Transcriptional regulation plays an important role in Drosophila circadian rhythms. The period promoter has been well studied, but the timeless promoter has not been analyzed in detail. Mutagenesis of the canonical E box in the timeless promoter reduces but does not eliminate timeless mRNA cycling or locomotor activity rhythms. This is because there are at least two other cis-acting elements close to the canonical E box, which can also be transactivated by the circadian transcription factor Clock. These E-box-like sequences cooperate with the canonical E-box element to promote high-amplitude transcription, which is necessary for wild-type rhythmicity (McDonald, 2001).

Noncanonical E-box-dependent transcription has been described extensively for the Myc family of binding proteins, whose canonical high-affinity binding site has been determined as CACGTG by sequential selection and amplification of binding sites. These studies have led to the identification of lower-affinity, noncanonical MYC-MAX binding sites, such as CATGTG, CACGCG, CATGCG, CACGAC, and CAACGTG. MYC-MAX dimers are able to bind a similar set of sequences in vivo, in a tumorigenic cell line. A similar observation has been reported for mCLOCK/CYC. Four E boxes have been identified in the first and second introns of the mammalian dbp gene (coding for PAR leucine zipper transcription factor family member that binds to mammalian Per promoter), whose gene product is important in generating the cycling transcription of several circadian genes in the liver. All four E boxes were shown to activate a luciferase reporter in cell culture assays upon transfection with mCLOCK and BMAL1, but only two of the E-box regions show circadian differences in DNase I-hypersensitive sites: one with a canonical CACGTG motif and the other with a noncanonical CACATG motif. This study provides definitive evidence that a noncanonical E box contributes to circadian transcription in Drosophila (McDonald, 2001).

The idea that multiple E boxes contribute to circadian transcription has been reaffirmed. Previously, the study of the mper1 promoter had demonstrated a role for multiple canonical E boxes. However, the tim promoter is unique: it possesses three functional E boxes within a short distance, about 150 bp. This combination of multiple elements likely allows important protein-protein interactions, which contribute to the stronger amplitude of tim than of per transcriptional cycling. The observed direct correlation between behavioral period length and tim mRNA levels suggests that this more robust tim transcription amplitude is crucial for proper 24-h behavioral rhythmicity. These interactions could also accelerate the kinetics of transcription factors binding to the tim promoter and be responsible for tim's earlier peak of transcription. This also has important implications for the phase of Per accumulation, since Tim counteracts the effects of Dbt on Per stability (McDonald, 2001).

The two TER boxes (11-bp Tim E-box-like repeats, TER1 and TER2) each have the identical sequence GCGGCACGTTG. In S2 cells, the TER1 mutations, CAAGTTG and CTCGAAG, cause the same eightfold deficit in transcription as does deletion of the entire sequence by transversion mutations, demonstrating the necessity of an intact noncanonical E box for proper function. These data suggest that TER1 and probably TER2 serve as additional binding sites for the Clk-Cyc transcription factor complex. It has been shown previously that the mammalian homolog of Cyc (BMAL1 or MOP3) can bind multiple members of the PAS domain family. Therefore, Clk or Cyc could also dimerize with another partner to bind the TER1 element with high affinity. Alternatively or in addition, another protein could bind to the 5' side of the TER motif (GCGG) and stabilize the Clk-Cyk heterodimer on the 3' adjacent noncanonical E box (McDonald, 2001).

An E box has also been identified in the first intron of the tim gene. No activity of this sequence could be detected in S2 cells, and a transgenic construct lacking the whole TER E-box region but containing this intronic E box failed to generate detectable tim mRNA levels. This indicates that the intronic E box is probably nonfunctional, for unknown reasons. Nevertheless, the possibility that the TERs and the E box collaborate with other sequence elements cannot be ruled out. However, transgenic flies with only the region of the tim promoter containing TER1 and the E box fused to luciferase exhibit robust transcriptional cycling. These two elements can therefore work independently of any other tim promoter sequence. TER1 alone may even be able to drive transcriptional cycling, since a mutation in the E box does not completely eliminate circadian oscillations of luciferase activity (McDonald, 2001).

Do the two TERs have different functions? TER1 appears to be more important than TER2. Both in vivo and in S2 cells, disrupting TER1 leads to strong effects on the transcriptional activity of the tim promoter. In addition, examination of the TER1/TER2 double mutant suggests that TER2 is somewhat redundant in regard to TER1. This could be due to a distance effect: TER1 is closer to the E box, and factors bound to these motifs could be interacting more strongly. Disruption of TER1 has a substantial effect on expression levels (decrease of ~50%) but little or no effect on cycling amplitude. In contrast, mutagenizing both TERs has a detrimental effect on cycling amplitude as well as expression levels. It is therefore proposed that both TERs are important determinants of tim mRNA levels and their oscillations and thus contribute to proper rhythmicity. The observation that transgenic flies lacking an E box but containing TER1 have less arrhythmicity than do flies without both elements further supports this notion (McDonald, 2001).

Another element conserved within the timeless and period promoters is also apparent. This PERR box has the sequence GTTCGCACAA, which does not correspond to any known transcription factor binding site described in the literature. Mutating this element in the context of the tim promoter-S2 cell assay leads to decreases in transcription comparable to those caused by a mutation in the TER1 box. Activation of the double TER1/PERR mutant is equal to that of the TER1 and PERR single mutants, suggesting that these two elements might collaborate to enhance tim transcription in S2 cells. Importantly, mutation of the PERR box leads to a similar ~10-fold decrease in transcription in the context of the period promoter (McDonald, 2001).

These data strongly suggest that the PERR box plays a role in per as well as tim transcription. However, PERR box mutant flies have at most subtle differences in luciferase activity compared to wild-type flies. Since S2 cells are derived from embryos, the PERR box might play a role during development rather than in adult flies. Alternatively, PERR could contribute to tissue-specific transcriptional regulation of per and tim. If this element were active in only a small subset of cells, it would explain why mutations have no observable effect in the luciferase assays (McDonald, 2001).

This study therefore shows the importance of tim regulation for proper 24-h rhythmicity. Circadian period is very sensitive to tim mRNA levels, with more tim mRNA generating periods closer to 24 h. per mRNA cycling follows the same trend, because per mRNA trough levels are progressively lowered with increasing tim mRNA. Near-wild-type TIM levels are therefore required for accurate negative feedback on Per transcription. Lower Tim levels probably delay Per stabilization and nuclear entry and therefore lengthen the period. However, the amount of tim mRNA required for rhythmicity is rather low, and the levels generated by the minimal promoter are probably very close to this threshold. It is somewhat surprising that these periods are not longer than ~29 to 30 h, suggesting that tim levels can affect period only within a narrow range. tim missense mutations can have a much larger effect on period. In the timpMIN line (containing the minimal amount of tim promoter rescuing behavior), it is also surprising that Per is not cycling under LD, whereas it cycles in DD. Perhaps Tim levels fall just below threshold in LD, because Tim is degraded by light. Per cycling in timpMIN.1 would therefore be due to the increased amount of Tim in DD, which is sufficient to generate Per stabilization and accumulation and yield a quasinormal cycling profile (McDonald, 2001).

It will be of considerable interest to monitor precisely the kinetics of Per and Tim nuclear entry in lines with very limited amounts of Tim, both in peripheral oscillators and in the circadian pacemaker cells (the ventral lateral neurons). This could solidify the relationship between Per and Tim relocalization on the one hand and period length on the other. It will also be important to verify that expression levels and cycling of Per and Tl in the lateral neurons (the cells responsible for behavioral rhythmicity), parallel what is observed in the biochemical assays (McDonald, 2001).

