BIOLOGICAL OVERVIEW

In 1971 when Konopka and Benzer identified the Drosophila clock gene, the news was met with both excitement and scepticism. As molecular biology has advanced, the scepticism regarding clock, or as it is now also known, period, has receded, thanks to parallel discoveries: the sequencing of the gene, the ability to study protein synthesis and movement between nucleus and cytoplasm, and the discovery of period's partner, Timeless.

The myriad phenotypic effects of period mutation, from determining the 24 hour diurnal rhythm of activity to modification of the courtship song all point to the tremendous importance of photoperiod response in the life of the fly. Incredible as it may seem, the fly does not need eyes to respond to light. A few cells in the brain are sufficient to independently sense the light-dark cycle and to respond by directing appropriate modifications of behavior.

Of central importance to the photoperiod response of the fly are a group of lateral neurons in the optic lobe. These cells have soma at the anterior margin of the medulla and arborize exptensively in the optic lobe and the brain. In addition to staining for PER protein, they are also positive for pigment-dispersing hormone (PDH), one of a family of octadecapeptides implicated in the circadian rhythm of pigmentary changes in the crustacean eye. These lateral neurons show arborizations that are always in close proximity to PER-containing glial cells (Helfrich-Förster, 1995). It has been suggested that PDH is the effector of circadian changes in insects, as it is in crustaceans. Injection of PDF in Musca, the housefly, mimics circadian fluctuations in the girth of lamina neurites (reviewed by Meinertzhagen, 1996).

Understanding of period biology is only partially complete. How does it function to regulate transcription? What are its targets, and what is period doing in all the other cells in which it is expressed? Has the biology of period been evolutionarily conserved? These are but a few of the questions that await advances in biology to further understanding of the Period protein. Many of these questions have been answered with the cloning of Clock. 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 has 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). Further characterization of the Drosophila Clk mutant phenotype 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).

Period protein can function in in a Timeless independent manner. The mutation timeless^UL (UL for ultralong) generates 33 hr rhythms, prolonged nuclear localization of Period/Timeless^UL protein complexes, and protracted derepression of period and timeless transcription. Light induced elimination of Tim^UL from nuclear Per/Tim^UL complexes gives strong downregulation of per and tim expression. Thus, in the absence of Tim, nuclear Per can function as a potent negative transcriptional regulator. Two additional studies support this role for Per: (1) Drosophila expressing Per that constitutively localizes to nuclei produce dominant behavioral arrhythmicity, and (2) constitutively nuclear Per represses Clock/Cycle-mediated transcription of per in cultured cells without Tim. Conversion of Per/Tim heterodimers to nuclear Per proteins appears to be required to complete transcriptional repression and terminate each circadian molecular cycle (Rothenfluh, 2000b).

What is the relevance of controlling the step from Per/Tim complex to Per? Toward the end of each molecular cycle, nuclear Per that is released from Per/Tim complexes becomes increasingly phosphorylated in a fashion dependent on the kinase Doubletime. When this phosphorylation is suppressed by dbt mutants, nuclear Per shows greatly increased stability. Thus, phosphorylation should regulate the duration of repression by nuclear Per. Since both Per/Tim complexes and nuclear Per can repress per and tim transcription, but only phosphorylated Per proteins are significantly degraded, termination of each molecular cycle should be triggered by the conversion of Per/Tim complexes to Per. While periodic degradation of Tim will be precisely set by an LD cycle, sustained molecular oscillations and behavioral rhythmicity close to 24 hr must be set in DD by light-independent turnover of Tim. This specific downregulation of Tim can be seen to occur in DD several hours before a corresponding diminution in the level of Per. Thus, a light-independent mechanism effecting nuclear Tim degradation should be a key determinant of period length. tim^UL may affect this mechanism (Rothenfluh, 2000b).

These studies raise the possibility that nuclear Per and the Per/Tim complex can perform distinct functions. For example, the observation that per and tim expression is decreased by removing Tim from Per/Tim^UL complexes indicates that quantitative or qualitative differences distinguish the activities of Tim-independent and Tim-associated forms of Per in vivo. This also suggests that, in vivo, full repression of per and tim expression requires the activity of nuclear Per at the end of each molecular cycle. There may also be different contributions to the regulation of Drosophila Clk expression. Clk protein negatively regulates CLK RNA accumulation, which cycles with a phase distinct from that of per and tim. Per and Tim block this Clk activity, such that Per/Tim nuclear translocation is associated with increased CLK RNA synthesis. However, CLK RNA levels fall at dawn, suggesting that the conversion of Per/Tim dimers to nuclear Per restores the autoregulatory activity of Clk. Accordingly, Per may regulate per and tim expression, while Per/Tim complexes control transcription of per, tim, and Clk. Such a mechanism could provide a general basis for establishing molecular oscillations with a variety of phases from a single clock (Rothenfluh, 2000b).

A model is proposed for the roles of Per and the Per/Tim Complex in transcriptional regulation. per and tim transcription promotes accumulation, with a delay, of heterodimeric complexes of Per and Tim proteins. The Per/Tim complex then translocates to the nucleus, initiates repression of per and tim transcription, and derepresses Clock. Per/Tim complexes are stable; however, specific degradation of Tim releases nuclear Per. In the absence of Tim, nuclear Per shows further repression of per and tim transcription, bringing PER and TIM RNA pools to their lowest levels. Phosphorylation of nuclear Per, regulated by Dbt, leads to Per degradation, and the cycle starts anew. Phosphorylation of nuclear Per may also promote its repressor function in the absence of Tim. In this model, no role for Tim without Per is proposed because Per-independent Tim proteins have not been observed in wild-type nuclei (Rothenfluh, 2000b).

PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription

Transcriptional activation by Clock-Cycle (Clk-Cyc) heterodimers and repression by Period-Timeless (Per-Tim) heterodimers are essential for circadian oscillator function in Drosophila. Per-Tim has been found to interact with Clk-Cyc to repress transcription, and this interaction is shown to inhibit binding of Clk-Cyc to E-box regulatory elements in vivo. Coincident with the interaction between Per-Tim and Clk-Cyc is the hyperphosphorylation of Clk. This hyperphosphorylation occurs in parallel with the Per-dependent entry of Double-time (Dbt) kinase into a complex with Clk-Cyc, where Dbt destabilizes both Clk and Per. Once Per and Clk are degraded, a novel hypophosphorylated form of Clk accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that Per-dependent rhythms in Clk phosphorylation control rhythms in E-box-dependent transcription and Clk stability, thus linking Per and Clk function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals (Yu, 2006).

ChIP studies demonstrate that Clk-Cyc is only bound to E-boxes when target genes are being actively transcribed. Since Per-Dbt/Per-Tim-Dbt complexes interact with Clk-Cyc to inhibit transcription (Darlington, 1998; Lee, 1998), these data imply that these Per-containing complexes inhibit transcription by removing Clk-Cyc from E-boxes. It is also possible that binding of these Per-containing complexes to Clk-Cyc effectively blocks Clk and Cyc antibody access, in which case Per complexes would inhibit transcription while Clk-Cyc is bound to E-boxes. Given that the polyclonal Clk and Cyc antibodies used in this study were raised against full-length proteins and have been used to immunoprecipitate Per-containing complexes (Lee, 1998), it is highly unlikely that all Clk and Cyc epitopes are fully blocked by Per complex binding. Thus, it is concluded that Clk-Cyc rhythmically binds E-boxes in concert with target gene activation (Yu, 2006).

Per complex binding could remove Clk-Cyc from E-boxes by directly altering their conformation or by promoting Clk phosphorylation. The region of Per that inhibits Clk-Cyc transcription, called the Clk-Cyc inhibitory domain or CCID, is near the C terminus. The CCID can act independently of the N terminus of Per, where the Dbt-binding domain resides. This observation argues that Per does not inhibit Clk-Cyc binding to E-boxes by promoting Dbt-dependent Clk phosphorylation. Dbt- and CK2-dependent phosphorylation nevertheless enhances transcriptional repression in S2 cells by potentiating Per inhibition or by inhibiting Clk activity directly. Unfortunately, these disparate results from S2 cells do not allow distinguishing between the different effects of Per complex binding to inhibit transcription outlined above (Yu, 2006).

In mammals, mCry complexes bind to CLOCK-BMAL1 and repress transcription without removing CLOCK-BMAL1 from E-boxes. This contrasts with the situation in flies, where Per complexes inhibit transcription by inhibiting Clk-Cyc E-box binding, and suggests that these Per and mCry complexes repress transcription via different mechanisms. Although mCry complexes do not remove CLOCK-BMAL1 from E-boxes, they repress transcription by inhibiting the CLOCK-BMAL1-induced acetylation of histones by blocking p300 histone acetyl transferase function or introducing a histone deacetylase. Even though Per complexes repress transcription by inhibiting Clk-Cyc binding to E-boxes, this does not exclude the possibility that rhythms in histone acetylation are also involved in regulating rhythmic transcription in flies. Since chromatin remodeling is generally accepted as a prerequisite for transcription initiation, it would be surprising if rhythms in transcription were not accompanied by rhythms in histone acetylation or some other form of chromatin remodeling (Yu, 2006).

A rhythm in Clk phosphorylation has been defined in which hyperphosphorylated Clk predominates during times of transcriptional repression and hypophosphorylated Clk predominates during times of transcriptional activation. This rhythm occurs in parallel to the rhythm in Per phosphorylation; hyperphosphorylated Per and Clk accumulate in nuclei during the late night and early morning, then these forms are degraded and hypophosphorylated forms of Per and Clk accumulate in the cytoplasm and nucleus, respectively, during the late day and early evening. The rhythm in Clk and Per phosphorylation are not merely coincidental; the accumulation of hyperphosphorylated Clk is Per dependent. Although Per is not itself a kinase, it is bound by Dbt kinase. Per brings Dbt into the nucleus, where Per-Dbt or Per-Tim-Dbt complexes bind Clk-Cyc to inhibit transcription (Yu, 2006 and references therein).

Since Dbt enters a complex containing Clk-Cyc at times when Clk becomes hyperphosphorylated, Dbt may also act to phosphorylate Clk. However, an in vitro assay for Dbt phosphorylation is not available, thus it iw not known whether Dbt directly phosphorylates Clk. Dbt acts to reduce Clk levels in S2 cells even though Per levels are very low. It is therefore possible that Dbt can act to destabilize Clk in a Per-independent manner, although it is believed this is unlikely to be the case since Clk hyperphosphorylation and complex formation with Dbt are both Per dependent (Yu, 2006).

