Protein Timeless Research Articles

Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation

Timeless was originally identified in Drosophila as an essential component of circadian cycle regulation, where its function is tightly controlled at the protein level by tyrosine phosphorylation and subsequent degradation. In mammals, Timeless has also been implicated in circadian rhythms as well as cell cycle control and embryonic development. Here we report that mammalian Timeless is an SH3 domain-binding protein and substrate for several members of the Src protein–tyrosine kinase family, including Fyn, Hck, c-Src and c-Yes. Co-expression of Tim with Fyn or Hck was followed by ubiquitylation and subsequent degradation in human 293T cells. While c-Src and c-Yes also promoted Tim ubiquitylation, in this case ubiquitylation correlated with Tim protein accumulation rather than degradation. Both c-Src and c-Yes selectively promoted modification of Tim through Lys63-linked polyubiquitin, which may explain the differential effects on Tim protein turnover. These data show distinct and opposing roles for individual Src-family members in the regulation of Tim protein levels, suggesting a unique mechanism for the regulation of Tim function in mammals.

CULLIN-3 Controls TIMELESS Oscillations in the Drosophila Circadian Clock

Author SummaryCircadian clocks adjust the physiology and behavior of organisms to the day/night cycle and rely on molecular feedback loops that generate daily oscillations of transcription. In the Drosophila fruit fly, the PERIOD (PER) and TIMELESS (TIM) proteins coordinate the clock–they accumulate during the night, form a complex, and repress their own gene expression in the early morning. The temporal control of this oscillation involves the phosphorylation, ubiquitination, and proteasomal degradation of the PER and TIM proteins. The SUPERNUMERARY LIMBS (SLMB) ubiquitin ligase is known to play a key role in controlling the degradation of phosphorylated PER and TIM. In this study we investigated the role of another ubiquitin ligase, CULLIN-3 (CUL-3). We found that inhibition of CUL-3 activity results in the abolition of rest/activity rhythms in flies and flattens the PER and TIM oscillations. CUL-3 physically interacts and forms a complex with a lowphosphorylated version of TIM in the absence of PER, thereby allowing its accumulation during the night. In contrast, when PER is present SLMB preferentially interacts with phosphorylated TIM, favoring its degradation. The results suggest that CUL-3 and SLMB share the work to control the oscillations of the PER and TIM proteins during the day/night cycle.

The Circadian Clock Protein Timeless Regulates Phagocytosis of Bacteria in Drosophila

Survival of bacterial infection is the result of complex host-pathogen interactions. An often-overlooked aspect of these interactions is the circadian state of the host. Previously, we demonstrated that Drosophila mutants lacking the circadian regulatory proteins Timeless (Tim) and Period (Per) are sensitive to infection by S. pneumoniae. Sensitivity to infection can be mediated either by changes in resistance (control of microbial load) or tolerance (endurance of the pathogenic effects of infection). Here we show that Tim regulates resistance against both S. pneumoniae and S. marcescens. We set out to characterize and identify the underlying mechanism of resistance that is circadian-regulated. Using S. pneumoniae, we found that resistance oscillates daily in adult wild-type flies and that these oscillations are absent in Tim mutants. Drosophila have at least three main resistance mechanisms to kill high levels of bacteria in their hemolymph: melanization, antimicrobial peptides, and phagocytosis. We found that melanization is not circadian-regulated. We further found that basal levels of AMP gene expression exhibit time-of-day oscillations but that these are Tim-independent; moreover, infection-induced AMP gene expression is not circadian-regulated. We then show that phagocytosis is circadian-regulated. Wild-type flies exhibit up-regulated phagocytic activity at night; Tim mutants have normal phagocytic activity during the day but lack this night-time peak. Tim appears to regulate an upstream event in phagocytosis, such as bacterial recognition or activation of phagocytic hemocytes. Interestingly, inhibition of phagocytosis in wild type flies results in survival kinetics similar to Tim mutants after infection with S. pneumoniae. Taken together, these results suggest that loss of circadian oscillation of a specific immune function (phagocytosis) can have significant effects on long-term survival of infection.