Although transcriptional regulation is a major force in generating circadian oscillations of clock molecules, posttranscriptional mechanisms also contribute to molecular rhythms. Applying novel transgenic period-luciferase constructs in transgenic Drosophila, the authors show that sequences within per's 5'-untranslated region mediate posttranscriptional regulation at the RNA level. Further mapping suggests that the relevant sequences for the correct phasing of period mRNA expression are located within the first intron. The results are consistent with a clock-regulated temporal stabilization of period mRNA during its daily upswing in the morning. This process is inferred to depend on a function of the Period and Timeless proteins, and could further contribute to the observed delay between RNA and protein accumulation. Similarly, applying timeless-luciferase constructs led to the demonstration that regulatory elements for proper temporal timeless expression are present in a 4 kb promoter fragment and in sequences within the first intron. The results establish that, for normal rhythmicity, expression of clock genes requires regulation at the transcriptional, posttranscriptional, and posttranslational levels (Stanewsky, 2002).

Transcriptional Regulation

Drosophila Clock protein induces transcription of the circadian rhythm genes period and timeless. dClock functions as a heterodimer with a Drosophila homolog of BMAL1 termed Cycle. These proteins act through an E-box sequence in the period promoter. The timeless promoter contains an 18-base pair element encompassing an E-box, which is sufficient to confer Clock responsiveness to a reporter gene. Period and Timeless proteins block Clock's ability to transactivate their promoters via the E-box. Thus, Clock drives expression of period and timeless, which in turn inhibit Clock's activity and close the circadian loop. It is likely that either Per or Tim binds either Clock or Cycle, giving rise to a nonfunctional complex (Darlington, 1998).

The low Per and Tim levels in Jrk flies, mutant for Clock, could be due to reduced protein stability or to reduced protein synthesis in the mutant strains. To distinguish between these possibilities, PER and TIM mRNA levels were measured. Low and noncycling RNA levels were revealed, suggesting reduced synthesis rather than stability. Consistent with this notion, Jrk heterozygotes have a low amplitude of RNA cycling, which parallels the reduced amplitude of the protein rhythms and semidominance of the behavioral rhythm defect. To measure transcription rates directly, nuclear run-on assays were performed in homozygous Jrk flies. per and tim transcription rates are found to be temporally constant and approximately equal to the very low trough levels of wild-type flies. It is concluded that the behavioral arrhythmicity of Jrk mutants is largely due to a defect in the transcription of clock genes, including per and tim (Allada, 1998).

The basic region, involved in sequence-specific DNA contacts, is remarkably conserved between Drosophila and Mouse Clock proteins, with 11 out of 13 amino acids being identical; this suggests that the two proteins bind to similar if not identical DNA targets. In fact, 6 out of 9 amino acids are identical to a consensus generated for bHLH proteins that bind the CAC/GTG E box half-site, including the critical R residue at position 15; this is consistent with the dramatic effect of the Clk mutant on per E box-mediated transcription. As expected, the tim gene also has an E box in its 5' noncoding region. In-vitro experiments indicate that human Clock preferentially binds and activates transcription from DNA targets very similar to the Drosophila per E box (Allada, 1998).

Period and Timeless were examined in wild-type, heterozygous, and homozygous cycle flies under LD conditions. Western analysis with an anti-Per antibody reveals very little protein in cyc0/cyc0 fly heads at any time of day. As predicted from the robust rhythms, cyc0/+ heterozygotes show normal Per cycling, with normal levels and a normal temporal phosphorylation program. For all genotypes, similar results were obtained for Tim. The low Per and Tim levels could be due to reduced protein stability or to reduced protein synthesis in the homozygous mutant strain. To distinguish between these possibilities, per and tim mRNA levels were measured. Low RNA levels and little or no cycling are found in the cyc0/cyc0 head extracts, suggesting reduced synthesis rather than reduced stability. The cyc effect on per and tim RNA levels and cycling could be transcriptional or posttranscriptional. To directly measure transcription rates, nuclear run-on assays were performed in homozygous cyc flies. In this genotype, per and tim transcription rates show no evidence of cycling and are approximately equal to the very low trough levels of wild-type flies observed at ZT1. The result is essentially identical to that observed in homozygous Clock flies (Allada, 1998). Taken together, the data suggest that cyc, like Clk, affects the transcription of the clock genes per and tim (Rutila, 1998).

The accumulation of TIMmRNA follows a circadian rhythm whose phase and period are indistinguishable from those of PER mRNA. TIM mRNA oscillations are dependent on PER and TIM proteins, demonstrating a feedback control of tim expression by a mechanism that also regulates per expression (Sehgal, 1995)

A recessive third chromosomal mutation that abolishes bioluminescence rhythms has been identified, cryb. cryb is an apparent null mutation in a gene encoding Cryptochrome, Drosophila's version of the blue light receptor cryptochrome. To determine the mutation's effects on per and tim transcription, a per-luc or a newly generated tim-luc fusion gene (each encoding luciferase sequences only) were introduced into homozygous mutant genetic backgrounds. luc-reported expression in both cases is arrhythmic. In contrast to other recently identified mutations affecting per and tim expression (Allada, 1998 and Rutila, 1998), the new mutation does not give rise to profound subnormalities in overall levels of per and tim expression in mutant flies. Nevertheless, western blot analyses using head extracts of mutant flies maintained in LD show that the levels of Tim and Per protein remain at high levels throughout the day and night, relative to the very low troughs observed during the daytime in wild type. In addition, Tim and Per proteins are anomalously present in both hypo- and hyperphosphorylated forms in a temporally unchanging manner. That Tim stays at the same levels during the day and night in the mutant is especially interesting, because the rapid disappearance of this protein in response to light is the earliest response to this stimulus of a known component of Drosophila's rhythm system. Yet the absence of rhythmic clock gene transcription indicates that the mutant is doubly defective. This is because either of two regulatory phenomena is sufficient to drive Tim cycling: oscillating tim expression (which occurs in the absence of environmental fluctuations) or light suppression of TIM (in the absence of tim mRNA cycling). Against this background, the absence of effects of some (but not all) orthodox visual mutations on light-induced Tim degradation is notable (Yang, 1998), as is the fact that peak sensitivity for this light effect is in the blue range (Suri, 1998). Thus, the new mutation might uniquely affect elements of the light entrainment pathway, which would include extraocular reception and processing of blue light inputs. Alternatively, the mutation could affect a protease whose targets include Tim and Per (Stanewsky, 1998).

If that is not the case, and the new mutation causes a specific defect in the light entrainment pathway, protein oscillations in temperature cycles should not be affected. Western blots of extracts from mutant and normal heads showed that Per and Tim fluctuated robustly in 12 hr:12 hr, 25°C:20°C cycles; such cyclings continued in constant conditions. The daily mobility shifts of Per and Tim are apparent in both wild type and mutant genetic backgrounds, indicating that the phosphorylation program can function in the mutant (Stanewsky, 1998).

The behavioral changes seen upon lowering the gene dose of vrille or upon increasing the gene dosage of vri could result from an aberrant circadian oscillator or a block in an output pathway from the clock. Clock gene cycling in the LNs was examined. LNs in the brains of third instar larvae (lvLNs) were studied, since it is simple to see all of the LNs at this stage in whole-mount preparations. The lvLNs have functional clocks and have been used to determine the phenotype of a hypomorphic dbt mutant. These cells persist to form a subset of the adult LNs and can retain the memory of larval light-dark cycles and pulses (Blau, 1999 and references).

Wild-type control larvae [tim(UAS)-gal4 heterozygotes with no UAS transgene] were entrained to light-dark cycles and then held in constant darkness for 1 day. They show strong cycling of TIM mRNA with low levels at CT3 and high levels at CT15 in the four to five lvLNs at the center of each brain lobe. Tim and Per proteins also oscillate with low levels at CT9-10 and high levels at CT22. There are also tim- and per-expressing cells anterior to the lvLNs whose oscillations are reversed relative to the pacemaker cells. tim RNA can be detected at CT3, but not CT15, and Tim protein can be seen in these cells at CT10, but not CT22. In contrast to the patterns of per and tim expression detected in wild-type larvae, all of the UAS-vri lines show abnormal cycling of clock gene products, with a perfect correlation between the severity of the molecular phenotypes observed and the behavioral phenotypes recorded. In line V1, Tim RNA levels at CT15 are lower than in wild type, and Tim protein is predominantly cytoplasmic at CT22 in contrast to wild type, which shows nuclear staining at this time. In V1, Per protein is present at CT22, but weaker and largely cytoplasmic. Line V2 produces very low levels of TIM mRNA and Tim protein, which was also cytoplasmic, and Per protein is undetectable. In line V3, there is no detectable tim RNA, nor any Tim or Per protein at any time point in constant darkness. In a separate experiment, TIM mRNA could not be detected in V3 larval brains at any of the time points taken every 4 hr between CT4 and CT24, while there is robust TIM RNA cycling in wild-type controls. Clearly, blocking the normal cycle of vri activity affects clock gene expression in lvLNs (Blau, 1999).

