InteractiveFly: GeneBrief

vrille: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Vrille is a bZIP transcription factor related to other leucine zipper proteins found in all vertebrate species. vrille plays a role in two complex Drosophila pathways. Initially described as a maternal effect lethal mutation, various alleles produce impaired zygotic development resulting in ventralization. vrille acts as enhancer of decapentaplegic (ddp) mutations during the development of dorsal/ventral polarity in the embryo (George, 1997). vrille was later found in a screen for genes whose expression is controlled by the biological clock of adult flies (Blau, 1999). The closest mammalian homolog to VRI is E4BP4, a transcriptional repressor (Cowel, 1992).

Mutants in dpp result in the ventralization of the embryo. In such mutants the dorsal region, where dpp is normally expressed, is ventralized and develops characteristics of the ventral neuroectoderm. Genetic screens have been performed to identify genes interacting with dpp. Genetic interactors might act (1) upstream of dpp, regulating dpp expression either directly or indirectly, or (2) downstream, regulating genes coding for protein involved in the transmission of the dpp signal, or regulating various targets of the dpp pathway. Genes discovered in such screens include Mothers against dpp (Mad) and Medea, a MAD homolog: both genes appear to be essential components of the dpp pathway. vrille is one such dpp interactor, acting as an enhancer of dpp and easter mutations. Easter is involved in triggering a protease cascade that leads to the activation of Dorsal, which functions to repress dpp in the ventral portion of the embryo during early development.

Putative truncated Vri proteins with no bZIP domain, as well as P element induced mutations and P excision secondary mutations, are able to maternally aggravate the embryonic ventralized ea and dpp phenotypes; this suggests a maternal participation of vrille in early establishment of dorsoventral polarity of the embryo. vri mutations alter wing vein differentiation, which leads to a phenotype very similar to that of shortvein alleles of dpp. Furthermore, vri enhances dpp phenotypes in wing and Mad mutation acts as a dominant enhancer of the vri wing phenotype. vri is expressed both in embryonic-specific regions of the gut, and in the larval gut and imaginal discs. Although the function of the gene remains to be elucidated, vri appears to be active during various stages of development and in different cell types (George, 1997).

bZIP transcription factors can act as heterodimers, acting together with partner bZIP proteins. Genetical analysis suggests that Vri acts in concert with another factor. It is noteworthy that all the vri alleles tested behave like antimorphs, since they show a dominant enhancement of dpp phenotypes stronger than a deficiency. In other words, an altered Vri protein shows a stronger effect than the complete absence of Vri. Therefore, a non-functional Vri protein, which is still able to bind to another factor, might be expected to have a stronger effect than the simple absence of one copy of the gene. The same observation has been made with tkv. Df(2L)tkv^Sz2, which totally deletes the nearby tkv genes and vri as well, does not enhance dpp, whereas tkv point mutations do. The occurrence of a large number of antimorphic alleles is consistent with the nature of the two proteins, a bZIP transcription factor and a serine-threonine kinase receptor, which both act as dimers (George, 1997).

What other protein could partner with Vri in regulating the Dpp pathway? Three of the known D. melanogaster bZIP transcription factors are implicated in the dpp pathway. The homolog of the mammalian fos oncogene, Drosophila Fos related antigen (Fra or Kayak), is expressed in embryos (in the head, dorsal ectoderm, amnioserosa, a subset of cells of the peripheral nervous system, a portion of the midgut, the hindgut and in the anal pads). The Fra partner, Jun related antigen (Jra or Djun), is expressed throughout development. It is noteworthy that, in mammals, these genes are induced in response to TGFbeta. A Drosophila homolog of CREB, Cyclic-AMP response element binding protein A (dCREB-A), is expressed in ovarian columnar follicular cells and in male reproductive organs, embryonically in salivary glands and the brain, in the optic lobe, and in the midgut of the adult. It is interesting to note that it has been shown that dCREB-A is required for dorsoventral patterning of the larval cuticle; it was proposed that it functions near the end of both the DPP-and SPI-signaling cascades. Some localizations of these bZIP factors coincide with the Vri expression pattern. Furthermore, putative Fos/Jun and CREB binding sites have been identified close to the Zerknullt (a Hox transcription factor of the dpp pathway) binding sites in the RACE (an amnioserosa specific gene) promoter. It has been suggested that Hox-bZIP synergy is a common feature of dpp signaling. It will therefore be interesting to test for a genetic and molecular interaction between these factors and vri (George, 1997 and references therein).

Several other genes in the dpp pathway exhibit maternal ventralizing effects. These include schnurri, which encodes a zinc finger transcription factor, and punt , a type II TGFbeta receptor. However, the level of ventralization is stronger with vri than with punt, schnurri or thick veins. Therefore, partial reduction of vri function in mothers enhances the slight ventralization induced by a weakly ventralizing easter allele. No additional increased ventralization is observed when a vri allele is also provided zygotically. Furthermore, the maternal effect of the vri gene enhances dpp ventralizing embryonic phenotypes. This is observed with all the available lethal vri alleles and two different dpp alleles. In the strongest interaction, no vri/dpp embryos survive and moderately ventralized embryos are observed, whereas in the same context with tkv, only the more moderately ventralized V4 embryos are recovered. With some vri alleles a zygotic effect is observed (George, 1997).

Thus, vri plays a role in dorsal/ventral patterning. Its exact position in this pathway is not known, particularly because there are likely to be many targets of Dpp signaling and because Vri is likely to act with a partner in regulating multiple genes both maternally and zygotically.

vrille, in its role as a clock gene, was cloned in a differential display protocol designed to discover genes expressed in the adult head. The circadian clock runs using a transcriptional negative feedback loop involving Period/Timeless and Clock/Cycle dimeric complexes. It was hypothesized that other genes with important clock roles may be regulated by this loop. Differential display was used to search for genes whose RNA levels in adult Drosophila heads respond to nuclear entry of the Per/Tim complex. A comparison was made of the profiles, over time, of wild-type flies and per⁰¹ mutants to select for clock-controlled rather than light-regulated transcripts. per⁰¹ mutants make no Per protein, and therefore no Per/Tim dimeric complex enters the nucleus (Blau, 1999).

vri is expressed in circadian pacemaker cells in the brain; VIR mRNA oscillates in phase with per and tim expression in wild-type flies, and vri is regulated by the same transcriptional loop that controls PER and TIM mRNA levels. Flies with altered levels of vri show a range of behavioral rhythm phenotypes. Reducing vri gene dosage causes period shortening. Suppression of the normal cycle of vri expression generates long-period rhythms or arrhythmicity. These latter phenotypes are associated with a block in per and tim expression, indicating that vri regulates the central clock. Accumulation of pigment dispersing factor (PDF), a neuroactive peptide hormone, is also suppressed, suggesting that vri additionally connects the clock to behavior (Blau, 1999).

Since VRI mRNA oscillates, is expressed in pacemaker cells, and encodes a transcription factor, it seemed possible that vri would regulate patterns of gene activity involved in behavioral rhythmicity. Effects of Vrille on behavior were monitored either by decreasing the gene dosage of vri, or increasing gene dosage. vri homozygotes are lethal, dying late in embryogenesis, thereby preventing testing of the phenotypes of adult flies with no functional Vri. To test whether vri mutations affect rhythmic behavior, the locomotor activity rhythms of adult Drosophila heterozygous for null mutations of vri were examined under three conditions: (1) flies heterozygous for vri¹, a null allele with a single point mutation that introduces a stop codon upstream of the bZIP domain; (2) alleles of vri⁵, a lethal P element inserted in the vri gene; and (3) Df(2L)-cl(h3), in which the vri chromosomal region is deleted. In all cases, reducing the dosage of vri by half shortens the period length of the locomotor activity rhythm by 0.4 to 0.8 hr. These effects on period length are comparable in magnitude to those associated with a heterozygous deletion or a duplication of the per locus. These results show that a particular level of vri activity is required to set the period of the Drosophila clock to 24 hr (Blau, 1999).

