Targets of Activity

PER might be responsible for the regulation of its own transcription. Levels of PER RNA do not cycle in per mutant flies (Hardin, 1990). PER mRNA follows a diurnal rhythm, peaking hours earlier in the light-dark cycle than PER protein, which peaks some time after the beginning of the dark period. TIM mRNA peaks at the same time as PER, indicating that the two are co-regulated, perhaps by the PER-TIM heterodimer (Reppert, 1995).

Dreg-5 is a gene whose mRNA is expressed in fly heads with a circadian rhythm nearly identical to that of the per gene, continuing to phase with PER mRNA even in conditions of total darkness and also when the daily feeding time is altered. The phase of DREG-5 protein oscillation, however, is different from that of PER protein, suggesting that the two genes have a common transcriptional control but different post-transcriptional control mechanisms (Van Gelder, 1996).

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

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

Circadian regulation of gene expression systems in the Drosophila head

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 ClkJrk), 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 ClkJrk) 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 ClkJrk 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).

The genes in a second cluster (Clock cluster are primarily grouped together based on their peak phase (average phase ZT2). By virtue of the mutant expression data, several subclusters within this phase group can be identified. The known circadian genes Clock and takeout (to) are part of this cluster. Clk is found in a clustered pair with the leucyl aminopeptidase gene CG9285. In terms of chromosomal organization, to, CG11891, and CG10513 map closely together on chromosome 3R. Two additional circadian genes in this chromosomal region (CG11852, CG10553). Interestingly, the Clk cluster contains three pairs of homologous genes with very similar expression patterns: the UDP-glycosyl transferase genes Ugt35a and Ugt35b, the enteropeptidase genes CG9645 and CG9649, and the long-chain fatty acid transporter genes CG6178 and CG11407. In the first two cases, the homologous genes are also directly adjacent to each other on the chromosome. An overview of the map positions of all circadian genes in this study is available as supplemental information online ( Apart from Ugt35a and Ugt35b, several other genes with a predicted function in detoxification are members of the Clk cluster (CG17524, CG8993, CG3174, Cyp6a21). It may also be noteworthy that the genes for three oxidoreductases found in this group [Photoreceptor dehydrogenase (Pdh), CG15093, CG12116] have almost identical phases (ZT3) (Claridge-Chang, 2001).

All genes of the apterous (ap) cluster are defined by both the oscillatory phase of their expression pattern (average phase ZT17) and by a distinct expression profile in the three clock mutants. Although the 6 hr sampling interval in the mutant data makes it difficult to reliably detect oscillations, it seems that the majority of the genes in this cluster shows some degree of periodicity in the three mutant light-dark regime (LD) time courses. Although it cannot be ruled out that there are circadian oscillations independent from the known clock genes, the hypothesis that there may be a light-driven response underlying the observed mutant expression pattern is favored. The genes in this group may, therefore, be regulated not only by the circadian clock, but also by a direct light-dependent mechanism. It should be mentioned that evidence of gene expression patterns that are purely light-driven in wild-type flies was sought, but little indication was found of such regulation. Instead, genes with both a strong light-driven oscillation and a weak circadian component were encountered. apterous (ap) encodes a LIM-homeobox transcription factor, which is known to act both in neural development and in neuropeptide expression. The ap cluster includes the genes for the transcription factor moira, the synaptic regulator syndapin, two septins (Sep1 and CG9699), and two ATP binding cassette (ABC) transporters (CG6162, CG9990). In terms of chromosomal organization, CG6166, the gene adjacent to CG6162 on chromosome 3R is homologous to CG9990 and coregulated with CG6162 and CG9990 (Claridge-Chang, 2001).

The founding member of the fourth cluster, ebony (e), encodes ß-alanyl-dopamine synthase and has roles in both cuticle tanning and regulating circadian locomotor behavior. Among the other cluster members are six genes that function in protein cleavage (CG9377, Ser99Da, SP1029, CG7828, CG11531, BcDNA:GH02435), three transcription factor genes (CG15632, CG17257, CG6755), as well as two genes each that act in signal transduction (prune, loco), the cytoskeleton (TpnC47D, Chd64), and lipid metabolism (ATPCL, CG1583) (Claridge-Chang, 2001).

The per0, tim01, and ClkJrk mutations affect genes that are essential for maintaining circadian rhythms and result in both molecular and behavioral arrhythmicity. In addition to abrogating the oscillations of the per, tim, vri, to, and Clk transcripts, these mutations also affect their absolute levels of expression. per0 and tim01 flies have intermediate or somewhat elevated levels of per, tim, vri, and to transcript, and decreased levels of Clk transcript whereas ClkJrk mutants have the opposite effect. Based on these observations, genome-wide expression data was gathered from per0, tim01, and ClkJrk mutant flies in three separate four-point time course experiments. A rank-sum Wilcoxon test was employed to determine if any of the interrogated transcripts were significantly up- or down-regulated in any of the mutants when compared to the total wild-type expression data set (Claridge-Chang, 2001).

