Within a 69-bp DNA fragment upstream of the per gene, a circadian transcriptional enhancer has been identified. This enhancer drives high-amplitude PER mRNA cycling under light-dark-cycling or constant-dark conditions; this activity is Per protein dependent. An E-box sequence within this 69-bp fragment is necessary for high-level expression, but not for rhythmic expression, indicating that Per mediates circadian transcription through other sequences in this fragment. Since Per protein is unlikely to bind DNA, the identification of clock control sequences should allow the identification of factors that confer cyclic expression (Hao, 1997).

A new regulatory element necessary for the correct temporal expression of the period gene was identified by monitoring real-time per expression in living individual flies carrying two different period-luciferase transgenes. luciferase RNA driven from only the per promoter is not sufficient to replicate the normal pattern of PERm RNA cycling; however, a per-luc fusion RNA driven from a transgene containing additional per sequences cycles identically to endogenous per. The results indicate the existence of at least two circadian-regulated elements--one within the promoter and one within the transcribed portion of the per gene. Phase and amplitude analysis of both per-luc transgenes reveals that normal per expression requires the regulation of these elements at distinct phases and suggests a mechanism by which biological clocks sustain high-amplitude feedback oscillations. When the amplitude and phases of the promoter-only oscillations (Amp = 5-fold, early phase) and the promoterless oscillations (Amp = 2.5 fold, late phase) are combined, one obtains the phase and amplitude of endogenous PER mRNA cycling (Amp = 14-fold, normal phase (Stanewsky, 1997a).

The period (per) gene is an essential component of the circadian timekeeping mechanism in Drosophila. This gene is expressed in a circadian manner, giving rise to a protein that feeds-back to regulate its own transcription. A 69 bp clock regulatory sequence (CRS) has been identified previously, upstream of the period gene. Drosophila Clock and Cycle encode proteins that activate per and tim transcription via E-boxes located in per and tim upstream sequences. One of the E-boxes targeted by dCLK and CYC is located within the 69 bp CRS upstream of per; this CRS is required for rhythmic transcription. The CRS confers wild-type mRNA cycling when used to drive a lacZ reporter gene in transgenic flies. To determine whether the CRS also mediates proper developmental and spatial expression and behavioral rescue, the ability of CRS to drive either lacZ or per was tested in transgenic flies. The results show that the CRS is able to activate expression in pacemaker neuron precursors in larvae and essentially all tissues that normally express per in pupae and adults. The CRS is sufficient to rescue circadian feedback loop function and behavioral rhythms in per01 flies. However, the period of locomotor activity rhythms shortens if a stronger basal promoter is used. This study shows that regulatory elements sufficient for clock-dependent and tissue-specific per expression in larvae, pupae, and adults are present in the CRS and that the period of adult locomotor activity rhythms is dependent, in part, on the overall level of per transcripts (Hao, 1999).

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).

Circadian oscillator networks rely on a transcriptional activator called Clock/Cycle (Clk/Cyc) in insects and CLOCK/BMAL1 or NPAS2/BMAL1 in mammals. Identifying the targets of this heterodimeric basic-helix-loop-helix (bHLH) transcription factor poses challenges and it has been difficult to decipher its specific sequence affinity beyond a canonical E-box motif, except perhaps for some flanking bases contributing weakly to the binding energy. Thus, no good computational model presently exists for predicting Clk/Cyc, CLOCK/BMAL1, or NPAS2/BMAL1 targets. This study used a comparative genomics approach and first studied the conservation properties of the best-known circadian enhancer: a 69-bp element upstream of the Drosophila melanogaster period gene. This fragment shows a signal involving the presence of two closely spaced E-box-like motifs, a configuration that is also detected in the other four prominent Clk/Cyc target genes in flies: timeless, vrille, Pdp1, and cwo. This allows for the training of a probabilistic sequence model that was tested using functional genomics datasets. The predicted sequences are overrepresented in promoters of genes induced in a study by a glucocorticoid receptor-CLK fusion protein. The mouse genome was then scanned with the fly model and many known CLOCK/BMAL1 targets were found harbor sequences matching this consensus. Moreover, the phase of predicted cyclers in liver agreed with known CLOCK/BMAL1 regulation. Taken together, a predictive model was built for CLK/CYC or CLOCK/BMAL1-bound cis-enhancers through the integration of comparative and functional genomics data. Finally, a deeper phylogenetic analysis reveals that the link between the CLOCK/BMAL1 complex and the circadian cis-element dates back to before insects and vertebrates diverged (Paquet, 2008; . Full text of article).

