Clock: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Clock

Synonyms -

Cytological map position - 66A5-66A12

Function - transcription factor

Keywords - photoperiod response, brain

Symbol - Clk

FlyBase ID:FBgn0023076

Genetic map position - 3-

Classification - bHLH and PAS domain

Cellular location - nuclear and cytoplasmic

NCBI links: Entrez Gene

Clock orthologs: Biolitmine
Recent literature
Liu, T., Mahesh, G., Houl, J. H. and Hardin, P. E. (2015). Circadian activators are expressed days before they initiate clock function in late pacemaker neurons from Drosophila. J Neurosci 35: 8662-8671. PubMed ID: 26041931
Circadian pacemaker neurons in the Drosophila brain control daily rhythms in locomotor activity. These pacemaker neurons can be subdivided into early or late groups depending on whether rhythms in period (per) and timeless (tim) expression are initiated at the first instar (L1) larval stage or during metamorphosis, respectively. Because CLOCK-CYCLE (CLK-CYC) heterodimers initiate circadian oscillator function by activating per and tim transcription, a Clk-GFP transgene was used to mark when late pacemaker neurons begin to develop. It was surprising to see that CLK-GFP was already expressed in four of five clusters of late pacemaker neurons during the third instar (L3) larval stage. CLK-GFP is only detected in postmitotic neurons from L3 larvae, suggesting that these four late pacemaker neuron clusters are formed before the L3 larval stage. A GFP-cyc transgene was used to show that CYC, like CLK, is also expressed exclusively in pacemaker neurons from L3 larval brains, demonstrating that CLK-CYC is not sufficient to activate per and tim in late pacemaker neurons at the L3 larval stage. These results suggest that most late pacemaker neurons develop days before novel factors activate circadian oscillator function during metamorphosis.
Lee, E., Cho, E., Kang, D. H., Jeong, E. H., Chen, Z., Yoo, S. H. and Kim, E. Y. (2016). Pacemaker-neuron-dependent disturbance of the molecular clockwork by a Drosophila CLOCK mutant homologous to the mouse Clock mutation. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27489346
Circadian clocks are composed of transcriptional/translational feedback loops (TTFLs) at the cellular level. In Drosophila TTFLs, the transcription factor Clock (Clk)/Cycle (Cyc) activates clock target gene expression, which is repressed by the physical interaction with Period (Per). This study shows that amino acids (AA) 657-707 of Clk, a region that is homologous to the mouse Clock exon 19-encoded region, is crucial for Per binding and E-box-dependent transactivation in S2 cells. Consistently, in transgenic flies expressing Clk with an AA657-707 deletion in the Clock (Clkout) genetic background (p{Clk-Δ};Clkout), oscillation of core clock genes' mRNAs displayed diminished amplitude compared with control flies, and the highly abundant dCLK&Delta657-707 showed significantly decreased binding to Per. Behaviorally, the p{dClk-Δ};Clkout flies exhibited arrhythmic locomotor behavior in the photic entrainment condition but showed anticipatory activities of temperature transition and improved free-running rhythms in the temperature entrainment condition. Surprisingly, p{dClk-Δ};Clkout flies showed pacemaker-neuron-dependent alterations in molecular rhythms; the abundance of dCLK target clock proteins was reduced in ventral lateral neurons (LNvs) but not in dorsal neurons (DNs) in both entrainment conditions. In p{dClk-Delta};Clkout flies, however, strong but delayed molecular oscillations in temperature cycle-sensitive pacemaker neurons, such as DN1s and DN2s, were correlated with delayed anticipatory activities of temperature transition. Taken together, this study reveals that the LNv molecular clockwork is more sensitive than the clockwork of DNs to dysregulation of dCLK by AA657-707 deletion. Therefore, it is proposed that the dCLK/CYC-controlled TTFL operates differently in subsets of pacemaker neurons, which may contribute to their specific functions.
Cho, E., Lee, E. and Kim, E. Y. (2016). Diversification of molecular clockwork for tissue specific function: insight from novel Drosophila Clock mutant homologous to mouse Clock allele. BMB Rep 49(11):587-589. PubMed ID: 27756446
The circadian clock system enables organisms to anticipate the rhythmic environmental changes and to manifest behavior and physiology at advantageous times of day. Transcriptional/translational feedback loop (TTFL) is the basic feature of eukaryotic circadian clock and is based on the rhythmic association of circadian transcriptional activator and repressor. In Drosophila, repression of dCLOCK/CYCLE (dCLK/CYC) mediated transcription by PERIOD (PER) is critical for inducing circadian rhythms of gene expression. Pacemaker neurons in the brain control specific circadian behaviors upon environmental timing cues such as light and temperature cycle. This study shows here that amino acids 657-707 of dCLK is important for the transcriptional activation and the association with PER both in vitro and in vivo. Flies expressing dCLK lacking AA657-707 in Clkout genetic background, homologous to the mouse Clock allele where exon 19 region is deleted, display pacemaker-neuron-dependent perturbation of the molecular clockwork. Namely, the molecular rhythms in light-cycle-sensitive pacemaker neurons such as ventral lateral neurons (LNvs) were significantly disrupted but those in temperature-cycle-sensitive pacemaker neurons such as dorsal neurons (DNs) were robust. These results suggest that the dCLK-controlled TTFL diversified in pacemaker-neuron-dependent manner which contribute to specific functions such as different sensitivities to entraining cues.
Vaccaro, A., Issa, A. R., Seugnet, L., Birman, S. and Klarsfeld, A. (2017). Drosophila Clock is required in brain pacemaker neurons to prevent premature locomotor aging independently of its circadian function. PLoS Genet 13(1): e1006507. PubMed ID: 28072817
Circadian clocks control many self-sustained rhythms in physiology and behavior with approximately 24-hour periodicity. This study examined the effects of clock disruptions on locomotor aging and longevity in Drosophila. Lifespan was found to be 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. 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. These 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.