If a VRI mRNA or protein oscillation of specified amplitude is required for wild-type function of the Drosophila clock, behavioral rhythms might also be disturbed by eliminating cyclical expression of vri. Since vri is essential for proper development, the GAL4-UAS system was used to continuously express vri only in clock cells. The driver P element contained the timeless promoter for clock cell specificity, with five GAL4-binding sites (UAS) inserted 333 bases upstream of the putative start site of transcription. This was fused to the yeast transcriptional activator GAL4 to make the P element tim(UAS)-gal4. It was reasoned that with such a construct, endogenous factors would activate expression from the tim promoter, and subsequently GAL4 proteins should positively influence their own expression. In this way, oscillations from the tim promoter might be negated. Flies were transformed with either this P element or with a UAS-vri cDNA P element, and one tim(UAS)-gal4 line was crossed to three independently isolated UAS-vri lines (V1, V2, and V3) (Blau, 1999).

Initially tested were the effects of these transgenes on vri oscillations. In the presence of driver but no UAS-vri transgene, VRI mRNA levels show a 9-fold oscillation in flies held in constant darkness for 1 day. In contrast, in flies that also contain the UAS-vri P element, vri levels are close to the peak of the wild-type oscillation at all times of day. There is still a 2.5-fold oscillation in VRI mRNA levels, but the phase of this weak oscillation is reversed with the peak now at CT2 (CT indicates circadian time: time in constant darkness). These results demonstrate that the normal vri oscillations have been removed. The reason for the weak antiphase oscillation is not known, but it may contribute to the behavioral phenotypes observed (Blau, 1999).

Next the behavioral rhythms of the three UAS-vri lines crossed to the tim(UAS)-gal4 driver were tested and a series of phenotypes were observed. Line V1 flies are all rhythmic, but the period of the behavioral rhythm is lengthened to 25.5 hr. Most flies in lines V2 and V3 are arrhythmic (78% and 92%, respectively). A few flies have either long-period rhythms of ~28 hr or weak rhythms of 26.5 to 29 hr. However, visual inspection of the records of V3 flies considered rhythmic by computer analysis indicate that these are atypical long-period rhythms. All of these mutant phenotypes require the tim(UAS)-gal4 driver, since control flies homozygous for the UAS-vri transgenes alone uniformly show wild-type rhythms. These rhythm phenotypes are not simply due to overexpression of any bZIP protein, since flies expressing either D-Jun or D-Fos from the tim(UAS)-gal4 transgene have wild-type rhythms (Blau, 1999).

Thus perturbing vri levels causes different kinds of rhythm phenotypes. Flies missing one functional copy of vri have short-period rhythms, while continuous vri expression causes long-period rhythms or arrhythmia. The latter phenotypes are associated with changes in the molecular cycles of per and tim, and the strengths of the molecular and behavioral phenotypes are perfectly correlated. It is concluded that a normal vri oscillation is essential for a 24 hr clock at both the molecular and behavioral levels. These results indicate that vri is a clock gene as well as a clock-controlled gene (Blau, 1999).

One of the most interesting questions in circadian biology is how a molecular cycle is translated into time of day information for the behaving organism. The neuropeptide vasopressin is directly regulated in the mouse by cycling activity of the CLK/BMAL-1 complex (Jin, 1999). Since vasopressin is a well known modulator of neuroendocrine function, its regulation by the CLK/BMAL-1 complex indicates how some physiological responses can be directly programmed by a circadian clock. This report shows that pdf expression is regulated by the Drosophila clock and requires cycling vri expression. pdf encodes a neuropeptide expressed in the axons of the pacemaker cells, and these projections connect the LNs with target cells in the dorsal brain. PDF protein has been shown to accumulate in the LN axons with a circadian rhythm. The period of this rhythm is shortened by the per^S mutation, and continuous accumulation of PDF in the dorsal brain is associated with arrhythmia and a variety of period changes in adult locomotor activity. PDF mRNA levels do not cycle in wild-type flies. Since continuous expression of vri suppresses PDF protein accumulation without affecting accumulation of pdf mRNA, cycling Vri expression in wild-type Drosophila is likely to contribute to the observed cycling of Pdf protein. Vri may affect Pdf levels by specifying rhythmic expression of a factor involved in translation, maturation, stabilization, transport, or release of the neuropeptide (Blau, 1999 and references therein).

All of these observations point to a likely role for Pdf in coupling a molecular clock to timed behavior; this study has demonstrated that vri conveys essential regulatory signals from the clock to Pdf. There is also evidence that Pdf can in turn influence function of the clock. In the cockroach, microinjection of PDF produces time-dependent shifts in the phase of the locomotor activity rhythm (Petri, 1997). The magnitude of these phase shifts (up to 4 hr) is similar to that produced by light (Petri, 1997). This indicates that a transient change in Pdf level will cause a stable change in molecular components of a clock that regulates behavior in at least some insects. Possibly, the novel pathway of per and tim suppression observed in V2 and V3 Drosophila lines is a direct consequence of eliminating Pdf (Blau, 1999).

A striking conservation of some features of vri expression is seen in flies when compared to members of the related PAR domain family of mice transcription factors. Drosophila vri and murine dbp, tef, and hlf RNAs all show circadian oscillations (Wuarin, 1990; Falvey, 1995; Fonjallaz, 1996); loss of dbp affects mouse locomotor activity rhythms (Lopez-Molina, 1997). Thus, a role in circadian rhythmicity may be conserved for this family of transcription factors throughout much of the animal kingdom (Blau, 1999).

REGULATION

Promoter Structure

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 Clk^Jrk 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).

vrille, Pdp1, and Clock form a second feedback loop in the Drosophila circadian clock

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 Clk^Jrk 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 Clk^Jrk 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 Clk^Jrk and cyc⁰ mutants? This question is especially relevant given the very low levels of Clk in Pdp1^P205 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 Clk^Jrk 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 Pdp1^P205 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).

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).

Targets of Activity

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 Clk^Jrk and V3 larvae produce little PDF, PDF mRNA levels were also measured. PDF mRNA is not detectable in Clk^Jrk 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).

DEVELOPMENTAL BIOLOGY

Embryonic

The maternal effect detected by the genetic interaction of vrille with easter and dpp clearly predicts maternal and early embryonic accumulation of VRI mRNA, but makes no prediction for other developmental functions. A developmental Northern analysis was carried out. Poly A+ RNA from Oregon R embryos, larvae, pupae and adults raised at 25° was hybridized to the 2.8 kb cDNA7. RNA expression is dynamic throughout development. In embryos aged from 0 to 4 hours (stages 1 to 8: germ band elongation completed) two major transcripts of 4.9 and 6.2 kb are present at a low level. In 4 to 8 hour embryos (stages 8 to 11: beginning of germ band retraction) two major types 3.3 and 3.8 kb long were detected along with minor transcripts with higher molecular weights (up to more than 10 kb). The long transcripts do not hybridize with a 15 kb genomic probe mapping upstream cDNA1 and therefore the vri locus extends over at least 20 kb. The large number of bands observed in embryos aged between 0 to 8 hours is also observed when using a genomic probe mapping 5' to cDNA7 with no bZIP domain and no OPA repeats. These bands are, therefore, not due to cross hybridization. In older embryos aged from 8 to 24 hours (stages 11 to 17), the transcripts are more abundant and are represented exclusively by the two 3.3 and 3.8 kb major types. In third instar larvae, the 3.3 and 3.8 kb transcripts are still present, together with an abundant 1.5 kb transcript. In pupae (6 to 7 days of development), and in female adult flies, only the 3.3 and 3.8 kb transcripts are present at a high level. In males, an abundant 1.6 kb transcript is present. The 3.3 and 3.8 kb transcripts probably correspond respectively to the 3340 and 3807 bp putative cDNAs previously described. cDNAs corresponding to longer or shorter RNAs were not recovered and the origin of these RNAs is thus unknown. The expression of RNAs throughout development implicates many other functions besides those giving early embryonic phenotypes (George, 1997).

