BIOLOGICAL OVERVIEW

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 (Clk^Jrk) 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 Clk^Jrk 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;Clk^Jrk 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;Clk^Jrk 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 Clk^Jrk or Cyc⁰ single mutants and in per⁰¹;Clk^Jrk 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 (dPer^δCBD) was evaluated in flies the clock mechanism was operational, albeit with longer periods. Intriguingly, the interaction between dPer^δCBD and dClk is Tim-dependent and modulated by light, revealing a novel and unanticipated in vivo role for Tim in circadian transcription. Finally, dPer^δCBD 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 dPer^δCBD to bind to dClk, p{dper^δCBD} 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 dPer^δCBD 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 dPer^δCBD 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 dPer^δCBD and dClk/Cyc and/or effects on the ability of dPer^δCBD 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{dper^δCBD} 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{dper^δCBD} 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{dper^δCBD} 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).

Systematic modeling-driven experiments identify distinct molecular clockworks underlying hierarchically organized pacemaker neurons

In metazoan organisms, circadian (∼24 h) rhythms are regulated by pacemaker neurons organized in a master-slave hierarchy. Although it is widely accepted that master pacemakers and slave oscillators generate rhythms via an identical negative feedback loop of transcription factor CLOCK (CLK) and repressor PERIOD (PER), their different roles imply heterogeneity in their molecular clockworks. Indeed, in Drosophila, defective binding between CLK and PER disrupts molecular rhythms in the master pacemakers, small ventral lateral neurons (sLN(v)s), but not in the slave oscillator, posterior dorsal neuron 1s (DN1_ps). This study developed a systematic and expandable approach that unbiasedly searches the source of the heterogeneity in molecular clockworks from time-series data. In combination with in vivo experiments, it was found that sLN_vs exhibit higher synthesis and turnover of PER and lower CLK levels than DN1_ps. Importantly, light shift analysis reveals that due to such a distinct molecular clockwork, sLN_vs can obtain paradoxical characteristics as the master pacemaker, generating strong rhythms that are also flexibly adjustable to environmental changes. These results identify the different characteristics of molecular clockworks of pacemaker neurons that underlie hierarchical multi-oscillator structure to ensure the rhythmic fitness of the organism (Jeong, 2022).

The circadian clock enables organisms to manifest about 24-h (circadian) rhythms of behavior and physiology coordinated with rhythmic environmental changes. The generic model of the circadian clock is composed of input, oscillator, and output, wherein the oscillator entrains to time cues (zeitgeber) and regulates the output rhythms. This system operates as a network in which the master pacemaker and slave oscillator are organized in a hierarchical manner. The master pacemaker receives the light signal, the prominent zeitgeber, and then drives the slave oscillator that regulates distinct outputs such as sleep, feeding, metabolic homeostasis, etc. In this hierarchy system, the master pacemaker can generate strong rhythms to yield clear signals to the slave oscillator while still being able to flexibly adjust their phase in response to changes in environmental lighting conditions. However, the molecular mechanisms underlying these somewhat paradoxical characteristics of the master pacemaker are poorly understood (Jeong, 2022).

In Drosophila, small ventral lateral neurons (sLN_vs) act as the master pacemaker. That is, sLN_vs maintain free-running rhythms under constant darkness. On the other hand, posterior dorsal neuron 1s (DN1_ps) act as the slave oscillator receiving neuropeptide pigment-dispersing factor (PDF) from sLN_vs, which is critical to maintain their rhythms. Without PDF signaling from sLN_vs, DN1_ps rapidly lose molecular oscillation, and DN1_ps follow the speed of genetically modified sLN_vs. Furthermore, DN1_ps harbor connections with output centers such as premotor, sleep, and neuroendocrine centers. Taken together, the circadian clock of Drosophila has a hierarchical organization, with sLN_vs being the master pacemaker receiving light signals and DN1_ps being the slave oscillator releasing output signals, although the organization can be potentially changed in the presence of environmental or genetic perturbations (Jeong, 2022).