Nippe, O. M., Wade, A. R., Elliott, C. J. H. and Chawla, S. (2017). Circadian rhythms in visual responsiveness in the behaviorally arrhythmic Drosophila clock mutant Clk(Jrk). J Biol Rhythms 32(6):583-592. PubMed ID: 29172879
An organism's biological day is characterized by a pattern of anticipatory physiological and behavioral changes that are governed by circadian clocks to align with the 24-h cycling environment. This study used flash electroretinograms (ERGs) and steady-state visually evoked potentials (SSVEPs) to examine how visual responsiveness in wild-type Drosophila melanogaster and the circadian clock mutant ClkJrk varies over circadian time. The ERG parameters of wild-type flies vary over the circadian day, with a higher luminance response during the subjective night. The SSVEP response that assesses contrast sensitivity also showed a time-of-day dependence, including 2 prominent peaks within a 24-h period and a maximal response at the end of the subjective day, indicating a tradeoff between luminance and contrast sensitivity. Moreover, the behaviorally arrhythmic ClkJrk mutants maintained a circadian profile in both luminance and contrast sensitivity, but unlike the wild-types, which show bimodal profiles in their visual response, ClkJrk flies show a weakening of the bimodal character, with visual responsiveness tending to peak once a day. It is concluded that the ClkJrk mutation mainly affects 1 of 2 functionally coupled oscillators and that the visual system is partially separated from the locomotor circadian circuits that drive bouts of morning and evening activity. As light exposure is a major mechanism for entrainment, this work suggests that a detailed temporal analysis of electrophysiological responses is warranted to better identify the time window at which circadian rhythms are most receptive to light-induced phase shifting.
Liu, T., Mahesh, G., Yu, W. and Hardin, P. E. (2017). CLOCK stabilizes CYCLE to initiate clock function in Drosophila. Proc Natl Acad Sci U S A 114(41): 10972-10977. PubMed ID: 28973907
The Drosophila circadian clock keeps time via transcriptional feedback loops. These feedback loops are initiated by CLOCK-CYCLE (CLK-CYC) heterodimers, which activate transcription of genes encoding the feedback repressors PERIOD and TIMELESS. Circadian clocks normally operate in approximately 150 brain pacemaker neurons and in many peripheral tissues in the head and body, but can also be induced by expressing CLK in nonclock cells. These ectopic clocks also require cyc, yet CYC expression is restricted to canonical clock cells despite evidence that cyc mRNA is widely expressed. This study shows that CLK binds to and stabilizes CYC in cell culture and in nonclock cells in vivo. Ectopic clocks also require the blue light photoreceptor CRYPTOCHROME (CRY), which is required for both light entrainment and clock function in peripheral tissues. These experiments define the genetic architecture required to initiate circadian clock function in Drosophila, reveal mechanisms governing circadian activator stability that are conserved in perhaps all eukaryotes, and suggest that Clk, cyc, and cry expression is sufficient to drive clock expression in naive cells.
Du, J., Zhang, Y., Xue, Y., Zhao, X., Zhao, X., Wei, Y., Li, Z., Zhang, Y. and Zhao, Z. (2018). Diurnal protein oscillation profiles in Drosophila head. FEBS Lett. PubMed ID: 30311939
Circadian clocks control daily rhythms in physiology, metabolism, and behavior in most organisms. Proteome-wide analysis of protein oscillations is still lacking in Drosophila. In this study, the total protein and phosphorylated protein in Drosophila heads in a 24-hour daily time-course were assayed by using the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) method, and 10 and 7 oscillating proteins as well as 19 and 22 oscillating phosphoproteins in the w(1118) wild type and Clk(Jrk) mutant strains were separately identified. Lastly, a mini screen was performed to investigate the functions of some oscillating proteins in circadian locomotion rhythms. This study provides the first proteomic profiling of diurnally oscillating proteins in fly heads, thereby providing a basis for further mechanistic studies of these proteins in circadian rhythm.
Cho, E., Kwon, M., Jung, J., Hyun Kang, D., Jin, S., Choi, S. E., Kang, Y. and Kim, E. Y. (2019). AMP-activated protein kinase regulates circadian rhythm by affecting CLOCK in Drosophila. J Neurosci. PubMed ID: 30819799
The circadian clock organizes the physiology and behavior of organisms to their daily environmental rhythms. The central circadian timekeeping mechanism in eukaryotic cells is the transcriptional-translational feedback loop (TTFL). In the Drosophila TTFL, the transcription factors CLOCK (CLK) and CYCLE (CYC) play crucial roles in activating expression of core clock genes and clock-controlled genes. Many signaling pathways converge on the CLK/CYC complex and regulate its activity to fine-tune the cellular oscillator to environmental time cues. This study aimed to identify factors that regulate CLK by performing tandem affinity purification (TAP) combined with mass spectrometry (MS) using Drosophila S2 cells that stably express HA/FLAG-tagged CLK and V5-tagged CYC. SNF4Agamma, a homolog of mammalian AMP-activated protein kinase gamma (AMPKgamma), was identified as a factor that co-purified with HA/FLAG-tagged CLK. The AMPK holoenzyme composed of a catalytic subunit AMPKalpha and two regulatory subunits, AMPKbeta and AMPKgamma, directly phosphorylated purified CLK in vitro. Locomotor behavior analysis in Drosophila revealed that knockdown of each AMPK subunit in pacemaker neurons induced arrhythmicity and long periods. Knockdown of AMPKbeta reduced CLK levels in pacemaker neurons, and thereby reduced pre-mRNA and protein levels of CLK downstream core clock genes such as period and vrille. Finally, overexpression of CLK reversed the long-period phenotype that resulted from AMPKbeta knockdown. Thus, it is concluded that AMPK, a central regulator of cellular energy metabolism, regulates the Drosophila circadian clock by stabilizing CLK and activating CLK/CYC-dependent transcription.
Mahesh, G., Rivas, G. B. S., Caster, C., Ost, E. B., Amunugama, R., Jones, R., Allen, D. L. and Hardin, P. E. (2020). Proteomic analysis of Drosophila CLOCK complexes identifies rhythmic interactions with SAGA and Tip60 complex component NIPPED-A. Sci Rep 10(1): 17951. PubMed ID: 33087840