In situ hybridizations performed in embryos show a uniformly distributed maternal product at preblastoderm stage. From stage 10 (germ band fully elongated), the transcripts begin to localize and can be seen at higher levels in the primordium of the foregut. At stage 13, transcripts are present at high levels in the hypopharyngal lobe at the ventral opening of the stomodeum, the foregut, the proventriculus primordium, the hindgut, anal pads and posterior spiracles. At stage 14, during head involution and dorsal closure, the transcripts are located in stripes along the epidermis in the anterior part of each segment. The stomodeum, hindgut and anal pads are still strongly labeled and a longitudinal stripe is observed dorsally along the epidermis. At stage 15 (end of dorsal closure) thin stripes are seen dorsally across the closing epidermis and the amnioserosa is weakly labeled. The ventral most regions of the epidermis and the central nervous system are not labeled, although 50% of the ventral epidermis shows vri expression. At stage 16, the transcripts are still present in the stomodeum, anal pads and in a network of lateral and dorsal cells probably corresponding to the tracheal track (George, 1997).

vri transcripts are not expressed in ovarian stem cells, oogonia or early cysts and are first detectable at stage 8 in the nucleus and cytoplasm of nurse cells, which is consistent with maternally provided RNAs, and in the columnar follicular epithelial cells. The transcripts are also expressed in gut, brain and imaginal discs of third instar larvae. Similar localizations or sub-patterns were observed with the betagal staining of all the PlacZ mutant alleles, the differences between the five alleles being only quantitative. In embryos, the foregut, posterior spiracles and anal pads express lacZ: in ovaries, the border cells and columnar epithelial cells are stained, but not the nurse cells. This could reflect the fact that the P elements are localized in transcripts provided only zygotically or, on the contrary, that these P elements completely abolish the production of the maternal RNAs. The staining in border cells is not observed with in situ hybridizations and it is not known whether this is an ectopic localization unrelated to the real expression of the transcripts. Staining is observed in larval gut in a pattern very similar to that observed by in situ hybridization and in the central region of imaginal discs. These results are consistent with the hypothesis whereby the P element mutations alter the bZIP function (George, 1997).

The coregulation of PER, TIM, and VRI mRNA levels makes it likely that all three genes are expressed in the same cells. In situ hybridizations to adult head sections reveal that vri and tim are expressed in identical regions of the head. vri and tim are both expressed in the photoreceptor cells, which contain functional clocks, and in two clusters of cells in the central brain corresponding in position to the ventral and dorsal lateral neurons (LNs), which are the pacemaker cells responsible for circadian locomotor behavior. Double labeling experiments were performed with third instar larval brains to confirm that vri is expressed in LNs. An antibody was used against crustacean pigment dispersing hormone, which cross-reacts with the highly related Drosophila pigment dispersing factor) (PDF) and labels only the LNs in each brain lobe. Coexpression of LacZ and Tim with PDF was found, although in the case of vri, the pattern is not limited to LNs, consistent with Vrille's role in development (George, 1997). vri expression in larval LNs suggests that vri may play a role in the clock during much of development (Blau, 1999).

Adult

Vrille RNA levels oscillate, cycling with a phase and amplitude (10- to 12-fold changes) comparable to tim. Like TIM, VRI mRNA accumulates constitutively and at an intermediate level in per⁰¹ mutants. Northern blots of head RNA have resolved two species of vri RNA of approximately 3.4 and 3.8 kb that differ only in their 3' untranslated regions, and which both oscillated in a clock-dependent manner. VRI mRNA also oscillates robustly in wild-type flies maintained in constant darkness following entrainment to light-dark cycles, further confirming vri regulation by the clock. Since oscillations of per promoter activity and Per and Tim gene products can be detected throughout the body of Drosophila, oscillations in RNA isolated from bodies of male flies were sought. Clock-dependent cycling is observed for VRI and TIM, with 4- to 5-fold oscillations detected for both RNAs in LD cycles (Blau, 1999).

Mechanisms composing Drosophila's clock are conserved within the animal kingdom. To learn how such clocks influence behavioral and physiological rhythms, the complement of circadian transcripts in adult Drosophila heads was determined. High-density oligonucleotide arrays were used to collect data in the form of three 12-point time course experiments spanning a total of 6 days. Analyses of 24 hr Fourier components of the expression patterns revealed significant oscillations for ~400 transcripts. Based on secondary filters and experimental verifications, a subset of 158 genes showed particularly robust cycling and many oscillatory phases. Circadian expression is associated with genes involved in diverse biological processes, including learning and memory/synapse function, vision, olfaction, locomotion, detoxification, and areas of metabolism. Data collected from three different clock mutants (per₀, tim₀₁, and Clk_Jrk), are consistent with both known and novel regulatory mechanisms controlling circadian transcription (Claridge-Chang, 2001).

A genome-wide expression analysis was performed aimed at identifying all transcripts from the fruit fly head that exhibit circadian oscillations in their expression. By taking time points every 4 hr, a data set was obtained that has a high enough sampling rate to reliably extract 24 hr Fourier components. Time course experiments spanning a day of entrainment followed by a day of free-running were performed to take advantage of both the self-sustaining property of circadian patterns and the improved amplitude and synchrony of circadian patterns found during entrainment. 36 RNA isolates from wild-type adult fruit fly heads, representing three 2 day time courses, were analyzed on high-density oligonucleotide arrays. Each array contained 14,010 probe sets (each composed of 14 pairs of oligonucleotide features) including ~13,600 genes annotated from complete sequence determination of the Drosophila genome. To identify different regulatory patterns underlying circadian transcript oscillations, four-point time course data was colleced from three strains of mutant flies with defects in clock genes (per⁰, tim⁰¹, and Clk^Jrk) during a single day of entrainment. Because all previously known clock-controlled genes cease to oscillate in these mutants but exhibit changes in their average absolute expression levels, the analysis of the mutant data was focused on changes in absolute expression levels rather than on evaluations of periodicity (Claridge-Chang, 2001).

To organize the 158 statistically significant circadian transcripts in a way that was informed by the data, hierarchical clustering was performed. Both the log ratio wild-type data (normalized per experiment) and the log ratios for each of the three clock mutants (normalized to the entire data set) were included to achieve clusters that have both a more or less uniform phase and a uniform pattern of responses to defects in the circadian clock. One of the most interesting clusters generated by this organization is the per cluster. This cluster contains genes that have an expression peak around ZT16 and a tendency to be reduced in expression in the Clk^Jrk mutant. Strikingly, all genes previously known to show this pattern of oscillation (per, tim, vri) are found in this cluster. In fact, the tim gene, which has multiple representations on the oligonucleotide arrays, has two independent representations in this cluster. Together with the novel oscillator CG5798, per, tim, and vri form a subcluster (average phase ZT14) that shows upregulation in both the per⁰ and tim⁰¹ mutants. The fact that per, tim, and vri all function in the central circadian clock raises the possibility that several other genes from this cluster, including the ubiquitin thiolesterase gene CG5798 and the gene coding for the channel modulator Slowpoke binding protein (Slob) may function in the circadian clock or directly downstream of it (Claridge-Chang, 2001).

EFFECTS OF MUTATION

The two initial vri mutations, l(2)jf23^Sz7 and l(2)jf23^Sz36, were recovered in a screen as lethal EMS induced mutations on the 2nd chromosome (Szidonya, 1988). They have been renamed vri¹ and vri², respectively. Five other vri alleles were recovered by testing the P induced lethal stocks. Df(2L)tkv^Sz2 totally deletes vri and represents a null allele for vri and the nearby tkv, l(2)03771 and probably l(2)jf24 genes. Df(2L)tkv^Sz2 homozygous embryos show the typical tkv null phenotype with no dorsal closure and head involution resulting in the absence of dorsal epidermis. In developing embryos, no defects are observed before dorsal closure. Principally, at the blastoderm stage the expression of the Zen protein is normal and no obvious gastrulation defects are observed. vri lethal mutants die as embryos and, except with vri⁸, no dominant maternal lethality is detectable when vri females are crossed to wild type males. vri¹, vri² as well as vri¹/vri² homozygous embryos have very similar phenotypes. The embryos are shortened and the dorsal epidermis often appears wrinkled and reduced, and tracheae are interrupted. This dorsal shortening leads to a slight 'tail up' phenotype. Filzkorper are often internal. Less frequently the head skeleton is abnormal and ventral denticles are fused or missing. The vri^P alleles result in similar phenotypes but these latter are stronger than those described above. The head skeleton is almost always defective. In some embryos the germ band remains extended, suggesting defects in germ band retraction. Some embryos are convoluted, with ventral denticles extended laterally and Filzkorper internal and presenting an abnormal morphology. This phenotype is similar to those observed in weakly ventralized embryos. These latter phenotypes have a low penetrance, however, and for this reason it could not be determined (using the expression of zen) whether they result from defects occurring in the early stages of establishment of dorsoventral polarity or later on. The vri^P alleles are probably not null alleles since the mutants show apparent defects in dorsoventral polarity, not observed with the tkv^Sz2 null allele.