Clk is phosphorylated to some extent in the absence of Per and is hyperphosphorylated in the absence of functional Dbt, indicating that other kinases act to phosphorylate Clk. The accumulation of hyperphosphorylated Clk in dbt^AR/dbt^P flies suggests that Dbt triggers Clk degradation subsequent to Clk hyperphosphorylation. A similar situation is seen for Per, where hyperphosphorylated Per accumulates in the absence of functional Dbt, and phosphorylation by CK2 precedes Dbt-dependent phosphorylation and Per destabilization. In addition, rhythmically expressed phosphatases may also contribute to Clk phosphorylation rhythms (Yu, 2006 and references therein).

Rhythms in Clk phosphorylation may function to modulate Clk stability, subcellular localization, and/or activity. Clk levels do not change appreciably throughout the daily cycle despite approximately fivefold higher levels of Clk mRNA at dawn than at dusk. If a less stable hyperphosphorylated form of Clk accumulates when Clk mRNA is high and a more stable hypophosphorylated form of Clk accumulates when Clk mRNA is low, they would tend to equalize total Clk levels over the daily cycle. This possibility is supported by results in ARK flies, which express Clk mRNA in the opposite circadian phase (i.e., Clk mRNA peak at dusk rather than dawn). The overall level of Clk cycles in ARK flies with a peak in (hypophosphorylated) Clk around dusk, consistent with hypophosphorylated Clk being more stable than hyperphosphorylated Clk. This possibility is also supported by Dbt-dependent destabilization of Clk in S2 cells since Dbt associates with Clk as hyperphosphorylated Clk accumulates in wild-type flies. If hypophosphorylated Clk is relatively stable, higher levels of Clk might be expected to accumulate in per⁰¹ flies. However, constant low levels of Clk mRNA likely limit Clk accumulation in per⁰¹ flies. Clock phosphorylation is coupled to its degradation in cultured mammalian cells, yet degradation of phosphorylated Clock does not lead to a rhythm in Clock abundance even though Clock mRNA levels are constant (Yu, 2006).

Studies in cultured mammalian cells also demonstrate that Clock phosphorylation promotes Clock-BMAL1 nuclear localization, although the significance of this nuclear localization is not clear given that Clock-BMAL1 binding to E-boxes is either constant or more robust during transcriptional repression in vivo. In contrast, Clk is nuclear throughout the daily cycle in flies (Yu, 2006).

The coincidence between Clock phosphorylation and transcriptional repression in mice supports the possibility that phosphorylation inhibits Clock-BMAL1 activity, perhaps by promoting HDAC binding or inhibiting HAT binding. Likewise, hypophosphorylated and hyperphosphorylated Clk accumulate in parallel with target gene activation and repression, respectively, in flies. This relationship suggests that the state of Clk phosphorylation may alter its ability to activate target genes. Given that target gene activation occurs when Clk-Cyc is bound to E-boxes and that E-box binding coincides with the accumulation of hypophosphorylated Clk, it is possible that Clk hyperphosphorylation compromises Clk-Cyc binding to E-boxes and, consequently, target gene transcription is repressed. Precedent for such a regulatory mechanism is seen in the Neurospora clock, where limiting levels of FREQUENCY (FRQ) promote phosphorylation of WHITE COLLAR 1 (WC1) and WHITE COLLAR 2 (WC2), thereby inhibiting WC1-WC2 binding to C-box regulatory elements and repressing transcription. In contrast to FRQ in Neurospora, Per is considerably more abundant than Clk in Drosophila and forms stable complexes with Clk-Cyc. In addition, Per/Per-Tim can release Clk-Cyc from E-boxes in vitro, thus demonstrating that Per/Per-Tim binding is sufficient to release Clk-Cyc from E-boxes independent of Clk phosphorylation. Taken together with the in vitro E-box binding results, the high levels of Per relative to Clk and the formation of stable Per-Tim-Clk-Cyc complexes in flies argue that Per/Per-Tim binding may also be sufficient to inhibit E-box binding by Clk-Cyc in vivo, although they do not rule out a role for Clk hyperphosphorylation in inhibiting Clk-Cyc E-box binding. For instance, Per/Per-Tim binding could function to initially remove Clk-Cyc from E-boxes, and subsequent Clk phosphorylation could maintain Clk-Cyc in a form that is incapable of binding E-boxes (Yu, 2006).

The constant levels and rhythmic phosphorylation of Clk defined in this study are similar to those previously characterized for mammalian Clock. This similarity extends beyond metazoans to fungi, where positive elements of the Neurospora circadian feedback loop; i.e., WC1 and WC2, are also rhythmically phosphorylated. In each of these organisms, phosphorylation of positive factors increases when they interact with their respective negative feedback regulators, and decreases when they activate target gene transcription in the absence of these feedback inhibitors. This remarkable similarity suggests that phosphorylation controls one or more critical aspects of positive element function, and consequently, the rhythm in the positive element phosphorylation has become a conserved feature of circadian feedback loops in eukaryotes (Yu, 2006 and references therein).