Out of the 14010 probe sets on the arrays, 4865 showed up- or down-regulation in one or more of the three mutants with a p value lower than 0.05; 2544 were significantly changed in the tim01 flies; 1810 were significantly different in per0, and 2181 in ClkJrk. It is unclear what proportion of these changes depends on the actual mutations themselves, because (1) the three mutant fly strains have different genetic backgrounds and (2) data was collected for only one population of each mutant strain. Although there are known examples of noncircadian genes whose expression is affected by clock mutations, it was decided that it would be more informative to consider effects of the clock mutations only with respect to the subset of 158 strong oscillators. Among this set, 72 genes were found with one or more significant expression changes in the three clock mutant strains (Claridge-Chang, 2001).

Included in the regulated set are tim (twice independently), vri, to, and Clk, and their patterns agree with previously published observations. The hierarchical clustergram shows four basic patterns of regulation: (type I) increased in per0 and tim01 but decreased in ClkJrk (e.g., vri, CG5798); (type II) decreased in per0 and tim01 but increased in ClkJrk (e.g., Clk, CG15447); (type III) decreased in all three mutants (e.g., Ugt36Bc/CG17932, ea), and (type IV) increased in all three mutants (e.g., Pdh, CG11891). Type I and II match the two known modes of regulation for circadian genes. The behavioral and molecular phenotypes of the per0 and tim01 mutations are almost identical. It may, therefore, be relevant that no circadian genes are found that are significantly upregulated in per0 and significantly downregulated in tim01 or vice versa. Apart from genes that were a priori predicted to have expression patterns of type I (vri, tim, to) or II (Clk), novel genes were found for each of these two expression patterns. The average phases for the type I and type II subclusters are, respectively, ZT12 and ZT7, but there is large variation in phase among the members of each of these subclusters. to is in the type I subcluster and has a phase peak at ZT2, whereas CG15447 is in the type II subcluster and peaks at ZT10. This phenomenon of phase differences among transcripts with a similar response to clock defects has been described previously for type I regulation in the case of to. Here, a similar phenomenon was detected for genes with Clk-like type II regulation. Type III and IV predict a novel and unexpected response to the circadian mutants (Claridge-Chang, 2001).

The promoter sequences of the set of 158 genes was tested for the presence of known and candidate circadian enhancer motifs. The results suggest that in fact this set is enriched in such elements. For example, the frequency of E boxes in the set of oscillators (42 hits in total) is significantly higher than the frequency in random selections of genes. The significance of this result does, however, depend on the inclusion of well-studied genes (per, tim, vri). Some novel oscillators with remarkable frequencies of 'circadian transcription elements' are loco (1 PDP1 site; 3 W boxes; 8 CRE elements; 3 TER elements); trpl (1 E box; 1 PDP1 site; 9 W boxes; 6 CRE elements; 12 TER elements); Rh5 (5 W boxes; 5 CRE elements; 6 TER elements), and Slob (1 E box; 1 PERR element; 2 PDP1 sites; 6 W boxes; 15 TER elements). It is noteworthy that many robustly cycling genes have no known circadian transcription elements in their promoters or first introns (Claridge-Chang, 2001).

The set of 158 circadian genes were organized according to annotated or predicted molecular function. Several of these functional classes may provide insights into pathways influencing rhythmic behavior (Claridge-Chang, 2001).

Synaptic Transmission and Plasticity
An emerging theory of the function of sleep postulates that it is required for neural plasticity, synaptic maintenance, and remodelling. Behaviorally defined sleep has been identified in the fly, with behavioral recordings in LD indicating increased rest during the dark phase. The assay for circadian expression uncovered a number of genes known to be involved in synaptic function and synaptic plasticity (Claridge-Chang, 2001).

Three oscillating trancripts encode synaptic vesicle endocytosis factors: ß-adaptin (Bap), AP-1gamma, and syndapin. The first two are adaptors between the budding membrane and clathrin lattice, while syndapin is thought to connect vesicle endocytosis to actin. Clock modulation of the synaptic vesicle pool is consistent with the idea of modulated synaptic function, although it is unclear what effect raising or lowering endocytotic factors would have on synaptic function (Claridge-Chang, 2001).