The zygotic phenotype of the total absence of the vri gene is difficult to detect, due to the presence of at least three other embryonic lethal genes within Df(2L)tkv^Sz2, the smallest available deficiency. Df(2L)tkv^Sz2 deleting mainly the thick veins gene itself presents strong head and dorsal cuticular defects. l(2)03771, represented by a single P induced mutation, shows weakly ventralizing embryonic phenotypes and is clearly not a null allele. In order to obtain a better view of the null vri phenotype, the Df(2L)tkv^Sz2 homozygous phenotype was observed in the presence of a tkv cDNA transgene, P[Ubi-tkv-2], which is capable of the total rescue of tkv null mutants. Such tkv rescued deficiency embryos die and possess a phenotype very similar to the homozygous phenotypes of vri¹ or vri², which can therefore be considered null alleles. However, vri/Df(2L)tkv^Sz2 hemizygous progeny hatch and die as larvae. Thus, the phenotype appears weaker when hemizygous than when homozygous. Although this delayed lethality could be due to a background effect, it would suggest that the alleles are not null or even hypomorphic, but rather neomorphic or antimorphic. Alternatively, it could be that one gene within the deficiency acts as a dominant suppressor of the embryonic lethality. The same results are observed with the P induced alleles. One hypothesis to explain why the hemizygous progeny die as larvae whereas homozygotes do not hatch is that the product of these alleles, including vri¹ and vri², is able to antagonize itself or the wild type maternal product and therefore has a stronger effect than the total absence of Vri (George, 1997).

Mad acts as a dominant enhancer of vri phenotypes in wing. About 10% of Mad⁶+/+ vri² flies show a wing phenotype. The L5 vein is shortened and sometimes the posterior cross vein is also shortened and extra vein material is observed along the L2 vein. The same phenotype is observed with vri¹ whereas with the other alleles the effect is weaker. Since this phenotype in not observed in the Mad/+ and vri/+ controls, it is concluded that it is due to the association of both genes. In order to investigate a possible interaction between vri and dpp in wing, a dominant effect of vri was sought in a dpp- context. The dpp^hr4/dpp^d6 phenotype consists of a reduction of wing to about one half of the wild type size and no defects in eyes or legs. When one dose of vri is associated with this genotype in dpp^hr4 vri²/dppd⁶ + flies, a further reduction in wing size is observed with reduction of veins. Furthermore, eyes are smaller with a rough aspect and legs are truncated. The enhancement of dpp phenotypes by vri² is always observed, although the strength of the enhancement is variable. The same phenotypes are observed with vri¹ (George, 1997).

Vri is closely related to bZIP transcription factors involved in growth or cell death. vri clonal and overexpression analyses reveal defects at the cellular level. vri clones in the adult cuticle contain smaller cells with atrophic bristles. The phenotypes are strictly cell autonomous. Clones induced in the eye precursor cells lead to individuals with smaller eyes and reduced number of ommatidia with an abnormal morphology and shorter photoreceptor cell stalks. Overexpression of vri is anti-proliferative in embryonic dorsal epidermis and in imaginal discs, and induces apoptosis. On the wing surface, larger cells with multiple trichomes are observed, suggesting cytoskeletal defects. In salivary glands, vri overexpression leads to smaller cells and organs. vri has been shown to be involved in locomotion and flight and interacts genetically with genes encoding actin-binding proteins. The phenotypes observed are consistent with the hypothesis that vri is required for normal cell growth and proliferation via the regulation of the actin cytoskeleton (Szuplewski, 2003).

The functional analysis of vri performed by mutant clone induction observed in adults indicates that vri is cell autonomous and involved in hair and cell growth. The fact that vri acts in a strict cell-autonomous manner suggests that it does not regulate the expression of a diffusive molecule such as a growth factor or a hormone. Smaller cells are recovered on the whole cuticle with shorter thinner or atrophic bristles. In the wing, clones have an abnormal shape and degenerative tissues are observed. These defects could be due to cytoskeleton defects. Clones induced in the eye precursor cells result in smaller eyes with a significantly reduced number of ommatidia with an atrophic morphology and a reduced size with the stronger allele. The photoreceptor cell stalks are shorter and atrophic. These results suggest that vri cells grow more slowly and are less viable than vri⁺ cells, even when they are not surrounded by wild-type cells (Szuplewski, 2003).

vri overexpression phenotypes suggest a role in cell cycle and proliferation. However, these phenotypes are not rescued by simultaneous overexpression of the genes encoding activators of proliferation, Drosophila E2F, cyclin E or string. Therefore, it is unlikely that Vri is either a direct repressor of genes that activate proliferation or an activator of those acting as inhibitors of proliferation like rbf or dacapo. It could act upstream in the Ras/MAPK or PI3K pathways regulating growth and involved in the regulation of the mammalian homolog of Vri (NFIL3A) acting mostly as a repressor. Genetic interactions have been tested in double-heterozygotes with available members of these pathways and vri, but neither reduction in viability nor any strong phenotypes were recovered. This could result from genes with non-limiting products and/or be due to the functional redundancy of vri. Alternatively, vri may control cell size independently of growth signals (Szuplewski, 2003).

vri loss-of-function and overexpression phenotypes, more probably, could result from primary defects in cytoskeletal actin network. Although cytoskeletal integrity and adhesion are altered in mutants of regulators of cell growth and proliferation, these effects are indirect. New vri phenotypes affect wing shape flight and locomotion. Locomotory defects could result from neurological or muscular alteration. Interaction was found with the alpha actn and bent genes involved in muscle actin function, which suggests that the effect is rather at the muscular level, although no gross defect was observed in indirect flight muscle. However these defects appear degenerative and must be studied in more detail. Hair atrophic phenotypes are observed in interaction with these two genes, suggesting an effect in other cell types. Although the locomotory and hair defects are not necessarily related, it is notable that the genes interacting with vri affect different types of actin, muscle and non-muscle actin. It will be interesting to search for the direct targets of Vri to understand its implication in locomotion and cytoskeletal integrity (Szuplewski, 2003).

EVOLUTIONARY HOMOLOGS

E4BP4: a vertebrate protein related to Vrille

A novel member of the bZIP family of DNA-binding proteins has been described and designated E4BP4. It displays an unusual DNA-binding specificity that overlaps that of the activating transcription factor family of factors (ATF/CREB). When expressed in a transient transfection assay with a suitable reporter plasmid, E4BP4 strongly represses transcription in a DNA-binding-site-dependent manner. Examination of a series of deletion mutants reveals that sequences responsible for the repressing potential of E4BP4 lie within the carboxyl-terminal region of the protein. No similarity has been found between this region and the repressing domains of other known eukaryotic transcriptional repressors (Cowell, 1992).

The bZIP factor E4BP4 overlaps in DNA binding site specificity with the transcriptional activator CREB and members of the ATF family of transcription factors, but is an active transcriptional repressor. The repressing activity of E4BP4 maps to a small 'domain' of 65 amino acids that retains its ability to repress transcription when transferred to the heterologous DNA binding domain of the yeast transcriptional activator GAL4. This segment of the E4BP4 polypeptide contains a high proportion of charged amino acids and does not resemble the repression domains that have been characterized so far from other active transcriptional repressors such as the Drosophila Kruppel, Engrailed and Even-skipped proteins. A mutation that changes the charge configuration of this repression module results in a complete loss of repressor activity. The E4BP4-GAL4 fusion protein is able to repress the residual transcription from minimal promoters containing the adenovirus E4 or E1b TATA box. This is consistent with a mechanism of action whereby E4BP4 interacts with some component of the general transcription machinery to cause repression of basal and activated transcription. Although a number of nuclear proteins are able to interact with the E4BP4 repression domain in vitro, these proteins do not appear to include the general transcription factors TFIIB or TBP (Cowell, 1994).