Despite the different roles of these pacemaker neurons, they share common molecular mechanisms to generate circadian rhythms that are well conserved in all life-forms: the interlocked multiple transcriptional-translational feedback loops (TTFLs) composed of core clock proteins. In the Drosophila core TTFL, CLK, and CYCLE (CYC) activate the transcription of per and timeless (tim); PER and TIM proteins, in turn, repress their own transcription in which PER is the core repressor. This core TTFL regulates the 24-h period rhythmic expression of clock genes and other clock-controlled genes. Although sLN_vs and DN1_ps generate circadian rhythms via identical TTFLs, unexpectedly, it was previously found that their rhythms are altered in a different way in p{Clk-Δ};Clk^out flies (here referred to as Clk-Δ flies), which express CLK defective in PER binding. Specifically, in Clk-Δ flies, PER oscillation was significantly dampened in sLN_vs but quasinormal in DN1_ps, demonstrating heterogeneity in their molecular clocks (Jeong, 2022).

This study took advantage of Clk-Δ flies to understand pacemaker neuron–specific molecular clockworks. Specifically, the neuron-specific alteration of a time series of PER in Clk-Δ flies was analyzed by developing a systematic modeling approach. This allowed systematic investigation of all possible molecular differences in the core TTFL between sLN_vs and DN1_ps with their mathematical models developed in this study. With a combination of in vivo experiments, essential differences were found in the molecular clockworks of sLN_vs and DN1_ps to reproduce their different rhythm alterations by Clk-Δ: CLK levels are higher in DN1_ps than in sLN_vs, the synthesis of PER is more efficient, and the degradation of PER is faster in sLN_vs than in DN1_ps. Furthermore, it was found that such distinct molecular mechanisms of the core TTFL in sLN_vs are critical for its ability to act as the master pacemaker, generating strong rhythms while flexibly adapting its phase to environmental changes (e.g., jet lag) via in silico experiments. In conclusion, this study presents pacemaker neuron–specific molecular clockworks that underlie the hierarchical organization of the circadian clock to ensure the rhythmic fitness of the organism (Jeong, 2022).

In the Drosophila circadian clock, some molecular differences in pacemaker neurons have been reported in addition to their different repertoire of transcripts. However, it has been poorly understood whether and why key molecular mechanisms for generating circadian rhythms differ among pacemaker neurons. This study found differences between the core TTFL of the circadian clock in sLN_vs and DN1_ps by using a combination of theoretical and experimental approaches. Furthermore, it was found that such distinct characteristics of the core TTFL in sLN_vs enables them to generate strong rhythms while flexibly adapting their phase upon changes to the environmental lighting conditions (Jeong, 2022).

To understand why PER rhythms are altered differently by the same Clk-Δ mutation between sLN_vs and DN1_ps, a systematic modeling approach was developed. That is, all possible differences in the core TTFL of sLN_vs and DN1_ps were investigated to identify key differences that explain their different alterations by the Clk-Δ mutation. This allowed identification of the parameter sets of mathematical models that can reproduce different time series of PER between sLN_vs and DN1_ps. Then, by analyzing the common patterns of the parameter sets, it was possible to identify key differences in molecular clockworks between sLN_vs and DN1_ps: higher synthesis and turnover of PER and lower CLK levels in sLN_vs than DN1_ps. While it was assumed that the dissociation constants are the same to avoid the identifiability issue of the parameter estimation, the dissociation constants could also differ in molecular clockworks between sLN_vs and DN1_ps (Jeong, 2022).

The patterns of parameter sets rather than a single best-fit parameter set to avoid overfitting, because the best-fit parameter may not yield the most meaningful parameters when models contain a large number of parameters. Such a systematic approach has also been successfully used to resolve the unexpected dynamics in biological systems. While these previous studies focused on identification of hidden regulation underlying a single system, this study investigated the difference between two systems, sLN_vs and DN1_ps. Thus, the regularization cost, penalizing the difference in the values of parameters between the LN and DN models, was used to avoid unnecessary differences. This systematic modeling framework is expandable to identify heterogeneity of other systems (Jeong, 2022).