Circadian clocks keep time via ~ 24 h transcriptional feedback loops. In Drosophila, CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors are feedback loop components whose transcriptional status varies over a circadian cycle. This study used mass spectrometry to identify proteins that interact with GFP-tagged CLK (GFP-CLK) in fly heads at different times of day. Many expected and novel interacting proteins were detected, of which several interacted rhythmically and were potential regulators of protein levels, activity or transcriptional output. Genes encoding these proteins were tested to determine if they altered circadian behavior via RNAi knockdown in clock cells. The NIPPED-A protein, a scaffold for the SAGA and Tip60 histone modifying complexes, interacts with GFP-CLK as transcription is activated, and reducing Nipped-A expression lengthens circadian period. RNAi analysis of other SAGA complex components shows that the SAGA histone deubiquitination (DUB; see Non-stop) module lengthened period similarly to Nipped-A RNAi knockdown and weakened rhythmicity, whereas reducing Tip60 HAT expression drastically weakened rhythmicity. These results suggest that CLK-CYC binds NIPPED-A early in the day to promote transcription through SAGA DUB and Tip60 HAT activity.
Xia, X., Fu, X., Du, J., Wu, B., Zhao, X., Zhu, J. and Zhao, Z. (2020). Regulation of circadian rhythm and sleep by miR-375-timeless interaction in Drosophila. Faseb J. PubMed ID: 33078445
MicroRNAs are important coordinators of circadian regulation that mediate the fine-tuning of gene expression. The global functional miRNA-mRNA interaction network involved in the circadian system remains poorly understood. This study used CLEAR (Covalent Ligation of Endogenous Argonaute-bound RNAs)-CLIP (Cross-Linking and Immuno-Precipitation) to explore the regulatory functions of miRNAs in the circadian system by comparing the miRNA-mRNA interactions between Drosophila wild-type strain W(1118) and a mutant of the key circadian transcriptional regulator Clock (Clk(jrk)). This experimental approach unambiguously identified tens of thousands of miRNA-mRNA interactions in both the head and body. The miRNA-mRNA interactome showed dramatic changes in the Clk(jrk) flies. Particularly, among ~300 miRNA-mRNA circadian relevant interactions, multiple interactions involving core clock genes pdp1, tim, and vri displayed distinct changes as a result of the Clk mutation. Based on the CLEAR-CLIP analysis, this study found a novel regulation of the circadian rhythm and sleep by the miR-375-timeless interaction. The results indicated that Clk disruption abolished normal rhythmic expression of miR-375 and the functional regulation occurred in the l-LNv neurons, where miR-375 modulated the circadian rhythm and sleep via targeting timeless. This work provides the first global view of miRNA regulation in the circadian rhythm.
Xiao, Y., Yuan, Y., Jimenez, M., Soni, N. and Yadlapalli, S. (2021). Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms. Proc Natl Acad Sci U S A 118(28). PubMed ID: 34234015
Circadian clocks regulate ∼24-h oscillations in gene expression, behavior, and physiology. This study has elucidated how spatiotemporal organization and dynamics of core clock proteins and genes affect circadian rhythms in Drosophila clock neurons. Using high-resolution imaging and DNA-fluorescence in situ hybridization techniques, it was demonstrate that Drosophila clock proteins (PERIOD and CLOCK) are organized into a few discrete foci at the nuclear envelope during the circadian repression phase and play an important role in the subnuclear localization of core clock genes to control circadian rhythms. Specifically, this study showed that core clock genes, period and timeless, are positioned close to the nuclear periphery by the PERIOD protein specifically during the repression phase, suggesting that subnuclear localization of core clock genes might play a key role in their rhythmic gene expression. Finally, loss of Lamin B receptor, a nuclear envelope protein, was shown to leads to disruption of PER foci and per gene peripheral localization and results in circadian rhythm defects. These results demonstrate that clock proteins play a hitherto unexpected role in the subnuclear reorganization of core clock genes to control circadian rhythms, revealing how clocks function at the subcellular level. The results further suggest that clock protein foci might regulate dynamic clustering and spatial reorganization of clock-regulated genes over the repression phase to control circadian rhythms in behavior and physiology.
Rivas, G. B. S., Zhou, J., Merlin, C. and Hardin, P. E. (2021). CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the putative Drosophila ortholog of CLOCK INTERACTING PROTEIN CIRCADIAN. Curr Biol. PubMed ID: 34331859
The Drosophila circadian clock is driven by a transcriptional feedback loop in which CLOCK-CYCLE (CLK-CYC) binds E-boxes to transcribe genes encoding the PERIOD-TIMELESS (PER-TIM) repressor, which releases CLK-CYC from E-boxes to inhibit transcription. CLOCKWORK ORANGE (CWO) reinforces PER-TIM repression by binding E-boxes to maintain PER-TIM bound CLK-CYC off DNA, but also promotes CLK-CYC transcription through an unknown mechanism. To determine how CWO activates CLK-CYC transcription, CWO target genes were identified that are upregulated in the absence of CWO repression, conserved in mammals, and preferentially expressed in brain pacemaker neurons. Among the genes identified was a putative ortholog of mouse Clock Interacting Protein Circadian (Cipc), which represses CLOCK-BMAL1 transcription. Reducing or eliminating Drosophila Cipc expression shortens period, while overexpressing Cipc lengthens period. Cipc represses CLK-CYC transcription in vivo, but not uniformly, as per is strongly repressed, tim less so, and vri hardly at all. Long period rhythms in cwo mutant flies are largely rescued when Cipc expression is reduced or eliminated, indicating that increased Cipc expression mediates the period lengthening of cwo mutants. These results suggest a mechanism for CWO-dependent CLK-CYC activation: CWO inhibition of CIPC repression promotes CLK-CYC transcription. This mechanism may be conserved since cwo and Cipc perform analogous roles in the mammalian circadian clock.