Kinetics of Doubletime Kinase-dependent Degradation of the Drosophila Period Protein

Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT(S)), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT(S) in cell culture resembles that with wild-type DBT and posits that, in flies DBT(S) likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation.

Post-Translational Regulation and Nuclear Entry of TIMELESS and PERIOD Are Affected in New timeless Mutant

The molecular circadian clock consists of a feedback loop in which canonical clock proteins negatively regulate transcription of their own genes. Timed nuclear entry of these proteins is critical, but regulation of this event is poorly understood. In Drosophila melanogaster, the idea that nuclear entry of PERIOD (PER) is controlled by its partner protein TIMELESS (TIM) has been challenged by several studies. We identify here a novel mutation in the tim gene that eliminates behavioral rhythms while allowing robust expression of TIM and PER. Mutant TIM can bind to and stabilize PER. However, neither protein is expressed cyclically, and phosphorylation of both is reduced. In addition, TIM and PER are localized in the cytoplasm at all times of day, and mutant TIM attenuates transcriptional feedback by PER in cultured cells, suggesting that it holds PER in the cytoplasm. In fact, much of the reduced phosphorylation of PER in the new tim mutant appears to result from the cytoplasmic localization of PER. Interestingly, mutating a threonine near the original mutation produces similar phenotypes, raising the possibility that defective phosphorylation is the basis of TIM dysfunction in the novel tim mutant. We also show that a stable form of PER is cytoplasmic in tim-null flies. These studies establish an essential role of TIM in the timed nuclear entry of PER.

A Key Temporal Delay in the Circadian Cycle ofDrosophilaIs Mediated by a Nuclear Localization Signal in the Timeless Protein

Regulated nuclear entry of the Period (PER) and Timeless (TIM) proteins, two components of the Drosophila circadian clock, is essential for the generation and maintenance of circadian behavior. PER and TIM shift from the cytoplasm to the nucleus daily, and the length of time that PER and TIM reside in the cytoplasm is an important determinant of the period length of the circadian rhythm. Here we identify a TIM nuclear localization signal (NLS) that is required for appropriately timed nuclear accumulation of both TIM and PER. Transgenic flies with a mutated TIM NLS produced circadian rhythms with a period of ∼30 hr. In pacemaker cells of the brain, PER and TIM proteins rise to abnormally high levels in the cytoplasm of timΔNLS mutants, but show substantially reduced nuclear accumulation. In cultured S2 cells, the mutant TIMΔNLS protein significantly delays nuclear accumulation of both TIM and wild-type PER proteins. These studies confirm that TIM is required for the nuclear localization of PER and point to a key role for the TIM NLS in the regulated nuclear accumulation of both proteins.

QUASIMODO, a Novel GPI-Anchored Zona Pellucida Protein Involved in Light Input to the Drosophila Circadian Clock

Circadian clocks are synchronized to the solar day via visual and specialized photoreceptors. In Drosophila, CRYPTOCHROME (CRY) is a major photoreceptor that mediates resetting of the circadian clock via light-dependent degradation of the clock protein TIMELESS (TIM). However, in the absence of CRY, this TIM-mediated resetting still occurs in some pacemaker neurons, resulting in synchronized behavioral rhythms when flies are exposed to light-dark cycles. Even in the additional absence of visual photoreception, partial molecular and behavioral light synchronization persists. Therefore, other important clock-related photoreceptive and synchronization mechanisms must exist. We identified a novel clock-controlled gene (quasimodo) that encodes a light-responsive and membrane-anchored Zona Pellucida domain protein that supports light-dependent TIM degradation. Whereas wild-type flies become arrhythmic in constant light (LL), quasimodo mutants elicit rhythmic expression of clock proteins and behavior in LL. QUASIMODO (QSM) can function independently of CRY and is predominantly expressed within CRY-negative clock neurons. Interestingly, downregulation of qsm in the clock circuit restores LL clock protein rhythms in qsm-negative neurons, indicating that qsm-mediated light input is not entirely cell autonomous and can be accessed by the clock circuit. Our findings indicate that QSM constitutes part of a novel and CRY-independent light input to the circadian clock. Like CRY, this pathway targets the clock protein TIM. QSM's light-responsive character in conjunction with the predicted localization at the outer neuronal membrane suggests that its function is linked to a yet unidentified membrane-bound photoreceptor.