The predicted lower CLK levels in sLN_vs than in DN1_ps were confirmed by in vivo experiments. Given the lower amount of the transcription factor CLK, one can imagine that de novo–synthesized nascent per transcript would be lower, producing a lower amount of PER in sLN_vs than in DN1_ps. But PER is more rapidly synthesized in sLN_vs than in DN1_ps, indicating that the production of PER in sLN_vs might be enhanced by posttranscriptional mechanisms. Intriguingly, the translation activation complex of ATX2 and TYF posttranscriptionally regulates per mRNA in an LN-specific manner. miRNAs are also important posttranscriptional regulators of gene expression that degrade target mRNA and/or inhibit its translation. Numerous miRNAs regulate the circadian rhythm by affecting Clk, clockwork orange, tim, or output genes. While no microRNA (miRNA) targeted toward per mRNA has been identified so far, the different repertoire of miRNA in pacemaker neurons might be responsible for the different kinetics of PER accumulation (Jeong, 2022).

In addition, the predicted higher turnover rate of PER in sLN_vs than in DN1_ps was also confirmed by in vivo experiments. What could cause these differences in the degradation rate of PER between sLN_vs and DN1_ps? Throughout the day, PER is progressively phosphorylated by several kinases including DBT (casein kinase I ortholog in flies), casein kinase 2 (CK2), NEMO, and Shaggy. Hyperphosphorylated PER is degraded by the ubiquitin-proteasome system via recognition of Ser47 phosphorylation by the ubiquitin ligase Supernumerary Limbs (SLIMB). Thus, different repertoires of kinase activity in each group of pacemaker neurons might result in different kinetics of PER hyperphosphorylation leading to Ser47 phosphorylation, which is the PER degradation mark. For instance, Ser47 phosphorylation and PER degradation is delayed by NEMO-dependent phosphorylation of the middle part of PER. Intriguingly, NEMO is expressed in sLN_vs, large ventral lateral neurons, dorsal lateral neurons, and DN1s but not in DN2s or DN3s. Furthermore, CK2 is expressed only in LN_vs. Collectively, it was reasoned that a unique repertoire of kinases cooperates to make PER more susceptible to degradation in sLN_vs than in DN1_ps. Of course, given that the phosphorylation status of a protein is regulated by phosphatases, the phosphatase repertoire and/or expression level could be another important determinant of degradation rate. Indeed, PP1 and PP2A affected PER phosphorylation and thus its stability (Jeong, 2022).

The mathematical model predicted that the CLK-Δ mutation induced different rhythm alterations in sLN_vs and DN1_ps due to the differences in molecular clockworks between sLN_vs and DN1_ps. Specifically, when PER levels are higher than CLK levels in the nucleus, the majority of CLK is sequestered and, thus, transcription of per mRNA is suppressed. As a result, only when PER levels are lower than CLK levels in the nucleus is the transcription of per mRNA promoted (i.e., the activation phase). However, as the binding between CLK and PER is disrupted due to the CLK-Δ mutation, even when PER levels are higher than CLK levels, free CLK is available, and thus, the transcription of per mRNA is weakly promoted. This weak transcription of per mRNA dramatically increases PER levels compared to CLK levels due to the high synthesis rate of PER and the low CLK levels in the LN-Δ model. As a result, the transcription of per mRNA cannot be fully promoted. This disruption of the transition from the suppression phase to the activation phase dampens circadian rhythms in the LN-Δ model. On the other hand, the weak transcription of per mRNA has little effect on the ratio between PER levels and CLK levels due to the high CLK levels and low synthesis rate of PER in the DN-Δ model. Thus, even in the presence of the CLK-Δ mutation, the transition between the activation and suppression phases occurs, leading to quasinormal circadian rhythms in the DN-Δ model. Taken together, the LN model shows a greater sensitivity of the transcription in response to the change in the level of PER than the DN model to generate stronger rhythms. However, due to such greater sensitivity, when the system is perturbed (i.e., mutation), the LN model shows a more sensitive response compared to the DN model, leading to the loss of rhythms. As the greater sensitivity is cooperatively generated by the high synthesis rate of PER and low level of CLK, the LN-Δ model predicts that changing a single parameter alone (i.e., synthesis rate of PER) will not rescue the rhythms disrupted by the CLK-Δ mutation in sLN_vs. It would be interesting in future work to investigate whether the disrupted rhythms can be rescued by simultaneously changing multiple parameters of the molecular clockworks in sLN_vs (Jeong, 2022).

