Since the timing and strength of the VRI mRNA oscillations are almost identical to those of timeless, it seemed possible that vri and tim transcription would be regulated by the same transcription factors. VRI mRNA levels were tested in adult heads of ClkJrk and cyc0 mutant flies, which show constitutively low transcription of the per and tim genes. VRI mRNA, like TIM mRNA, is produced at low levels in these mutants. Like TIM, VRI mRNA levels are high or intermediate in per01 and tim01 mutants, indicating that VRI levels are linked to the activities of period, tim, Clock , and cycle. The correspondence of vri, per, and tim regulation in wild-type and clock mutants suggests that the dCLK/Cyc complex might directly regulate vri transcription (Blau, 1999).
The vri promoter sequence was searched and four CACGTG motifs, that are potential Clk/Cyc-binding sites, were found. One of these is closely related to the functional Clk/Cyc-binding site in the per promoter. vri promoter sequence (2.8 kb), including all four sites, was fused to a luciferase reporter and transfected into Drosophila S2 cells either with or without an expression vector for Clk. This assay has been used to show that Clk can activate the per and tim promoters in cultured cells, and it relies on the endogenous production of Cyc in S2 cells. The vri, per, and tim promoters were all strongly activated by Clk (658-, 49-, and 271-fold, respectively). To test whether the most conserved of the potential Clk/Cyc-binding sites in the vri promoter is sufficient for activation by this complex, reporter constructs composed of four copies of either this wild-type sequence, or a mutant sequence in which the central CG is reversed, upstream of a basal promoter and a luciferase reporter gene, were generated. The wild-type vri sequence is strongly activated (190-fold) by Clk, while the mutant E box is activated only 3-fold. Therefore, Clk can bind and activate the vri promoter. A promoter fragment containing the remaining CACGTG sites also responds to Clk expression, suggesting further Clk/Cyc binding to one or more of these sites. The results indicate that the transcriptional loop that causes per and tim transcription to cycle with a 24 hr period also regulates other genes (Blau, 1999).
The Drosophila circadian clock consists of two interlocked transcriptional feedback loops. In one loop, Clock/Cycle activates period expression, and Period protein then inhibits Clock/Cycle activity. Clock is also rhythmically transcribed, but its regulators are unknown. vrille (vri) and Par Domain Protein 1 (Pdp1) encode related PAR family bZIP transcription factors whose expression is directly activated by Clock/Cycle. Vri and Pdp1 proteins are shown to feed back and directly regulate Clock expression. Repression of Clock by Vri is separated from activation by Pdp1 since Vri levels peak 3-6 hours before Pdp1. Rhythmic vri transcription is required for molecular rhythms, and the clock stops in a Pdp1 null mutant, identifying Pdp1 as an essential clock gene. Thus, Vri and Pdp1, together with Clock itself, comprise a second feedback loop in the Drosophila clock that gives rhythmic expression of Clock, and probably of other genes, to generate accurate circadian rhythms (Cyran, 2003).
vri and Pdp1 encode basic zipper transcription factors with highly conserved basic DNA binding domains, suggesting they bind the same set of target genes. vri and Pdp1 are both direct targets of Clk/Cyc. First, a test was performed to see which Pdp1 isoform(s) are clock-controlled since four alternative promoters and alternative splicing generate six Pdp1 isoforms in vivo. RNase protection probes specific for the different isoforms revealed that only Pdp1epsilon RNA levels oscillate in adult fly heads (Cyran, 2003).
Taking time points every three hours during a light-dark (LD) cycle revealed that vri and Pdp1epsilon RNA levels oscillate with similar phases to one another, but peak levels of Pdp1epsilon are not reached until 3-6 hr after the peak of vri RNA levels. Oscillating Pdp1epsilon RNA levels are also seen in constant darkness. Pdp1epsilon RNA levels were high at both ZT2 and ZT14 in per0 and tim01 mutants. Pdp1epsilon RNA is low at both ZT2 and ZT14 in ClkJrk and cyc0 mutants at levels close to the Pdp1epsilon RNA levels at ZT2 in wild-type flies. The phase of Pdp1epsilon RNA expression in wild-type flies, and the loss of rhythms in clock mutants, are both consistent with Pdp1epsilon transcription being regulated in a similar manner to per, tim, and vri transcription. Indeed, analysis of the first 4 kb of sequence upstream of the start site of Pdp1epsilon transcription reveals six perfect CACGTG E boxes, which are potential Clk/Cyc binding sites. This is similar to the vri promoter, which has 4 E boxes in 2.4 kb. Thus, Pdp1epsilon is the clock-regulated Pdp1 transcript (Cyran, 2003).