An Unexpected Role for the Clock Protein Timeless in Developmental Apoptosis

BackgroundProgrammed cell death is critical not only in adult tissue homeostasis but for embryogenesis as well. One of the earliest steps in development, formation of the proamniotic cavity, involves coordinated apoptosis of embryonic cells. Recent work from our group demonstrated that c-Src protein-tyrosine kinase activity triggers differentiation of mouse embryonic stem (mES) cells to primitive ectoderm-like cells. In this report, we identified Timeless (Tim), the mammalian ortholog of a Drosophila circadian rhythm protein, as a binding partner and substrate for c-Src and probed its role in the differentiation of mES cells.Methodology/Principal FindingsTo determine whether Tim is involved in ES cell differentiation, Tim protein levels were stably suppressed using shRNA. Tim-defective ES cell lines were then tested for embryoid body (EB) formation, which models early mammalian development. Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate. Cells retained within the centers of the failed cavities strongly expressed the pluripotency marker Oct4, suggesting that further development is arrested without Tim. Immunoblots revealed reduced basal Caspase activity in the Tim-defective EBs compared to wild-type controls. Furthermore, EBs formed from Tim-knockdown cells demonstrated resistance to staurosporine-induced apoptosis, consistent with a link between Tim and programmed cell death during cavitation.Conclusions/SignificanceOur data demonstrate a novel function for the clock protein Tim during a key stage of early development. Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate. As a consequence, further development was halted, and the cells present in the failed cavity remained pluripotent. These findings reveal a new function for Tim in the coordination of ES cell differentiation, and raise the intriguing possibility that circadian rhythms and early development may be intimately linked.

AKT and TOR Signaling Set the Pace of the Circadian Pacemaker

The circadian clock coordinates cellular and organismal energy metabolism. The importance of this circadian timing system is underscored by findings that defects in the clock cause deregulation of metabolic physiology and result in metabolic disorders. On the other hand, metabolism also influences the circadian clock, such that circadian gene expression in peripheral tissues is affected in mammalian models of obesity and diabetes. However, to date there is little to no information on the effect of metabolic genes on the central brain pacemaker which drives behavioral rhythms. We have found that the AKT and TOR-S6K pathways, which are major regulators of nutrient metabolism, cell growth, and senescence, impact the brain circadian clock that drives behavioral rhythms in Drosophila. Elevated AKT or TOR activity lengthens circadian period, whereas reduced AKT signaling shortens it. Effects of TOR-S6K appear to be mediated by SGG/GSK3beta, a known kinase involved in clock regulation. Like SGG, TOR signaling affects the timing of nuclear accumulation of the circadian clock protein TIMELESS. Given that activities of AKT and TOR pathways are affected by nutrient/energy levels and endocrine signaling, these data suggest that metabolic disorders caused by nutrient and energy imbalance are associated with altered rest:activity behavior.

Light-Mediated TIM Degradation within Drosophila Pacemaker Neurons (s-LNvs) Is Neither Necessary nor Sufficient for Delay Zone Phase Shifts

Circadian systems are entrained and phase shifted by light. In Drosophila, the model of light-mediated phase shifting begins with photon capture by CRYPTOCHROME (CRY) followed by rapid TIMELESS (TIM) degradation. In this study, we focused on phase delays and assayed TIM degradation within individual brain clock neurons in response to light pulses in the early night. Surprisingly, there was no detectable change in TIM staining intensity within the eight pacemaker s-LNvs. This indicates that TIM degradation within s-LNvs is not necessary for phase delays, and similar assays in other genotypes indicate that it is also not sufficient. In contrast, more dorsal circadian neurons appear light-sensitive in the early night. Because CRY is still necessary within the s-LNvs for phase shifting, the results challenge the canonical cell-autonomous molecular model and raise the question of how the pacemaker neuron transcription-translation clock is reset by light in the early night.