Per-dependent regulation of Clk-Cyc binding to E-boxes, Per-dependent formation of Per-Dbt and/or Per-Dbt-Tim complexes with Clk-Cyc, and Per-dependent rhythms in Clk phosphorylation suggest a model for the regulation of rhythmic transcription. During the late day and early evening, hypophosphorylated Clk-Cyc binds E-boxes to activate transcription of per, tim, and other genes within and downstream of the transcriptional feedback loop. Accumulating levels of per mRNA peak during the early evening, but Per accumulation is delayed due to Dbt-dependent (and possibly CK2-dependent) phosphorylation, which destabilizes Per. Per is subsequently stabilized via Tim binding, which inhibits further phosphorylation of Per by Dbt. Phosphorylation of Tim by SGG then promotes the translocation of Tim-Per-Dbt complexes into the nucleus, where they bind (hypophosphorylated) Clk-Cyc and repress transcription by inhibiting E-box binding and promoting Clk hyperphosphorylation and degradation. These transcriptional repression mechanisms are not mutually exclusive; Clk hyperphosphorylation may inhibit E-box binding as well as promote Clk degradation. Dbt is able to enter the nucleus in per⁰¹ flies, but does not associate with Clk in the absence of Per. This suggests that Per is required to either bring Dbt into a complex with Clk-Cyc, enable phosphorylation of Clk after Dbt enters the complex, or both. Once the Tim-Per-Dbt-Clk-Cyc complex has formed, hyperphosphorylated Per and Clk levels decline in a coordinated fashion by mid-day. Tim is eliminated prior to hyperphosphorylated Per and Clk via separate light-dependent and light-independent mechanisms. As hyperphosphorylated Clk and Clk mRNA decline during the day, hypophosphorylated Clk accumulates. This hypophosphorylated Clk forms complexes with Cyc and binds E-boxes in the absence of nuclear Tim-Per-Dbt complexes, thus initiating the next cycle of transcription (Yu, 2006 and references therein).

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).

PDF and cAMP enhance Per stability in Drosophila clock neurons

The neuropeptide PDF is important for Drosophila circadian rhythms: pdf⁰¹ (pdf-null) animals are mostly arrhythmic or short period in constant darkness and have an advanced activity peak in light-dark conditions. PDF contributes to the amplitude, synchrony, as well as the pace of circadian rhythms within clock neurons. PDF is known to increase cAMP levels in PDR receptor (PDFR)-containing neurons. However, there is no known connection of PDF or of cAMP with the Drosophila molecular clockworks. This study discovered that the mutant period gene per^S ameliorates the phenotypes of pdf-null flies. The period protein (Per) is a well-studied repressor of clock gene transcription, and the per^S protein (PerS) has a markedly short half-life. The result therefore suggests that the PDF-mediated increase in cAMP might lengthen circadian period by directly enhancing Per stability. Indeed, increasing cAMP levels and cAMP-mediated protein kinase A (PKA) activity stabilizes Per, in S2 tissue culture cells and in fly circadian neurons. Adding PDF to fly brains in vitro has a similar effect. Consistent with these relationships, a light pulse causes more prominent Per degradation in pdf⁰¹ circadian neurons than in wild-type neurons. The results indicate that PDF contributes to clock neuron synchrony by increasing cAMP and PKA, which enhance Per stability and decrease clock speed in intrinsically fast-paced PDFR-containing clock neurons. It is further suggested that the more rapid degradation of PerS bypasses PKA regulation and makes the pace of clock neurons more uniform, allowing them to avoid much of the asynchrony caused by the absence of PDF (Li, 2014).

Since the original observation that pdf⁰¹ flies have a highly reliable 1-2 h advanced activity phase in LD and short period in DD before they become arrhythmic, it has been assumed that PDF functions at least in part to lengthen the period of at least some brain oscillators that run too fast in its absence. Indeed, there is evidence in favor of this notion, and it is likely that the pdf⁰¹ strain arrhythmicity results from conflicts between neuronal oscillators that run too fast and others that maintain a ∼24-h pace or may even run more slowly without PDF. The substantial improvement of pdf⁰¹ rhythmicity by the per^S gene therefore suggests that per^S endows all oscillators with such a short period that they have a more uniform pace and substantially reduced oscillator asynchrony without PDF (Li, 2014).

Although there was no information on how PDF might function to lengthen the period of the fast oscillators, the effect of per^S implicates Per as a candidate molecular target. Because PerS is known to disappear rapidly in the nighttime, this further suggests that the Per degradation rate might be the biochemical target of PDF period lengthening. An even more specific version of this notion follows from the PDF-mediated increases in cAMP levels in PDFR-expressing clock neurons. Because PDFR is expressed in many clock neurons, including subsets of LNvs, LNds, and DN1s, this increase in cAMP may slow the pace of Per degradation in intrinsically fast-paced PDFR-expressing clock neurons. Indeed, the data indicate that increasing cAMP levels and PKA activity inhibits Per degradation in cell culture as well as in fly brains. Although these increases are probably in excess of what normally occurs in response to PDF, addition of PDF to brains in vitro has a similar effect. Because the additions of kinase inhibitors Rp-cAMPS and PKI increased the rate of Per degradation in S2 cells as well as in brains, it is suggested that PDF-induced up-regulation of cAMP level and PKA activity likely affect Per stability (Li, 2014).

A light pulse at night caused more prominent Per degradation in pdf⁰¹ mutant flies than in wild-type flies. As nighttime light also causes premature Tim degradation and a consequent advance in Per degradation in many clock neurons, some of these neurons could be the intrinsically fast (22- to 23-h period) oscillators that are impacted by PDF and experience enhanced cAMP levels to slow their rate of Per degradation and clock pace. These probably include the s-LNvs and the DN1s, many of which are PDFR-positive. Based on the behavioral phenotype of pdf⁰¹ flies in LD and DD, the effect of PDF on Per degradation probably occurs in the late night–early morning in a LD cycle and at the same (subjective) time in DD. This is also the time when Per degradation is most prominent (Li, 2014).

