InteractiveFly: GeneBrief

cycle: Biological Overview | References

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 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 (Clk^jrk) 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 Clk^jrk 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 Clk^jrk 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, Clk^jrk, 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 Clk^jrk 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, Clk^jrk 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 Clk^jrk 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 Clk^jrk 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 Hsp83^e6A flies. However, both Hsp83 heterozygotes exhibit a sleep rebound that is significantly different from that of Hsp60^RA75 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).

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 (Clk^Jrk) 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 dbt^AR/dbt^P 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).

Search PubMed for articles about Drosophila Cycle

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. Medline abstract: 9630223

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. 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. Medline abstract: 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. Medline abstract: 9144434

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.

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. 12015603

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. 16543224

date revised: 6 October 2007

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