Mammalian TIMELESS Is Required for ATM-dependent CHK2 Activation and G2/M Checkpoint Control

Timeless (Tim), a core circadian clock gene in Drosophila, is retained in mammals but has no apparent mammalian circadian clock function. Mammalian TIM is essential for ATR-dependent Chk1 activation and S-phase arrest. We report that TIM is likewise essential for ATM-dependent Chk2-mediated signaling of doxorubicin-induced DNA double strand breaks. TIM depletion attenuates doxorubicin-induced G(2)/M cell cycle arrest and sensitizes cancer cells to doxorubicin-induced cytotoxicity. TIM is, thereby, a potential novel anticancer drug target whose inhibition may enhance the therapeutic cytotoxicity of agents that activate DNA damage pathways as part of their mechanism.

A Constant Light-Genetic Screen Identifies KISMET as a Regulator of Circadian Photoresponses

Circadian pacemakers are essential to synchronize animal physiology and behavior with the day∶night cycle. They are self-sustained, but the phase of their oscillations is determined by environmental cues, particularly light intensity and temperature cycles. In Drosophila, light is primarily detected by a dedicated blue-light photoreceptor: CRYPTOCHROME (CRY). Upon light activation, CRY binds to the pacemaker protein TIMELESS (TIM) and triggers its proteasomal degradation, thus resetting the circadian pacemaker. To understand further the CRY input pathway, we conducted a misexpression screen under constant light based on the observation that flies with a disruption in the CRY input pathway remain robustly rhythmic instead of becoming behaviorally arrhythmic. We report the identification of more than 20 potential regulators of CRY-dependent light responses. We demonstrate that one of them, the chromatin-remodeling enzyme KISMET (KIS), is necessary for normal circadian photoresponses, but does not affect the circadian pacemaker. KIS genetically interacts with CRY and functions in PDF-negative circadian neurons, which play an important role in circadian light responses. It also affects daily CRY-dependent TIM oscillations in a peripheral tissue: the eyes. We therefore conclude that KIS is a key transcriptional regulator of genes that function in the CRY signaling cascade, and thus it plays an important role in the synchronization of circadian rhythms with the day∶night cycle.

A Role for the PERIOD:PERIOD Homodimer in the Drosophila Circadian Clock

Circadian clocks in eukaryotes rely on transcriptional feedback loops, in which clock genes repress their own transcription resulting in molecular oscillations with a period of ∼24 h. In Drosophila, the clock proteins Period (PER) and Timeless (TIM) operate in such a feedback loop, whereby they first accumulate in the cytoplasm of clock cells as a heterodimer. Nuclear translocation of the complex or the individual PER and TIM proteins is followed by repression of per and tim transcription, whereby PER seems to act as the prime repressor. We found that in addition to PER:TIM complexes, functional PER:PER homodimers exist in flies. Specific disruption of PER homodimers results in drastically impaired behavioral and molecular rhythmicity, pointing the biological importance of this clock protein complex. Analysis of PER subcellular distribution and repressor competence in the PER dimer mutant revealed defects in PER nuclear translocation and a disruption of rhythmic period transcription. The striking similarity of these phenotypes with that of reduced CKII activity suggests that the formation or function of the PER dimer is closely linked to this kinase. Our results confirm a previous structural model for PER and provide strong evidence that PER homodimers are important for circadian clock function.

Light-Dependent Interactions between the Drosophila Circadian Clock Factors Cryptochrome, Jetlag, and Timeless

Circadian clocks regulate daily fluctuations of many physiological and behavioral aspects in life. They are synchronized with the environment via light or temperature cycles [1]. Natural fluctuations of the day length (photoperiod) and temperature necessitate a daily reset of the circadian clock on the molecular level. In Drosophila, the blue-light photoreceptor Cryptochrome (Cry) mediates a rapid light-dependent degradation of the clock protein Timeless (Tim) via the F box protein Jetlag (Jet) and the proteasome, which initiates the resetting of the molecular clock [2, 3]. Cry is also degraded in the light but whereas the degradation of Tim is well characterized [4-8], the mechanism for light-dependent degradation of Cry is mostly unknown. Until now it was believed that these two degradation pathways are distinct [4, 9]. Here we reveal that Jetlag also interacts with Cry in a light-dependent manner. After illumination, Jetlag induces massive degradation of Cry, which can be prevented in vitro and in vivo by adding Tim as an antagonist. We show that the affinity of Tim for Cry and Jetlag determines the sequential order of Tim and Cry degradation and thus reveal an intimate connection between the light-dependent degradation of these two proteins by the same proteasomal pathway.