The different phases of vri and Pdp1epsilon RNAs may reflect subtly different transcriptional activities of their promoters and/or different mRNA half-lives. Thus, the vri promoter could be stronger than the Pdp1epsilon promoter, and vri RNA may have a shorter half-life than Pdp1epsilon RNA. Indeed, the vri 3' UTR contains seven copies of an AATAA element, likely to be associated with mRNA instability (Cyran, 2003).
Direct regulation of Pdp1epsilon expression by Clk/Cyc make it likely that Pdp1epsilon protein would be found in clock cells as shown for vri. Pdp1 protein is detected at night (ZT21) but not by day (ZT10) in larval pacemaker cells, marked by the neuropeptide pigment dispersing factor (PDF). Oscillation of Pdp1 protein continues in constant darkness in wild-type pacemaker cells but is blocked by null or dominant-negative mutations in the per, tim, Clk, and cyc clock genes (Cyran, 2003).
A robust oscillation in Pdp1 levels is also visible in photoreceptor cells of the adult eye, which contain functional clocks. Low Pdp1 levels are seen during the day at ZT9, and high levels in the middle of the night at ZT18. The oscillation is especially clear in the outer photoreceptor cell nuclei. Pdp1 at ZT18 colocalizes with ELAV, which marks the nuclei of neurons. Although antibodies to Pdp1 do not distinguish between the different Pdp1 isoforms, RNase protection data and Western blots detect rhythmic expression of only Pdp1epsilon in fly heads. Pdp1 protein is thus rhythmically detectable in both central and peripheral clock cells and it is a nuclear protein, as predicted by its ability to activate transcription (Cyran, 2003).
Current models of the Drosophila circadian oscillator are based upon rhythmic activation of per/tim transcription by cycling levels of Clk/Cyc, and rhythmic repression of per/tim transcription by cycling levels of Per/Tim. While these models explain Per and Tim oscillations, the molecular mechanisms underlying cycling of Clk/Cyc have been unknown. This study identifies Vri as a rhythmically expressed Clk repressor and Pdp1epsilon as a rhythmically expressed Clk activator. Vri and Pdp1epsilon are shown to directly regulate Clk transcription by binding the same site in the Clk promoter. Pdp1 is required for circadian clock oscillation and for Clk expression, thus establishing it as a novel and essential clock gene. Vri and Pdp1epsilon proteins accumulate with a phase delay that presumably underlies sequential repression and activation of Clk transcription. Thus, Vri, Pdp1epsilon, and Clk form a second feedback loop in the circadian oscillator responsible for regulating rhythms in Clk/Cyc levels (Cyran, 2003).
A second feedback loop in the Drosophila clock, interlocked to the first feedback loop, has been predicted to explain antiphase rhythms of Clk and per expression. Direct regulation of vri and Pdp1epsilon transcription by Clk/Cyc, and direct regulation of Clk expression by Vri and Pdp1epsilon proteins establishes the existence of this second loop and identifies its components (Cyran, 2003).
The first loop of this model starts with activation of per and tim expression by Clk/Cyc at about noon. Per/Tim then feeds back to inhibit Clk/Cyc activity during the second half of the night. In the second loop, Clk/Cyc also activates vri and Pdp1epsilon transcription at about noon. vri and Pdp1epsilon RNAs and proteins accumulate with different kinetics such that Vri protein accumulates first and represses Clk expression. Pdp1epsilon protein then accumulates and activates Clk transcription after Vri-mediated repression ends in the middle of the night. However, newly produced Clk protein is inactive due to the presence of Per repressor. Once Per is degraded, Clk/Cyc reactivates per/tim and vri/Pdp1epsilon transcription to start a new cycle. The two loops are linked together by Clk/Cyc and restart simultaneously (Cyran, 2003).