In Drosophila, there are two well-characterized photoperiod response genes: period (per) and timeless (tim). The protein levels, RNA levels, and transcription rates of these two genes undergo robust circadian oscillations. In addition, mutations in the two proteins (Per and Tim) alter or abolish the periodicity and phase of these rhythms, demonstrating that both proteins regulate their own transcription. Although there is no evidence indicating that the effects on transcription are direct, Per contains a PAS domain, which has been shown to mediate interactions between transcription factors. Most of these PAS-containing transcription factors also contain well-characterized basic helix-loop-helix (bHLH) DNA-binding domains. However, Per lacks any known DNA-binding domain, and there is no evidence that Per interacts directly with DNA. Therefore it was proposed that Per regulates transcription by interacting with DNA-binding transcription factors of the bHLH-PAS family and how Per transcription is regulated remained an open question (Allada, 1998 and references).

Recent data have extended this model in two ways: (1) an enhancer has been identified in the per promoter capable of driving cycling transcription of a reporter gene (Hao, 1997). Notably, the activity of this 69-base pair element requires an E box (CACGTG), a known binding site for some bHLH transcription factors, including bHLH-PAS transcription factors. (2) The cloning of the mouse circadian rhythm gene, mClock, revealed a bHLH-PAS transcription factor involved in circadian rhythms. Recently, mouse per genes have been identified and found to undergo circadian oscillation in mammalian clock tissues. Thus, mouse CLOCK may drive the cycling transcription of mouse per genes through evolutionarily conserved E box elements in mouse per promoters. If so, one might expect to find a Drosophila orthologs of mClock, which would drive cycling of the Drosophila per gene (Allada, 1998).

Jrk, a novel arrhythmic Drosophila mutant, has been identified which severely disrupts cycling transcription of the per and tim genes. The cloning and identification of the Jrk gene reveals that it is the apparent homolog of the mouse Clock gene; it has therefore been named Drosophila Clock (Clk). The mutation in Clk results in a premature stop codon that truncates the protein, deleting most of the putative C-terminal activation domain. This truncation is consistent with the semidominant mutant phenotype, similar to the original mouse Clock mutant (Allada, 1998).

A second Drosophila clock gene, cycle (cyc) has also been cloned. Homozygous mutant cyc flies have a behavioral and molecular phenotype that resembles closely that of homozygous Clk flies: they are behaviorally arrhythmic and exhibit little per and tim transcription. Further phenogenetic analyses indicate that, like Clk, the cyc locus has a dosage effect on period. It is suggested that cycle is a nonvital, dedicated clock gene. Cloning of cyc indicates that, like Clk, it encodes a bHLH-PAS transcription factor and is a Drosophila homolog of the human gene BMAL1 (MOP3) (Ikeda, 1997; Hogenesch, 1997 and 1998). Biochemical work (Hogenesch, 1998) indicates that BMAL1 is the partner of mammalian CLOCK and that the heterodimer binds to and activates transcription from per-like E boxes. Based on all of these results, it is proposed that the CYC:CLK heterodimer binds to per and tim E boxes and makes a major contribution to the circadian transcription of Drosophila clock genes (Rutila, 1998). Further characterization of the Drosophila Clk mutant phenotype and a second study (Hogenesch, 1998) suggest that the wild-type Drosophila protein (Clk) interacts directly with the per and tim E boxes and makes a major contribution to the circadian transcription of clock genes. The similar mouse mutant phenotype and the remarkable sequence conservation strongly support the presence of similar clock mechanisms and components in the common ancestor of Drosophila and mammals more than 500 million years ago (Allada, 1998).

Placed in the context of the current understanding of the Drosophila circadian oscillator, these results indicate that Clock closes the feedback loop. A Clock-Cycle complex drives expression of per and tim by binding an E-box that is present in their promoters. With time, Per and Tim heterodimers accumulate, translocate to the nucleus, and act as dominant negative inhibitors of Clock-Cycle. As mRNA and protein levels fall, the inhibition is relieved, which allows Clock-Cycle to initiate a new round of synthesis (Darlington, 1998).

Drosophila Clock (Clk) is rhythmically expressed, with peaks in mRNA and protein (Clk) abundance early in the morning. Clk mRNA cycling is regulated by Period-Timeless (Per-Tim)-mediated release of Clk- and Cycle (Cyc)-dependent repression. Lack of both Per-Tim derepression and Clk-Cyc repression results in high levels of Clk mRNA, which implies that a separate Clk activator is present. These results demonstrate that the Drosophila circadian feedback loop is composed of two interlocked negative feedback loops: a per-tim loop, which is activated by Clk-Cyc and repressed by Per-Tim, and a Clk loop, which is repressed by Clk-Cyc and derepressed by Per-Tim (Glossop, 1999).

Comparatively little is known about the regulation of Clk mRNA cycling. The levels of Clk mRNA are low in mutants lacking Per (per01) or Tim (tim01) function, which suggests that Per and Tim activate Clk transcription in addition to their roles as transcriptional repressors. The mechanism of Per-Tim-dependent activation is not known, but three models have been proposed to account for this activation. In the first two models, Per and Tim promote Clk transcription by shuttling transcriptional activators into the nucleus or by coactivating a transcriptional complex. In the third model, Per or Tim or both inhibit the activity of a transcriptional repressor complex (Glossop, 1999).

To distinguish among these alternative models, Clk mRNA levels were measured in different clock gene mutant combinations. Because Clk and Cyc are both required for per and tim activation, it was predicted that mutants lacking functional Clk (ClkJrk) or Cyc (Cyc0) would exhibit low levels of Clk mRNA because the concentrations of the Per and Tim activators (of Clk) would be low. It was surprising to find that the level of Clk mRNA is indistinguishable from the wild-type peak in both mutants. The levels of Clk mRNA do not vary significantly over the circadian cycle in these mutants, which is consistent with the lack of a functional circadian oscillator (Glossop, 1999).