The COP9 Signalosome Is Required for Light-Dependent Timeless Degradation andDrosophilaClock Resetting

The ubiquitin-proteasome system plays a major role in the rhythmic accumulation and turnover of molecular clock components. In turn, these approximately 24 h molecular rhythms drive circadian rhythms of behavior and physiology. In Drosophila, the ubiquitin-proteasome system also plays a critical role in light-dependent degradation of the clock protein Timeless (TIM), a key step in the entrainment of the molecular clocks to light-dark cycles. Here, we investigated the role of the COP9 signalosome (CSN), a general regulator of protein degradation, in fly circadian rhythms. We found that null mutations in the genes encoding the CSN4 and CSN5 subunits prevent normal TIM degradation by light in the pacemaker lateral neurons (LNs) as does LN-specific expression of a dominant-negative CSN5 transgene. These defects are accompanied by strong reductions in behavioral phase shifts of adult flies lacking normal CSN5 activity in LNs. Defects in TIM degradation and resetting of behavioral phases were rescued by overexpression of Jetlag (JET), the F-box protein required for light-mediated TIM degradation. Flies lacking normal CSN activity in all clock neurons are rhythmic in constant light, a phenotype previously associated with jet mutants. Together, these data indicate that JET and the CSN lie in a common pathway leading to light-dependent TIM degradation. Surprisingly, we found that manipulations that strongly inhibit CSN activity had minimal effects on circadian rhythms in constant darkness, indicating a specific role for the CSN in light-mediated TIM degradation.

TIMELESS Is an Important Mediator of CK2 Effects on Circadian Clock FunctionIn Vivo

Circadian oscillations in clock components are central to generation of self-sustained 24-h periodicity. In the Drosophila molecular clock, accumulation, phosphorylation, and degradation of PERIOD (PER) and TIMELESS (TIM) proteins govern period length. Yet little is known about the kinases that phosphorylate TIM in vivo. It has been shown previously that the protein kinase CK2 phosphorylates TIM in vitro. Here, we identify a role for CK2 in TIM regulation in vivo. Induction of a dominant-negative CK2alpha, CK2alpha(Tik) (Tik), increases TIM protein and tim transcript levels, reduces oscillation amplitude, and results in persistent cytoplasmic TIM localization. Exposure to light and subsequent TIM degradation results in an increase in the fraction of the transcriptional repressor PER that is nuclear and suppression of per and tim RNA levels. TIM protein, but not tim transcript, levels are elevated in Tik mutants in a per(01) background. In contrast, Tik effects on PER are undetectable in a tim(01) background, suggesting that TIM is required for CK2 effects on PER. To identify potential CK2 target sites, we assayed TIM phosphorylation rhythms in a deletion mutant that removes a conserved serine-rich domain and found that TIM protein does not show robust rhythmic changes in mobility by Western blotting, a hallmark of rhythmic phosphorylation. The period lengthening effects in Tik heterozygotes are reduced in a tim(UL) mutant that disrupts a putative CK2 phosphorylation site. Together, these data indicate that TIM is an important mediator of CK2 effects on circadian rhythms.

Identification of novel genes involved in light-dependent CRY degradation through a genome-wide RNAi screen

Circadian clocks regulate many different physiological processes and synchronize these to environmental light:dark cycles. In Drosophila, light is transmitted to the clock by a circadian blue light photoreceptor CRYPTOCHROME (CRY). In response to light, CRY promotes the degradation of the circadian clock protein TIMELESS (TIM) and then is itself degraded. To identify novel genes involved in circadian entrainment, we performed an unbiased genome-wide screen in Drosophila cells using a sensitive and quantitative assay that measures light-induced degradation of CRY. We systematically knocked down the expression of approximately 21,000 genes and identified those that regulate CRY stability. These genes include ubiquitin ligases, signal transduction molecules, and redox molecules. Many of the genes identified in the screen are specific for CRY degradation and do not affect degradation of the TIM protein in response to light, suggesting that, for the most part, these two pathways are distinct. We further validated the effect of three candidate genes on CRY stability in vivo by assaying flies mutant for each of these genes. This work identifies a novel regulatory network involved in light-dependent CRY degradation and demonstrates the power of a genome-wide RNAi approach for understanding circadian biology.

