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: | Entrez Gene |
Recent literature
Cao, W. and Edery, I. (2017). Mid-day siesta in natural populations of D. melanogaster from Africa exhibits an altitudinal cline and is regulated by splicing of a thermosensitive intron in the period clock gene. BMC Evol Biol 17(1): 32. PubMed ID: 28114910
Many diurnal animals exhibit a mid-day 'siesta', generally thought to be an adaptive response aimed at minimizing exposure to heat on warm days, suggesting that in regions with cooler climates mid-day siestas might be a less prominent feature of animal behavior. Drosophila exhibits thermal plasticity in its mid-day siesta that is partly governed by the thermosensitive splicing of the 3'-terminal intron (termed dmpi8) from the key circadian clock gene period (per). For example, decreases in temperature lead to progressively more efficient splicing, which increasingly favors activity over sleep during the mid-day. This study sought to determine if the adaptation of Drosophila from its ancestral range in the lowlands of tropical Africa to the cooler temperatures found at high altitudes involved changes in mid-day sleep behavior and/or dmpi8 splicing efficiency. Using natural populations of Drosophila from different altitudes in tropical Africa, flies from high elevations were shown to have a reduced mid-day siesta and less consolidated sleep. A single nucleotide polymorphism (SNP) in the per 3' UTR has strong effects on dmpi8 splicing and mid-day sleep levels in both low and high altitude flies. Intriguingly, high altitude flies with a particular variant of this SNP exhibit increased dmpi8 splicing efficiency compared to their low altitude counterparts, consistent with reduced mid-day siesta. Thus, a boost in dmpi8 splicing efficiency appears to have played a prominent but not universal role in how African flies adapted to the cooler temperatures at high altitude. These findings point towards mid-day sleep behavior as a key evolutionary target in the thermal adaptation of animals.
Beckwith, E. J., Hernando, C. E., Polcownuk, S., Bertolin, A. P., Mancini, E., Ceriani, M. F. and Yanovsky, M. J. (2017). Rhythmic behavior is controlled by the SRm160 splicing factor in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 28801530
Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kD (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period (per) levels. Moreover, rhythmic accumulation of the neuropeptide Pigment Dispersing Factor (PDF) in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160 downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model.
Fujii, S., Emery, P. and Amrein, H. (2017). SIK3-HDAC4 signaling regulates Drosophila circadian male sex drive rhythm via modulating the DN1 clock neurons. Proc Natl Acad Sci U S A 114(32): E6669-e6677. PubMed ID: 28743754
The physiology and behavior of many organisms are subject to daily cycles. In Drosophila melanogaster the daily locomotion patterns of single flies are characterized by bursts of activity at dawn and dusk. Two distinct clusters of clock neurons-morning oscillators (M cells) and evening oscillators (E cells)-are largely responsible for these activity bursts. In contrast, male-female pairs of flies follow a distinct pattern, most notably characterized by an activity trough at dusk followed by a high level of male courtship during the night. This male sex drive rhythm (MSDR) is mediated by the M cells along with DN1 neurons, a cluster of clock neurons located in the dorsal posterior region of the brain. This study reports that males lacking Salt-inducible kinase 3 (SIK3) expression in M cells exhibit a short period of MSDR but a long period of single-fly locomotor rhythm (SLR). Moreover, lack of Sik3 in M cells decreases the amplitude of Period (Per) cycling in DN1 neurons, suggesting that SIK3 non-cell-autonomously regulates DN1 neurons' molecular clock. This study also shows that Sik3 reduction interferes with circadian nucleocytoplasmic shuttling of Histone deacetylase 4 (HDAC4), a SIK3 phosphorylation target, in clock neurons and that constitutive HDAC4 localization in the nucleus shortens the period of MSDR. Taking these findings together, it is concluded that SIK3-HDAC4 signaling in M cells regulates MSDR by regulating the molecular oscillation in DN1 neurons.
Long, D. M. and Giebultowicz, J. M. (2017). Age-related changes in the expression of the circadian clock protein PERIOD in Drosophila glial cells. Front Physiol 8: 1131. PubMed ID: 29375400
Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER) declines in the heads of aged Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Immunocytochemistry experiments have been conducted to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. The data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain.