Conceptually, a molecular clock must separate the phases of clock gene transcription and repression otherwise clock components reach a stable steady state. The delay separating active transcription and repression of per/tim is controlled by the Double-time and Shaggy/GSK3 protein kinases that regulate Per/Tim accumulation and nuclear transport. The phases of Clk transcription and repression are separated by two mechanisms: (1) accumulation of Vri protein before Pdp1epsilon, which ensures that repression of Clk precedes activation; and (2) Per inhibition of Clk/Cyc activity in the early morning which prevents reactivation of vri and Pdp1epsilon transcription even when Clk levels are high (Cyran, 2003).
Does the model fit the data? The model explains the observation that Vri represses Clk independently of nuclear Per/Tim. It also suggests that in a per0 background, Clk expression is repressed because of high Vri protein levels. High levels of Vri must therefore dominate over high Pdp1epsilon levels and suppress Clk expression in per0 flies. Indeed, overexpression of vri is dominant and stops the clock in an otherwise wild-type background with constantly low Clk expression (Cyran, 2003).
However, this model does not immediately explain why Clk RNA levels are high in ClkJrk and cyc0 mutants. In the absence of Clk/Cyc function, vri RNA levels are low, and the consequently low levels of Vri protein would not be sufficient to repress Clk expression. But how can expression of Clk RNA be maximal with very low Pdp1epsilon levels in ClkJrk and cyc0 mutants? This question is especially relevant given the very low levels of Clk in Pdp1P205 homozygous mutant larvae in constant darkness, which makes the existence of additional factors that positively regulate Clk expression in constant conditions unlikely. The simplest explanation is that the very low levels of Pdp1epsilon RNA present in ClkJrk and cyc0 mutants are still sufficient to give enough Pdp1epsilon protein to activate Clk when competition from Vri is minimal due to very low Vri protein levels. Indeed, Clk RNA levels are close to their peak at ZT3 and ZT6 in wild-type flies when both Vri and Pdp1epsilon levels are very low. In contrast, Pdp1epsilon protein is totally absent in Pdp1P205 null mutants because the Pdp1 gene is deleted and thus, Clk is at low levels. However, further work is required to test this hypothesis (Cyran, 2003).
The model can also be used to explain how clock-controlled genes are expressed with different phases. Genes activated by Clk/Cyc will reach maximum RNA levels at ~ZT14 and these include per, tim, vri, and Pdp1epsilon. Genes regulated by Vri and Pdp1epsilon will peak at ~ZT2 and Clk is one example. Another candidate Vri/Pdp1epsilon regulated gene is cryptochrome (cry), whose RNA levels oscillate in phase with Clk RNA and follow the same pattern as Clk in clock mutants. Indeed, overexpression of Vri represses cry expression, and the cry promoter contains functional Vri (and therefore probably also Pdp1epsilon) binding sites (Cyran, 2003).
It is also conceivable that certain DNA sequences bind Vri with higher affinity than Pdp1epsilon or vice versa. One could then imagine two promoters, one with 5 optimal Vri and another with 5 optimal Pdp1epsilon binding sites, that would give RNA expression profiles differing by ~2-4 hr. Such a mechanism may help to explain the multiple peaks of rhythmically expressed genes found in Drosophila (Cyran, 2003).
Most clock genes are conserved between Drosophila and mammals, and they function in a broadly similar mechanism. For example, peak levels of Bmal1 and Clock RNAs are antiphase to mPer1 and mPer2 in mice just as Clk RNA peaks in antiphase to Drosophila per. A recent study identified the clock-controlled Bmal1 repressor, which parallels the Vri repression of Clk data presented in this study. The data extend the similarities of the Drosophila and mammalian clocks and suggest the existence of a rhythmically transcribed Bmal1 transcriptional activator that plays an analogous role to Drosophila Pdp1 in the second mammalian feedback loop (Cyran, 2003).
However, the Bmal1 repressor is REV-ERBα, an orphan nuclear receptor, which is unrelated to Vri. Perhaps even more surprising is that REV-ERBα is dispensable for rhythmicity in mice, although it adds robustness and precision to the circadian clock. Posttranscriptional regulation of clock proteins in the first loop presumably compensates for the loss of rhythmic Bmal1 expression in the second loop in rev-erbα-/- mice. Posttranscriptional regulation of Clk protein also plays an important part in the Clk protein cycle. However, the magnitude of the period alterations in vri and Pdp1 heterozygous flies are comparable to those seen in mice homozygous for a rev-erbα knockout. Therefore, the Drosophila clock may rely more heavily on transcriptional control than the mammalian clock, especially in the second loop (Cyran, 2003).