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).

To identify specific sequence elements mediating the mutant effects on transcription, the effects of the cycle mutation were examined on a minimal per promoter element. This 69 bp enhancer contains a critical per-derived E box and drives rhythmic expression of a reporter gene (lacZ). To this end, the E box/lacZ construct was crossed into cyc0/cyc0 mutant flies and lacZ RNA levels were assayed for cycling by RNase protection. The results are dramatic and indicate that there is little or no cycling lacZ RNA transcription in the homozygous mutant flies, suggesting that Cyc affects the transcriptional activity of the per circadian transcriptional enhancer. These features of Cyc are similar to those of Clk, which probably binds to and activates transcription at the per E box (Rutila, 1998).

The mammalian protein BMAL1 was cloned as an "orphan" protein of the bHLH-PAS transcription factor family with no known biological function. However, there are recent biochemical experiments indicating that it may play a role in circadian rhythm-relevant transcription in mammals: it can act as a heterodimeric partner of mouse Clock (mClock) in DNA binding and transcriptional activation. The BMAL1:mClock heterodimer selects a DNA-binding sequence that resembles the critical E box sequence within the cycling element in the Drosophila per upstream region, and there are features of this enhancer in addition to the central CACGTG hexamer that provide specificity for the BMAL1:mCLOCK heterodimer. As transcripts from mouse per genes undergo circadian oscillations in level, these genes may contain a similar target cycling element to that of Drosophila per. The BMAL1:mCLOCK heterodimer could be the heterodimeric factor that binds to this cycling element and activates clock-relevant transcription (Hogenesch, 1998 and Rutila, 1998 and references).

By analogy, it is proposed that Cyc and Clk heterodimerize, bind to Drosophila clock gene E boxes, and function to drive the circadian-regulated transcription of these genes. This makes cyc and Clk the first Drosophila circadian rhythm genes with a known biochemical role and a defined place in the clock circuit; it also places them upstream of per and tim. RNA and transcription experiments in cyc0 and in Clk mutant flies (Allada, 1998) fully support such an assignment (Rutila, 1998).

Cyc is approximately half as big as Clk; the difference appears to be largely the extensive glutamine-rich C-terminal half of Clk (Allada, 1998). This may indicate that Clk brings the transcriptional activation domain to the complex. The dominant phenotype of the Clk mutant and the elimination of the Q-rich region by the mutation (Allada, 1998) are consistent with this notion. The mutant protein would then be able to dimerize with Cyc and bind DNA but would be unable to activate transcription. This would explain its recessive as well as its dominant features (Rutila, 1998).

per transcription is regulated by a PER-TIM heterodimer. PER first accumulates in the cytoplasm, increasing during the day and reaching peak levels at night (Hardin, 1990). It then enters the nucleus during a restricted part of the circadian cycle (near the middle of the dark period in the light-dark cycle). The delay between PER synthesis and entry into the nucleus is critical to the timing of the circadian cycle (Curtin, 1995). By the end of the night period, transcripts reach their lowest level. Transfer of wild type flies to conditions of constant light suppresses the cycling of PER abundance and phosphorylation, and produces constituatively low levels of PER (Price, 1995).

The peak of PER mRNA abundance occurs 4 hours after lights-off, indicating that there is a fixed phase relationship between the PER mRNA cycle and lights-off transition. When day length is changed, the peak levels of PER mRNA shifts within a day to match the new light-dark cycle. Kept in constant light, flies exhibit a behavioral arrhythmicity and PER levels become constitutively low, at about 50% of peak values under a normal light-dark cycle. Shortening the length of night reveals that a period of darkness is necessary for per feedback loop function. A period of darkness of 6 to 8 hours is required for wild-type feedback loop function (as judged by mRNA cycling amplitude) (Qui, 1996).

