period: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity and Post-transcriptional Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - period

Synonyms - clock (clk)

Cytological map position - 3B1-2

Function - photoperiod regulation - transcription factor

Keywords - neural, photoperiod response, transcriptional repressor

Symbol - per

FlyBase ID:FBgn0003068

Genetic map position - 1-1.2

Classification - PAS protein

Cellular location - nuclear and cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene |

Recent literature
Kang, S. W., Lee, E., Cho, E., Seo, J. H., Ko, H. W. and Kim, E. Y. (2015). Drosophila peptidyl-prolyl isomerase Pin1 modulates circadian rhythms via regulating levels of PERIOD. Biochem Biophys Res Commun. PubMed ID: 25998391.
Summary:
In animal circadian clock machinery, the phosphorylation program of Period (Per) leads to the spatio-temporal regulation of diverse Per functions, which are crucial for the maintenance of approximately 24-hr circadian rhythmicity. The peptidyl-prolyl isomerase PIN1 modulates the diverse functions of its substrates by inducing conformational changes upon recognizing specific phosphorylated residues. This study shows that overexpression of Drosophila pin1, dodo (dod), lengthens the locomotor behavioral period. Using Drosophila S2 cells, it was demonstrated that Dod associates preferentially with phosphorylated species of Per, which delays the phosphorylation-dependent degradation of Per. Consistent with this, Per protein levels are higher in flies overexpressing dod. Taken together, it is suggested that Dod plays a role in the maintenance of circadian period by regulating Per metabolism.

Medina, I., Casal, J. and Fabre, C.C. (2015). Do circadian genes and ambient temperature affect substrate-borne signalling during Drosophila courtship? Biol Open [Epub ahead of print]. PubMed ID: 26519517
Summary:
Courtship vibratory signals can be air-borne or substrate-borne. They convey distinct and species-specific information from one individual to its prospective partner. This study focuses on the substrate-borne vibratory signals generated by the abdominal quivers of the Drosophila male during courtship; these vibrations travel through the ground towards courted females and coincide with female immobility. It is not known which physical parameters of the vibrations encode the information that is received by the females and induces them to pause. The intervals between each vibratory pulse were examined, a feature that was reported to carry information for animal communication. However, evidence of periodic variations in the lengths of these intervals could not be found, as has been reported for fly acoustical signals. Because it has been suggested that the genes involved in the circadian clock may also regulate shorter rhythms, effects of period on the interval lengths were determined. Males that were mutant for the period gene produce vibrations with significantly altered interpulse intervals; also, treating wild type males with constant light results in similar alterations to the interpulse intervals. These results suggest that both the clock and light/dark cycles have input into the interpulse intervals of these vibrations. By altering the interpulse intervals by other means, it was found that ambient temperature also has a strong effect. However, behavioural analysis suggests that only extreme ambient temperatures can affect the strong correlation between female immobility and substrate-borne vibrations.

Chiu, J. C. and Edery, I. (2015). Identification of light-sensitive phosphorylation sites on PERIOD that regulate the pace of circadian rhythms in Drosophila. Mol Cell Biol [Epub ahead of print]. PubMed ID: 26711257
Summary:
The main components regulating the pace of circadian clocks in animals are Period (Per) proteins, transcriptional regulators that by means of complex multi-site phosphorylation programs undergo daily changes in levels and nuclear accumulation. This study investigated the function of two phosphorylation sites at Ser826 and Ser828 located in a putative nuclear localization signal (NLS) on the Drosophila melanogaster Per protein. These sites are phosphorylated by Doubletime (Dbt; Drosophila homolog of CK1delta/), the key circadian kinase regulating the daily changes in Per stability and phosphorylation. Mutant flies where phosphorylation at Ser826/Ser828 is blocked manifest behavioral rhythms with periods slightly longer then 1 hour and altered temperature compensation properties. Intriguingly, although phosphorylation at these sites does not influence Per stability, timing of nuclear entry or transcriptional autoinhibition, the phospho-occupancy at Ser826/Ser828 is rapidly stimulated by light and blocked by Timeless (Tim), the major photosensitive clock component in Drosophila and a crucial binding partner of Per. These findings identify the first phosphorylation sites on core clock proteins that are acutely regulated by photic cues and suggest that some phospho-sites on Per proteins can modulate the pace of downstream behavioral rhythms without altering central aspects of the clock mechanism.