Homologs of Vri and Pdp1 do exist in mammals and are even expressed with a circadian rhythm in pacemaker cells. However, genetic loss-of-function experiments suggest that none of the three mammalian Pdp1 homologs, either alone or in combination, affects the period length of circadian locomotor activity by more than 30 min. Similar loss-of-function experiments have yet to be performed for E4BP4, the mammalian homolog of Vri. The mammalian homologs of vri and Pdp1 may thus play only an ancillary regulatory role in the mammalian central clock, with their primary role being the regulation of rhythmic clock outputs (Cyran, 2003).
Tightly regulated and interconnected feedback loops are conserved in the circadian clocks of all the model organisms so far studied. A second interconnected loop adds robustness to oscillators. Two transcription loops also provide the potential for multiple inputs to the clock such as light, temperature, membrane potential, and redox state. Additionally, a second transcriptional loop provides a mechanism to regulate a novel phase of rhythmic expression of clock output genes. Such downstream genes presumably allow an organism to anticipate a constantly changing, but relatively predictable, environmental cycle, and adjust its behavior and physiology accordingly. The identification of downstream genes that link the molecular ticking of a central clock to changes in whole animal behavior and physiology is clearly the next major challenge in circadian biology (Cyran, 2003).
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).
The behavioral changes seen upon lowering the gene dose of vri 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).
It is possible that the strongest behavioral and molecular clock phenotypes are derived from elimination of pacemaker cells in response to continuous expression of vri. The clock is functional in line V1 -- it just has a longer cycle (26 hr) in adults, and the pacemaker cells in lines V1 and V2 are present, since they both show cytoplasmic staining of Tim. However, there is no available molecular evidence that line V3 retains lvLNs, since per and tim expression is undetectable. To determine the fate of the lvLNs in V3, expression of PDF, whose expression in the brain lobes is restricted to the LNs, was monitored. PDF immunoreactivity was found in line V3 that marked the expected number of pacemaker cells, but PDF accumulation was strongly reduced in each cell compared to wild type. Line V2 also showed lower levels of PDF immunoreactivity, while V1 PDF levels were close to wild type. Therefore, the lvLNs are still present in line V3, but continuous vri expression downregulates PDF. Reductions in PDF levels were also examined in other clock mutants and it was found that mutations in dClk and cyc reduce PDF staining, while null mutations in tim and per do not. There is also a cluster of eight cells at the tip of the ventral ganglion that express PDF but not Per or Tim. PDF levels are constant in these cells among the different mutants tested (Blau, 1999).
Since ClkJrk and V3 larvae produce little PDF, PDF mRNA levels were also measured. PDF mRNA is not detectable in ClkJrk lvLNs, whereas wild-type accumulation of PDF mRNA is found in V3 lvLNs. This indicates a unique clock defect in Drosophila that continuously express vri. The results also show that dClk and vri independently contribute to pdf regulation, with vri affecting a posttranscriptional stage of pdf expression (Blau, 1999).
The Drosophila circadian oscillator consists of interlocked period/timeless and Clock (Clk) transcriptional/translational feedback loops. Within these feedback loops, Clk and Cycle (Cyc) activate per and tim transcription at the same time as they repress Clk transcription, thus controlling the opposite cycling phases of these transcripts. Clk-Cyc directly bind E box elements to activate transcription, but the mechanism of Clk-Cyc-dependent repression is not known. This study shows that Clk-Cyc-activated gene, vrille (vri), encodes a repressor of Clk transcription, thereby identifying vri as a key negative component of the Clk feedback loop in Drosophila's circadian oscillator. The blue light photoreceptor encoding cryptochrome (cry) gene is also a target for Vri repression, suggesting a broader role for Vri in the rhythmic repression of output genes that cycle in phase with Clk (Glossop, 2003).
Overexpression of Vri in larval oscillator cells leads to low/absent levels of per and tim mRNA. The simplest explanation of this result is that Vri binds per and tim regulatory elements and represses transcription directly. However, given that per and tim share a common mode of activation, by CLK-CYC, low levels of per and tim mRNA would also result if Vri repressed Clk. Given these alternatives, it is of paramount importance to determine the daily phase of cycling, since Vri, which acts as a repressor, is predicted to cycle in the opposite phase as the mRNAs of the gene(s) it represses (Glossop, 2003).