Entrainment of the Drosophila Circadian Clock: More Heat Than Light

Circadian rhythms are produced by a biological clock that is synchronized (or entrained) by cycles of light and temperature. In Drosophila, light triggers the interaction of the photoreceptor cryptochrome (CRY) with the circadian clock protein timeless (TIM). The absence of this interaction in cryb mutants eliminates this entrainment mechanism. The abundance of TIM and period (PER) oscillate throughout the day, and they form a complex that moves to the nucleus to rhythmically repress transcription of the per and tim genes. Because the CRY:TIM interaction triggers rapid degradation of TIM, the phase of these molecular oscillations is reset by light, which thereby entrains the circadian clock. A study now shows that heat pulses trigger an association between CRY and PER:TIM, which suggests that CRY:PER:TIM also contributes to entrainment by temperature. In wild-type flies, CRY:PER:TIM formation requires high temperatures and is only triggered by heat pulses in the early night, but in per(L) mutants, which exhibit a temperature-sensitive lengthening of circadian periods, CRY:PER(L:)TIM formation is triggered by lower temperatures and throughout the night. Because CRY:PER:TIM is formed under the same conditions that entrain circadian behavior, formation of the complex is likely to mediate entrainment by heat pulses. Whereas per(L )flies exhibit longer periods at higher temperatures, per(L);cry(b) flies exhibit similar periods at different temperatures, which suggests that an altered interaction between CRY and PER(L):TIM contributes to a lack of temperature compensation. Future work should determine how the interaction between CRY and PER:TIM entrains rhythms to temperature and affects temperature compensation.

PER-TIM interactions with the photoreceptor cryptochrome mediate circadian temperature responses in Drosophila.

Drosophila cryptochrome (CRY) is a key circadian photoreceptor that interacts with the period and timeless proteins (PER and TIM) in a light-dependent manner. We show here that a heat pulse also mediates this interaction, and heat-induced phase shifts are severely reduced in the cryptochrome loss-of-function mutant cryb. The period mutant perL manifests a comparable CRY dependence and dramatically enhanced temperature sensitivity of biochemical interactions and behavioral phase shifting. Remarkably, CRY is also critical for most of the abnormal temperature compensation of perL flies, because a perL; cryb strain manifests nearly normal temperature compensation. Finally, light and temperature act together to affect rhythms in wild-type flies. The results indicate a role for CRY in circadian temperature as well as light regulation and suggest that these two features of the external 24-h cycle normally act together to dictate circadian phase.

A mathematical model of the Drosophila circadian clock with emphasis on posttranslational mechanisms

Experimental evidence points increasingly to the importance of posttranslational processes such as phosphorylation and translocation in the molecular circadian clocks of many organisms. We develop a mathematical model of the Drosophila circadian clock that incorporates the emerging details of the timing of nuclear translocation of the PERIOD and TIMELESS proteins. Most models assume that these proteins enter the nucleus as a complex, but recent experiments suggest that they in fact enter the nucleus separately. Our model reproduces observed patterns of intracellular localization of PERIOD and TIMELESS during light–dark cycles and in constant darkness, as well as phenotypes of several clock mutants. We also use the model to demonstrate how the Drosophila clock can exhibit robust oscillations with constant mRNA levels of period or timeless, and propose a possible mechanism for oscillations in double-rescue experiments of per 01 – tim 01 mutants. The model also explains (via posttranslational processes) the counter-intuitive observation that total dCLOCK levels are at their lowest at the circadian time when active nuclear dCLOCK must be peaking in order to activate transcription of other clock genes, implying that for dCLOCK a posttranslationally generated rhythm is more important than the transcriptionally generated rhythm. These results support the idea that posttranslational processes play key roles in generating as well as modulating robust circadian oscillations. While it appears that posttranslational mechanisms alone are not sufficient to generate rhythms in Drosophila, posttranslational mechanisms can greatly amplify a very weak transcriptional rhythm.