Clock and cycle limit starvation-induced sleep loss in Drosophila.
ABSTRACT: Neural systems controlling the vital functions of sleep and feeding in mammals are tightly interconnected: sleep deprivation promotes feeding, whereas starvation suppresses sleep. Here we show that starvation in Drosophila potently suppresses sleep, suggesting that these two homeostatically regulated behaviors are also integrated in flies. The sleep-suppressing effect of starvation is independent of the mushroom bodies, a previously identified sleep locus in the fly brain, and therefore is regulated by distinct neural circuitry. The circadian clock genes Clock (Clk) and cycle (cyc) are critical for proper sleep suppression during starvation. However, the sleep suppression is independent of light cues and of circadian rhythms as shown by the fact that starved period mutants sleep like wild-type flies. By selectively targeting subpopulations of Clk-expressing neurons, we localize the observed sleep phenotype to the dorsally located circadian neurons. These findings show that Clk and cyc act during starvation to modulate the conflict of whether flies sleep or search for food.
Project description:Circadian oscillators are autonomous molecular rhythms that reside in cells to align whole-organism physiology and behavior to the 24-h day. In flies, as in mammals, the oscillator operates in cells that coexpress CLOCK (CLK) and CYCLE (CYC). Recent work in Drosophila has shown that CLK is unique in its ability to generate heterologous oscillators, indicating that Clk gene expression defines the circadian cell fate. Here, using standard in vitro and in vivo techniques, we show that TWIN-OF-EYELESS (TOY; dPax6) regulates Clk expression in small ventrolateral neurons (s-LNvs) that coordinate sleep-wake cycles. Crucially, toy binds multiple sites at the Clk locus, is expressed independent of CLK-CYC in LNvs, regulates CLK protein levels under optimal photoperiodic conditions, and sets clock-speed during endogenous free-run. Furthermore, TOY is necessary for the onset of Clk expression in LNvs during embryogenesis. We propose that TOY contributes to a transcription complex that functions upstream of the oscillator to promote Clk expression in s-LNvs.
Project description:The Clock-Cycle (CLK-CYC) heterodimer constitutes a key circadian transcription complex in Drosophila. CYC has a DNA-binding domain but lacks an activation domain. Previous experiments also indicate that most of the transcriptional activity of CLK-CYC derives from the glutamine-rich region of its partner CLK. To address the role of transcription in core circadian timekeeping, we have analyzed the effects of a CYC-viral protein 16 (VP16) fusion protein in the Drosophila system. The addition of this potent and well-studied viral transcriptional activator (VP16) to CYC imparts to the CLK-CYC-VP16 complex strongly enhanced transcriptional activity relative to that of CLK-CYC. This increase is manifested in flies expressing CYC-VP16 as well as in S2 cells. These flies also have increased levels of CLK-CYC direct target gene mRNAs as well as a short period, implicating circadian transcription in period determination. A more detailed examination of reporter gene expression in CYC-VP16-expressing flies suggests that the short period is due at least in part to a more rapid transcriptional phase. Importantly, the behavioral effects require a period (per) promoter and are therefore unlikely to be merely a consequence of generally higher PER levels. This indicates that the CLK-CYC-VP16 behavioral effects are a consequence of increased per transcription. All of this also suggests that the timing of transcriptional activation and not the activation itself is the key event responsible for the behavioral effects observed in CYC-VP16-expressing flies. The results taken together indicate that circadian transcription contributes to core circadian function in Drosophila.
Project description:The Drosophila circadian oscillator controls daily rhythms in physiology, metabolism and behavior via transcriptional feedback loops. CLOCK-CYCLE (CLK-CYC) heterodimers initiate feedback loop function by binding E-box elements to activate per and tim transcription. PER-TIM heterodimers then accumulate, bind CLK-CYC to inhibit transcription, and are ultimately degraded to enable the next round of transcription. The timing of transcriptional events in this feedback loop coincide with, and are controlled by, rhythms in CLK-CYC binding to E-boxes. PER rhythmically binds CLK-CYC to initiate transcriptional repression, and subsequently promotes the removal of CLK-CYC from E-boxes. However, little is known about the mechanism by which CLK-CYC is removed from DNA. Previous studies demonstrated that the transcription repressor CLOCKWORK ORANGE (CWO) contributes to core feedback loop function by repressing per and tim transcription in cultured S2 cells and in flies. Here we show that CWO rhythmically binds E-boxes upstream of core clock genes in a reciprocal manner to CLK, thereby promoting PER-dependent removal of CLK-CYC from E-boxes, and maintaining repression until PER is degraded and CLK-CYC displaces CWO from E-boxes to initiate transcription. These results suggest a model in which CWO co-represses CLK-CYC transcriptional activity in conjunction with PER by competing for E-box binding once CLK-CYC-PER complexes have formed. Given that CWO orthologs DEC1 and DEC2 also target E-boxes bound by CLOCK-BMAL1, a similar mechanism may operate in the mammalian clock.