Interestingly, the firing rate of PDF-containing neurons, the l-LNvs as well as the s-LNvs, is also maximal near the beginning of the day, in DD as well as LD; this is also the likely time of maximal PDF release from s-LNv dorsal projections. In addition, the l-LNvs promote light-mediated arousal, also mediated at least in part by PDF. Taken together with the fact that light has been shown to increase the firing rate of l-LNvs in a CRY-dependent manner, it is likely that lights on in the morning also potentiates the PDF-cAMP system. Note that the end of the night-beginning of the day is the time in the circadian cycle dominated by clock protein turnover, i.e., this is when there is little per or tim RNA or protein synthesis. This further supports a focus on clock protein turnover regulation at these times (Li, 2014).

Because the mammalian neuropeptide VIP contributes to oscillator synchrony within the SCN in a manner that resembles at least superficially the contribution of PDF to oscillator synchrony within the fly brain circadian network, VIP might function similarly to PDF. However, VIP probably connects differently to the mammalian clock system. For example, morning light almost certainly up-regulates clock protein transcription in mammals, for example, per1 transcription. Therefore, VIP-mediated up-regulation of cAMP levels probably activates CREB and clock gene transcription through CRE sites in mammalian clock gene promoters rather than influencing clock protein turnover like in flies (Li, 2014).

The stabilization effect of PDF and cAMP on Per requires PKA activity within circadian neurons. The effect could be indirect, through unknown PKA targets including other clock proteins. However, Per is known to be directly phosphorylated by multiple kinases; they include Nemo, which stabilizes Per. In addition, a study in Neurospora shows that PKA directly phosphorylates and stabilizes FRQ. Because FRQ and Per have similar roles, protein turnover in the two clock systems may be similar beyond the shared role of the CK1 kinase. Based also on the S2 cell experiments, it is suggested that PKA directly phosphorylates Per and enhances its stability. This could occur by inhibiting a conformational switch to a less stable structure, a possibility that also applies to NEMO-mediated Per stabilization. PKA could also phosphorylate other clock proteins; this is by analogy to the known Per kinases Nemo and Doubletime (Dbt), which also phosphorylate Clk (Li, 2014).

The more rapid intrinsic degradation of PerS may at least partially bypass the effect of PKA phosphorylation and therefore PDFR stimulation. This may endow all circadian neurons with a more uniform period, which can maintain synchrony and therefore rhythmicity without PDF. The fact that PerS is less sensitive than Per to increases in cAMP levels is consistent with this interpretation, although an earlier phase of PerS degradation might also influence this result (Li, 2014).

One further consideration is the 0.5-h period difference between the per^S and the per^S;;pdf⁰¹ strains. A residual period-lengthening effect of PDF suggests that per^S does not endow all oscillators with the identical period, i.e., that there is still some asynchrony between different per^S neurons without PDF. This may reflect an incomplete bypass of PKA by PerS or an additional effect of cAMP or PKA on other clock proteins. Nonetheless, several per^S neuronal oscillators maintain a strong amplitude without PDF. Although this is commonly taken to reflect an effect on synchrony, another possibility is based on data indicating that PDF normally enhances oscillator amplitude as well as synchrony; weak amplitudes may then be the more proximal cause of behavioral arrhythmicity. With this notion in mind, it is suggested that PerS-containing oscillators are not only short period but also more robust, i.e., that the more rapid turnover of PerS makes the clock stronger. More robust rhythmicity is also apparent in the behavioral records of all per^S-containing strains. In this view, the stronger degradation 'drive' of PerS makes these oscillators more cell autonomous and therefore less dependent on neuronal mechanisms like firing and PDF release, which enhance oscillator synchrony and amplitude. The general notion is that discrete differences in clock molecule properties can change the relationship of the transcriptional cycle to the circadian brain network (Li, 2014).

An ecdysone-responsive nuclear receptor regulates circadian rhythms in Drosophila

Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and Clock/Cycle (Clk/Cyc) (in Drosophila) drive the expression of the period (per) family genes. The Per-containing complexes inhibit the activity of CLOCK/BMAL1 or Clk/Cyc, thereby forming a negative feedback loop. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. This study shows that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor Unfulfilled (Hr51) are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and Unf bind to per regulatory sequences and act together to enhance the Clk/Cyc-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. These results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks (Jaumouille, 2015).

This study has identified the nuclear receptors E75 and UNF as components of the molecular clocks in the s-LNvs. E75 is the closest homolog of mammalian REV-ERB α and REV-ERB β, which play important roles in the molecular clock feedback loops. In contrast with Rev-Erb α/β, which represses transcription, the results demonstrated that E75 is neither a potent repressor nor a strong activator but potentiates the activation of per transcription by UNF. Despite these mechanistic divergences, the notion that Rev-Erb homologs are integral to the molecular oscillators in both Drosophila and mammals highlights the significance of transcriptional regulations via nuclear receptors in metazoan circadian clocks (Jaumouille, 2015).