Zhang, Z., Cao, W. and Edery, I. (2018). The SR protein B52/SRp55 regulates splicing of the period thermosensitive intron and mid-day siesta in Drosophila. Sci Rep 8(1): 1872. PubMed ID: 29382842
Similar to many diurnal animals, Drosophila melanogaster exhibits a mid-day siesta that is more robust as temperature increases, an adaptive response that aims to minimize the deleterious effects from exposure to heat. This temperature-dependent plasticity in mid-day sleep levels is partly based on the thermal sensitive splicing of an intron in the 3' untranslated region (UTR) of the circadian clock gene termed period (per). This study evaluated a possible role for the serine/arginine-rich (SR) splicing factors in the regulation of the 3’-terminal intron (termed dmpi8) from period splicing efficiency and mid-day siesta. Using a Drosophila cell culture assay B52/SRp55 increases dmpi8 splicing efficiency, whereas other SR proteins have little to no effect. The magnitude of the stimulatory effect of B52 on dmpi8 splicing efficiency is modulated by natural variation in single nucleotide polymorphisms (SNPs) in the per 3' UTR that correlate with B52 binding levels. Down-regulating B52 expression in clock neurons increases mid-day siesta and reduces dmpi8 splicing efficiency. These results establish a novel role for SR proteins in sleep and suggest that polymorphisms in the per 3' UTR contribute to natural variation in sleep behavior by modulating the binding efficiencies of SR proteins.
Fropf, R., Zhou, H. and Yin, J. C. P. (2018). The clock gene period differentially regulates sleep and memory in Drosophila. Neurobiol Learn Mem [Epub ahead of print]. PubMed ID: 29474956
Circadian regulation is a conserved phenomenon across the animal kingdom, and its disruption can have severe behavioral and physiological consequences. Core circadian clock proteins are likewise well conserved from Drosophila to humans. While the molecular clock interactions that regulate circadian rhythms have been extensively described, additional roles for clock genes during complex behaviors are less understood. This study showed that mutations in the clock gene period (per) result in differential time-of-day effects on acquisition and long-term memory of aversive olfactory conditioning. Sleep is also altered in period mutants: while its overall levels don't correlate with memory, sleep plasticity in different genotypes correlates with immediate performance after training. This study further describes distinct anatomical bases for Period function by manipulating Period activity in restricted brain cells and testing the effects on specific aspects of memory and sleep. In the null mutant background, different features of sleep and memory are affected when a form of the period gene is reintroduce in glia, lateral neurons, and the fan-shaped body. The results indicate that the role of the clock gene period may be separable in specific aspects of sleep or memory; further studies into the molecular mechanisms of these processes suggest independent neural circuits and molecular cascades that mediate connections between the distinct phenomena.
Gorska-Andrzejak, J., Chwastek, E. M., Walkowicz, L. and Witek, K. (2018). On variations in the level of PER in glial clocks of Drosophila optic lobe and its negative regulation by PDF signaling. Front Physiol 9: 230. PubMed ID: 29615925
The level of the core protein of the circadian clock Period (PER) expressed by glial peripheral oscillators depends on their location in the Drosophila optic lobe. It appears to be controlled by the ventral lateral neurons (LNvs) that release the circadian neurotransmitter Pigment Dispersing Factor (PDF). Glial cells of the distal medulla neuropil (dMnGl) that lie in the vicinity of the PDF-releasing terminals of the LNvs possess receptors for PDF (PDFRs) and express PER at significantly higher level than other types of glia. Surprisingly, the amplitude of PER molecular oscillations in dMnGl is increased twofold in PDF-free environment, that is in Pdf0 mutants. The Pdf0 mutants also reveal an increased level of glia-specific protein REPO in dMnGl. The photoreceptors of the compound eye (R-cells) of the PDF-null flies, on the other hand, exhibit de-synchrony of PER molecular oscillations, which manifests itself as increased variability of PER-specific immunofluorescence among the R-cells. Moreover, the daily pattern of expression of the presynaptic protein Bruchpilot (BRP) in the lamina terminals of the R-cells is changed in Pdf0 mutant. Considering that PDFRs are also expressed by the marginal glia of the lamina that surround the R-cell terminals, the LNv pacemakers appear to be the likely modulators of molecular cycling in the peripheral clocks of both the glial cells and the photoreceptors of the compound eye. Consequently, some form of PDF-based coupling of the glial clocks and the photoreceptors of the eye with the central LNv pacemakers must be operational.
Yang, Y. and Edery, I. (2018). Parallel clinal variation in the mid-day siesta of Drosophila melanogaster implicates continent-specific targets of natural selection. PLoS Genet 14(9): e1007612. PubMed ID: 30180162
Similar to many diurnal animals, Drosophila melanogaster exhibits a mid-day siesta that is more robust as ambient temperature rises, an adaptive response aimed at minimizing exposure to heat. Mid-day siesta levels are partly regulated by the thermosensitive splicing of a small intron (termed dmpi8) found in the 3' untranslated region (UTR) of the circadian clock gene period (per). Using the well-studied D. melanogaster latitudinal cline along the eastern coast of Australia, this study showed that flies from temperate populations sleep less during the day compared to those from tropical regions. Combinations of four single nucleotide polymorphisms (SNPs) were identified in the 3' UTR of per that yield several different haplotypes. The two most abundant of these haplotypes exhibit a reciprocal tropical-temperate distribution in relative frequency. Intriguingly, transgenic flies with the major tropical isoform manifest increased daytime sleep and reduced dmpi8 splicing compared to those carrying the temperate variant. These results strongly suggest that for a major portion of D. melanogaster in Australia, thermal adaptation of daily sleep behavior included spatially varying selection on ancestrally derived polymorphisms in the per 3' UTR that differentially control dmpi8 splicing efficiency. Prior work showed that African flies from high altitudes manifest reduced mid-day siesta levels, indicative of parallel latitudinal and altitudinal adaptation across continents. However, geographical variation in per 3' UTR haplotypes was not observed for African flies, providing a compelling case for inter-continental variation in factors targeted by natural selection in attaining a parallel adaptation. It is proposed that the ability to calibrate mid-day siesta levels to better match local temperature ranges is a key adaptation contributing to the successful colonization of D. melanogaster beyond its ancestral range in the lowlands of Sub-Saharan Africa.
Schlichting, M., Menegazzi, P., Rosbash, M. and Helfrich-Forster, C. (2019). A distinct visual pathway mediates high light intensity adaptation of the circadian clock in Drosophila. J Neurosci. PubMed ID: 30606757
In order to provide organisms a fitness advantage, circadian clocks have to react appropriately to changes in their environment. High light intensities (HI) play an essential role in the adaptation to hot summer days, which especially endanger insects of desiccation or prey visibility. This study shows that solely increasing light intensity leads to an increased midday siesta in Drosophila behavior. Interestingly, this change is independent of the fly's circadian photoreceptor cryptochrome (CRY), and solely caused by a small visual organ, the Hofbauer-Buchner (HB) eyelets. Using receptor knockdowns, immunostaining, as well as recently developed calcium tools, the eyelets were shown to activate key core clock neurons, namely the s-LNvs, at HI. This activation delays the decrease of PER in the middle of the day and propagates to downstream target clock neurons that prolong the siesta. Together a new pathway is shown for integrating light intensity information into the clock network, suggesting new network properties and surprising parallels between Drosophila and the mammalian system.
Grima, B., Papin, C., Martin, B., Chelot, E., Ponien, P., Jacquet, E. and Rouyer, F. (2019). Period-controlled deadenylation of the timeless transcript in the Drosophila circadian clock. Proc Natl Acad Sci U S A 116(12): 5721-5726. PubMed ID: 30833404
The Drosophila circadian oscillator relies on a negative transcriptional feedback loop, in which the Period (Per) and Timeless (Tim) proteins repress the expression of their own gene by inhibiting the activity of the Clock (Clk) and Cycle (Cyc) transcription factors. A series of posttranslational modifications contribute to the oscillations of the Per and Tim proteins but few posttranscriptional mechanisms have been described that affect mRNA stability. This study reports that down-regulation of the POP2 deadenylase, a key component of the CCR4-NOT deadenylation complex, alters behavioral rhythms. Down-regulating POP2 specifically increases Tim protein and tim mRNA but not tim pre-mRNA, supporting a posttranscriptional role. Indeed, reduced POP2 levels induce a lengthening of tim mRNA poly(A) tail. Surprisingly, such effects are lost in per (0) mutants, supporting a Per-dependent inhibition of tim mRNA deadenylation by POP2. This study reports a deadenylation mechanism that controls the oscillations of a core clock gene transcript.
Yang, Y. and Edery, I. (2019). Daywake, an anti-siesta gene linked to a splicing-based thermostat from an adjoining clock gene. Curr Biol 29(10): 1728-1734. PubMed ID: 31080079
Sleep is fundamental to animal survival but is a vulnerable state that also limits how much time can be devoted to critical wake-dependent activities. Although many animals are day-active and sleep at night, they exhibit a midday nap, or "siesta," that can vary in intensity and is usually more prominent on warm days. In humans, the balance between maintaining the wake state or sleeping during the day has important health implications, but the mechanisms underlying this dynamic regulation are poorly understood. Using the well-established Drosophila melanogaster animal model to study sleep, this study identified a new wake-sleep regulator that was termed daywake (dyw). dyw encodes a juvenile hormone-binding protein that functions in neurons as a day-specific anti-siesta gene, with little effect on sleep levels during the nighttime or in the absence of light. Remarkably, dyw expression is stimulated in trans via cold-enhanced splicing of the dmpi8 intron from the reverse-oriented but slightly overlapping period (per) clock gene. The functionally integrated dmpi8-dyw genetic unit operates as a "behavioral temperate acclimator" by increasingly counterbalancing siesta-promoting pathways as daily temperatures become cooler and carry reduced risks from daytime heat exposure. While daily patterns of when animals are awake and when they sleep are largely scheduled by the circadian timing system, dyw implicates a less recognized class of modulatory wake-sleep regulators that primarily function to enhance flexibility in wake-sleep preference, a behavioral plasticity that is commonly observed in animals during the midday, raising the possibility of shared mechanisms.
Bu, B., He, W., Song, L. and Zhang, L. (2019). Nuclear envelope protein MAN1 regulates the Drosophila circadian clock via Period. Neurosci Bull. PubMed ID: 31230212
Almost all organisms exhibit ~24-h rhythms, or circadian rhythms, in a plentitude of biological processes. These rhythms are driven by endogenous molecular clocks consisting of a series of transcriptional and translational feedback loops. Previously, it has been shown that the inner nuclear membrane protein MAN1 regulates this clock and thus the locomotor rhythm in flies, but the mechanism remains unclear. This study further confirmed the previous findings and found that knocking down MAN1 in the pacemaker neurons of adult flies is sufficient to lengthen the period of the locomotor rhythm. Molecular analysis revealed that knocking down MAN1 led to reduced mRNA and protein levels of the core clock gene period (per), likely by reducing its transcription. Over-expressing per rescued the long period phenotype caused by MAN1 deficiency whereas per mutation had an epistatic effect on MAN1, indicating that MAN1 sets the pace of the clock by targeting per.
Roessingh, S., Rosing, M., Marunova, M., Ogueta, M., George, R., Lamaze, A. and Stanewsky, R. (2019). Temperature synchronization of the Drosophila circadian clock protein PERIOD is controlled by the TRPA channel PYREXIA. Commun Biol 2: 246. PubMed ID: 31286063
Circadian clocks are endogenous molecular oscillators that temporally organize behavioral activity thereby contributing to the fitness of organisms. To synchronize the fly circadian clock with the daily fluctuations of light and temperature, these environmental cues are sensed both via brain clock neurons, and by light and temperature sensors located in the peripheral nervous system. This study demonstrates that the TRPA channel PYREXIA (PYX) is required for temperature synchronization of the key circadian clock protein PERIOD. A molecular synchronization defect was observed explaining the previously reported defects of pyx mutants in behavioral temperature synchronization. Surprisingly, surgical ablation of pyx-mutant antennae partially rescues behavioral synchronization, indicating that antennal temperature signals are modulated by PYX function to synchronize clock neurons in the brain. These results suggest that PYX protects antennal neurons from faulty signaling that would otherwise interfere with temperature synchronization of the circadian clock neurons in the brain.

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).

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

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

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

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

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

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

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

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

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

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).


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


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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