A repression domain from the active transcriptional repressor E4BP4 maps to a 65 amino acid segment near the C-terminus of the polypeptide. The E4BP4 repression domain interacts specifically with the TBP binding repressor protein Dr1. Mutants that affect the ability of E4BP4 to bring about transcriptional repression are also deficient in their binding of Dr1. The results are discussed in the light of evidence for squelching of a 'global' repressor by a DNA binding defective E4BP4 mutant (Cowell, 1996).

The transcription factor E4BP4 has been isolated by lambda gt11 expression cloning using a probe containing the CRE/ATF-like sequence located between -2764 bp and -2753 bp in the upstream regulatory region for the human IL-1 beta gene. DNaseI protection, gel mobility shift analysis, and cotransfection studies were performed to investigate the binding and functional properties of E4BP4 using IL-1 beta promoter sequences. By DNaseI footprinting, a protection pattern was generated over the CRE/ATF-like site and the flanking sequences by bacterially produced E4BP4. Competition experiment by gel shift assay indicates that E4BP4 binds specifically to a CRE/ATF-like site, not an NF kappa B-like site. In cotransfection studies, E4BP4 represses promoter activity and this repression is mediated through the CRE/ATF-like site. Mutational analysis of E4BP4 suggests that the DNA binding as well as repression activities require the leucine heptad repeat domain. Analysis of E4BP4 produced in Escherichia coli and Sf9 cells infected with recombinant baculovirus indicates that baculovirus produced protein shows enhanced binding to the CRE/ATF-like site compared to the E. coli-produced protein. Analysis of posttranslational modifications indicates that E4BP4 produced in Sf9 cells is phosphorylated and this phosphorylation is important for the DNA binding activity of E4BP4 (Chen, 1995).

The E2A-HLF (hepatic leukemia factor) oncoprotein, generated in pro-B lymphocytes by fusion of the trans-activation domain of E2A to the basic region/leucine zipper (bZIP) domain of HLF, functions as an anti-apoptotic transcription factor in leukemic cell transformation. When introduced into interleukin 3 (IL-3)-dependent mouse pro-B lymphocytes, E2A-HLF prevents apoptosis induced by growth factor deprivation, suggesting that IL-3 mediates cell survival through activation of a transcription factor whose activity can be constitutively replaced by the chimeric oncoprotein. Four bZIP transcription factors have been considered as candidates for this putative IL-3-regulated factor, each of which binds avidly to the DNA consensus sequence recognized by E2A-HLF and is related to the Caenorhabditis elegans CES-2 (cell death specification protein) neuron-specific mediator of cell death. The expression and binding activity of the Nfil3 protein (also called E4bp4), but not of Hlf, Dbp, or Tef, was found to be regulated by IL-3 in mouse pro-B cell lines (Baf-3 and FL5.12). Northern blot analysis shows that Nfil3/E4bp4 is regulated as a 'delayed-early' IL-3-responsive gene, requiring de novo protein synthesis. In the absence of IL-3, enforced expression of the human NFIL3/E4BP4 cDNA promotes the survival but not the growth of IL-3-dependent pro-B cells. These results implicate NFIL3/E4BP4 (nuclear factor regulated by IL-3/adenovirus E4 promoter binding protein) in a distinct growth factor-regulated signaling pathway that is responsible for the survival of early B-cell progenitors, and whose alteration by E2A-HLF leads to childhood B lineage leukemia (Ikushima, 1997).

Hematopoietic cells require cytokine-initiated signals for survival as well as proliferation. The pathways that transduce these signals, ensuring timely regulation of cell fate genes, remain largely undefined. The NFIL3 (E4BP4) transcription factor, Bcl-xL, and constitutively active mutants of components in Ras signal transduction pathways have been identified as key regulation proteins affecting murine interleukin-3 (IL-3)-dependent cell survival. Expression of NFIL3 is regulated by oncogenic Ras mutants through both the Raf-mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. NFIL3 inhibits apoptosis without affecting Bcl-xL expression. By contrast, Bcl-xL levels are regulated through the membrane proximal portion in the cytoplasmic domain of the receptor (betac chain), that is shared by IL-3 and granulocyte-macrophage colony-stimulating factor. Activation of either pathway alone is insufficient to ensure cell survival, indicating that multiple independent signal transduction pathways mediate the survival of developing B-lymphoid cells (Kuribara, 1999).

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. Chicken pineal genes were sought that are induced by light in a time-of-day-dependent manner. Isolated was the chick homolog of bZIP transcription factor E4bp4 (cE4bp4), which shows high similarity to vrille, one of the Drosophila clock genes. cE4bp4 is expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase is nearly opposite that of the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays show that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5'-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies show that the prolongation of the light period to early subjective night maintains the high level expression of the pineal cE4bp4, and presumably as a consequence delays the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 are followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene (Doi, 2001).

PAR domain family: Cloning and cyclic expression

The treatment of cultured rat-1 fibroblasts or H35 hepatoma cells with high concentrations of serum induces the circadian expression of various genes whose transcription also oscillates in living animals. Oscillating genes include rper1 and rper2 (rat homologs of the Drosophila clock gene period), and the genes encoding the transcription factors Rev-Erb alpha, DBP, and TEF. In rat-1 fibroblasts, up to three consecutive daily oscillations with an average period length of 22.5 hr have been recorded. The temporal sequence of the various mRNA accumulation cycles is the same in cultured cells and in vivo. The serum shock of rat-1 fibroblasts also results in a transient stimulation of c-fos and rper expression and thus mimics light-induced immediate-early gene expression in the suprachiasmatic nucleus (Balsalobre, 1998).

The expression of dbp, a putative clock-controlled transcription factor, was investigated in the mouse brain by in situ hybridization using antisense cRNA probe. Positive dbp mRNA signals were detected in various parts of the brain, with the highest expression in the suprachiasmatic nucleus (SCN). The circadian expression profile was investigated in SCN and extra-SCN areas. In the SCN, dbp mRNA signals show a peak at early daytime (ZT/CT4) and a trough at early nighttime (ZT/CT16) in both light-dark and constant dark conditions. In the cerebral cortex and the caudate-putamen, dbp mRNA is also expressed in a circadian manner, but the phase of dbp mRNA expression in these structures shows a 4-8 hr delay compared to that in the SCN. These findings indicate that the circadian expression profile of dbp in the extra-SCN brain areas is different from that in the SCN (Yan, 2000).

PAR domain family: Transcriptional targets

A chicken liver cDNA expression library was screened with a probe spanning the distal region of the chicken vitellogenin II (VTGII) gene promoter and clones for a transcription factor have been isolated and named VBP (for vitellogenin gene-binding protein). VBP binds to one of the most important positive elements in the VTGII promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP was deduced from a nearly full length cDNA copy and is found to contain a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. Moreover, VBP is a stable dimer free in solution. A data base search reveals that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control (Iyer, 1991).

Hepatic cytochrome P450 cholesterol 7 alpha-hydroxylase, CYP7, is regulated in vivo at the protein and the mRNA level in response to multiple physiological factors, including liver cholesterol synthesis, bile acid feedback inhibition, and diurnal rhythm. An investigation was carried out to see whether the liver transcription factor DBP (albumin promoter D-site binding protein), which undergoes a striking diurnal rhythm in rat liver (DBP levels during evening/morning approximately 100:1), contributes to the diurnal regulation of CYP7 gene expression. DNase I footprinting analysis using bacterially expressed DBP and a cloned 5'-flanking DNA segment of the rat CYP7 gene revealed five distinct DBP-binding sites, designated A-E, distributed between nucleotides (nts) -41 and -295 relative to the CYP7 transcription start site. CYP7-directed gene transcription in HepG2 cells transfected with a 5'-CYP7 promoter-chloramphenicol acetyl-transferase reporter is activated up to 12-fold upon cotransfection of a DBP expression vector. 5'-Deletion analyses and site-specific mutagenesis reveals that this stimulating effect of DBP can in part be ascribed to its functional interaction with three different DBP binding sites. C/EBP beta (LAP), another liver-enriched basic-leucine zipper transcription factor, binds to these same sites but effects a more modest increase in CYP7-directed gene transcription (up to 3-4-fold) when expressed in HepG2 cells. Competition for CYP7 promoter-binding sites between C/EBP, which undergoes an approximately 2-fold diurnal change in rat liver, and the diurnally regulated DBP, is proposed to determine the relative rates of basal versus diurnally regulated CYP7 gene transcription and thus may be a primary mechanism for setting the 3-6-fold amplitude that characterizes the circadian rhythm of liver CYP7 expression. Moreover, since DBP is first expressed in rat liver 3-4 weeks after birth, these findings may account for both the enhanced expression and the onset of the diurnal pattern of CYP7 enzyme levels at this stage of development (Lee, 1994).