Allen, V.W., O'Connor, R.M., Ulgherait, M., Zhou, C.G., Stone, E.F., Hill, V.M., Murphy, K.R., Canman, J.C., Ja, W.W. and Shirasu-Hiza, M.M. (2015). period-regulated feeding behavior and TOR signaling modulate survival of infection. Curr Biol [Epub ahead of print]. PubMed ID: 26748856
Summary:
Most metazoans undergo dynamic, circadian-regulated changes in behavior and physiology. Currently, it is unknown how circadian-regulated behavior impacts immunity against infection. This study of behaviorally arrhythmic Drosophila circadian period mutants identifies a novel link between nutrient intake and tolerance of infection with B. cepacia, a bacterial pathogen of rising importance in hospital-acquired infections. Infection tolerance in wild-type animals was found to be stimulated by acute exposure to dietary glucose and amino acids. Glucose-stimulated tolerance was induced by feeding or direct injection; injections reveal a narrow window for glucose-stimulated tolerance. In contrast, amino acids stimulate tolerance only when ingested. The role of a known amino-acid-sensing pathway, the TOR (Target of Rapamycin) pathway, was investigated in immunity. TORC1 is circadian regulated and inhibition of TORC1 decreases resistance, as in vertebrates. Surprisingly, inhibition of the less well-characterized TOR complex 2 (TORC2) dramatically increases survival, through both resistance and tolerance mechanisms. This work suggests that dietary intake on the day of infection by B. cepacia can make a significant difference in long-term survival. TOR signaling mediates both resistance and tolerance of infection, and TORC2 was identified as a novel potential therapeutic target for increasing survival of infection.

Liao, J., Seggio, J. A. and Ahmad, S. T. (2016). Mutations in the circadian gene period alter behavioral and biochemical responses to ethanol in Drosophila. Behav Brain Res 302: 213-219. PubMed ID: 26802726
Summary:
Clock genes, such as period, which maintain an organism's circadian rhythm, can have profound effects on metabolic activity, including ethanol metabolism. In turn, ethanol exposure has been shown in Drosophila and mammals to cause disruptions of the circadian rhythm. Previous studies from our labs have shown that larval ethanol exposure disrupted the free-running period and period expression of Drosophila. In addition, a recent study has shown that arrhythmic flies show no tolerance to ethanol exposure. As such, Drosophila period mutants, which have either a shorter than wild-type free-running period (perS) or a longer one (perL), may also exhibit altered responses to ethanol due to their intrinsic circadian differences. This study tested the initial sensitivity and tolerance of ethanol exposure on Canton-S, perS, and perL, and then measured their Alcohol Dehydrogenase (ADH) and body ethanol levels. perL flies had slower sedation rate, longer recovery from ethanol sedation, and generated higher tolerance for sedation upon repeated ethanol exposure compared to Canton-S wild-type flies. Furthermore, perL flies had lower ADH activity and had a slower ethanol clearance compared to wild-type flies. The findings of this study suggest that period mutations influence ethanol induced behavior and ethanol metabolism in Drosophila and that flies with longer circadian periods are more sensitive to ethanol exposure.

Parisky, K.M., Agosto Rivera, J.L., Donelson, N.C., Kotecha, S. and Griffith, L.C. (2016). Reorganization of sleep by temperature in Drosophila requires light, the homeostat, and the circadian clock. Curr Biol [Epub ahead of print]. PubMed ID: 26972320
Summary:
Increasing ambient temperature reorganizes the Drosophila sleep pattern in a way similar to the human response to heat, increasing daytime sleep while decreasing nighttime sleep. Mutation of core circadian genes blocks the immediate increase in daytime sleep, but not the heat-stimulated decrease in nighttime sleep, when animals are in a light:dark cycle. The ability of per01 flies to increase daytime sleep in light:dark can be rescued by expression of PER in either LNv or DN1p clock cells and does not require rescue of locomotor rhythms. Prolonged heat exposure engages the homeostat to maintain daytime sleep in the face of nighttime sleep loss. In constant darkness, all genotypes show an immediate decrease in sleep in response to temperature shift during the subjective day, implying that the absence of light input uncovers a clock-independent pro-arousal effect of increased temperature. Interestingly, the effects of temperature on nighttime sleep are blunted in constant darkness and in cryOUT mutants in light:dark, suggesting that they are dependent on the presence of light the previous day. In contrast, flies of all genotypes kept in constant light sleep more at all times of day in response to high temperature, indicating that the presence of light can invert the normal nighttime response to increased temperature. The effect of temperature on sleep thus reflects coordinated regulation by light, the homeostat, and components of the clock, allowing animals to reorganize sleep patterns in response to high temperature with rough preservation of the total amount of sleep.