Project description:Many organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila, the master clock gene Clock promotes the transcription of several key target genes. Two of these gene products, PER and TIM, repress CLK-CYC-mediated transcription. To recognize additional direct CLK target genes, we designed a genome-wide approach and identified clockwork orange (cwo) as a new core clock component. cwo encodes a transcriptional repressor that synergizes with PER and inhibits CLK-mediated activation. Consistent with this function, the mRNA profiles of CLK direct target genes in cwo mutant flies manifest high trough values and low amplitude oscillations. Because behavioral rhythmicity fails to persist in constant darkness (DD) with little or no effect on average mRNA levels in flies lacking cwo, transcriptional oscillation amplitude appears to be linked to rhythmicity. Moreover, the mutant flies are long period, consistent with the late repression indicated by the RNA profiles. These findings suggest that CWO acts preferentially in the late night to help terminate CLK-CYC-mediated transcription of direct target genes including cwo itself. The presence of mammalian homologs with circadian expression features (Dec1 and Dec2) suggests that a similar feedback mechanism exists in mammalian clocks.
Project description:Circadian oscillator networks rely on a transcriptional activator called CLOCK/CYCLE (CLK/CYC) in insects and CLOCK/BMAL1 or NPAS2/BMAL1 in mammals. Identifying the targets of this heterodimeric basic-helix-loop-helix (bHLH) transcription factor poses challenges and it has been difficult to decipher its specific sequence affinity beyond a canonical E-box motif, except perhaps for some flanking bases contributing weakly to the binding energy. Thus, no good computational model presently exists for predicting CLK/CYC, CLOCK/BMAL1, or NPAS2/BMAL1 targets. Here, we use a comparative genomics approach and first study the conservation properties of the best-known circadian enhancer: a 69-bp element upstream of the Drosophila melanogaster period gene. This fragment shows a signal involving the presence of two closely spaced E-box-like motifs, a configuration that we can also detect in the other four prominent CLK/CYC target genes in flies: timeless, vrille, Pdp1, and cwo. This allows for the training of a probabilistic sequence model that we test using functional genomics datasets. We find that the predicted sequences are overrepresented in promoters of genes induced in a recent study by a glucocorticoid receptor-CLK fusion protein. We then scanned the mouse genome with the fly model and found that many known CLOCK/BMAL1 targets harbor sequences matching our consensus. Moreover, the phase of predicted cyclers in liver agreed with known CLOCK/BMAL1 regulation. Taken together, we built a predictive model for CLK/CYC or CLOCK/BMAL1-bound cis-enhancers through the integration of comparative and functional genomics data. Finally, a deeper phylogenetic analysis reveals that the link between the CLOCK/BMAL1 complex and the circadian cis-element dates back to before insects and vertebrates diverged.
Project description:Circadian clocks control many self-sustained rhythms in physiology and behavior with approximately 24-hour periodicity. In many organisms, oxidative stress and aging negatively impact the circadian system and sleep. Conversely, loss of the clock decreases resistance to oxidative stress, and may reduce lifespan and speed up brain aging and neurodegeneration. Here we examined the effects of clock disruptions on locomotor aging and longevity in Drosophila. We found that lifespan was similarly reduced in three arrhythmic mutants (ClkAR, cyc0 and tim0) and in wild-type flies under constant light, which stops the clock. In contrast, ClkAR mutants showed significantly faster age-related locomotor deficits (as monitored by startle-induced climbing) than cyc0 and tim0, or than control flies under constant light. Reactive oxygen species accumulated more with age in ClkAR mutant brains, but this did not appear to contribute to the accelerated locomotor decline of the mutant. Clk, but not Cyc, inactivation by RNA interference in the pigment-dispersing factor (PDF)-expressing central pacemaker neurons led to similar loss of climbing performance as ClkAR. Conversely, restoring Clk function in these cells was sufficient to rescue the ClkAR locomotor phenotype, independently of behavioral rhythmicity. Accelerated locomotor decline of the ClkAR mutant required expression of the PDF receptor and correlated to an apparent loss of dopaminergic neurons in the posterior protocerebral lateral 1 (PPL1) clusters. This neuronal loss was rescued when the ClkAR mutation was placed in an apoptosis-deficient background. Impairing dopamine synthesis in a single pair of PPL1 neurons that innervate the mushroom bodies accelerated locomotor decline in otherwise wild-type flies. Our results therefore reveal a novel circadian-independent requirement for Clk in brain circadian neurons to maintain a subset of dopaminergic cells and avoid premature locomotor aging in Drosophila.