A recessive third chromosomal mutation that abolishes bioluminescence rhythms has been identified, cryb. cryb was isolated based on its elimination of period-controlled luciferase cycling. 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. 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 role of dCREB2 (CrebB-17A) in circadian rhythms has been examined. dCREB2 activity cycles with a 24 hr rhythm in flies, both in a light:dark cycle and in constant darkness. A mutation in dCREB2 shortens circadian locomotor rhythm in flies and dampens the oscillation of period, a known clock gene. Cycling dCREB2 activity is abolished in a period mutant, indicating that dCREB2 and Period affect each other and suggesting that the two genes participate in the same regulatory feedback loop. It is proposed that dCREB2 supports cycling of the Period/Timeless oscillator. These findings support CREB's role in mediating adaptive behavioral responses to a variey of environmental stimuli (stress, growth factors, drug addiction, circadian rhythms, and memory formation) in mammals and long-term memory formation and circadian rhythms in Drosophila (Belvin, 1999).

To measure dCREB2 activity in vivo, transgenic Drosophila lines were constructed carrying the luciferase reporter gene driven by an enhancer element comprised of consensus CREB binding sites. Three cAMP response elements (CREs), 5'-TGACGTCA-3', were placed upstream of the TATA box region of the hsp70 gene promoter, followed by the luciferase reporter gene. This sequence was flanked by the scs and scs' insulator elements to reduce potential positional effects caused by the random insertion site of the transgene. The transfected lines are referred to as CRE-luc lines. A mutant CRE-luc reporter construct (mCRE-luc) was also generated in which the consensus CRE sites were mutated to TGAAATCA. dCREB2 protein binds this mutant CRE site with at least 20-fold lower affinity in gel shift experiments. This construct is otherwise identical to wild-type CRE-luc (Belvin, 1999).

The expression of luciferase in the wild-type CRE-luc flies oscillates in a 24 hr rhythm, both in a light:dark cycle and in constant darkness. The main peak of activity occurs just after lights out, with the nadir just before the main peak. Since this rhythmic transcription pattern is sustained in constant darkness, it is regulated by the circadian system, rather than simply being a response to light. In light:dark conditions, a second peak is observed in the middle of the day; however, these two peaks gradually blend together under conditions of constant darkness. This pattern is very similar to that seen for per activity. The period-luciferase reporter also exhibits a similar secondary peak under light:dark conditions, even though per RNA peaks only once per cycle. It is likely that the secondary peaks of both reporters, which occur during the day, are due to a light response of luciferase rather than a circadian response. The expression level of the mCRE-luc reporter is drastically reduced relative to the wild-type reporter, indicating that the CRE sites mediate the high-level expression of the wild-type reporter (Belvin, 1999).

If dCREB2 mutation S162 is indeed in the clock, then it should affect the per clock gene. The effects of S162 on two different per-dependent reporters were examined. The first is a transcriptional fusion with a 4.2 kb fragment of the per promoter upstream of the luciferase reporter gene, referred to as per-luc. The second is a translational fusion containing the same promoter fragment, plus the 5' untranslated region and the first 2.4 kb of the per coding region fused in frame to the luciferase gene, referred to as BG-luc. When the expression of these reporters was compared, it was found that the BG-luc reporter cycles much more robustly than the per-luc reporter, consistent with the interpretation that there are at least two mechanisms contributing to the cycling of Per: one mediated by the promoter, and the other(s) mediated by sequences in either the per transcript or Per protein itself. S162 affects the two reporters differently. The S162 mutation reduces both the expression level and cycling pattern of the per-luc reporter. However, its effect on the BG-luc reporter is weaker. In S162 flies, the BG-luc reporter maintains a robust cycling pattern, although its expression level and amplitude are reduced. The peak in the mutant background also occurs in advance of the peak in wild-type flies, consistent with the short period phenotype of these flies (Belvin, 1999).