Rev-Erb α and Rev-Erb α are rhythmically transcribed by the CLOCK/BMAL1 transcriptional activators, and REV-ERBs periodically repress the transcription of Bmal1, thereby forming a feedback loop to ensure robust molecular oscillations of the mammalian clock. A previous study demonstrated that E75 is a cycling target of Clk/Cyc in the fly head (Kumar, 2014). Because E75 has three isoforms, it was not possible to determine whether any of the isoforms were rhythmically expressed in the LNvs from the RNA profiles of the isolated LNvs. Nonetheless, the results indicate that E75 together with Unf (which is not a Clk/Cyc target) reinforces the main loop of the core fly clock composed of Clk/Cyc and Per/Tim through a feedforward mechanism, showcasing the mechanistic parallels between fly and mammalian clocks (Jaumouille, 2015).

E75 has been demonstrated to covalently bind to heme, and its binding appears to stabilize the E75 and facilitates the binding of nitric oxide (NO) and carbon monoxide (CO). The NO/CO binding to E75 modulates the transcriptional activity of its known heterodimeric partner DHR3. To test whether similar mechanisms are involved in the action of E75 in the s-LNvs, attempts were made to disrupt cellular heme metabolism by knocking down the enzymes in the heme biosynthesis pathway, coproporphyrinogen oxidase (Coprox) and protoporphyrinogen oxidase (Ppox), and the key enzyme in the heme degradation pathway, heme oxygenase (Ho). These experiments were inconclusive, as no effect on the behavioral rhythms were observed by any knockdown with Pdf-GAL4, and knockdown with Tim-GAL4 was lethal (Jaumouille, 2015).

S2 cell experiments showed that Unf is a transcriptional activator of per, and concurrent expression of E75 and Unf increases the turnover of Unf binding to per regulatory sequences. This high turnover is correlated with higher transcriptional activity. The finding that E75 acts through Unf on transcription is consistent with in vivo data: (1) depletion of both Unf and E75 in adult LNvs abolishes the behavioral rhythms; (2) E75 overexpression has no effect on the behavioral rhythms; and (3) E75 overexpression does not rescue Unf knockdown. Although unf mRNA levels do not oscillate, Unf protein levels cycle in the s-LNvs, peaking at zeitgeber time (ZT)2 and lowest at ZT14. Low Unf levels may reflect the degradation as a consequence of higher transcriptional activity. Indeed, per is most actively transcribed around ZT13 when Unf levels are minimum in the s-LNvs. Nonetheless, downregulation and arrhythmia of Per levels in the s-LNvs is most probably not the sole cause of the altered locomotor rhythms in the Unf knockdown, E75 knockdown, and Unf/E75 double knockdown. A recent study showed the implication of E75 in the repression of Clk transcription, although the current results are not in concordance with this observation probably due to the differences in the reagents used for E75 knockdown and the timing of knockdown. Deciphering whether E75 and Unf heterodimerize or bind to adjacent sequences, how they cooperate with Clk/Cyc, and whether any ligand is involved in their transcriptional regulation will yield new insights into the diverse mode of nuclear receptor crosstalk and their critical roles in circadian biology (Jaumouille, 2015).

Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity

Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian cellular clocks continue to tick away in darkness without intercellular communication. To investigate these issues in Drosophila, behavior as well as molecular rhythms were assayed within individual brain clock neurons while blocking communication within the ca. 150 neuron clock network. CRISPR-mediated neuron-specific circadian clock knockouts were also generated. The results point to two key clock neuron groups: loss of the clock within both regions but neither one alone has a strong behavioral phenotype in darkness; communication between these regions also contributes to circadian period determination. Under these dark conditions, the clock within one region persists without network communication. The clock within the famous PDF-expressing s-LNv neurons however was strongly dependent on network communication, likely because clock gene expression within these vulnerable sLNvs depends on neuronal firing or light (Schlichting, 2019).

Neuronal networks make myriad contributions to behavior and physiology. By definition, individual neurons within a network interact, and different networks also interact to coordinate specialized functions. For example, the visual cortex and motor output centers must coordinate to react properly to environmental changes. In a less immediate fashion, sleep centers and circadian clocks are intertwined to properly orchestrate animal physiology. The brain clock is of special interest: it not only times and coordinates physiology within neuronal tissues but also sends signals to the body to keep the entire organism in sync with the cycling external environment (Schlichting, 2019).

The small, circumscribed Drosophila clock network is ideal to address circadian communication issues. The comparable region in mammals, the suprachiasmatic nucleus, is composed of thousands of cells depending on the species. There are in contrast only 75 clock neurons per hemisphere in Drosophila. These different clock neurons can be divided into several subgroups according to their location within the fly brain. There are 4 lateral and three dorsal neuron clusters, which have different functions in controlling fly physiology (Schlichting, 2019).

The four small ventro-lateral neurons (sLNvs) are arguably the most important of the 75 clock neurons. This is because ablating or silencing these neurons abolishes rhythms in constant darkness (DD). They reside in the accessory medulla region of the fly brain, an important pacemaker center in many insects, and express the neuropeptide PDF. In addition, they are essential for predicting dawn. A very recent study suggests that the sLNvs are also able to modulate the timing of the evening (E) peak of behavior via PDF. The other ventral-lateral group, the four large-ventro-lateral neurons (lLNvs), also express PDF and send projections to the medulla, the visual center of the fly brain; they are important arousal neurons. Consistent with the ablation experiments mentioned above, the absence of pdf function or reducing PDF levels via RNAi causes substantial arrhythmic behavior in DD (Schlichting, 2019).