Project description:The Drosophila circadian oscillator is comprised of transcriptional feedback loops that are activated by CLOCK (CLK) and CYCLE (CYC) and repressed by PERIOD (PER) and TIMELESS (TIM) . The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM [2-4]. Although kinases that control PER and TIM levels and subcellular localization have been identified [5-10], additional kinases are predicted to target PER, TIM, and/or CLK to promote time-specific transcriptional repression. We screened for kinases that alter circadian behavior via clock cell-directed RNA interference (RNAi) and identified the proline-directed kinase nemo (nmo) as a novel component of the circadian oscillator. Both nmo RNAi knockdown and a nmo hypomorphic mutant shorten circadian period, whereas nmo overexpression lengthens circadian period. CLK levels increase when nmo expression is knocked down in clock cells, whereas CLK levels decrease and PER and TIM accumulation are delayed when nmo is overexpressed in clock cells. These data suggest that nmo slows the pace of the circadian oscillator by altering CLK, PER, and TIM expression, thereby contributing to the generation of an ~24 hr circadian period.
Project description:Insect circadian rhythms are generated by a circadian clock consisting of transcriptional/translational feedback loops, in which CYCLE and CLOCK are the key elements in activating the transcription of various clock genes such as timeless (tim) and period (per). Although the transcriptional regulation of Clock (Clk) has been profoundly studied, little is known about the regulation of cycle (cyc). Here, we identify the orphan nuclear receptor genes HR3 and E75, which are orthologs of mammalian clock genes, Ror? and Rev-erb?, respectively, as factors involved in the rhythmic expression of the cyc gene in a primitive insect, the firebrat Thermobia domestica. Our results show that HR3 and E75 are rhythmically expressed, and their normal, rhythmic expression is required for the persistence of locomotor rhythms. Their RNAi considerably altered the rhythmic transcription of not only cyc but also tim. Surprisingly, the RNAi of HR3 revealed the rhythmic expression of Clk, suggesting that this ancestral insect species possesses the mechanisms for rhythmic expression of both cyc and Clk genes. When either HR3 or E75 was knocked down, tim, cyc, and Clk or tim and cyc, respectively, oscillated in phase, suggesting that the two genes play an important role in the regulation of the phase relationship among the clock genes. Interestingly, HR3 and E75 were also found to be involved in the regulation of ecdysis, suggesting that they interconnect the circadian clock and developmental processes.
Project description:Circadian clocks have evolved as internal time keeping mechanisms that allow anticipation of daily environmental changes and organization of a daily program of physiological and behavioral rhythms. To better examine the mechanisms underlying circadian clocks in animals and to ask whether clock gene expression and function during development affected subsequent daily time keeping in the adult, we used the genetic tools available in Drosophila to conditionally manipulate the function of the CYCLE component of the positive regulator CLOCK/CYCLE (CLK/CYC) or its negative feedback inhibitor PERIOD (PER). Differential manipulation of clock function during development and in adulthood indicated that there is no developmental requirement for either a running clock mechanism or expression of per. However, conditional suppression of CLK/CYC activity either via per over-expression or cyc depletion during metamorphosis resulted in persistent arrhythmic behavior in the adult. Two distinct mechanisms were identified that may contribute to this developmental function of CLK/CYC and both involve the ventral lateral clock neurons (LN(v)s) that are crucial to circadian control of locomotor behavior: (1) selective depletion of cyc expression in the LN(v)s resulted in abnormal peptidergic small-LN(v) dorsal projections, and (2) PER expression rhythms in the adult LN(v)s appeared to be affected by developmental inhibition of CLK/CYC activity. Given the conservation of clock genes and circuits among animals, this study provides a rationale for investigating a possible similar developmental role of the homologous mammalian CLOCK/BMAL1 complex.
Project description:The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tiling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation.