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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

GENE STRUCTURE

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

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

Bases in 5' UTR - 497

Exons - eight

Bases in 3' UTR - 368

PROTEIN STRUCTURE

Amino Acids - 1218

Structural Domains and Evolutionary homologs

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

date revised: 28 MAY 97 

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