The high level of Clk mRNA in the absence of Clk-dependent Per accumulation indicates that Per-dependent Clk activation does not occur by nuclear localization of an activator or by coactivation. However, the possibility remains that low levels of per and tim transcripts in ClkJrk or Cyc0 mutants lead to some active Per-Tim dimer formation and subsequent activation of Clk transcription. To eliminate this possibility, Clk mRNA levels were measured in per01;ClkJrk and per01;Cyc0 double mutants. In both cases, the levels of Clk mRNA observed under light-dark (LD) or constant dark (DD) conditions are close to the peak level in wild-type flies, indicating that Per-Tim activates Clk transcription through derepression (Glossop, 1999).

The Clk repressor that is removed as a result of Per-Tim accumulation appears to be either Clk-Cyc itself or a repressor that is activated by Clk-Cyc. When comparing the levels of Clk between per01 flies and per01;ClkJrk or per01;Cyc0 double mutants, the presence of active Clk and Cyc results in the repression of Clk transcript accumulation. In per01 mutants, Clk mRNA is at low but detectable levels. This suggests that in the absence of Per-Tim derepression, Clk transcription reaches a steady state in which activation and Clk-Cyc-dependent repression equilibrate to produce low levels of Clk mRNA transcripts and, hence, of Clk protein. In per01 and tim01 mutants, per and tim transcription is constitutive and per and tim transcripts are relatively low in abundance. This result can be explained by the partial activation of per and tim by low levels of Clk-Cyc dimers in the absence of Per-Tim repression (Glossop, 1999).

On the basis of these observations, it is proposed that interlocked negative feedback loops mediate circadian oscillator function in Drosophila (see reviews by Hardin, The Circadian Timekeeping System of Drosophila and Vallone, Start the clock! Circadian rhythms and development). Late at night, Per-Tim dimers in the nucleus bind to and sequester Clk-Cyc dimers. This interaction effectively inhibits Clk-Cyc function, which leads to the repression of per and tim transcription and the derepression of Clk transcription. As Per-Tim levels fall early in the morning (ZT 0-3), Clk-Cyc dimers are released and repress Clk expression, thereby decreasing Clk mRNA levels so that they are low by the end of the day (ZT 12). Concomitant with the drop in Clk mRNA levels (through Clk-Cyc-dependent repression) is the accumulation of per and tim mRNA (through E-box-dependent Clk-Cyc activation). As Clk mRNA falls to low levels early in the evening (ZT 15), the levels of Clk-Cyc also fall, leading to a decrease in per and tim transcription and an increase in Clk mRNA accumulation. A new cycle then begins as high levels of Per and Tim enter the nucleus and Clk starts to accumulate late at night (Glossop, 1999).

These observations also fit well with the regulation of Drosophila cryptochrome (cry), whose mRNA cycles in phase with that of Clk. Like Clk, CRY mRNA transcripts are constitutively low in per01 mutants and constitutively high in ClkJrk or Cyc0 single mutants and in per01;ClkJrk or per01;Cyc0 double mutants. These striking similarities between Clk and CRY mRNA phases (in the wild type) and Clk and CRYmRNA levels in circadian mutants suggest that the cry locus may be regulated by the same Per-Tim release of Clk-Cyc repression mechanism as Clk (Glossop, 1999).

These results reveal the existence of a Clk feedback loop and its regulatory interactions with the well-characterized per-tim feedback loop. One clear prediction from these experiments is that there is a separate activator of Clk expression. Such an activator is indicated by the high levels of Clk mRNA in the absence of Per and of either Clk or Cyc. This observation is somewhat surprising because the presence of this activator is independent of factors that control the expression of other clock genes (that is, Per, Clk, and Cyc) (Glossop, 1999).

Data supporting the existence of interlocked per-tim and Clk feedback loops were obtained from whole heads, raising the possibility that Clk expression in small subsets of 'clock-specific' cells such as the locomotor activity pacemaker cells (that is, lateral neurons) could be masked by Clk expression in other tissues. However, the autonomy and synchrony of per expression in diverse tissues in the head and body suggest that the circadian feedback loop mechanism is the same in all tissues and argue against fundamental tissue-specific differences in the feedback loop mechanism (Glossop, 1999).

An important aspect of circadian biology is how the clock regulates clock-controlled genes (CCGs). In mammals, it has been shown in vitro that CLOCK and BMAL-1 (the mammalian ortholog of Cyc) activate vasopressin gene transcription and that all three mouse Pers and Tim repress this activation, resulting in peak vasopressin mRNA transcripts by midmorning (ZT 6). Although this mode of regulation may be more general for CCGs whose mRNA transcripts peak in phase with per (or mPer), it does not explain how CCGs that cycle in antiphase are regulated. The results presented here provide a possible mechanism by which the clock regulates CCGs whose mRNAs cycle in antiphase to those of per. The similarities between Clk and cry mRNA profiles in the wild type and in several single and double circadian mutants suggest that Per-Tim release of Clk-Cyc repression may serve a more general role in regulating CCG mRNAs that cycle in antiphase to per mRNA (Glossop, 1999).

Two distinct modes of PERIOD recruitment onto dCLOCK reveal a novel role for TIMELESS in circadian transcription

Negative transcriptional feedback loops are a core feature of eukaryotic circadian clocks and are based on rhythmic interactions between clock-specific repressors and transcription factors. In Drosophila, the repression of dClock (dClk)-Cycle (Cyc) transcriptional activity by dPeriod (dPer) is critical for driving circadian gene expression. Although growing lines of evidence indicate that circadian repressors such as dPer function, at least partly, as molecular bridges that facilitate timely interactions between other regulatory factors and core clock transcription factors, how dPer interacts with dClk-Cyc to promote repression is not known. This study identified a small conserved region on dPer required for binding to dClk, termed CBD (for dClk binding domain). In the absence of the CBD, dPer is unable to stably associate with dClk and inhibit the transcriptional activity of dClk-Cyc in a simplified cell culture system. CBD is situated in close proximity to a region that interacts with other regulatory factors such as the Doubletime kinase, suggesting that complex architectural constraints need to be met to assemble repressor complexes. Surprisingly, when dPer missing the CBD (dPerδCBD) was evaluated in flies the clock mechanism was operational, albeit with longer periods. Intriguingly, the interaction between dPerδCBD and dClk is Tim-dependent and modulated by light, revealing a novel and unanticipated in vivo role for Tim in circadian transcription. Finally, dPerδCBD does not provoke the daily hyperphosphorylation of dClk, indicating that direct interactions between dPer and dClk are necessary for the dClk phosphorylation program but are not required for other aspects of dClk regulation (Sun, 2010).