Daily scheduled feeding is a potent Zeitgeber that elicits anticipatory activity in mammals. Recent studies have revealed that daytime feeding of nocturnal laboratory rodents completely inverts the phase of circadian gene expression in peripheral tissues such as heart, liver and kidney, independently of environmental light cycles. To investigate whether feeding is a potent time cue for Drosophila, the behavioral activity rhythm and peripheral expression profile of clock genes were examined in Drosophila under 12 h of night-time restricted feeding. Flies could not exhibit food-anticipatory activity rhythms under restricted feeding. Expression profiles of the clock genes period and timeless were not affected by either the phase or the amplitude in the periphery. These results suggest that feeding is not a more potent Zeitgeber than the light/dark cycle at either the individual behavioral level or at the peripheral molecular clock levels in Drosophila (Oishi, 2004).

Repression of tim by Period and Cryptochrome

Although most circadian clock components are conserved between Drosophila and mammals, the roles assigned to the Cryptochrome (Cry) proteins are very different: Drosophila Cry functions as a circadian photoreceptor, whereas mammalian Cry proteins (mCry1 and 2) are transcriptional repressors essential for molecular clock oscillations. This study demonstrates that Drosophila Cry also functions as a transcriptional repressor. RNA levels of genes directly activated by the transcription factors Clock (Clk) and Cycle (Cyc) are derepressed in cryb mutant eyes. Conversely, while overexpression of Cry and Period (Per) in the eye repressed Clk/Cyc activity, neither Per nor Cry repressed individually. Drosophila Cry also represses Clk/Cyc activity in cell culture. Repression by Cry appears confined to peripheral clocks, since neither cryb mutants nor overexpression of Per and Cry together in pacemaker neurons significantly affected molecular or behavioral rhythms. Increasing Clk/Cyc activity by removing two repressors, Per and Cry, leads to ectopic expression of the timeless clock gene, similar to overexpression of Clk itself. It is concluded that Drosophila Cry functions as a transcriptional repressor required for the oscillation of peripheral circadian clocks and for the correct specification of clock cells (Collins, 2006).

Several pieces of evidence point to Drosophila Cry, like its mammalian counterparts, functioning as a repressor of Clk/Cyc-activated transcription: (1) expression of four Clk/Cyc target genes is derepressed in cryb mutants; (2) overexpression of cry together with per is sufficient to repress tim and vri expression in the eye, and this is supported by Cry repressing Clk/Cyc-activated transcription in transfected cells, either alone or in conjunction with Per; and (3) removing both Cry and Per leads to ectopic tim expression in the brain (Collins, 2006).

Although Cry and Per seem to function together to repress Clk/Cyc activity, the results do not imply a direct interaction between Cry and Per proteins. Drosophila Cry-Per interactions have been detected in yeast, but Cry and Per appear to interact only via Tim in vivo. Furthermore, Per continues to repress Clk/Cyc activity in vivo during the first half of the day, presumably after Cry has been degraded by light. Thus, Cry and Per seem to control distinct steps in repression of Clk/Cyc activity, with Cry probably initiating, and Per maintaining, repression. Further experiments will be required to test whether Tim also facilitates repression. While in vitro studies indicated that Tim helps remove Clk/Cyc from DNA, in vivo studies of the timUL mutant suggests that Tim does not participate in repression of per and tim transcription and instead stabilizes Per and facilitates its nuclear entry. Given that Drosophila Tim interacts with both Per and Cry in vivo, it will be interesting to test whether the Per-Cry interactions detected in mammalian clock cells are mediated via mTim (Collins, 2006).

Very little is known about the developmental specification of clock neurons. Per and Cry normally restrict tim expression to cells that adopt a circadian cell fate. The results complement experiments in which overexpression of Clk led to ectopic tim expression, since they reveal that cells not normally destined to develop as clock cells repress Clk/Cyc activity during development. However, there must be additional factors that contribute to clock cell fate, since the ectopic Tim+ve cells in per01; cryb double mutant larvae did not produce PDP1. Similarly, there must be unidentified factors that maintain repression of tim in nonclock cells, since repression of Clk/Cyc activity will prevent further per expression. The presence of extra Tim-expressing cells may also explain the Tim-dependent rhythmic behavior of per01; cryb in LD cycles, since ectopic Tim expression influences LD behavior (Collins, 2006).

The findings that Cry functions as a repressor in Drosophila are supported by the high conservation across species of the “core” photolyase-like domain of Cry, which is sufficient for repression in Xenopus. TheDrosophila crym mutation removes most of the Cry C terminus and interferes with Cry's response to light. However, CryM still supports a functional clock in the eyes, suggesting that the remaining core of CryM functions as a transcriptional repressor (Collins, 2006).

Cry's homology with DNA photolyases has led to the suggestion that Cry was the original molecule that allowed organisms to respond to light -- primitive organisms could detect light and regulate gene expression with one molecule (Cry) to avoid damage by sunlight during light-sensitive processes such as DNA replication. While ancestral Cry may have acted as both a light sensor and repressor, non-Drosophilid insects such as the monarch butterfly Danaus plexippus have two cry genes and divide repressor/light sensor function between them. Thus, circadian clocks may well have their origins in rapid responses to light, and the anticipatory clock gene networks could have subsequently been built around Cry, a light-responsive protein and a transcriptional repressor, the function of which has gradually become specialized (Collins, 2006).

Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component

Many organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila, the master clock gene Clock promotes the transcription of several key target genes. Two of these gene products, Per and Tim, repress Clk-Cyc-mediated transcription. To recognize additional direct Clk target genes, a genome-wide approach was designed and clockwork orange (cwo) was identified as a new core clock component. cwo encodes a transcriptional repressor functioning downstream of Clk that synergizes with Per and inhibits Clk-mediated activation. Consistent with this function, the mRNA profiles of Clk direct target genes in cwo mutant flies manifest high trough values and low amplitude oscillations. Impaired activity of Cwo leads to an elevated trough of per, tim, vri, and Pdp1 mRNA at ZT3 (three hours into the morning) in cwo RNAi transgenic flies compared with those of wild-type flies. Because behavioral rhythmicity fails to persist in constant darkness (DD) with little or no effect on average mRNA levels in flies lacking cwo, transcriptional oscillation amplitude appears to be linked to rhythmicity. Moreover, the mutant flies are long period, consistent with the late repression indicated by the RNA profiles. These findings suggest that Cwo acts preferentially in the late night to help terminate Clk-Cyc-mediated transcription of direct target genes including cwo itself. The presence of mammalian homologs with circadian expression features (Dec1 and Dec2) suggests that a similar feedback mechanism exists in mammalian clocks (Kadener, 2007). To other studies similarly identified Clockwork orange an a transcriptional repressor that inhibits Clk-mediated activation (Matsumoto, 2007; Lim, 2007).