Fu, J., Murphy, K.A., Zhou, M., Li, Y.H., Lam, V.H., Tabuloc, C.A., Chiu, J.C. and Liu, Y. (2016). Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD. Genes Dev 30: 1761-1775. PubMed ID: 27542830
Summary:
Codon usage bias is a universal feature of all genomes, but its in vivo biological functions in animal systems are not clear. To investigate the in vivo role of codon usage in animals, this study took advantage of the sensitivity and robustness of the Drosophila circadian system. By codon-optimizing parts of Drosophila period (per), a core clock gene that encodes a critical component of the circadian oscillator, per codon usage was shown to be important for circadian clock function. Codon optimization of per results in conformational changes of the PER protein, altered PER phosphorylation profile and stability, and impaired PER function in the circadian negative feedback loop, which manifests into changes in molecular rhythmicity and abnormal circadian behavioral output. The study provides an in vivo example that demonstrates the role of codon usage in determining protein structure and function in an animal system. These results suggest a universal mechanism in eukaryotes that uses a codon usage "code" within genetic codons to regulate cotranslational protein folding.

Zhou, J., Yu, W. and Hardin, P. E. (2016). CLOCKWORK ORANGE enhances PERIOD mediated rhythms in transcriptional repression by antagonizing E-box binding by CLOCK-CYCLE. PLoS Genet 12: e1006430. PubMed ID: 27814361
Summary:
The Drosophila circadian oscillator controls daily rhythms in physiology, metabolism and behavior via transcriptional feedback loops. CLOCK-CYCLE (CLK-CYC) heterodimers initiate feedback loop function by binding E-box elements to activate per and tim transcription. PER-TIM heterodimers then accumulate, bind CLK-CYC to inhibit transcription, and are ultimately degraded to enable the next round of transcription. The timing of transcriptional events in this feedback loop coincide with, and are controlled by, rhythms in CLK-CYC binding to E-boxes. PER rhythmically binds CLK-CYC to initiate transcriptional repression, and subsequently promotes the removal of CLK-CYC from E-boxes. However, little is known about the mechanism by which CLK-CYC is removed from DNA. Previous studies demonstrated that the transcription repressor CLOCKWORK ORANGE (CWO) contributes to core feedback loop function by repressing per and tim transcription in cultured S2 cells and in flies. This study shows that CWO rhythmically binds E-boxes upstream of core clock genes in a reciprocal manner to CLK, thereby promoting PER-dependent removal of CLK-CYC from E-boxes, and maintaining repression until PER is degraded and CLK-CYC displaces CWO from E-boxes to initiate transcription. These results suggest a model in which CWO co-represses CLK-CYC transcriptional activity in conjunction with PER by competing for E-box binding once CLK-CYC-PER complexes have formed. Given that CWO orthologs DEC1 and DEC2 also target E-boxes bound by CLOCK-BMAL1, a similar mechanism may operate in the mammalian clock.
Harper, R. E., Dayan, P., Albert, J. T. and Stanewsky, R. (2016). Sensory conflict disrupts activity of the Drosophila circadian network. Cell Rep 17: 1711-1718. PubMed ID: 27829142
Summary:
Periodic changes in light and temperature synchronize the Drosophila circadian clock, but the question of how the fly brain integrates these two input pathways to set circadian time remains unanswered. This study explored multisensory cue combination by testing the resilience of the circadian network to conflicting environmental inputs. Misaligned light and temperature cycles can lead to dramatic changes in the daily locomotor activities of wild-type flies during and after exposure to sensory conflict. This altered behavior is associated with a drastic reduction in the amplitude of Period (Per) oscillations in brain clock neurons and desynchronization between light- and temperature-sensitive neuronal subgroups. The behavioral disruption depends heavily on the phase relationship between light and temperature signals. These results represent a systematic quantification of multisensory integration in the Drosophila circadian system and lend further support to the view of the clock as a network of coupled oscillatory subunits.
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 (see reviews by Hardin, The Circadian Timekeeping System of Drosophila and Vallone, Start the clock! Circadian rhythms and development). 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 timelessUL (UL for ultralong) generates 33 hr rhythms, prolonged nuclear localization of Period/TimelessUL protein complexes, and protracted derepression of period and timeless transcription. Light induced elimination of TimUL from nuclear Per/TimUL 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. timUL 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/TimUL 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 dbtAR/dbtP 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).

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 perS ameliorates the phenotypes of pdf-null flies. The period protein (Per) is a well-studied repressor of clock gene transcription, and the perS 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 perS gene therefore suggests that perS 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 perS 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 perS and the perS;;pdf01 strains. A residual period-lengthening effect of PDF suggests that perS does not endow all oscillators with the identical period, i.e., that there is still some asynchrony between different perS 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 perS 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 perS-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).


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).

cDNA clone length - There are three species of the cDNA, the most common being 4.5 kb

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).


period: Evolutionary Homologs | Regulation | Targets of Activity and Post-transcriptional Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised: 28 MAY 97 

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