A shared feature of eukaryotic circadian pacemaker mechanisms is that daily cycles in gene expression involve the phase-specific interaction of one or more repressors with core clock transcription factors. Initial findings identified Per proteins in animals and Frequency (Frq) in Neurospora as key components underlying the main negative feedback loops operating in their respective clocks. Early models, mostly based on work in Drosophila and Neurospora, suggested that the direct binding of Per or Frq to their relevant transcription factors was the biochemical mode-of-action underlying these repressors. More recent work is beginning to refine this view and it is now thought that these 'repressors' function, at least partly, by acting as molecular bridges to ensure the timely assembly and/or delivery of larger repressor complexes that inhibit elements functioning in the positive arms of circadian transcriptional feedback loops. Intriguingly, Per and Frq proteins also share another role in that phosphorylation driven changes in their daily levels are central to setting clock pace. Thus, repressors such as Per and Frq act as a critical nexus in clock mechanisms by connecting phosphorylation-based biochemical timers to the regulation of transcription, yielding appropriately phased daily cycles in gene expression. How the binding of these period-setting repressors to core clock transcription factors leads to inhibition in transactivation potential is not well understood (Sun, 2010).

This study identified the C4 region on dPer as the sole or major dClk binding domain (termed CBD). Despite the inability of dPerδCBD to bind to dClk, p{dperδCBD} flies manifest quasi-normal feedback circuitry within the core oscillator mechanism, almost certainly as a result of Tim facilitating the close interaction of dPer with dClk, enabling temporal repression of dClk-Cyc-mediated transcription. Thus, Tim is a bona fide component of the in vivo circadian repressor complex, and presumably plays a role in modulating the interaction between dClk and dPer. Moreover, this study reports that direct binding between dClk and dPer is not necessary for dPer's repressor activity, but is likely necessary for normal hyperphosphorylation of dClk (Sun, 2010).

An approach that is providing insights into how these 'phospho-timing repressor' clock proteins function is the identity of regions required for promoting transcriptional inhibition. In Drosophila, early work mapped a region on dPer necessary for strong inhibition of dClk/Cyc activity in an S2 cell transcription assay. This region, termed the CCID domain, contains two highly conserved regions, namely C3 and C4. The C3 region contains the sole or major domain on dPer required for stable interaction with Dbt, termed the dPDBD, and is critical not only for hyperphosphorylation of dPer but also for inhibiting dClk-Cyc-mediated transcription, despite the fact that eliminating this region does not abrogate the ability of dPer to stably interact with dClk in vivo. In contrast, the newly identified CBD is required for the physical interaction of dPer with dClk. Thus, the CCID is comprised of at least two distinct regions with different biochemical modes-of-action; a region required for physical interaction with dClk and another that functions as a scaffold to promote binding of regulatory factors that modulate the activity/metabolism of dClk/Cyc (Sun, 2010).

An unanticipated aspect of this work is that tim can promote the binding of dPerδCBD to dClk in a simplified cell culture system and in flies, an event that rescues dPer's repressor function. Earlier work suggested that Tim is dispensable for repression of dClk/Cyc transactivity. However, although Tim exhibits little to no repressor activity toward dClk-Cyc-mediated transcription in S2 cells, it enhances that of dPer. This enhancement was largely attributed to the fact that Tim stimulates the nuclear localization of dPer. The current findings suggest a physiological role for Tim in modulating circadian transcription by regulating the interaction between dPer and dClk. Although dPer can bind dClk in the absence of Tim, it is possible that by interacting with both dPer and dClk, Tim influences the properties of the repressor complex. For example, Tim might modulate the conformation of dPer, enhancing the assembly/activity of the repressor complex. In support of this view, brief light stimulation in the night leads to subtle but noticeable effects on dper/tim RNA levels that precede significant changes in the abundance of dPer. Of particular interest, dper/tim RNA levels were induced by light exposure in flies expressing a dPer variant (named dPer-δC2) missing a short stretch of conserved amino acids (515-568). Although the basis for the impaired function of dPer-δC2 was not clear, based on the current results it is possible that its interaction with dClk/Cyc is defective in the absence of Tim (Sun, 2010).

The findings that Tim has a more pivotal role in transcriptional regulation might also be relevant to a recent study suggesting a two-step mechanism for dPer-mediated inhibition of dClk-Cyc activity, whereby during the first phase of inhibition dPer is bound to dClk at the chromatin, followed by a second off-DNA sequestration of dClk by dPer (Menet, 2010). The switch from an on-DNA to an off-DNA mechanism is thought to occur around ZT18, around the time tim begins to accumulate in the nucleus. It is speculated that Tim might regulate progression from an on-DNA to an off-DNA inhibitory mechanism by modulating the interaction between dPer and dClk/Cyc (Sun, 2010).

Although dPerδCBD in the presence of Tim can suppress dClk-Cyc-mediated transcription, the efficiency is lower compared with that of wild-type dPer. Several different scenarios could account for this, including less favorable spatial alignment between dPerδCBD and dClk/Cyc and/or effects on the ability of dPerδCBD to bind and/or deliver regulatory factors to dClk/Cyc. Perhaps a more interesting possibility is suggested by the less extensive phosphorylation of dClk in p{dperδCBD} flies. Hyperphosphorylated dClk is mainly detected in the late night/early day during times when dClk-Cyc transcriptional activity is inhibited, suggesting that highly phosphorylated dClk is less active. The absence of hyperphosphorylated isoforms of dClk in p{dperδCBD} flies suggests that direct association between dPer and dClk is required to provoke dPer-dependent dClk phosphorylation. The lack of hyperphosphorylated isoforms of dClk in p{dperδCBD} flies might also contribute to the higher overall levels of dper/tim transcripts. In this context it is noteworthy that Frq is thought to play a major role in repressing the positive limb of the circadian transcriptional circuits in Neurospora by regulating the phosphorylated state of the WCC complex, the key clock transcription factor driving cyclical gene expression in that system. Future studies will be required to better understand the biochemical function of dClk phosphorylation (Sun, 2010).