Why, then, do sLN_vs have such different molecular properties from DN1_ps? Due to the fast synthesis and turnover rates of PER, sLN_vs can generate rhythms with high amplitudes, which is critical to yield clear signals to slave oscillators. Unexpectedly, although typically strong oscillators with high amplitudes have difficulty in adapting to environmental changes (e.g., jet lag), this study found that sLN_vs can be reentrained to the new LD cycle as rapidly as DN1_ps. Interestingly, before the reentrainment, unlike DN1_ps, the phases of sLN_vs are greatly dispersed. This in silico study proposes that the phase dispersion stems from the distinct property of TTFL in sLN_vs from DN1_ps due to its spike-like and sensitive transcription yielding strong oscillation. Another study also showed that the sensitive response of per mRNA in response to the environmental change, yielding phase plasticity, is the feature of robust oscillators. That is, the sensitive change of per mRNA, and thus sensitive phase shifts under environmental change, counterbalance the change of the other reaction rates and lead to period robustness. Taken together, due to the distinct molecular clockworks of sLN_vs compared to those of DN1_ps, sLN_vs could obtain both robustness (i.e., high amplitude and period robustness) and plasticity (i.e., fast entrainment and a wide range of entrainment), which are critical characteristics for a master pacemaker (Jeong, 2022).

Dietary restriction and the transcription factor Clock delay eye aging to extend lifespan in Drosophila Melanogaster

Many vital processes in the eye are under circadian regulation, and circadian dysfunction has emerged as a potential driver of eye aging. Dietary restriction is one of the most robust lifespan-extending therapies and amplifies circadian rhythms with age. This study demonstrates that dietary restriction extends lifespan in Drosophila melanogaster by promoting circadian homeostatic processes that protect the visual system from age- and light-associated damage. Altering the positive limb core molecular clock transcription factor, CLOCK, or CLOCK-output genes, accelerates visual senescence, induces a systemic immune response, and shortens lifespan. Flies subjected to dietary restriction are protected from the lifespan-shortening effects of photoreceptor activation. Inversely, photoreceptor inactivation, achieved via mutating rhodopsin or housing flies in constant darkness, primarily extends the lifespan of flies reared on a high-nutrient diet. These findings establish the eye as a diet-sensitive modulator of lifespan and indicates that vision is an antagonistically pleiotropic process that contributes to organismal aging (Hodge, 2022).

Progressive declines in circadian rhythms are one of the most common hallmarks of aging observed across most lifeforms. Quantifying the strength, or amplitude, of circadian rhythms is an accurate metric for predicting chronological age. Many cellular processes involved in aging (e.g., metabolism, cellular proliferation, DNA repair mechanisms, etc.) display robust cyclic activities. Both genetic and environmental disruptions to circadian rhythms are associated with accelerated aging and reduced longevity. These observations suggest that circadian rhythms may not merely be a biomarker of aging; rather, declines in circadian rhythms might play a causal role. The observation that DR and DR-memetics, such as calorie restriction and time-restricted feeding, improve biological rhythms suggests that clocks may play a fundamental role in mediating their lifespan-extending benefits (Kato, 2022).

This study identified circadian processes that are selectively amplified by DR. The findings demonstrate that DR amplifies circadian homeostatic processes in the eye, some of which are required for DR to delay visual senescence and improve longevity in Drosophila. Disrupting CLK function within photoreceptors accelerates visual declines and shortens lifespan, while overexpressing wild-type CLK protects against age-associated declines in vision and rescues AL-dependent declines in photoreceptor function. These data also demonstrate that photoreceptor stress has deleterious effects on organismal health; overstimulation of the photoreceptors induced a systemic immune response and reduced longevity (Kato, 2022).