The two highly related PAR basic region leucine zipper proteins TEF and DBP accumulate according to a robust circadian rhythm in liver and kidney. In liver nuclei, the amplitude of daily oscillation has been estimated to be 50-fold and 160-fold for TEF and DBP, respectively. While DBP mRNA expression is the principal determinant of circadian DBP accumulation, the amplitude of TEF mRNA cycling is insufficient to explain circadian TEF fluctuation. Conceivably, daily variations in TEF degradation or nuclear translocation efficiency may explain the discrepancy between mRNA and protein accumulation. In vitro, TEF and DBP bind the same DNA sequences. Yet, in co-transfection experiments, these two proteins exhibit different activation potentials for the two reporter genes examined. While TEF stimulates transcription from the albumin promoter more potently than DBP, only DBP is capable of activating transcription efficiently from the cholesterol 7 alpha hydroxylase (C7alphaH) promoter. However, a TEF-DBP fusion protein, carrying N-terminal TEF sequences and the DNA binding/dimerization domain of DBP, enhances expression of the C7alphaH-CAT reporter gene as strongly as wild-type DBP. These results suggest that the promoter environment, rather than the affinity with which PAR proteins recognize their cognate DNA sequences in vitro, determines the promoter preferences of TEF and DBP (Fonjallaz, 1996).

To study the molecular mechanisms of circadian gene expression, attempts were made to identify genes whose expression in mouse liver is regulated by the transcription factor DBP (albumin D-site-binding protein). This PAR basic leucine zipper protein accumulates according to a robust circadian rhythm in nuclei of hepatocytes and other cell types. The Cyp2a4 gene, encoding the cytochrome P450 steroid 15alpha-hydroxylase, is a novel circadian expression gene. This enzyme catalyzes one of the hydroxylation reactions leading to further metabolism of the sex hormones testosterone and estradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displays circadian kinetics indistinguishable from those of the highly related CYP2A5 gene. Proteins encoded by both the Cyp2a4 and Cyp2a5 genes also display daily variation in accumulation, though this is more dramatic for CYP2A4 than for CYP2A5. Biochemical evidence, including in vitro DNase I footprinting on the Cyp2a4 and Cyp2a5 promoters and cotransfection experiments with the human hepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 genes are indeed regulated by DBP. These conclusions are corroborated by genetic studies, in which the circadian amplitude of CYP2A4 and CYP2A5 mRNAs and protein expression in the liver are significantly impaired in a mutant mouse strain homozygous for a dbp null allele. These experiments strongly suggest that DBP is a major factor controlling circadian expression of the Cyp2a4 and Cyp2a5 genes in the mouse liver (Lavery, 1999).

PAR domain family proteins: Regulation of behaviorial circadian rhythms

DBP, a PAR leucine zipper transcription factor, accumulates according to a robust circadian rhythm in liver and several other tissues of mouse and rat. DBP mRNA levels also oscillate strongly in the suprachiasmatic nucleus (SCN) of the hypothalamus, believed to harbor the central mammalian pacemaker. However, peak and minimum levels of DBP mRNA are reached about 4 h earlier in the SCN than in liver, suggesting that circadian DBP expression is controlled by different mechanisms in SCN and in peripheral tissues. Mice homozygous for a DBP-null allele display less locomotor activity and free-run with a shorter period than otherwise isogenic wild-type animals. The altered locomotor activity in DBP mutant mice and the highly rhythmic expression of the DBP gene in SCN neurons suggest that DBP is involved in controlling circadian behavior. However, since DBP-/- mice are still rhythmic and since DBP protein is not required for the circadian expression of its own gene, dbp is more likely to be a component of the circadian output pathway than a master gene of the clock (Lopez-Molina, 1997).

Albumin D-binding protein (DBP) is a PAR leucine zipper transcription factor that is expressed according to a robust circadian rhythm in the suprachiasmatic nuclei, harboring the circadian master clock, and in most peripheral tissues. Mice lacking DBP display a shorter circadian period in locomotor activity and are less active. Thus, although DBP is not essential for circadian rhythm generation, it does modulate important clock outputs. The role of DBP in the circadian and homeostatic aspects of sleep regulation have been studied by comparing DBP deficient mice (dbp-/-) with their isogenic controls (dbp+/+) under light-dark (LD) and constant-dark (DD) baseline conditions, as well as after sleep loss. Whereas total sleep duration was similar in both genotypes, the amplitude of the circadian modulation of sleep time, as well as the consolidation of sleep episodes, was reduced in dbp-/- under both LD and DD conditions. Quantitative EEG analysis demonstrates a marked reduction in the amplitude of the sleep-wake-dependent changes in slow-wave sleep delta power and an increase in hippocampal theta peak frequency in dbp-/- mice. The sleep deprivation-induced compensatory rebound of EEG delta power is similar in both genotypes. In contrast, the rebound in paradoxical sleep is significant in dbp+/+ mice only. It is concluded that the transcriptional regulatory protein DBP modulates circadian and homeostatic aspects of sleep regulation (Franken, 2000).

E4BP4, a basic leucine zipper transcription factor structurally related to Vrille, contains a DNA-binding domain closely related to DBP, HLF, and TEF, which are PAR proteins. The phase of e4bp4 mRNA rhythm is opposite that of the dbp, hlf, and tef rhythms in the suprachiasmatic nucleus (SCN), the mammalian circadian center, and the liver. The protein levels of E4BP4 and DBP also fluctuate in almost the opposite phase. Moreover, all PAR proteins activate, whereas E4BP4 suppresses, the transcriptional activity of the reporter gene containing a common binding sequence in transcriptional assays in vitro. An electrophoretic mobility shift assay has demonstrated that E4BP4 is not able to dimerize with the PAR proteins, but is able to compete for the same binding sites with them. Furthermore, sustained low e4bp4 and high dbp mRNA levels are found in mCry-deficient mice. These results indicate that the E4BP4 and PAR proteins are paired components of a reciprocating mechanism wherein E4BP4 suppresses the transcription of target genes during the time of day when E4BP4 is abundant, and the PAR proteins activate them at another time of day. E4BP4 and the PAR proteins may switch back and forth between the on-off conditions of the target genes (Mitsui, 2001).

On the basis of these results, two working models are proposed that can explain the relationship between the e4bp4 gene and the putative core feedback loop including CLOCK/BMAL1, mPERs, and mCRYs. It is hypothesized that the e4bp4 gene is regulated by an unidentified transcriptional repressor (X) that is regulated by CLOCK/BMAL1 and the negative elements, mPERs and mCRYs, as in the case of the dbp, hlf, and tef genes. When X mRNA is translated rapidly and the produced X protein accumulates in the nuclei of the SCN cells with little delay and depresses expression of e4bp4, the phase of the e4bp4 rhythm thus is expected to be opposite that of the dbp, hlf, and tef rhythms. Rapidly translated E4BP4 suppresses the transcriptions of target genes during the time of day when E4BP4 is abundant, and the PAR proteins activate them at another time of day. Thereby, the E4BP4 and PAR proteins increase the amplitude of the rhythmically expressed transcript levels of the target genes. In a second model, the e4bp4 gene is regulated by an unidentified repressor (Y) that is indirectly regulated by CLOCK/BMAL1, mPERs, and mCRYs via DBP-, HLF-, or TEF-mediated regulation. In addition to these two models, it also is conceivable that the rhythmic expression of the e4bp4 gene is controlled by the cycling presence of an unidentified positive element that drives the rhythmic expression of the bmal1 gene, depending on mPER2. It is noteworthy that in all three models, the existence of an unidentified activator or repressor is indicated (Mitsui, 2001).