Polyclonal antisera were generated against full-length Vri and used for Western blot analysis. In wild-type flies, two Vri-specific bands are detected: a weak band of ~98 kDa and a strong broad band of ~82-89 kDa. Phosphatase treatment reduces the molecular weight of the weak band to ~95 kDa and collapses the strong broad band to a tight band of ~80 kDa, which matches the predicted molecular weight of Vri. As expected, Vri levels are low in cyc01 mutant flies since vri mRNA expression is CLK-CYC dependent. In addition, Vri levels are lower at ZT 3 than at ZT 15 in wild-type flies, suggesting that Vri levels cycle (Glossop, 2003).
To more precisely determine the phase of Vri cycling, Vri was measured in wild-type flies collected every 2 hr in LD. Vri levels peak during the early night (ZT 13-17) and reach trough levels during the early day (ZT 01-05;. A similar cycling phase for Vri persists in constant darkness. This cycling profile closely follows that of per, tim, and vri mRNA transcripts and is opposite that of Clk mRNA, which peaks between ZT 22 and 04 and falls to low levels between ZT 10 and 16. Vri protein therefore cycles in opposite phase to Clk mRNA; this supports a role for Vri as the Clk repressor (Glossop, 2003).
Transcription from the Clk gene is initiated at two sites: a minor site, which has been detected only through 5' RACE and RT-PCR, and a major start site that accounts for the vast majority of Clk transcription in heads. Based on the available Clk cDNAs, the minor transcript includes an additional 5' exon, plus the entire major transcript due to the removal of a ~5 kb intron between the exons initiated at the minor (first) and major (second) transcription start sites. To identify a region that mediates Clk circadian transcription, a genomic DNA fragment from -8000 to +40 (the major Clk transcription start = +1) was used to drive Gal4 mRNA transcription in transgenic flies containing a functional clock. This fragment mediates rhythmic Gal4 mRNA transcription having the same phase and amplitude as Clk transcripts in wild-type flies, thus demonstrating that all the necessary circadian regulatory elements are present (Glossop, 2003).
For Vri to be a direct repressor of Clk, the 8.0 kb Clk genomic fragment should contain binding sites for Vri. The consensus binding site for Vri has not been determined; however, the basic (DNA binding) domain shares >85% homology with that of mammalian E4BP4, and hence, Vri should bind similar DNA sequences as E4BP4. The optimal binding site for E4BP4 has been determined as 5'-(A/G)TTAC: (A/G)T(A/C)A(A/T/C)-3'. A search for E4BP4-consensus sites at the Clk locus identified multiple sites on both DNA strands upstream of the major transcription start site. This density of E4BP4 sites is much higher than predicted by chance. Searches of the per and tim loci (i.e., 4 kb upstream through intron 1) identified no 10 E4BP4 sites. This evidence is in line with the number of sites predicted by chance. The lack of E4BP4 binding sites and the phase of Vri cycling are inconsistent with Vri directly repressing per and tim, but do support the possibility that Vri directly represses Clk (Glossop, 2003).
Thus the b-ZIP transcription factor Vri feeds back to control circadian transcription of Clk within the oscillator mechanism. Clk-Cyc heterodimers activate per and tim transcription at the same time that they repress Clk transcription, thus providing a bidirectional switch that mediates the opposing cycling phases of these transcripts within the circadian oscillator. Since there are no canonical (CACGTG) E boxes within the region known to mediate Clk mRNA cycling, the simplest interpretation of these results is that Clk-Cyc also activate an intermediate that feeds back to repress Clk transcription. A model has been developed to explain this regulatory mechanism: it proposes that Vri functions to repress Clk transcription. Positive drive by Clk-Cyc and negative drive by Per and Tim confer vri mRNA and protein rhythms, and rhythms in Vri accumulation, in turn, mediate the rhythmic repression of Clk (Glossop, 2003).