Among the more interesting and unexpecting findings of this study is the observation that the Drosophila eye influences systemic immune responses, as elevated AMP expression was observed in the bodies of flies overexpressing CLK-Δ pan-neuronally and in flies with forced photoreceptor degeneration (ATPα-RNAi). It is possible that GAL4 misexpression may promote inflammatory responses in the fly bodies, although this study found a reduction in systemic inflammation in the rhodopsin-null lines suggesting that this phenomenon can originate at the photoreceptor. These systemic immune responses correlated with lifespan changes (increased body AMP expression is associated with declines in longevity and vice versa), similar to what is observed with chronic inflammation or “inflammaging” in other models. However, it cannot be concluded whether neuronal or eye-mediated increases in systemic inflammation are causal to aging in other tissues. Furthermore, the mechanisms by which the Drosophila eye, and, more specifically, the photoreceptor influence systemic immune responses are unclear. It is speculated that photoreceptor degeneration may disrupt the retinal-blood barrier such that damage signals from the eye propagate through to the hemolymph and activate AMP expression in distal tissues. Future studies are aimed at elucidating this mechanism, and its effect on longevity (Kato, 2022).

The findings of this study establish the eye as a diet-sensitive regulator of lifespan. DR's neuroprotective role in the photoreceptors appears to be mediated via the transcription factor CLK, which promotes the rhythmic oscillation of genes involved in the suppression of phototoxic cell stress. Given that CLK transcriptionally regulates circadian and non-circadian transcripts, future investigations may determine whether the time-of-day regulation of these genes by CLK is germane to promoting eye health with age. These studies may also examine whether the DR-mediated benefits on visual senescence and photoreceptor viability are mediated solely by CLK as a transcription factor (as demonstrated here) or whether circadian clock function (rhythmic output) is required. The findings also support the notion that age-related declines in the visual system impose a high cost on an organism's physiology. Perhaps this provides an alternative hypothesis for why several cave-dwelling animals, whose visual systems have undergone regressive evolution (e.g., cave-dwelling fish and naked-mole rats), are especially long-lived. Failing to develop a visual system may act as a pro-survival mechanism allowing organisms to avoid the damage and inflammation triggered by age-related retinal degeneration. Ultimately, developing a visual system, which is critical for reproduction and survival, may be detrimental to an organism later in life. Thus, vision may be an example of an antagonistically pleiotropic mechanism that shapes lifespan (Kato, 2022).

Dissecting the daily feeding pattern: Peripheral CLOCK/CYCLE generate the feeding/fasting episodes and neuronal molecular clocks synchronize them

A 24-h rhythm of feeding behavior, or synchronized feeding/fasting episodes during the day, is crucial for survival. Internal clocks and light input regulate rhythmic behaviors, but how they generate feeding rhythms is not fully understood. This study aimed to dissect the molecular pathways that generate daily feeding patterns. By measuring the semidiurnal amount of food ingested by single flies,it was demonstrated that the generation of feeding rhythms under light:dark conditions requires quasimodo (qsm) but not molecular clocks. Under constant darkness, rhythmic feeding patterns consist of two components: CLOCK (CLK) in digestive/metabolic tissues generating feeding/fasting episodes, and the molecular clock in neurons synchronizing them to subjective daytime. Although CLK is a part of the molecular clock, the generation of feeding/fasting episodes by CLK in metabolic tissues was independent of molecular clock machinery. These results revealed novel functions of qsm and CLK in feeding rhythms in Drosophila (Maruko, 2023).

Many organisms, including insects, fish, birds, rodents, and primates, show particular feeding patterns during the day. This study investigated the roles of molecular clock genes in food intake in Drosophila. In LD, light stimuli were sufficient to induce feeding via the QSM-mediated pathway, while feeding rhythms in DD can be dissected into two components: generation of feeding/fasting episodes by Clk/cyc in metabolic tissues and their synchronization by molecular clocks in neurons. These results suggest novel roles of qsm and clock molecules regulating feeding behavior (Maruko, 2023).