The genes albumin, cholesterol 7alpha hydroxylase, and cytochrome P450 (Cyp2c6, Cyp2a4, and Cyp2a5) are thought to be candidates for the target genes of the PAR proteins in the liver. Conversely, the interleukin 3 gene is thought to be a target of E4BP4 in T lymphocytes. The consensus-binding site for E4BP4 and the PAR proteins is different from the CLOCK/BMAL1 E-box binding site. Therefore, E4BP4 and the PAR proteins potentially could regulate a set of output genes that do not also possess an E-box. Moreover E4BP4 and the PAR proteins regulate the transcriptional activity of the mPer1 promoter in a transcriptional assay in vitro. Thus, the cyclic activities of E4BP4 and the PAR proteins may feed back onto the central clock mechanism (Mitsui, 2001).

E4BP4 has been shown to behave as an active transcriptional repressor that directly suppresses the transcriptional activities of genes whose promoters it binds. This active repression is mainly because of a small transferable repression domain of 65 amino acids in the C-terminal half of the protein. However, in the case of DBP the PAR (proline and acidic amino acid-rich) domain, which resides amino-terminal to the basic region, has been shown to act as an activation domain. Some other examples of the combination of activator and active repressor that bind the same site are found in genes controlling segmentation during early Drosophila development (e.g., Fushi tarazu and Engrailed). It has been proposed that competition between an activator and repressor is not only required, but also is sufficient to establish all-or-none switches in gene expression. E4BP4 and the PAR proteins may switch back and forth between the on-off conditions (Mitsui, 2001).

The Drosophila transcription factor Vrille contains a DNA-binding domain closely related to mammalian E4BP4, but lacks a PAR domain. It therefore is comparable to E4BP4. Recently, Vri was shown to be required for a functional Drosophila clock: reducing vri gene dosage caused period shortening and elimination of the normal vri cycle generated long-period rhythms or arrhythmicity. Therefore, this family of transcription factors may have an important role in circadian clocks in both Drosophila and mammals. However the phase of the e4bp4 oscillation is opposite that of the mPer1, mPer2, and mPer3 rhythms, which are regulated directly by CLOCK/BMAL1. In contrast, the cycling of vri mRNA in Drosophila is regulated directly by dCLOCK/dBMAL1, and vri mRNA thereby oscillates with the same phase as per mRNA in adult heads. Thus, there may be a difference in the way E4BP4 and Vri are utilized in the mammalian/Drosophila clocks (Mitsui, 2001 and references therein).

Negative control of circadian clock regulator E4BP4 by Casein kinase I-mediated phosphorylation

Light-dependent transcriptional regulation of clock genes is a crucial step in the entrainment of the circadian clock. E4bp4, a vertebrate ortholog of Drosophila Vrille, is a light-inducible gene in the chick pineal gland, and it encodes a bZIP protein that represses transcription of cPer2, a chick pineal clock gene. Prolonged light period-dependent accumulation of E4BP4 protein is temporally coordinated with a delay of the rising phase of cPer2 in the morning. E4BP4 is phosphorylated progressively and then disappears in parallel with induced cPer2 expression. Characterization of E4BP4 revealed Ser182, a phosphoacceptor site located at the amino-terminal border of the Ser/Thr cluster, which forms the phosphorylation motifs for casein kinase 1 (CK1). This serine/threonine cluster is evolutionarily conserved from vertebrate E4BP4 to Drosophila Vrille. CK1 physically associates with E4BP4 and phosphorylates it. CK1-catalyzed phosphorylation of E4BP4 results in proteasomal proteolysis-dependent decrease of E4BP4 levels, while E4BP4 nuclear accumulation is attenuated by CK1 in a kinase activity-independent manner. CK1-mediated posttranslational regulation is accompanied by reduction of the transcriptional repression executed by E4BP4. These results not only demonstrate a phosphorylation-dependent regulatory mechanism for E4BP4 function but also highlight the role of CK1 as a negative regulator for E4BP4-mediated repression of cPer2 (Doi, 2004).

The loss of circadian PAR bZip transcription factors results in epilepsy

DBP (albumin D-site-binding protein), HLF (hepatic leukemia factor), and TEF (thyrotroph embryonic factor) are the three members of the PAR bZip (proline and acidic amino acid-rich basic leucine zipper) transcription factor family. All three of these transcriptional regulatory proteins accumulate with robust circadian rhythms in tissues with high amplitudes of clock gene expression, such as the suprachiasmatic nucleus (SCN) and the liver. However, they are expressed at nearly invariable levels in most brain regions, in which clock gene expression only cycles with low amplitude. Mice deficient for all three PAR bZip proteins are highly susceptible to generalized spontaneous and audiogenic epilepsies that frequently are lethal. Transcriptome profiling revealed pyridoxal kinase (Pdxk) as a target gene of PAR bZip proteins in both liver and brain. Pyridoxal kinase converts vitamin B6 derivatives into pyridoxal phosphate (PLP), the coenzyme of many enzymes involved in amino acid and neurotransmitter metabolism. PAR bZip-deficient mice show decreased brain levels of PLP, serotonin, and dopamine, and such changes have previously been reported to cause epilepsies in other systems. Hence, the expression of some clock-controlled genes, such as Pdxk, may have to remain within narrow limits in the brain. This could explain why the circadian oscillator has evolved to generate only low-amplitude cycles in most brain regions (Gachon, 2004).

To elucidate the molecular link between PAR bZip transcription factors and epileptic seizures, PAR bZip-regulated genes were sought that could provide insight into the control of brain electrical activity. To this end brain mRNA populations from double-knockout mice, which are not prone to epileptic attacks, and triple-knockout animals, which are susceptible to audiogenic and spontaneous seizures, were compared by Affymetrix high-density oligonucleotide microarray hybridization. Three pools of brain RNA, composed of equivalent amounts of RNA from five males and five females each, were prepared for the two genotypes and compared by hybridization with Affymetrix oligonucleotide microarrays containing probe features for ~12,000 genes (representing 30%-50% of all genes). This resulted in a total of nine comparisons (Gachon, 2004).

Animals homozygous for the disrupted Tef allele contained considerably less Tef transcripts than animals homozygous for a Tef wild-type allele. Interestingly, NFIL3/E4BP4, a putative antagonist of PAR bZip transcription factors, is also down-regulated in TEF-deficient animals, suggesting that TEF stimulates the production of its own competitive inhibitor. In the brain, TEF also appears to stimulate the expression of mPer2 and Dec1, two transcriptional regulators that had been implicated in the negative limb of the circadian oscillator (Gachon, 2004).

Among the genes positively regulated by TEF was Pdxk. Pdxk mRNA levels scored about twofold less in Tef-deficient mice in nine out of nine comparisons. PDXK performs the last step in the conversion of B6 vitamers into pyridoxal phosphate (PLP), a coenzyme of numerous decarboxylases and transaminases involved in amino acid and neurotransmitter metabolism. Even moderate reductions in PLP levels have previously been associated with a susceptibility to epileptic seizures and an increase in delta EEG activity (Sharma, 1994), and thus the transcription of Pdxk was examined in greater detail to evaluate whether this gene might be a direct target gene of TEF. As a first step, the transcriptional start sites within the promoter of the murine Pdxk gene were determined using RACE technology. The sequencing of 13 RACE products yielded two major and several minor cap sites, located between 65 bp and 91 bp upstream of the translation initiation codon. A short conserved sequence block encompassing a PAR bZip recognition sequence (PARRE) could be discerned in the first intron, 12-20 bp downstream of the exon-intron splice junction. A double-stranded oligonucleotide containing this putative binding site was used in electromobility shift assays (EMSA) with liver and brain nuclear extracts harvested at 4-h intervals around the clock to investigate whether this sequence element indeed binds PAR bZip proteins. This element forms a prominent protein:DNA complex with liver nuclear proteins prepared from wild-type mice at times when DBP and TEF reach zenith levels (ZT10). A protein:DNA complex reflecting a liver nuclear protein with circadian accumulation is still discerned in double-knockout mice but not in triple-knockout mice, strongly suggesting that this complex contains TEF. Similar complexes were observed in EMSA experiments with brain nuclear extracts prepared from animals of the three genotypes. As expected on the basis of the temporal mRNA accumulation profiles, the PAR bZip proteins revealed by these EMSA studies accumulate at high levels throughout the day in the brain of wild-type mice. Likewise, TEF levels do not appear to follow a circadian rhythm in the brain of double-knockout animals (Gachon, 2004).