Integration of light and temperature in the regulation of circadian gene expression in Drosophila: Alternative splicing and differential regulation of per and tim are responsible for finely tuning the clock in response to changing environmental conditions

Circadian clocks are aligned to the environment via synchronizing signals, or Zeitgebers, such as daily light and temperature cycles, food availability, and social behavior. This study found that genome-wide expression profiles from temperature-entrained flies show a dramatic difference in the presence or absence of a thermocycle. Whereas transcript levels appear to be modified broadly by changes in temperature, there is a specific set of temperature-entrained circadian mRNA profiles that continue to oscillate in constant conditions. There are marked differences in the biological functions represented by temperature-driven or circadian regulation. The set of temperature-entrained circadian transcripts overlaps significantly with a previously defined set of transcripts oscillating in response to a photocycle. In follow-up studies, all thermocycle-entrained circadian transcript rhythms also responded to light/dark entrainment, whereas some photocycle-entrained rhythms did not respond to temperature entrainment. Transcripts encoding the clock components Period, Timeless, Clock, Vrille, PAR-domain protein 1, and Cryptochrome were all confirmed to be rhythmic after entrainment to a daily thermocycle, although the presence of a thermocycle resulted in an unexpected phase difference between period and timeless expression rhythms at the transcript but not the protein level. Generally, transcripts that exhibit circadian rhythms both in response to thermocycles and photocycles maintained the same mutual phase relationships after entrainment by temperature or light. Comparison of the collective temperature- and light-entrained circadian phases of these transcripts indicates that natural environmental light and temperature cycles cooperatively entrain the circadian clock. This interpretation is further supported by comparative analysis of the circadian phases observed for temperature-entrained and light-entrained circadian locomotor behavior. Taken together, these findings suggest that information from both light and temperature is integrated by the transcriptional clock mechanism in the adult fly head (Boothroyd, 2007; full text of article).

Transcriptional regulation of per and tim appears to be different in light and temperature entrainment. Whereas in light entrainment per and tim RNA expression is tightly coupled at all times, in 18°C/25°C temperature entrainment per RNA levels peak before tim RNA levels. This is a result of a temperature-induced advance in per expression and delay in the expression of the predominant tim transcript. Differences in per and tim regulation have been suggested based on the observation that these transcripts show different rates of degradation in response to a light pulse in the context of the long period mutant timul. In addition, while at lower temperatures per expression is upregulated in LD and DD, tim has been reported to be downregulated in LD and barely oscillatory in DD. Further, while the phases of both per and tim appeared advanced at lower temperatures, the advance in per was interpreted as a result of faster accumulation, while the advance in tim was thought to represent more rapid degradation. It has also very recently been reported that tim, but not per, transcript levels are upregulated in response to light pulses at cold temperatures. It is noteworthy, however, that the probe used in several previous studies to evaluate tim transcript expression with RNase protection assays may not have efficiently detected the timcold isoform since it spans the exons flanking the intron maintained in timcold. Additional analyses that take into account the contribution of the timcold isoform will, therefore, be needed to complement previous studies in order to more fully explore tim transcript responses (Boothroyd, 2007).

One of the factors involved in the reported differential expression of per and tim may be the alternative splicing of both transcripts. Much of the recent molecular work on temperature and the circadian clock has focused on the alternative splicing of an 89-bp intron in the 3' UTR of per, an event thought to be important in seasonal adaptation. Short, cold days lead to increased amounts of the spliced per variant, resulting in an earlier increase in PER protein abundance and an advanced phase of locomotor activity. Warmer temperatures result in less of the spliced variant, especially during the day. This appears to be a clock-dependent effect that results in the fly moving its behavior to the later (cooler) part of the day. Thus, per splicing allows the fly to adapt to changes in both temperature and photoperiod by regulating the amount of available PER protein. per alternative splicing is thought to be important in seasonal adaptation, as long photoperiods counteract the cold-induced behavioral advances by delaying the accumulation of TIM, in turn rendering prematurely produced PER unstable. Thus, the fly is able to integrate information from both light and temperature to generate behavior that is aligned to the environmental day. Regulation of per splicing in the presence of an environmental temperature cycle as compared to constant temperature needs to be investigated (Boothroyd, 2007).

Temperature-dependent alternative splicing of tim is described in this study. At 18°C, the last intron of tim is preferentially retained, resulting in a premature stop codon and a truncated protein. Although the expression of the predominant tim transcript is delayed relative to per, timcold cycles in phase with per. The differential expression of the two tim transcripts could reflect temperature-dependent control of splicing or of the stability of one of the splice forms. The functional significance of the production of timcold transcript is still being ascertained. It does, however, appear that the alternative splicing and differential regulation of per and tim are responsible for finely tuning the clock in response to changing environmental conditions, thus adding an additional level of complexity to the clock (Boothroyd, 2007).

Different groups of clock-bearing cells in the fly have been shown to regulate different rhythmic processes. For example, locomotor activity and eclosion rhythms, arguably the best-characterized rhythmic behaviors in Drosophila, require the ventral lateral neurons (LNvs) and the neuropeptide, Pigment Dispersing Factor. Cyclic olfactory responses do not depend on the LNs or Pigment Dispersing Factor, but instead depend on the antennal neurons. Egg-laying rhythms also appear to be regulated independently of the LNvs and Pigment Dispersing Factor. Thus, the image of the circadian clock as a single entity is transforming into a more complex model (Boothroyd, 2007).

A system of two coupled oscillators was proposed for the Drosophila clock almost 50 y ago (Pittendrigh, 1958). In this model, the master or A oscillator is autonomous, light-sensitive, and temperature-compensated. The slave or B oscillator, which is coupled to and driven by A, is responsive to temperature but not light. The evidence for this two-oscillator model came from the different responses in eclosion rhythms to light and temperature. Whereas light pulses administered at different times of day resulted in steady-state phase advances or delays, the phase changes resulting from temperature pulses were transient. The researchers concluded that the steady-state phase changes in response to light were a result of the eventual realignment of the A oscillator to the light signal. The transient responses to temperature pulses were proposed to be a result of temporary temperature-induced disturbances in B, with the return to the previous phase reflecting the A oscillator's resumption of control over B (Boothroyd, 2007).

A system of coupled oscillators has recently been demonstrated in the regulation of the morning and evening peaks of locomotor activity in the fly. The morning oscillator requires the presence of the LNvs, while the evening oscillator requires the dorsal lateral neurons. It was further shown that the evening oscillator is set by the morning oscillator by generating flies in which the morning and evening oscillators have different free-running periods. However, despite the parallels to Pittendrigh's original model, there is no published evidence that these or other oscillators would differentially respond to temperature, as opposed to light, as a Zeitgeber. So while it appears there is a multicellular clock network in Drosophila that is reflected by coordinate yet independently regulated outputs, the data presented in this study suggest that the response to multiple inputs, such as light and temperature, would still be integrated by a single autonomous clock mechanism. In today's jargon Pittendrigh's B oscillator would be describe as a circadian output pathway that can show direct clock-independent responses to temperature (Boothroyd, 2007).

The following observations support the hypothesis of a single, integrative transcriptional oscillator. First, the same set of core clock components (including PER, TIM, CLK, and CYC) appears to be required for producing both light-entrained and temperature-entrained oscillations. The global transcriptional signatures of arrhythmic tim01 flies that were found after thermocycle treatment resemble those found after photocycle treatment and do not exhibit obvious circadian rhythms. In addition, the results confirm the absence of circadian oscillations for core clock gene transcripts in the tim01 fly heads. Second, it is likely that the set of transcripts entrainable by thermocycles is closely related to the set of transcripts entrainable by light. Although the existence of circadian rhythms that specifically require temperature entrainment cannot be formally excluded, none have been found so far. Third, the phases of the transcripts that oscillate in response to both photo- and thermocycles maintain the same mutual phase relationships after entrainment by light or temperature. The phase observed at the onset of the thermophase is systematically advanced by about 6 h relative to the phase at the onset of light. Given the size of the delay that is commonly found between the environmental profiles for temperature relative to that of daylight, these results indicate cooperative entrainment by light and temperature under common natural circumstances. A response to temperature would be well integrated with the expected light cycle were it also supplied, and vice versa. Fourth, the temperature- and light-entrained phases of PER and TIM protein expression reflect the same relationship observed for the genome-wide circadian transcript signatures. This observation is consistent with the hypothesis that both light and temperature act via the same PER/TIM-dependent oscillator to generate circadian transcript profiles. Fifth, the entrained phase of locomotor activity behavior appears to follow the molecular circadian phase observed in temperature or light entrainment. The ability to accurately predict the phase of clock neuron-controlled circadian locomotor behavior based on the analysis of circadian transcript rhythms in a preparation of whole heads, which mostly represents peripheral clock cells, suggests that temperature entrainment just as light entrainment produces similar phases in peripheral clock cells and clock neurons. This result can be verified and extended in a future study by direct examination of the temperature-entrained molecular phase in the various subsets of clock neurons (Boothroyd, 2007).

