are intimately linked. This study reveals that a high-fat diet (HFD) generates a profound reorganization of specific metabolic pathways, leading to widespread remodeling of the liver clock. Strikingly, in addition to disrupting the normal circadian cycle, HFD causes an unexpectedly large-scale genesis of de novo oscillating transcripts, resulting in reorganization of the coordinated oscillations between coherent transcripts and metabolites. The mechanisms underlying this reprogramming involve both the impairment of CLOCK:BMAL1 chromatin recruitment and a pronounced cyclic activation of surrogate pathways through the transcriptional regulator PPARγ. Finally, it was demonstrated that the specifically of the nutritional challenge, and not the development of obesity, that causes the reprogramming of the clock and that the effects of the diet on the clock are reversible (Eckel-Mahan, 2013).

Metabolic and circadian processes are tightly linked, but the mechanisms by which altered nutrients influence the circadian clock have not been deciphered. This study has explored the effects of nutrient challenge in the form of HFD on the circadian metabolome and transcriptome and found that HFD induces transcriptional reprogramming within the clock that reorganizes the relationships between the circadian transcriptome and the metabolome. At least three mechanisms by which this reprograming occurs have been unraveled: (1) loss of oscillation of a large number of normally oscillating genes; (2) a phase advance of an additional subset of oscillating transcripts; and (3) a massive induction of de novo oscillating gene transcripts (Eckel-Mahan, 2013).

This study demonstrates that HFD-induced changes in the circadian clock implicate a reprogramming of the transcriptional system that relies on at least two key mechanisms. The first mechanism is the lack of proper CLOCK:BMAL1 chromatin recruitment to genes that would normally be considered as clock controlled. This results in a decrease or abrogation of oscillation in transcription. The second, illustrated by the de novo oscillations in transcriptional networks otherwise considered arrhythmic, relies in large part on the robust, circadian accumulation in the nucleus and on chromatin of the transcription factor PPARγ. Although it is predicted that other transcriptional pathways would contribute to clock reprogramming, including SREBP1, the role of PPARγ appears prominent. This nuclear receptor has been linked to circadian control during adipogenesis and osteogenesis, whereas its role in the liver clock is not fully understood. This study has determined that PPARγ circadian function in HFD-fed mice relies on a clock-controlled nuclear translocation of the protein and rhythmic chromatin recruitment to target genes (Eckel-Mahan, 2013).

In contrast to the PPARγ scenario, HFD does not affect CLOCK:BMAL1 nuclear translocation but impedes their specific chromatin recruitment. It is speculated that additional regulatory pathways are implicated that might interplay with the ones described in this study. In conclusion, the remarkable induction of de novo oscillation in both metabolites and transcripts under HFD indicates that a diet high in fat has previously unsuspected, potent, and pleiotropic effects on the circadian clock. Furthermore, the rapid influence of the diet on the clock (as demonstrated by the 3 day HFD experiment) reveals that this type of nutritional challenge-and not merely the development of diet-associated complications such as obesity-is capable of remodeling the clock. Further work will elucidate how the molecular composition of CLOCK:BMAL1 and PPARγ chromatin complexes may be influenced by nutritional challenges, possibly leading to modulation of enzymatic activities of specific coregulators and modifiers (Eckel-Mahan, 2013).

An intriguing concept that may be derived from this study relates to the potential of specific genes to be circadian or not. Indeed, the transcriptional remodeling in the HFD raises the hypothesis that, given the 'right' molecular environment, an extended array of transcripts and metabolites can oscillate. It is speculated that this may be achieved through the coordinated harmonics of energy balance, transcriptional control, and epigenetic state. In summary, nutrients have powerful effects on the cellular clock, revealing its intrinsic plasticity. These effects consist not only of the abrogation of pre-existing rhythms but the genesis of rhythms where they do not normally exist. This induction is rapid and does not require the onset of obesity, and it is also reversible. The reversible nature of these effects gives hope for novel nutritional and pharmaceutical strategies (Eckel-Mahan, 2013).

CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms

Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca(2+) in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. This study shows that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIalpha weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca(2+)-calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of CLOCK and BMAL1. These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities (Kon, 2014).

Class IIa histone deacetylases are conserved regulators of circadian function

Class IIa histone deacetylases (HDACs) regulate the activity of many transcription factors to influence liver gluconeogenesis and the development of specialized cells including muscle, neurons and lymphocytes. This study describes a conserved role for class IIa HDACs in sustaining robust circadian behavioral rhythms in Drosophila and cellular rhythms in mammalian cells. In mouse fibroblasts, over-expression of HDAC5 severely disrupts transcriptional rhythms of core clock genes. HDAC5 over-expression decreases BMAL1 acetylation on Lys537 and pharmacological inhibition of Class IIa HDACs increases BMAL1 acetylation. Furthermore, cyclical nucleocytoplasmic shuttling of HDAC5 was observed in mouse fibroblasts that is characteristically circadian. Mutation of the Drosophila homolog HDAC4 impairs locomotor activity rhythms of flies and decreases period mRNA levels. RNAi-mediated knockdown of HDAC4 in Drosophila clock cells also dampens circadian function. Given that the localization of Class IIa HDACs is signal-regulated and influenced by Ca2+ and cAMP signals, these findings offer a mechanism by which extracellular stimuli that generate these signals can feed into the molecular clock machinery (Fogg, 2014).