Several lines of evidence support this model. (1) vri transcription is activated by Clk-Cyc via E box elements in its upstream sequence, showing that vri could act as a Clk-Cyc-dependent intermediate factor. (2) Vri overexpression leads to the repression of per and tim. Since per and tim both rely upon Clk for their activation, Vri-dependent inhibition of Clk could readily account for their coordinate repression. That Vri acts as a repressor within the oscillator mechanism is further supported by genetic analysis: Vri overexpression leads to long period activity rhythms (via reductions in per and tim expression), and reduced vri copy number leads to short period activity rhythms (presumably through increases in per and tim expression). (3) vri mRNA and protein cycle in the same phase. With this phase relationship, Vri accumulates to high levels as Clk mRNA drops to low levels. After a substantial (~6 hr) delay, Per and Tim inhibit Clk-Cyc activation of vri, consequently reducing Vri to low levels as Clk mRNA accumulates to high levels. Once Per and Tim are degraded, the next cycle of vri expression is initiated. (4) Vri binds to sequence elements within the Clk circadian control region in vitro, which suggests that Vri action is direct. (5) Vri overexpression reduces Clk mRNA levels in vivo. This Vri-dependent repression preferentially affects the peak levels of Clk mRNA in wild-type animals, which suggests that normal peak levels of Vri are maximally active, since additional Vri cannot further reduce trough levels of Clk. Vri also represses the peak levels of Clk mRNA present in cyc01 animals. Since cyc01 flies lack a functional feedback mechanism due to the absence of Cyc, Per, Tim, and Vri, this result indicates that Vri represses Clk transcription directly in vivo rather than through other components of the feedback loop. The repression of Clk by Vri indicates that rhythmic Clk transcription occurs through the circadian repression of Clk. This is different from the situation with per, tim, and vri, where circadian transcription is mediated by rhythmic Clk-Cyc-dependent activation and PER-TIM-dependent repression (Glossop, 2003).
Vri overexpression in wild-type or cyc01 flies produces more Vri than the wild-type peak, yet Clk and cry mRNAs are not fully repressed to wild-type trough levels. The inability of high Vri levels to fully repress Clk and cry suggests that Vri may act in concert with another factor to repress transcription. Constant high levels of Vri do not fully repress Clk and cry expression in wild-type flies during the late evening and early morning. This temporal difference in the ability of Vri to repress implies that another repressor is present in limiting amounts at these times of the circadian cycle, i.e., a second rhythmically expressed repressor. Likewise, the inability of high levels of Vri to repress Clk completely in cyc01 flies suggests that the complimentary repressor is only present in limiting amounts in this genotype. Given that Vri is a bZIP transcription factor, it is tempting to speculate that Vri forms a heterodimer with another bZIP transcription factor to fully repress Clk and cry transcription. However, no such factor has been identified and it is also possible that another Clk and cry repressor acts independently of Vri. Although Vri alone cannot fully repress Clk, the ability of Vri to repress Clk activation by two-thirds indicates that Vri is the major Clk repressor (Glossop, 2003).
With the identification of Vri as a repressor of Clk transcription, it is apparent that Clk and Cyc function to activate a set of repressors that act on different targets at different times in the circadian cycle. Activation of vri by Clk-Cyc leads to the immediate production of Vri, which represses Clk transcription and consequently reduces the levels of Clk (and necessarily Clk-Cyc). Activation of per and tim by Clk-Cyc leads to the delayed accumulation of Per-Tim heterodimers. This delayed PER-TIM accumulation allows Vri to repress Clk from midday to early evening (ZT4 to ZT16) and inhibits the ability of newly generated Clk to activate per and tim expression until early morning (ZT4). This difference in accumulation between Vri and Per/Tim is therefore critical for controlling the opposite cycling phases of Clk and per/tim/vri within the interlocked feedback loop mechanism (Glossop, 2003).
The mammalian circadian oscillator is also comprised of interacting transcriptional/translational feedback loops that share many components with their Drosophila counterparts. In mammals, the mPer/mCry feedback loop is analogous to the per/tim feedback loop in flies in that CLOCK and BMAL1 (a Cyc homolog) activate transcription of the mPers (i.e., mPer1, mPer2, and mPer3) and mCrys (i.e., mCry1 and mCry2), which feed back to inhibit CLOCK-BMAL1 activation. Likewise, the mammalian Bmal1 loop is analogous to the Clk loop in flies: BMAL1 and CLOCK lead to the repression of Bmal1 transcription and the mPERs and mCRYs activate Bmal1 transcription. As in flies, CLOCK-BMAL1-dependent activation occurs via E box binding, but CLOCK-BMAL1-dependent repression has not been characterized (Glossop, 2003).
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