Other important clock neurons include the dorso-lateral neurons (LNds), which are essential for the timing of the E peak and adjustment to long photoperiods. Two other clock neuron groups, the lateral-posterior neurons (LPN and a subset of the dorsal neurons (DN1s), were recently shown to connect the clock network to sleep centers in the fly central complex. The DN2 neurons are essential for temperature preference rhythms, whereas no function has so far been assigned to the DN3s (Schlichting, 2019).

Despite these distinct functions, individual clock neuron groups are well-connected to each other. At the anatomical level, all lateral neuron clusters and even DN1 dorsal neurons send some of their projections into the accessory medulla, where they can interact. A second area of common interaction is the dorsal brain; only the lLNvs do not project there (Schlichting, 2019).

Several studies have investigated interactions between different clock neurons. Artificially expressing kinases within specific clock neurons causes their clocks to run fast or slow and also changes the overall free-running period of the fly, indicating that network signaling adjusts behavior. Similarly, speeding up or slowing down individual neurons is able to differentially affect behavioral timing in standard light-dark (LD) cycles. A high level of neuronal plasticity within the network also exists: axons of individual cells undergo daily oscillations in their morphology, and neurons change their targets depending on the environmental condition (Schlichting, 2019).

How neuronal communication influences the fly core feedback loop is not well understood. The latter consists of several interlocked transcriptional-translational feedback loops, which probably underlie rhythms in behavior and physiology. A simplified version of the core feedback loop consists of the transcriptional activators Clock (CLK) and Cycle (CYC) and the transcriptional repressors Period (PER) and Timeless (TIM). CLK and CYC bind to E-boxes within the period (per) and timeless (tim) genes (among other clock-controlled genes) and activate their transcription. After PER and TIM synthesis in the cytoplasm, they form a heterodimer and enter the nucleus toward the end of the night. There they interact with CLK and CYC, release them from their E-box targets and thereby inhibit their own transcription. All 75 pairs of clock neurons contain this canonical circadian machinery, which undergoes daily oscillations in level. Indeed, the immunohistochemical cycling of PER and TIM within these neurons is a classic assay to visualize these molecular oscillations (Schlichting, 2019).

Silencing PDF neurons stops their PER cycling, indicating an important role of neuronal firing in maintaining circadian oscillations. However, only two time points were measured, and the results were possibly confounded by developmental effects. PDF neuron silencing also phase advances PER cycling in downstream neurons, suggesting that PDF normally serves to delay cycling in target neurons. This is consistent with experiments showing that PDF signaling stabilizes PER. In addition, neuronal activation is able to mimic a light pulse and phase shift the clock due to firing-mediated TIM degradation (Schlichting, 2019).

To investigate more general features of clock neuron interactions on the circadian machinery, the majority of the fly brain clock neurons were silenced, and behavior and clock protein cycling within the circadian network was examined in a standard light-dark cycle (LD) as well as in constant darkness (DD). Silencing abolished rhythmic behavior but had no effect on clock protein cycling in LD, indicating that the silencing affects circadian output but not oscillator function in a cycling light environment. Silencing similarly abolished rhythmic behavior in DD but with very different effects on clock protein cycling. Although protein cycling in the LNds was not affected by neuronal silencing in DD, the sLNvs dampened almost immediately. Interestingly, this differential effect is under transcriptional control, suggesting that some Drosophila clock neurons experience activity-regulated clock gene transcription. Cell-specific CRISPR/Cas9 knockouts of the core clock protein Per further suggests that network properties are critical to maintain wild-type activity-rest rhythms. The data taken together show that clock neuron communication and firing-mediated clock gene transcription are essential for high amplitude and synchronized molecular rhythms as well as rhythmic physiology (Schlichting, 2019).

The central clock of animals is essential for dictating the myriad diurnal changes in physiology and behavior. Knocking out core clock components such as period or Clock severely disrupts circadian behavior as well as molecular clock properties in flies and mammals. This study show that similar behavioral effects occur when the central clock neurons are silenced and thereby abolish communication within this network and with downstream targets, that is fly behavior becomes arrhythmic in LD as well as DD conditions and resembles the phenotypes of core clock mutant strains (Schlichting, 2019).

Despite the loss of all rhythmic behavior, silencing did not impact the molecular machinery in LD conditions: PER and PDP1 protein cycling was normal. These findings suggest that (1) rhythmic behavior requires clock neuron output, which is uncoupled from the circadian molecular machinery by network silencing, and (2) synchronized molecular rhythms of clock neurons do not require neuronal activity. These findings are in agreement with previous work showing that silencing the PDF neurons had no effect on Per cycling within these neurons. The results presumably reflect the strong effect of the external light-dark cycle on these oscillators (Schlichting, 2019).

In DD however, the individual neurons change dramatically: the different neurons desynchronize, and their protein cycling damps to different extents. Interestingly, sLNv cycling relies most strongly on neuronal communication: these neurons cycle robustly in controls but apparently not at all in the silenced state. sLNvs were previously shown to be essential for DD rhythms. Unfortunately, the sensitivity of immunohistochemistry precludes determining whether the molecular clock has actually stopped or whether silencing has only (dramatically) reduced cycling amplitude. However, a simple interpretation of the adult-specific silencing experiment favors a stopped clock: decreasing the temperature to 18 degrees after a week at high temperature failed to rescue rhythmic behavior. A similar experiment in mammals gave rise to the opposite result, suggesting an effect of firing on circadian amplitude in that case. However, different explanations cannot be excluded, for example chronic effects of neuronal silencing or a too large phase difference between the different neuronal subgroups to reverse after a week without communication (Schlichting, 2019).