In summary, these findings demonstrate that the direct interaction of a key repressor to its target transcription factors is not necessary for its ability to engage in transcriptional inhibition. Moreover, Tim can promote the close association of dPer to dClk in a manner that sustains dPer's repressor capability, revealing a more direct role for tim in functional interactions between the negative and positive limbs of the circadian transcriptional feedback circuits operating in Drosophila. The dClk interaction domain on dPer is situated very close to the dPDBD region that functions, at least partly, by acting as a bridge to promote close interactions between regulatory factors (such as Dbt) and the dClk/Cyc complex. The close spacing on dPer between these two functional regions suggests that complex architectural constraints need to be met to assemble highly efficient repressor complexes. It will be of interest to determine whether other repressors, such as Per proteins in mammals and Frq in Neursopora, also have similar spatial arrangements. Intriguingly, in mammals the C-terminal region of mPer2, which is downstream of the casein kinase binding (CKB) domain, has been reported to be involved in directly binding to BMAL1 (Chen, 2009). Finally, the current findings strongly suggest that direct interactions between dPer and dClk/Cyc are required for dClk hyperphosphorylation. It is possible that some regulatory factors stay tightly bound to key clock repressors (such as Dbt to dPer) and thus require very close contact with central clock transcription factors to modulate them, whereas other factors are 'delivered' and establishing a high local concentration is sufficient to promote efficient transfer from the repressors to circadian-relevant transcription complexes (Sun, 2010).

CLOCK deubiquitylation by USP8 inhibits Clk/Cyc transcription in Drosophila

A conserved transcriptional feedback loop underlies animal circadian rhythms. In Drosophila, the transcription factors Clock (Clk) and Cycle (Cyc) activate the transcription of direct target genes like period (per) and timeless (tim). They encode the proteins Per and Tim, respectively, which repress Clk/Cyc activity. Previous work indicates that repression is due to a direct Per-Clk/Cyc interaction as well as Clk/Cyc phosphorylation. This study describes the role of ubiquitin-specific protease 8 (USP8; FlyBase term, UBPY ortholog) in circadian transcriptional repression as well as the importance of Clk ubiquitylation in Clk/Cyc transcription activity. usp8 loss of function (RNAi) or expression of a dominant-negative form of the protein (USP8-DN) enhances Clk/Cyc transcriptional activity and alters fly locomotor activity rhythms. Clock protein and mRNA molecular oscillations are virtually absent within circadian neurons of USP8-DN flies. Furthermore, Clk ubiquitylation cycles robustly in wild-type flies and peaks coincident with maximal Clk/Cyc transcription. As USP8 interacts with Clk and expression of USP8-DN increases Clk ubiquitylation, the data indicate that USP8 deubiquitylates Clk, which down-regulates Clk/Cyc transcriptional activity. Taken together with the facts that usp8 mRNA cycles and that its transcription is activated directly by Clk/Cyc, USP8, like Per and Tim, contributes to the transcriptional feedback loop cycle that underlies circadian rhythms (Luo, 2012).

This study addressed the function of the deubiquitylating enzyme USP8 in Drosophila circadian rhythms. Although usp8 mRNA was one of the top cyclers identified in head RNA more than a decade ago, it was only recently identified as one of the few non-clock gene mRNAs that cycles in circadian neuron RNA as well as in head RNA. This revived interest in understanding its role in circadian rhythms. The data in this study indicate that USP8 deubiquitylates Clk, which likely contributes to inhibiting clock gene transcription at the appropriate trough times in the circadian cycle. This regulation appears particularly important for clock neurons because interference of USP8 function by either RNAi knockdown or expression of USP8-DN dramatically alters fly locomotor activity, ranging from long periods to complete arrhythmicity (Luo, 2012).

The expression of USP8-DN also had dramatic effects on the oscillations of both tim and per mRNAs and proteins. For example, expression of USP8-DN under the control of the tim driver increases the RNA levels of per, tim, and vri at almost all times of the circadian cycle. Effects on pre-mRNA were similar and included the usual trough times, from late night to early morning (CT22-CT6). The increased levels of mRNA presumably contribute to the increase in Per and Tim in head extracts at CT6-CT10, a time when there is no more than a trace visible in wild-type extracts. However, it is noted that per and tim RNAs and proteins are still cycling in the USP8-DN-expressing flies, in apparent conflict with the stronger behavioral phenotypes of these mutant flies. It is suspected that this inconsistency reflects at least in part the heterogeneity of fly head tissues; i.e., a substantial circadian contribution from the eyes might mask a quantitatively more important contribution of USP8 within circadian neurons. Indeed, expression of USP8-DN has a much more dramatic effect on the levels of Per and Tim at CT10 and tim mRNA at CT22 in PDF neurons, reflecting and perhaps even causing the long period or arrhythmicity of these flies (Luo, 2012).

Most USP family proteins stabilize their substrates by preventing proteasome-mediated degradation and therefore enhancing protein levels. Yet USP8-DN increases Clk activity without affecting Clk levels. The same conclusion results from the S2 cell experiments: RNAi knockdown of endogenous USP8 or transfection of USP8-DN enhanced Clk/Cyc transcription activity, which is still sensitive to Per repression. Based on the results from flies as well as S2 cells, USP8 probably functions as an inhibitor of Clk/Cyc transcriptional activation (Luo, 2012).

Inhibition of USP8 causes an increase in Clk ubiquitylation, mostly monoubiquitylation. This presumably reflects the interaction of Clk with USP8 in S2 cells and fly heads; i.e., the direct deubiquitylation of Clk. Interestingly, Clk ubiquitylation levels cycle and peak at ZT10-ZT14, when maximal transcription occurs. Moreover, expression of USP8-DN dramatically increased the level of Clk ubiquitylation at ZT10 and ZT22, especially at ZT22. Because Clk binding at the E-box of the tim promoter did not change dramatically between the two strains (and perhaps even in the opposite direction), it is likely that Clk monoubiquitylation does not enhance DNA binding, but rather potentiates transcriptional activity. Monoubiquitylation of a transcription factor has been shown to help recruit other transcription factors and even Pol II to a promoter (Luo, 2012).