In order to demonstrate a direct effect of the S162 mutation on the clock, its effects on the Per protein itself were examined. Wild-type and S162 flies were entrained on a 12 hr light:12 hr dark cycle and aliquots were frozen every 2 hr throughout the cycle. Head extracts were prepared and analyzed by Western blot using an antibody directed against Per. Per is present at very low levels at ZT 6 and ZT 8 (Zeitgeber time; 6 and 8 hours after lights on), increasing through the lights of period to peak levels before lights on, which occurs at ZT 0. A corresponding change in phosphorylation, and protein mobility, accompanies the change in absolute levels, with Per becoming more highly phosphorylated as it accumulates. This temporal pattern of Per is altered in the S162 mutant background, where Per is present at more equal levels throughout the circadian cycle. At the peak time, ZT 20, the amount of Per is at least comparable to that in wild-type flies; however, it decreases less at ZT 6 and ZT 8, when Per is virtually absent in wild-type flies. At these trough periods of Per expression, a discrete doublet protein band persists in S162, perhaps representing preservation of certain phosphorylated forms. There also seems to be a general increase in the amount of Per protein throughout the cycle in the mutant flies. The change in both per-luc expression and Per protein levels in the S162 mutant background demonstrates that Per activity is under the influence of the dCREB2 gene. The effects of S162 on Tim protein were assayed in the same experiment. The effect of S162 on Tim is much more subtle than the effect on Per. The Tim protein appears to accumulate slightly sooner in the mutant than in the wild type (ZT 12 versus ZT 14); however, its overall oscillation remains fairly normal (Belvin, 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).

The novel Drosophila timblind mutation affects behavioral rhythms but not periodic eclosion: Transcriptional regulation of per

Circadian clock function depends on the tightly regulated exclusion or presence of clock proteins within the nucleus. A newly induced long-period timeless mutant, timblind, encodes a constitutively hypophosphorylated Tim protein. The mutant protein is not properly degraded by light, and timblind flies show abnormal behavioral responses to light pulses. This is probably caused by impaired nuclear accumulation of TimBLIND protein, that is observed in brain pacemaker neurons and photoreceptor cells of the compound eye. timblind encodes two closely spaced amino acid changes compared to the wild-type Tim protein; one of them is within a putative nuclear export signal of Tim. Under constant conditions, timblind flies exhibit 26-hr free-running locomotor rhythms, which are not correlated with a period lengthening of eclosion rhythms and period-luciferase reporter-gene oscillations. Therefore it seems possible that Tim -- in addition to its well-established role as core clock factor -- functions as a clock output factor, involved in determining the period length of adult locomotor rhythms (Wulbeck, 2005).

What are the consequences of the faulty Tim phosphorylation observed in timblind flies? They largely seem to be restricted to Tim itself, because cyclic expression, nuclear accumulation, as well as temporal mobility changes of PER are affected to a lesser extent. In contrast, the TimBLIND protein shows drastic defects in nuclear accumulation and almost no abundance fluctuation during the circadian cycle, in addition to the phosphorylation defects described above. This is another example of a newly emerging picture that Per and Tim can function independently of each other (Wulbeck, 2005).

If Per expression and function is not strongly affected by timblind, how is it that the mutant flies free-run with a 26-hr period? Although the free-running period of Per oscillations in the behavior controlling clock neurons has not been recorded directly, recordings of luminescence rhythms of flies expressing a PER-LUC fusion protein predominantly in clock neurons of the dorsal brain suggest that the circadian clock in timblind flies may tick with a 24-hr period and not with a 26-hr one, as would be predicted from their behavioral rhythm. Moreover, eclosion rhythms free-run with a 24-hr period in timblind flies. This is intriguing, because other tim alleles increase the period length of locomotor rhythms and eclosion to a similar extent. Both pupal and adult brains contain the same set of pacemaker neurons: the ventrally located small and large lateral neurons (LNv's, the more dorsally located LNd's, and three groups of neurons in the dorsal brain (DN1-3). Nevertheless, eclosion and adult locomotion could be controlled by different subsets of pacemaker neurons, because clock gene cycling has not been determined in all of these groups under free-running conditions. Therefore, it is possible that some of these cells indeed show a period of 26 hr (Wulbeck, 2005).

Alternatively, the discrepancy between the apparently normal clock function and lengthened free-running period of locomotor rhythms of timblind flies could be explained by a novel function of Tim in the nucleus in addition to its well-established role as crucial clock factor. If the circadian clock in timblind flies runs in a globally slow manner, all clock outputs would have longer-than-normal periods. But this is clearly not the case, given the normal eclosion rhythms and PER-LUC oscillations observed in the mutant flies. Therefore, it seems that the timblind defect is not at the level of the central oscillator but rather at the interface between the pacemaker and the output mediating locomotor rhythms. Although Tim alone is not able to function as a repressor of Clk/Cyc-activated transcription in vitro, it is possible that Tim acts alone or together with other proteins to regulate clock-controlled-genes (CCGs) downstream of the core molecular clock. A Per-independent function of Tim in regulating CCGs has been inferred from in vitro studies in which high levels of Tim (without Per) resulted in the activation (rather than the expected suppression) of E-box-driven reporter-gene expression. That this might indeed be the case is also indicated by the distinct effects of timblind on per-luc vs. tim-luc expression. Whereas per transcription occurs with an advanced peak and reduced cycling amplitude compared with control flies, tim expression levels are drastically increased, and the cycling amplitude is also blunted. This indicates differences in the regulation of the per and tim promoters and different functions for Tim in the feedback regulation acting on these regulatory sequences (Wulbeck, 2005).