In summary, this analyses revealed that thermocycle entrainment and photocycle entrainment produce very similar circadian expression profiles in fly heads, and that under common natural conditions light and temperature are expected to entrain both molecular and behavioral circadian rhythms cooperatively. As pointed out above, the results are in agreement with the notion that a single transcriptional clock is responsible for producing all light-entrained and temperature-entrained circadian rhythms. Nevertheless, the existence of a specialized temperature-entrained oscillator that is coupled to the general transcriptional clock circuits cannot be formally excluded. Such a theoretical temperature-entrained oscillator could have eluded detection in this analyses if it was located outside the head or in a small subset of the cells in the head or if it produced non-transcriptional circadian signals. Elucidation of the mechanisms of thermocycle entrainment will constitute an important next step in defining the temperature-entrained circadian oscillator(s) (Boothroyd, 2007).

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression

Clock (Clk) is a master transcriptional regulator of the circadian clock in Drosophila. To identify Clk direct target genes and address circadian transcriptional regulation in Drosophila, chromatin immunoprecipitation (ChIP) tiling array assays (ChIP-chip) were performed with a number of circadian proteins. Clk binding cycles on at least 800 sites with maximal binding in the early night. The Clk partner protein Cycle (Cyc) is on most of these sites. The Clk/Cyc heterodimer is joined 4-6 h later by the transcriptional repressor Period (Per), indicating that the majority of Clk targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. Clk has specific targets in different tissues, implying that important Clk partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation (Abruzzi, 2011).

Previous circadian models in Drosophila suggested a transcriptional cascade in which Clk directly controls a limited number of genes, including core clock genes, which then drive the oscillating expression of many different output genes. The results of this study indicate that Clk directly regulates not only the five core clock genes (i.e., pdp1, vri, tim, per, and cwo), but also many output genes, including ~60 additional transcription factors. Some of these are tissue-specific; e.g., lim1 and crp. In addition, Clk direct target gene regulation may impact timekeeping in yet unforeseen ways. For example, Clk, Per, and Cyc bind to three of the four known circadian kinases; i.e., dbt, nmo, and sgg. Although none of these mRNAs have been previously reported to cycle, both dbt and sgg have cycling Pol II, and dbt shows weak oscillations via qRT-PCR. nmo expression is enriched in circadian neurons and has been shown to cycle in l-LNvs. The data, taken together, indicate that this simple ChIP-chip strategy has uncovered important relationships and suggest that the transcriptional regulation of some of these new target genes warrants further investigation (Abruzzi, 2011).

Most of the 1500 Clk direct target genes are also bound by two other circadian transcription factors: Cyc and Per. Because a previous study showed that there is a progressive, ordered recruitment of Clk, Pol II, and Per on per and tim (Menet, 2010), the same basic mechanism is conserved on most Clk direct targets. Clk binding increases in late morning and gives rise to an increase in Pol II signal where detectable (ZT6-ZT10). Clk binding is maximal in the early night (ZT14), and both Clk binding and Pol II occupancy decrease rapidly after the repressor Per is bound to chromatin 4-6 h later, at ZT18. Interestingly, Per binds to nearly all Clk direct targets at the identical Clk/Cyc locations, suggesting Per recruitment via protein-protein interactions (Abruzzi, 2011).

The identical binding sites for Clk, Cyc, and Per suggest that binding is not background binding or 'sterile' binding with no functional consequence. This is because three components of the circadian transcription machinery are present with proper temporal regulation. Pol II cycling on ~30% of cycling Clk targets further supports this interpretation. The Pol II signal is maximal from mid- to late morning (ZT6-ZT10), which slightly anticipates the maximal transcription times of core circadian genes like per and tim. Most Pol II signals are promoter-proximal and may reflect poised Pol II complexes often found on genes that respond quickly to environmental stimuli (Abruzzi, 2011).

To address RNA cycling, ten direct target genes with Pol II cycling were examined. Eight of these genes show oscillating mRNA with >1.5-fold amplitude, suggesting that oscillating Pol II indeed reflects cycling transcription. Because this assay may underestimate cycling transcription due to tissue heterogeneity (i.e., masking by noncycling gene expression elsewhere in the head), ~30% is a minimal estimate of Clk direct targets with cyclical mRNA (Abruzzi, 2011).

Interestingly, previous microarray studies did not detect many of these genes. One possibility is that the alternative start sites that characterize 55% of Clk direct targets are not detectable on microarrays; e.g., moe and mnt. However, several mRNAs that cycle robustly by qRT-PCR are not isoform-specific and are also not consistently identified in microarray studies. A second possibility is that the relatively low cycling amplitude of many target genes -- twofold or less, compared with the much greater amplitudes of core clock genes such as tim, per, and pdp1, assayed in parallel -- may be more difficult to detect on microarrays (Abruzzi, 2011).

Low-amplitude cycling may result from relatively stable mRNA, which will dampen the amplitude of cycling transcription. Alternatively, or in addition, low-amplitude cycling may reflect cycling in one head location and noncycling elsewhere within the head, which will dampen head RNA cycling amplitude. This is likely for many eye-specific Clk targets, which appear expressed elsewhere in the head via a Clk-independent mechanism (Abruzzi, 2011).

A third and arguably more interesting explanation for low-amplitude cycling is that Clk binds on promoters with other transcription factors within single tissues. These could include chromatin modifiers and would function together with Clk in a gene- and tissue-specific fashion. For example, a gene could be constitutively expressed at a basal level by one transcription factor, with temporal Clk binding causing a modest boost to transcription. For example, gol is a Clk target exclusively in the eye, and gol mRNA cycles with a fourfold amplitude. Rather than cycling from 'OFF' (no or very low mRNA levels) to 'ON,' however, gol mRNA levels are quite high even at the trough or lowest time points. This suggests that gol cycles from a substantial basal level in the late night and daytime to an even higher level of expression in the evening and early night. Since mRNA levels decrease by >10-fold in GMR-hid flies, trough transcription levels are not likely from other tissues. Therefore, Clk probably acts on gol and other targets not as an 'ON/OFF switch,' but rather in concert with other factors to boost a basal level of gene expression at a particular time of day and cause low-amplitude cycling within a single tissue (Abruzzi, 2011).

The large number of Clk target genes in fly heads is explained in part by tissue-specific Clk binding. Transcription assays that measure the cycling of mRNA and Pol II binding in one head tissue can be masked by noncycling expression in another. The ChIP assays, in contrast, are not plagued with the same problem. They can identify a gene bound by the cycling circadian transcription machinery even if the same gene is constitutively expressed elsewhere in the head. Surprisingly 44% of Clk direct targets were no longer detected when eyes were ablated with GMR-hid. Because many of these mRNAs are not particularly eye-enriched, it is inferred that their genes are constitutively expressed under the control of other transcription factors elsewhere in the head (Abruzzi, 2011).

The large number of target genes is also explained by the efficiency and sensitivity of the ChIP assay. It is inferred that it can detect Clk binding from a relatively low number of cells within the fly head. Lim1 is one example and is expressed predominantly in a subset of circadian neurons (l-LNvs; enriched more than four times relative to head). Preliminary cell-specific Clk ChIP-chip experiments from LNvs confirm that lim1 is an enriched Clk direct target in these cells, suggesting that this is the source of a large fraction of the binding signal in the head ChIP-chip experiments. Experiments are under way to more clearly define circadian neuron-specific Clk-binding patterns (Abruzzi, 2011).