Ubiquitylation may be mechanistically related to the 'black widow' model of transcription activation, in which transcription factors on chromatin are actively degraded by the proteasome. This may apply to mammalian BMAL1, the ortholog of Cyc, suggesting that the same turnover mechanism may be relevant to Clk/Cyc in flies. A maximally active chromatin-bound Clk may therefore be monoubiquitylated, which can then experience two inhibitory fates. It can be further ubiquitylated, perhaps directly on chromatin, which may then require proteasome-mediated degradation to allow replacement by another maximally active monoubiquitylated Clk, or it can be deubiquitylated by USP8. The former may predominate at times of maximal transcription so that rapid recycling of Clk/Cyc by the proteasome maintains maximal levels of monoubiquitylated Clk and maximal transcription rates. In the late night-early morning, deubiquitylation by USP8 may predominate and help minimize clock gene transcription. It is notable that these are circadian times when cycling usp8 mRNA levels are maximal. However, it is possible that regulation of an E3 ligase also contributes to the cycling of Clk ubiquitylation (Luo, 2012).

The importance of differential ubiquitylation is reinforced by the different ubiquitylated Clk-binding patterns on some clock genes; i.e., vri and pdp1. The fact that ubiquitylated Clk occurs preferentially close to the transcription start sites of these genes suggests that Clk monoubiquitylation may help recruit factors to drive transcription; Pol II or factors associated with transcription initiation are good candidates. In this view, the eye-specific vri and pdp1 Clk-binding sites with poor ubiquitylation may reflect Clk-binding sites without these cofactors, allowing deubiquitylation to predominate. The relatively poor Clk ubiquitylation at these sites may also indicate a relationship with other transcription factors. Put otherwise, deubiquitylated Clk may play a more modest role at these sites and partner with (unidentified) factors that contribute most of the transcriptional activation potential, perhaps within certain eye cell types (Luo, 2012).

Nonetheless, the fact that Clk deubiquitylation by USP8 appears maximal at the end of the transcriptional cycle suggests that deubiquitylated Clk is associated with changes in chromatin structure and/or transcription complexes at this time. Indeed, most Clk is sequestered by Per in an off-DNA inhibitory complex at the end of the cycle, suggesting that this complex contains substantial levels of deubiquitylated Clk. Per may enhance USP8 activity on Clk or inhibit Clk monoubiquitylation within the Per-Clk complex. Alternatively, Per may function strictly to inhibit DNA binding, suggesting that Clk monoubiquitylation and even deubiquitylation are predominantly on-DNA events (Luo, 2012).

It is interesting that the original analysis of cycling mRNAs within PDF neurons indicated that the circadian amplitudes of most clock gene mRNAs were dramatically enhanced compared with their amplitudes in head RNA (Kula-Eversole, 2010). This is consistent with the enhanced effect of USP8 inhibition of mRNA cycling in PDF neurons compared with the effects on head RNA, suggesting that USP8 function may contribute to this enhanced amplitude of clock gene cycling. A challenge for the future will be to understand how and why USP8 functions differentially within circadian neurons. It will also be important to integrate this goal with the ability to assay Clk/Cyc binding and even ubiquitylated Clk binding as a function of circadian time within PDF neurons (Luo, 2012).

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).


Within a Drosophila Clock intron, a second ORF is found with significant similarity to the mammalian gene EB1, implicated in binding the adenomatosis polyposis coli (APC) C terminus (Allada, 1998).


Amino Acids - 1015 (Allada, 1998); 1023 (Darlington, 1998)

Structural Domains

Sequencing of multiple cDNAs indicates one open reading frame. The ORF has at least two forms: one corresponds to the full-length protein of 1015 amino acids and the other to a protein missing the bHLH and PAS A regions. Several transcript forms are found, none of which demonstrate robust circadian oscillations. The full-length Drosophila Clock protein contains all of the known subregions of mouse Clock, including bHLH, PAS A, PAS B, and prominent Q-rich activation domain (Allada, 1998).

Alignment of the full-length Drosophila Clock protein with mouse Clock reveals 35% identity over the entire overlap (>800 amino acids). Drosophila Clock also shows substantial sequence similarity to the mouse and human bHLH-PAS proteins NPAS2/MOP4, but the latter has no polyQ regions. There is no functional evidence, however, linking NPAS2/MOP4 to circadian rhythms. The sequence identity of Drosophila and Mouse Clock proteins is even more impressive in the three subregions where one can infer a biochemical function. The bHLH domains, involved in DNA binding and protein dimerization, have 71% similarity and 60% identity. The basic region, involved in sequence-specific DNA contacts, is remarkably conserved with 11 out of 13 amino acids being identical; this suggests that the two proteins bind to similar if not identical DNA targets. In fact, 6 out of 9 amino acids are identical to a consensus generated for bHLH proteins that bind the CAC/GTG E box half-site, including the critical R residue at position 15 (Allada, 1998).

The PAS region, implicated in protein dimerization, is also strikingly conserved between the insect and murine genes. The PAS B repeat and the region within PAS just C-terminal to PAS B are particularly conserved; 79% identical and 91% similar over a span of 107 amino acids (amino acids 262-368). The conservation of PAS and its demonstrated role in dimerization suggest that Drosophila Clock and mouse Clock may have conserved heterodimeric partners. Indeed, it appears that another Drosophila clock gene, cycle, encodes the relevant bHLH-PAS partner and that the same heterodimeric complex is functionally relevant in both systems. The third conserved region is the glutamine (Q)-rich C terminus of the protein. Glutamine-rich regions, especially polyglutamine repeats, are known to function in transcriptional activation (Allada, 1998).

Clock: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 17 November 2021

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