In either case, a stopped clock or an effect on clock protein oscillation amplitude, these results make another link to the mammalian literature: modeling of the clock network suggests that different neurons resynchronize more easily if the most highly-connected cells are intrinsically weak oscillators. The sLNvs are essential for DD rhythms, known to communicate with other clock neurons and are situated in the accessory medulla; this is an area of extensive neuronal interactions in many insects. These considerations rationalize weak sLNv oscillators (Schlichting, 2019).

An important role of interneuron communication in DD is in agreement with previous work showing that altering the speed of individual neuron groups can change the phase of downstream target neurons. An important signaling molecule is the neuropeptide PDF: its absence changes the phase of downstream target neurons, and silencing PDF neurons causes an essentially identical phenotype to the lack of PDF. However, the effects reported in this study are much stronger and show different levels of autonomy than PDF ablation, suggesting that other signaling molecules and/or the neuronal activity of additional clock neurons are essential to maintain proper rhythmic clock protein expression (Schlichting, 2019).

To address these possibilities, this study took two approaches. First, clock gene RNA levels were investigated after silencing. The goal was to assess whether the damping of silenced neurons is under gene expression control, likely transcriptional control. Indeed, tim mRNA profiles nicely reproduced the protein cycling profiles: robust cycling of all (assayed) clock neurons was maintained in LD even with silencing, but tim-mRNA levels in the sLNvs stopped cycling in DD; in contrast, robust cycling was maintained in the LNds. This suggests that the changes in protein cycling amplitude and also possibly phase are under transcriptional control. Importantly, the tim signal in the sLNvs disappeared upon silencing, suggesting that neuronal activity promotes clock gene transcription at least in this subset of neurons. This recapitulates for the first time in Drosophila the robust positive relationship between neuronal firing and clock gene transcription in mammals. To date, Drosophila neuronal firing had only been connected to post-transcriptional clock protein regulation, namely TIM degradation. Conceivably, these two effects are connected: TIM degradation might be required to relieve transcriptional repression and maintain cycling (Schlichting, 2019).

The second approach was a cell-specific knockout strategy, applied to the clock neuron network. Three guides were generated targeting the CDS of per and also CAS9 was expressed in a cell-specific manner. The guides caused double strand breaks in the per gene, which in turn led to cell-specific per mutations. This adult brain knockout strategy worked reliably and specifically, in glial cells as well as neurons, with high efficiency and with no apparent background effects. This strategy was successfully used to knock out most if not all Drosophila GPCRs and it is believed to be superior to RNAi for most purposes. Importantly, expression of the guides with the clk856-GAL4 driver phenocopied per⁰¹ behavior. To focus on individual clock neurons, cell-specific knockouts were generated in different clock neurons. PERKO in the PDF cells did not increase the level of arrhythmicity, only a PERKO in most lateral neurons, E cells as well as PDF cells, generated high levels of arrhythmic behavior (Schlichting, 2019).

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).

GENE STRUCTURE

The first and last exons of the commonest cDNA are non-coding. Exon 5 encodes a thronine-glycine repeat with 17 pairs of alternating residues (Citri, 1987).

The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila. The per gene is thought to encode three transcripts via differential splicing (types A, B, and C) that give rise to three proteins. Since the three PER mRNA types are based on the analysis of cDNA clones, RNase protection assays and reverse transcriptase-mediated PCR were carried out to see if these mRNA types are present in vivo. The results show that per generates two transcript types that differ only by the presence (type A) or absence (type B') of an alternative intron in the 3' untranslated region. The proteins produced by the mRNAs are identical. Type A and type B' transcripts are present in different tissues at various ratios: 2.0 ± 0.09 in eyes, 0.82 ± 0.14 in the brain, 0.41 ± 0.10 in the thorax, and 0.32 ± 0.09 in the abdomen. In wild-type fly heads, type A and type B' transcripts cycle with identical phases but different cycling amplitudes; the average cycling amplitude of type A transcripts in three experiments is 8.1-fold; that of type B' transcripts is 5.5-fold. Type A transcripts on the average are 1.2-fold more abundant than type B' transcripts. In fly bodies type A and type B' transcripts cycle with identical phases but with different amplitudes: the average cycling amplitude of type A transcripts from three experiments is 7.5-fold, and that of type B' transcripts is 5.5-fold. In male bodies, type B' transcripts are ~2.0-fold more abundant than type A transcripts, consistent with the levels seen in different body parts. Transgenic flies containing transgenes that produce only type B' transcripts, type A transcripts, or both transcripts rescue locomotor activity rhythms with average periods of 24.7, 25.4, and 24.4 h, respectively. Although no appreciable differences in type A and type B' mRNA cycling are observed, a slower accumulation of Per protein in flies making only type A transcripts suggests that the intron affects the translation of PER mRNA (Cheng, 1998).

Bases in 5' UTR - 497

Exons - eight

Bases in 3' UTR - 368

PROTEIN STRUCTURE

Amino Acids - 1218

Structural Domains and Evolutionary homologs

The PER protein contains an amino acid motif known as the PAS domain, also found in single-minded and the mammalian dioxin receptor. PER contains no known DNA-binding motif (Citri, 1987 and Huang, 1993). PER protein has a threonine-glycine repeat involved in temperature adaptation (Costa, 1992).

date revised: 1 March 2024 

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