This tissue specificity also suggests the existence of factors and/or chromatin modifications that help regulate Clk-mediated gene expression. They could enable Clk binding to specific genes in one tissue or inhibit binding in another tissue. These tissue-specific factors are strongly indicated by the pdp1 and lk6 Clk-binding patterns, which change so strikingly and specifically in GMR-hid. Although not unprecedented, tissue-specific factors that enable or inhibit specific DNA-binding locations are intriguing and warrant further investigation and identification (Abruzzi, 2011).

Differentially timed extracellular signals synchronize pacemaker neuron clocks

Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, the minimal clock neural circuit in Drosophila larvae were studied, manipulating either the four master pacemaker neurons (LNvs) or two dorsal clock neurons (DN1s). Unexpectedly, it was found that the PDF Receptor (PdfR) is required in both LNvs and DN1s to maintain synchronized LNv clocks. It was also found that glutamate is a second synchronizing signal that is released from DN1s and perceived in LNvs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LNvs severely dampened Timeless clock protein oscillations, it is concluded that the master pacemaker LNvs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LNvs. Finally it was found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LNvs. It is proposed that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species (Collins, 2014: PubMed).

Targets of Activity

The TIM-PER heterodimer regulates transcription of tim and per (Sehgal, 1995).

The Timeless protein is a central component of the circadian pacemaker machinery of the fruitfly. Both Tim and its partner protein, the Period protein Per, show robust circadian oscillations in mRNA and protein levels. Yet the role of Tim in the rhythm generation mechanism is largely unknown. To analyze Tim function, transgenic flies were constructed that carry a heat shock-inducible copy of the timeless gene in an arrhythmic tim loss-of-function genetic background. When heat shocked, Tim levels in these flies rapidly increases and initiates a molecular cycle of Per accumulation and processing with dynamics very similar to the Per cycle observed in wild-type flies. Analysis of Period mRNA levels and transcription has uncovered a novel role for Tim in clock regulation: Tim increases PER mRNA levels through a post-transcriptional mechanism. These results suggest positive as well as negative autoregulation in the Drosophila circadian clock (Suri, 1999).

As part of this regulatory loop, PER and TIM mRNA levels oscillate in a circadian fashion. Other cycling transcripts may participate in this central pacemaker mechanism or represent outputs of the clock. Crg-1 is a newly isolated circadianly regulated gene. Like PER and TIM transcript levels, Crg-1 transcript levels oscillate with a 24 h period in light:dark (LD) conditions, with a maximal abundance at the beginning of the night. These oscillations persist in complete darkness and depend upon Per and Tim proteins. The putative CRG-1 proteins show some sequence similarity with the DNA-binding domain of the HNF3/fork head family of transcription factors. In the adult head, in situ hybridization analysis reveals that per and Crg-1 have similar expression patterns in the eyes and optic lobes. Crg-1 is expressed in all photoreceptors; in the optic lobes expression is detected in the regions between neuropils. Strong staining is seen in the distal lamina and the region between lamina and medulla. The same subsets of cells express per. Crg-1 labeling is observed in the PER-expressing dorsal neurons and in regions surrounding neuropils within the central brain (Rouyer, 1997).

The Clock gene plays an essential role in the manifestation of 24 h circadian rhythms in mice and is a member of the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) superfamily of transcription factors. A novel Drosophila bHLH-PAS protein that is highly homologous to mammalian CLOCK has been characterized. Transcripts from this putative Clock ortholog (designated dClock) undergo daily rhythms in abundance that are antiphase to the cycling observed for the RNA products from the Drosophila melanogaster circadian clock genes period (per) and timeless (tim). Furthermore, dClock RNA cycling is abolished and the levels are at trough values in the absence of either PER or TIM, suggesting that these two proteins can function as transcriptional activators, a possibility which is in stark contrast to their previously characterized role in transcriptional autoinhibition. Finally, the temporal regulation of dClock expression is quickly perturbed by shifts in light-dark cycles, indicating that this molecular rhythm is closely connected to the photic entrainment pathway. The isolation of a Drosophila homolog of Clock together with the recent discovery of mammalian homologs of per indicate that there is high structural conservation in the integral components underlying circadian oscillators in Drosophila and mammals. Nevertheless, because mammalian Clock mRNA is constitutively expressed, these findings are a further example of striking differences in the regulation of putative circadian clock orthologs in different species (Bae, 1998).

Seasonal behavior in Drosophila melanogaster requires the photoreceptors, the circadian clock, and phospholipase C: Regulation of Per splicing by Tim

Drosophila locomotor activity responds to different seasonal conditions by thermosensitive regulation of splicing of a 3' intron in the period mRNA transcript. The control of locomotor patterns by this mechanism is primarily light-dependent at low temperatures. At warmer temperatures, when it is vitally important for the fly to avoid midday desiccation, more stringent regulation of splicing is observed, requiring the light input received through the visual system during the day and the circadian clock at night. During the course of this study, it was observed that a mutation in the no-receptor-potential-A(P41) (norpA(P41)) gene, which encodes phospholipase-C, generates an extremely high level of 3' splicing. This cannot be explained simply by the mutation's effect on the visual pathway and suggests that norpA(P41) is directly involved in thermosensitivity (Collins, 2004).

The proportion of per transcripts that were spliced at 18°C and 29°C, averaged over several LD 12:12 cycles was examined in Canton-S WT and per01, tim01, cryb, and per01; cryb mutant backgrounds. In all backgrounds splicing levels fall as the temperature rises, with 40%-60% of transcripts spliced at 18°C and 20%-45% at 29°C. However, not all genotypes react in the same way to temperature changes (Collins, 2004).

The smallest but nevertheless significant effect of temperature on splicing levels is observed in per01; cryb, suggesting that the temperature-sensing system for splicing may be compromised in the double mutant. A significant temperature x time effect reveals that the temporal patterns of cycling differ among temperatures, and the absence of any other significant interactions suggests that all genotypes respond similarly. There is very little evidence for a significant day/night cycle in the proportion of per transcripts that are spliced at 18°C, but at 29°C, all genotypes reveal a higher level of splicing post lights off (ZT12) compared to the trough at ZT8. At 18°C, the per01 and tim01 mutations have no significant effect on the level of splicing of per mRNA compared to WT. However in cryb flies, splicing levels are significantly elevated, particularly after lights off. This is also the case when per01; cryb is compared to WT. At 29°C, splicing levels are generally 5%-10% higher in per01, tim01, and cryb mutants compared to WT in the light, but 15%-20% higher after lights off at ZT12. This suggests that in the presence of light, splicing levels are reduced due largely to a clock-independent mechanism. In darkness, the clock and Cry become critical for maintaining this low splicing level at high temperatures (Collins, 2004).

The double mutant per01; cryb shows a highly significant increase in splicing of ~20% throughout the day/night cycle compared to WT. Thus, at high temperature, either the presence of the circadian photoreceptor Cry or a functional circadian clock is sufficient to largely repress daytime splicing. With both eliminated, daytime splicing levels are elevated. In contrast, repression of splicing in the absence of light requires the circadian clock plus Cry. It seems somewhat counterintuitive that Cry, which is activated by light, plays a more prominent role in repressing splicing at night than it does during the day (Collins, 2004).

Cry is likely to be a dedicated circadian photoreceptor yet at 29°C, splicing is repressed during the light phase even in cryb. This suggests that the light input to the splicing machinery cannot be primarily mediated by Cry. To confirm that light represses splicing, the effects that short photoperiods and constant darkness (DD) have on splicing levels in WT was investigated. There is a significant effect of reducing photoperiod on the splicing level with an elevated level of splicing in DD compared to LD 12:12, and similarly in LD 6:18, splicing levels are enhanced. Because the repression of splicing by light in LD 12:12 at 29°C does not require the presence of Cry, whether the visual system plays a role in setting the splicing level was investigated by examining the splicing of per transcripts in the mutants gl60j and norpAP41 (Collins, 2004).