Search PubMed for articles about Drosophila Cycle

Abruzzi, K. C., et al. (2011). Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression. Genes Dev. 25(22): 2374-86. PubMed ID: 22085964

Allada, R., et al. (1998). A mutant Drosophila homolog of mammalian Clock disrupts circadian rhythms and transcription of period and timeless. Cell 93: 791-804. PubMed ID: 9630223

Bouchard-Cannon, P., Mendoza-Viveros, L., Yuen, A., Kaern, M. and Cheng, H. Y. (2013). The circadian molecular clock regulates adult hippocampal neurogenesis by controlling the timing of cell-cycle entry and exit. Cell Rep 5(4): 961-73. PubMed ID: 24268780

Eckel-Mahan, K. L., Patel, V. R., de Mateo, S., Orozco-Solis, R., Ceglia, N. J., Sahar, S., Dilag-Penilla, S. A., Dyar, K. A., Baldi, P. and Sassone-Corsi, P. (2013). Reprogramming of the circadian clock by nutritional challenge. Cell 155: 1464-1478. PubMed ID: 24360271; Graphical Abstract

Fogg, P. C., O'Neill, J. S., Dobrzycki, T., Calvert, S., Lord, E. C., McIntosh, R. L., Elliott, C. J., Sweeney, S. T., Hastings, M. H. and Chawla, S. (2014). Class IIa histone deacetylases are conserved regulators of circadian function. J Biol Chem [Epub ahead of print]. PubMed ID: 25271152

Hendricks, J. C., et al. (2003). Gender dimorphism in the role of cycle (BMAL1) in rest, rest regulation, and longevity in Drosophila melanogaster. J. Biol. Rhythms. 18(1): 12-25. PubMed ID: 12568241

Hogenesch, J.B., Gu, Y.-Z., Jain, S., and Bradfield, C.A. (1998). The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc. Natl. Acad. Sci. 95: 5474-5479. PubMed ID: 9576906

Ikeda, M. and Nomura, M. (1997). cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS protein (BMAL1) and identification of alternatively spliced variants with alternative translation initiation site usage. Biochem. Biophys. Res. Com. 233: 258-264. PubMed ID: 9144434

Kon, N., Yoshikawa, T., Honma, S., Yamagata, Y., Yoshitane, H., Shimizu, K., Sugiyama, Y., Hara, C., Kameshita, I., Honma, K. and Fukada, Y. (2014). CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms. Genes Dev 28: 1101-1110. PubMed ID: 24831701

Meireles-Filho, A. C., Bardet, A. F., Yanez-Cuna, J. O., Stampfel, G. and Stark, A. (2013). cis-regulatory requirements for tissue-specific programs of the circadian clock. Curr Biol 24(1): 1-10. PubMed ID: 24332542

Menet, J. S., et al. (2010). Dynamic PER repression mechanisms in the Drosophila circadian clock: From on-DNA to off-DNA. Genes Dev 24: 358-367. PubMed ID: 20159956

Nguyen, K. D., Fentress, S. J., Qiu, Y., Yun, K., Cox, J. S. and Chawla, A. (2013). Circadian gene Bmal1 regulates diurnal oscillations of Ly6Chi) inflammatory monocytes. Science 341: 1483-1488. PubMed ID: 23970558

Rutila, J. E., et al. (1998). CYCLE is a second bHLH-PAS Clock protein essential for circadian rhythmicity and transcription of Drosophila period and timeless. Cell 93: 805-814. PubMed ID:

Shaw, P. J., Tononi, G., Greenspan, R. J. and Robinson, D. F. (2002). Stress response genes protect against lethal effects of sleep deprivation in Drosophila. Nature 417: 287-291. PubMed ID: 12015603

Thimgan, M. S., Seugnet, L., Turk, J. and Shaw, P. J. (2014). Identification of genes associated with resilience/vulnerability to sleep deprivation and starvation in Drosophila. Sleep [Epub ahead of print]. PubMed ID: 25409104

Yu, W., Zheng, H., Houl, J. H., Dauwalder, B. and Hardin. P. E. (2006). PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription. Genes Dev. 20(6): 723-33. PubMed ID: 16543224

Biological Overview

date revised: 17 November 2021

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