There has been a significant advance in understanding the circuits and neurotransmitters that mediate central clocks in the brain that control feeding behavior. As for the peripheral clocks, their functions in feeding behavior seem more diverse than those in the central clocks. In addition to contributing to the regulation of 24-h feeding rhythms, it was also reported that peripheral clocks negatively regulate food intake amount and feeding rhythm strength. However, it was elucidated whether the increase in food intake amount is caused by the disruption of the molecular clock machinery or other functions of CLK/CYC. The results suggest that fat body CLK regulates food intake amount independent of molecular clocks. Since flies with Clk/cyc disruption with various genotypes consistently show a reduction in fluctuation in food intake compared to their control (i.e., ClkJrk, cycout, cyc02, to>dnClk, cyc02, to > cyc), it is less likely that fluctuation in food intake is due to differences in the body size or other background effects. The suppression of CLK function in the fat body reduced fluctuations of feeding amount in per⁰¹ background, indicating that CLK/CYC in the metabolic tissues contributes to rhythmic feeding behavior in addition to its function as components of the molecular clock. It explains the different effects of clock genes on patterns of feeding rhythms: per- and tim-null mutants showed a shift in the peak of the feeding timing, and Clk- and cyc-null mutants ingest food constantly without peak of the feeding timing (Maruko, 2023).

The results suggest that the effect of CLK/CYC in the metabolic tissues to reduce feeding rhythm strength is likely to be mediated by suppression of feeding during the fasting period. Several factors that act on suppression of food intake in peripheral tissues have been reported, and expression of some of these genes, such as allatostatin A and its receptor, are regulated by peripheral CLK/CYC. In addition, rhythmic expression of the allatostatin A receptor-2 gene is affected by the disruption of the fat body clock. CLK is also associated with cAMP-responsive element binding protein (CREB), which is involved in the energy homeostasis of insects and mammals. Blocking CREB activity in the fat body increases food intake in flies, and Nejire, a homolog of CREB-binding protein (CBP)/p300, has been reported as a regulator of CLK/CYC-dependent transcription. Further studies on these pathways may reveal a novel signaling axis that constitute the feeding/fasting cycle (Maruko, 2023).

This study found the novel role of qsm in feeding behavior in LD. It has been reported that cry-null mutants do not display the morning peak in LD. It was also observed that cry-null mutants do not display the early morning peak in LD, while these flies still showed the synchronized feeding/fasting episodes in LD. In addition, the feeding/fasting episodes in DD were observed without the morning peak. Thus, the morning peak is not associated with the daytime feeding pattern that was the focus of this study. The results revealed that qsm is indispensable in the daytime feeding pattern. qsm encodes a ZP (Zona Pellucida) domain and constitutes part of CRY-independent light input to the circadian clock. QSM is expressed in many cells in the immediate proximity of clock neurons, and it is not clear in which cells QSM influences the feeding pattern for now. Further study to understand where and how the QSM regulates feeding rhythms in LD would help understand light-induced regulation of feeding behavior (Maruko, 2023).

In summary, these results revealed novel pathways that regulate the formation of feeding rhythms in Drosophila. Feeding/fasting rhythms coordinate metabolism and affect aging and life span. Further studies of these axes may contribute to human health (Maruko, 2023).

This study has successfully dissected the molecular pathways that regulate feeding patterns. However, these analyses are limited to several key components, and their upstream and downstream molecules remain to be elucidated. It was found that qsm regulates feeding rhythms under light:dark conditions, while its downstream signaling is unknown. This study also found that, under constant darkness conditions, CLK/CYC in digestive/metabolic tissues generates feeding/fasting episodes, and the molecular clock in neurons synchronizes them. However, molecular mechanisms that link those two components remain to be elucidated. Further studies are needed to understand the entire picture of the molecular pathways that generate feeding patterns (Maruko, 2023).

GENE STRUCTURE

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).

PROTEIN STRUCTURE

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).

date revised: 20 September 2023

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