The proportion of per mRNA transcripts that are spliced at both temperatures is increased in both the norpAP41 and gl60j backgrounds compared to WT. At 18°C, ~65% of per transcripts are spliced in norpAP41 and ~60% in gl60j, whereas at 29°C, these levels fall to ~55% and ~40%, respectively. Apart from a marginal difference between norpAP41; cryb, and norpAP41 at 18°C, there are no significant effects for either norpAP41 or gl60j when combined with cryb. These results indicate that the visual system rather than Cry is primarily responsible for the light-dependent repression of splicing. Unlike WT, per splicing levels do not rise after lights off at 29°C in either norpAP41 or gl60j (Collins, 2004).

Interestingly, in gl60j, there is a 20% difference between the splicing levels at different temperatures (60%-40%), whereas in norpAP41, this difference is reduced to 10% (65%-55%). The difference in gl60j is similar to that seen in WT (45%-25%). Thus per splicing in norpAP41 is relatively insensitive to temperature changes. It is also clear that the level of splicing in norpAP41 is significantly higher at all times and temperatures than gl60j. Therefore, the effect of norpAP41 on splicing is greater than that of gl60j, despite gl60j being the more severe visual mutant (Collins, 2004).

Locomotor activity profiles of all genotypes were also monitored at 18°C and 29°C. Because each genotype shows a higher level of splicing at 18°C than at 29°C, it would be predicted that this would generate an earlier evening activity peak at 18°C. This is the case for WT, cryb and norpAP41, but not for norpAP41; cryb or gl60j, where despite elevated splicing levels at higher temperatures, there is no difference in the phase of activity. gl60j cryb does not entrain to LD cycles at 25°C, so was not included in this analysis (Collins, 2004).

The average proportion of per transcripts that are spliced at 18°C rises from WT (45%) to cryb (50%) to gl60j (60%) to norpAP41 (65%), and at 29°C from WT (25%) to gl60j and cryb (~35%) to norpAP41 (55%). If the per splicing level is the only determinant of evening locomotor peak position, then a similar progression in the timing of this peak would be expected. The evening activity peaks of these different genotypes at 18°C and 29°C were compared. For norpAP41, cryb, and WT, there is an inverse relationship between average splicing levels and the position of the activity peak at 18°C, with norpAP41 and cryb having similarly advanced activity peaks compared to WT. At 29°C, the same inverse relationship holds, with norpAP41 advanced compared to cryb, which is in turn earlier than WT. Thus, those genotypes that show temperature-dependent changes in their evening activity generally display a correlation between average per splicing levels and the timing of the evening activity peak of the following day. Conversely, norpAP41; cryb and gl60j,, which show no significant differences in the phase of evening activity at different temperatures, have high splicing levels but relatively delayed evening activity peaks (Collins, 2004).

These observations raise the question of why the splicing level does not always relate to the timing of the evening locomotor activity peak, as in gl60j and norpAP41; cryb. Thus the per RNA profiles of gl60j and norpAP41 were compared to WT. Because per does not cycle in cryb whole head homogenates, the underlying cycle in this background was not examined. WT and norpAP41 show similar profiles, with an earlier per mRNA peak and higher overall level of per at the lower temperature. In contrast, there is no cycle in gl60j at either temperature, and levels of per are significantly different from WT and norpAP41 (Collins, 2004).

Therefore, to entrain locomotor behavior to different seasons, the fly's clock must respond to changes in both light and temperature. This is mediated through a molecular switch, whereby increases in temperature repress the splicing of an intron within the 3' UTR of per, delaying the onset of evening locomotor activity. Light also represses splicing, with higher splicing levels seen in shorter photoperiods, allowing locomotor activity to be fine-tuned to any given set of photoperiodic and temperature conditions. During the first day of DD, the level of splicing rises continuously. This is presumably because at the beginning of DD, the level of splicing is set low from the previous day's light input. Normally the light from the next day maintains this repressed level of splicing, but because this light input is absent, the repression of splicing is lifted, leading to a gradual rise in splicing levels (Collins, 2004).

The most obvious source for light input into the splicing machinery is the circadian photoreceptor Cry. However, analysis of the splicing levels in cryb shows that, although this mutation has an effect on splicing levels at 18°C, this effect is marginal and is seen only after lights off. This implies (1) that any function of Cry in the repression of splicing is not via the activation of this molecule by light; (2) because Cry is relatively dispensable for circadian locomotor rhythmicity per se, it also suggests that any minor role in splicing at low temperature is unrelated to the functioning of the clock. As the temperature rises, Per, Tim, and Cry all become involved in the regulation of per mRNA splicing. At 29°C, all three mutants show the same splicing phenotype, with ~30% of transcripts spliced during the day, but at night splicing is enhanced to ~45%. Although Per, Tim, and Cry are known to associate in light conditions, Cry and Tim can also associate in darkness, so it is not unexpected that the elimination of any one of the three proteins has a similar effect. Night time is also when the levels of these proteins are at their highest, and therefore any effects would be maximal (Collins, 2004).

At 29°C and in the presence of light, the levels of splicing in per01; cryb are elevated above those of either single mutant, which are themselves similar to WT. This suggests that the presence of either Per or Cry is required for light to repress splicing at 29°C. After lights off, the elevated levels of splicing of per are very similar in per01, tim01, cryb, and per01; cryb. Therefore Per, Tim, and Cry probably work together to repress splicing in the dark at 29°C. An alternative view for the virtually identical per01, tim01, and cryb splicing levels at 29°C is that this reflects a masking effect of light, so that exogenous LD cycles have a greater effect on splicing at night compared to WT, which shows a modest but significant day-night rhythm. Such stronger masking effects on locomotor behavior have also been observed in cryb mutants, but any mechanism that might relate or explain these observations remains obscure (Collins, 2004).

The examination of whole head homogenates means that the majority of biological material is derived from the eyes so may not represent exactly what occurs in the pacemaker neurons. The eyes are peripheral clocks, and the cryb mutation stops the cycling of the clock in whole head homogenates, although cycling continues in the pacemaker cells. One possibility is that the splicing observed in cryb does not truly reflect the role of Cry in setting splicing levels but is instead a consequence of the clock having stopped in the eyes, thus explaining why per01, tim01, and cryb all show the same splicing phenotype. However, if this splicing phenotype is simply what happens when the clock stops, then per01; cryb should show the same splicing phenotype as either single mutant. This is not the case, because the daytime splicing in per01; cryb at 29°C is dramatically elevated compared to either single mutant. Thus the splicing phenotypes of per01, tim01, and cryb cannot simply be a result of the clock having stopped. This means that it is the presence of these proteins, rather than their clock-dependent cycling, that is important to the regulation of per splicing levels (Collins, 2004).

In gl60j, there is no per mRNA cycle in whole head homogenates. This means that in the majority of cells in the gl60j head, the clock has either stopped or cells have become desynchronized. If the former is true, then splicing levels of gl60j should resemble those of per01 or tim01, and this is clearly not the case. If the latter is true, this could prevent the observation of any splicing rhythm, but the level of splicing observed should still represent the average level of splicing in this mutant background, which is clearly significantly different from WT. In any case, splicing levels observed in all visual mutants are likely to represent the effect of removing visual photoreception, because these elevated levels are similar to those observed in WT in DD (Collins, 2004).

norpAP41 and gl60j have considerably higher splicing levels than WT and cryb mutants at both temperatures, indicating that information received via the visual system rather than Cry drives this repression of splicing, which is borne out by analysis of gl60j cryb and norpAP41; cryb double mutants. The splicing levels of gl60j and gl60j cryb are similar at both temperatures, which is also true of norpA and norpAP41; cryb at 29°C. At 18°C, there is slightly more spliced per RNA in norpAP41; cryb than in the norpAP41 single mutant, reflecting the earlier result where cryb showed a marginal enhancement of splicing at cooler temperatures. These results also demonstrate that unspecific genetic background effects are not responsible for this marginal effect of cryb, because the double mutant background should make any interacting loci heterozygous. This lack of significant background effects in determining overall splicing levels has been confirmed by examining several natural European D. melanogaster lines. All mutants studied here show the same significantly enhanced splicing patterns when compared to any of the wild-caught isolates (Collins, 2004).