In summary, analysis of the novel timblind mutant underscores the importance of Tim nuclear accumulation for proper regulation of locomotor behavior during light entrainment and under constant conditions. Possibly as a consequence of constitutive nuclear export, Tim levels are subnormal within cellular nuclei of timblind flies, and Tim phosphorylation is impaired. This results in a diminished behavioral light response but not in a failure to respond to light. Moreover, judged by normal oscillations of per gene products and 24-hr eclosion rhythms, pacemaker function under constant conditions seems normal, indicating a novel role for Tim in clock output (Wulbeck, 2005).

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 Pdp1mRNA 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).

Natural variation in the Drosophila melanogaster clock gene period modulates splicing of its 3'-terminal intron and mid-day siesta

Drosophila melanogaster exhibits circadian (congruent with 24 hr) regulated morning and evening bouts of activity that are separated by a mid-day siesta. Increases in daily ambient temperature are accompanied by a progressively longer mid-day siesta and delayed evening activity. Presumably, this behavioral plasticity reflects an adaptive response that endows Drosophila melanogaster with the ability to temporally optimize daily activity levels over a wide range of physiologically relevant temperatures. For example, the shift in activity towards the cooler nighttime hours on hot days might minimize the risks associated with exposure to mid-day heat, whereas on cold days activity is favored during the warmer daytime hours. These temperature-induced shifts in the distribution of daily activity are partly based on the thermal sensitive splicing of an intron found in the 3' untranslated region (UTR) of the circadian clock gene termed period (per). As temperature decreases, splicing of this 3'-terminal intron (termed dmpi8) is gradually increased, which is causally linked to a shorter mid-day siesta. This study identified several natural polymorphisms in the per 3' UTR from wild-caught populations of flies originating along the east coast of the United States. Two non-intronic closely spaced single nucleotide polymorphisms (SNPs) modulate dmpi8 splicing efficiency, with the least efficiently spliced version associated with a longer mid-day siesta, especially at lower temperatures. Although these SNPs modulate the splicing efficiency of dmpi8 they have little to no effect on its thermal responsiveness, consistent with the notion that the suboptimal 5' and 3' splice sites of the dmpi8 intron are the primary cis-acting elements mediating temperature regulation. These results demonstrate that natural variations in the per gene can modulate the splicing efficiency of the dmpi8 intron and the daily distribution of activity, providing natural examples for the involvement of dmpi8 splicing in the thermal adaptation of behavioral programs in D. melanogaster (Low, 2012).

An ecdysone-responsive nuclear receptor regulates circadian rhythms in Drosophila

Little is known about molecular links between circadian clocks and steroid hormone signalling, although both are important for normal physiology. This study reports a circadian function for a nuclear receptor, ecdysone-induced protein 75 (Eip75/E75), which was identified through a gain-of-function screen for circadian genes in Drosophila melanogaster. Overexpression or knockdown of E75 in clock neurons disrupts rest:activity rhythms and dampens molecular oscillations. E75 represses expression of the gene encoding the transcriptional activator, Clock (Clk), and may also affect circadian output. Per inhibits the activity of E75 on the Clk promoter, thereby providing a mechanism for a previously proposed de-repressor effect of Per on Clk transcription. The Ecdysone receptor is also expressed in central clock cells and manipulations of its expression produce effects similar to those of E75 on circadian rhythms. E75 protects rhythms under stressful conditions, suggesting a function for steroid signalling in the maintenance of circadian rhythms in Drosophila (Kumar, 2014).

An ecdysone-responsive nuclear receptor regulates circadian rhythms in Drosophila

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

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

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

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

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

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

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