TaqMan RT-PCR technology was used to investigate the differences in Pdxk expression in the liver and brain of double- and triple-knockout mice. In the liver of double-knockout mice, Pdxk mRNA accumulates in a circadian fashion, with a phase compatible with that of TEF accumulation. In triple-knockout mice, Pdxk transcript levels are nearly constant throughout the day and amount to about one-third of the zenith levels attained in double-knockout mice. The Pdxk mRNA levels are also lower in the brain of triple-knockout mice (about two-fold on average). While the Pdxk mRNA concentrations determined in double-knockout mice are similar at most times of the day, the value obtained at ZT8 appears to be somewhat lower. However, in contrast to the clearly circadian expression pattern of liver Pdxk mRNA, the temporal accumulation profile of brain Pdxk transcripts did not score as circadian by the ANOVA test. The daily accumulation of Pdxk mRNA in liver and brain of homozygous Tef single-knockout mice was determined and it was found to be intermediate between double- and triple-knockout mice. This is in keeping with the low but significant seizure activities observed in the EEGs of Tef single-knockout animals (Gachon, 2004).

Several brain regions show significantly lower in situ hybridization signals in triple-knockout mice as compared with double-knockout mice. These include structures that have previously been implicated in the development of spontaneous and/or audiogenic epilepsies, such as cerebral cortex, amygadala, hippocampal formation, inferior colliculus, and periaqueductal gray (Gachon, 2004).

PLP is synthesized in the liver via the phosphorylation of B6 vitamers by PDXK, and a portion of the hepatic PLP is then exported into the bloodstream and transported to other sites, such as the brain. Before crossing the blood-brain barrier and the plasma membrane of target cells, PLP has to be dephosphorylated to pyridoxal (PL) by plasma phosphatases. In the target cells, PL is then rephosphorylated to PLP, which is the active coenzyme form of vitamin B6 (Gachon, 2004).

Whether the reduced Pdxk expression in the liver and brain of PAR bZip-deficient mice indeed affected the levels of PLP was examined. Both brain and liver levels of PLP are significantly reduced. Whereas PLP accumulation appears to follow a circadian rhythm in the liver of Tef wild-type mice, it is nearly invariable in the liver of PAR bZip triple-knockout mice and in the brain of double- and triple-knockout mice. Because Pdxk expression is more strongly dependent on TEF in a subset of brain structures, it is thought that in these structures the difference in PLP levels between TEF-proficient and TEF-deficient animals is considerably higher than the difference measured in extracts from total brains (Gachon, 2004).

In the brain, PLP is the coenzyme of several enzymes participating in the synthesis of neurotransmitters, such as gamma-aminobutyric acid (GABA), serotonin, and dopamine, which had previously been associated with epileptic seizures. Moreover, changes in PLP levels can also influence the levels of glutamate (Glu) and histamines by determining the rate of conversion of these neurotransmitters to other compounds. Whether the reduced levels of PLP in the brains of PAR bZip triple-knockout mice affected the brain concentrations of GABA, serotonin, dopamine, and histamine, was examined. The results revealed significantly reduced levels for serotonin and dopamine and increased levels of histamine in triple-knockout as compared with double-knockout animals (Gachon, 2004).

Since all three PAR bZip genes show circadian expression in the SCN and in peripheral tissues, whether these transcription factors are required for sustained rhythmicity in constant darkness (DD) was examined. The relevance of this issue is reinforced by the recent finding that PAR domain protein 1 (Pdp1), the only Drosophila PAR bZip domain protein, is an essential clock component in fruit flies. The circadian locomotor activity of triple-knockout mice was recorded using wheel running assays. PAR bZip-deficient mice are rhythmic in DD, suggesting that they still harbor a functional circadian pacemaker. Moreover, the period length of triple-knockout mice measured in DD is nearly identical to that measured for wild-type mice. Interestingly, Dbp single-knockout mice free-run with a period ~30 min shorter than that of wild-type mice, while Tef and Hlf single knockout mice free-run with periods ~-25 min longer than wild-type. Apparently, these positive and negative period differences counteract each other in triple-knockout animals, yielding a period length that is close to that observed for wild-type mice. Although triple-knockout animals display a rhythmic behavior in constant darkness, it was noticed that a large proportion of these animals show a spate of wheel running activity several hours before the onset of the dark phase in LD or the onset of the subjective night in DD (Gachon, 2004).

The circadian expression of core clock components in the livers of triple-knockout and wild-type mice were compared. The rhythmic expression of the four clock genes Bmal1, Per1, RevErbalpha, and Cry1 is nearly identical in animals of these two genotypes. It is thus concluded that PAR bZip proteins are dispensable for circadian rhythm generation, and hence that they are regulators of outputs of the mammalian circadian timing system rather than core components of the clock (Gachon, 2004).

By controlling Pdxk expression, PAR bZip transcription factors may play an important role in the fine-tuning of neurotransmitter homeostasis in the brain. PLP, the product of the PDXK reaction, serves as the coenzyme for a number of enzymes involved in the synthesis and degradation of neurotransmitters, and changes in PLP levels can result in severe neurotransmitter disequilibria and brain pathologies characterized by epilepsy. For example, the disruption of the gene encoding tissue-nonspecific alkaline phosphatase (TNAP) causes an approximately threefold reduction in brain PLP levels, and 100% of the Tnap mutant mice succumb to lethal epileptic seizures before 20 d of age. gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter of the central nervous system, probably plays the most important role in preventing the uncontrolled activation of epileptic foci and the propagation/amplification of seizure waves through downstream regions. The function of PLP in determining the concentration of GABA is complex, as PLP is the coenzyme of both glutamate decarboxylase (GAD), which converts glutamate to GABA, and GABA transaminase (GABA-T), which degrades GABA to succinic semialehyde. Moreover, two GAD isoforms, GAD65 and GAD67, with possibly different functions are expressed in most glutamatergic neurons. Whereas GAD65 is believed to be mostly responsible for the synthesis of GABA used for neurotransmission in nerve endings, GAD67 may produce GABA that is used to a large extent for metabolic purposes involving the tricarboxylic acid cycle. GAD67 has a higher affinity for PLP than GAD65, and exists primarily in the holoenzyme form at physiological PLP levels. In contrast, a large fraction of GAD65 is found in the apoenzyme form, and the activity of this GAD isoform is thus more dramatically affected by changes in PLP concentration. It appears that the bulk of brain GABA is synthesized by GAD67, because Gad65^-/- mice (genetic backgound 129xC57BL/6) contain normal basal levels of brain GABA. Since these mice are susceptible to spontaneous epileptic attacks, local reductions in GABA concentrations that are not revealed by measuring total brain GABA may be sufficient to provoke seizures. Hence, in spite of the virtually identical levels of total brain GABA measured in double- and triple-knockout mice, it remains possible that the susceptibility of PAR bZip triple-knockout animals to seizure is enhanced by a local reduction of GABA levels in key brain structures (Gachon, 2004 and references therein).

PLP also serves as the coenzyme of aromatic amino acid decarboxylase (AADC), an enzyme involved in the synthesis of the monoamines serotonin and dopamine. Both of these neurotransmitters have been associated with epileptic attacks. For example, young adults of a mouse strain homozygous for a serotonin receptor 5-HT2C null allele develop audiogenic seizures and mice homozygous for a dopamine receptor 2 (D2R) null allele develop epilepsies after injection of kainic acid at doses that do not provoke seizures in D2R wild-type littermates. Given these observations, it is suspected that the significantly reduced serotonin and dopamine concentrations in the brains of PAR bZip triple-knockout mice contribute to the susceptibility of these mice to epileptic attacks (Gachon, 2004 and references therein).

Two PLP-dependent enzymes, histidine decarboxylase and histamine oxidase, are involved in histamine synthesis and catabolism, respectively. Interestingly, high levels of PLP have been shown to result in a decrease of histamine levels, probably by promoting histamine degradation. Therefore, it is speculated that the high level of histamine found in the brain of PAR bZip triple-knockout mice is caused by a decrease in histamine catabolism, due to diminished PLP levels. Whether or not histamine imbalances play a role in the generation of epilepsies is still somewhat controversial (Gachon, 2004).

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Search PubMed for articles about Drosophila vrille

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