Unlike the clock and cryb mutants, there is no day-night difference in splicing levels at 29°C in either gl60j or norpAP41. One possibility is that visual system structures are required for the repression of splicing even in the dark, hence the overall elevated splicing levels in norpAP41 and gl60j at all times. This would be surprising, because such a role would obviously have to be light independent. More likely, the light input received through the eyes sets the splicing level during the day, and the clock maintains this repression at night. Thus, if the visual input is removed or reduced, as in DD, gl60j, or norpAP41 mutants, or in shorter photoperiods, then the subsequent splicing level is set higher. The difference in roles between cry and the visual system on per splicing levels may also partly explain recent observations that cryb mutants are able to adapt the timing of locomotor activity to long and short photoperiods, whereas flies with defective visual photoreception, including gl60j, are not (Collins, 2004).

Interestingly, although gl60j is the more severe visual mutant, norpAP41 has significantly higher per splicing levels than gl60j at both 18°C and 29°C. Additionally, whereas the difference between splicing levels at 18°C and 29°C is maintained in gl mutants (~65% and ~45% of transcripts spliced vs. ~45% and 25% in WT at 18°C and 29°C, respectively), this is greatly reduced in norpAP41 (65% and 55%). One possible explanation for this is that norpA may be a signaling molecule in the temperature-sensing pathway for the clock. The patterns of locomotor activity support a role for norpA in temperature sensing, with the norpAP41 fly's locomotor patterns seemingly more sensitive to high temperatures than WT. Additionally, norpAP41 evening locomotor activity peaks early at both 18°C and 29°C, and per mRNA splicing shows a corresponding elevation compared to WT. These are responses associated with low temperatures in WT D. melanogaster, and therefore norpAP41 mutants behave as if they have an impaired ability to detect high temperatures. norpAP41 flies still detect temperature changes (witness the altered evening peaks and splicing levels); they just react as if the temperature is colder than it actually is (Collins, 2004).

Thus, the enhanced per splicing seen in norpAP41 may reflect a direct link between norpA-encoded PLC signaling and the temperature sensitivity of the splicing mechanism, independent of norpA visual function. In the phototransduction cascade, rhodopsin activates a G-protein isoform that in turn activates the PLC encoded by norpA. As a result of this activation, Ca2+ permeable light-sensitive channels are opened, including members of the transient receptor potential (TRP) class. Recently it has been demonstrated that dANKTM1, a D. melanogaster TRP channel, is activated by temperatures from 24°C to 29°C. In addition, D. melanogaster painless mutant larvae have a disrupted TRP channel and display defective responses to thermal stimuli. Because several TRP family members act as thermal sensors in mammals, TRP channels appear to have an ancient heat-sensing function that is retained in both vertebrates and invertebrates. Given that this study has identified a heat-sensing role for norpA, and norpA is known to activate TRP channels in photoreception, it is not unreasonable to suppose that norpA plays a general role in responses to temperature stimuli (Collins, 2004).

per splicing levels may also impact on aspects of behavior other than the timing of evening locomotor activity. For instance, the free-running period of norpAP41 is ~1 h shorter than WT. The splicing levels of per mRNA are greatly elevated in this background, and elevated splicing is predicted to advance the Per protein cycle and thus speed up the clock. In fact, the splicing mechanism should have the effect of speeding up the clock at colder temperatures and slowing it down at high temperatures, thereby providing a potential basis for temperature compensation (Collins, 2004).

The position of the evening activity peak at different temperatures moves in different mutant backgrounds. For WT, norpAP41, and cryb, the level of splicing appears to correlate with the position of the evening activity peak at different temperatures. At 18°C, there is a small but significantly greater relative amount of spliced per RNA in cryb than in WT, resulting in the earlier evening activity peak seen in cryb flies. This difference in per splicing is greatest after lights off at both temperatures. This is when Per levels will be rising, because Tim is present for Per stabilization, so enhancement of Per accumulation by elevated per splicing is likely to have its most noticeable effect around dusk or early evening. A similarly consistent situation is seen in norpAP41: there is more spliced per mRNA present at 18°C (65%) than 29°C (55%), accounting for the earlier peak of evening activity at 18°C. Additionally these levels are higher than those seen in either WT (45% and 25% per transcripts spliced at each temperature) or cryb (55% and 40%) and relates to the earlier phases of locomotor activity seen in norpAP41 compared to the other genotypes. However, at 18°C there is more spliced per in norpAP41 than in cryb, but the evening activity peak occurs at the same time. The simplest explanation is that there is a limit to how early the evening activity peak can occur, no matter what the per splicing level, because splicing alters the accumulation of Per protein; this is limited by the light-dependent degradation of Tim. Therefore, in general, the level of splicing determines when the peak level of locomotor activity will occur (Collins, 2004).

The level of splicing of the per intron cannot be the only determinant of evening peak position, because the relationship between the per splicing level and evening activity peak position breaks down in norpAP41; cryb and gl60j, where there are different levels of splicing at the two temperatures but no corresponding difference in the evening peak position. When the underlying per mRNA cycles of gl60j, norpAP41, and WT flies were analyzed at 18°C and 29°C, it was found that whereas per levels cycle in norpAp41 and WT, this cycle is lost in gl60j. If there is no underlying per RNA cycle, then there is no mRNA peak to be advanced or delayed by splicing (Collins, 2004).

At the cellular level, although gl is not a clock component, when mutated, it eliminates a number of clock-expressing cells within the head, including the eyes, ocelli, Hofbauer-Buchner (H-B) eyelet, and the dorsal neuron 1 (DN1) cells. Despite this, the primary effect on the clock is to remove most of the visual entrainment pathway, but the clock in the key pacemaker cells of gl60j mutants must still be functional, because behavior still entrains to LD cycles and remains rhythmic in DD. It is significant that the crosstalk between different classes of clock cells is essential for the generation of robust behavioral rhythms. Thus loss of the overall per mRNA rhythm may be a consequence of disrupting this network in gl60j, and, while leaving the basic system intact, this affects the more subtle temperature-sensitive aspects of entrainment. A similar argument based on an interruption of the entrainment network can also be proposed to explain the corresponding results with norpAP41; cryb double mutants, because in this case per mRNA is assumed to be noncycling because of the cryb background. However, the locomotor behavior of cryb single mutants remains thermosensitive even though overall per mRNA is noncycling. Thus, only when the photoreceptive pathway and mRNA cycle are both compromised (as in gl and norpAP41; cryb) is locomotor behavior insensitive to temperature-dependent changes in per splicing levels (Collins, 2004).

A model is presented of how light and temperature may set the splicing level of the clock. How temperature is detected by the splicing machinery is not yet clear, but there is compelling evidence that norpA plays a role. At low temperatures, the splicing level is primarily set by light via the visual system rather than Cry, which is then remembered during the night. In longer periods of darkness such as in DD, this memory decays, and splicing levels begin to rise. Thus the visual system represses splicing by enhancing the effects of an unknown repressor molecule(s) that is sensitive to temperature change and the norpA PLC. At high temperatures, the regulation of splicing is more stringent and complex and recruits the circadian clock. Again, the light input received through the visual system sets the low splicing level during the day. This appears to also depend on the presence of at least two of the three molecules, Per, Tim, or Cry, because elimination of any one of these gives a barely detectable daytime rise in splicing, reflecting the very low levels of Per, Tim, and Cry at this time. However, elimination of both Per and Cry in the per01; cryb double mutant lifts all light-dependent repression during the day (Collins, 2004).

At night, the level of splicing set during the day by the visual system is again remembered and maintained by the clock at night. If per, tim, or cry is eliminated, then this repression of splicing is lost at night, generating the day/night difference in splicing levels. In gl60j cryb or norpAP41; cryb, because there is no visual light input during the day, there is no splicing level for the clock to remember, and therefore there is no day/night difference in splicing levels. Thus at high temperature, the visual system activates the repressor molecule during the day, and the clock maintains this activation at night. It is assumed that recruiting the clock at high temperature to inhibit per splicing is required to ensure that the fly's locomotor/foraging behavior is adaptive and does not encroach on those times of the day when there would be a significant risk of desiccation (Collins, 2004).

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

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