The Slob transcript peaks at ZT15 and is downregulated in the ClkJrk mutant, suggesting that CLK acts as an activator of Slob. There is an E box 5.4 kb upstream of the Slob transcriptional start site raising the possibility that CLK acts directly on the Slob promoter (Claridge-Chang, 2001).

If the cycling transcript can be shown to produce oscillating Slob protein, this could indicate a potent mechanism for rhythmic control of synaptic function, including synaptic plasticity: Slob protein binds the calcium-dependent potassium channel Slowpoke (Slo) and has been shown to increase Slo activity and voltage sensitivity. Slob is in turn bound by a second channel regulator, Leonardo. Hypomorphic mutations of leonardo produce defects in learning, and electrophysiological analyses of the larval neuromuscular junction (NMJ) in these mutants show presynaptic function and plasticity is greatly impaired in these animals. In contrast to Slob, Leonardo is a strong inhibitor of Slo, but requires Slob for this interaction. All three proteins colocalize to the presynaptic bouton at larval NMJs. Thus, Slob may contribute to a switching mechanism that ultimately places Slo channel activity under circadian control (Claridge-Chang, 2001).

Slo channels are widely expressed in the adult fly head, including the eye, lamina, medulla, central brain, and mushroom bodies, but it is not known which subset of these areas contain oscillating Slob expression. In situ hybridization was performed with larval brains to localize Slob RNA expression. Prominent staining was observed in a restricted region consistent with placement of the developing mushroom body. The staining also corresponds well with that region of the larval brain receiving PDF-rich projections of the circadian pacemaker cells, the lateral neurons. In future studies, it will be important to determine whether presence of the innervating LNs is required for cycling Slob expression (Claridge-Chang, 2001).

leonardo was initially implicated in presynaptic function by the effect of mutations on learning. Mutations of latheo also cause learning defects, and this protein is also found at larval NMJs. Lowered latheo function has been associated with elevated synaptic transmission and reduced synaptic plasticity. latheo shows cycling expression with peak accumulation at ZT12-15. Rhythmicity was detected in the expression of dunce and Calpain-B genes involved in learning and synaptic long-term potentiation, respectively (Claridge-Chang, 2001).

Amine Neurotransmitter-Related Functions
Two serotonin receptor transcripts, 5-HT2 and 5-HT1A, were found to oscillate with phases of ZT15 and ZT18, respectively. Serotonin is known to be involved in a variety of neuronal processes in animals, including synaptic plasticity, clock entrainment, and mating behavior. The 104 serotonergic neurons in the adult CNS have been mapped, but no studies have been done of either 5-HT receptor localization or receptor mutant phenotypes. Neither of these receptors are orthologs of the mammalian 5-HT receptor implicated in photic clock entrainment; this would be represented by theDrosophila 5-HT7 gene. 5-HT1A belongs in a class of receptors that respond to agonists by decreasing cellular cAMP, while 5-HT2 is homologous to mammalian receptors whose main mode of action involves activation of phospholipase C, a function involved in synaptic plasticity (Claridge-Chang, 2001).

The ebony transcript was found to oscillate, showing a peak of expression around ZT5. ebony oscillation connects with a body of earlier evidence linking ebony to circadian activity rhythms. ebony hypomorphs show severe defects in circadian rhythm including arrhythmicity/aberrant periodicity in the free-running condition, as well as abnormal activity patterns in LD conditions. Ebony is a putative ß-alanyl dopamine synthetase, and hypomorphs show elevated levels of dopamine. Dopamine has been implicated in control of motor behavior, since it induces reflexive locomotion in decapitated flies, and this response is under circadian control. The results suggest that oscillations of ebony contribute to the assembly of rhythmic locomotor behavior. Other evidence points to a role in clock resetting. In addition to impaired entrainment in LD, ebony flies show an abnormal ERG, and ebony is strongly expressed in the lamina and the medulla optic neuropile, a region associated with vision rather than motor control (Claridge-Chang, 2001).

The Drosophila eye is both a likely target of clock control and partly responsible for photic input to the central pacemaker. Several genes found to oscillate by microarray assay are components of visual processes (Claridge-Chang, 2001).

Photoreceptor cells contain peripheral clocks, suggesting that visual function may be regulated by the clock. In vertebrates, the synthesis of various visual components is known to be under circadian control. In Drosophila, electroretinogram (ERG) measurements of visual sensitivity reveal a 4-fold cycle in sensitivity, with a minimum at ZT4 and a broad peak around lights off (ZT12). This suggests that some of the fly visual components are clock controlled. However, a previous study of five major phototransduction components found no cycling of either mRNA or protein. In this genome-wide assay, the trpl transcript was found to oscillate with peak expression at ZT11. TRPL is one of two ion channels in the visual transduction pathway, along with TRP, a paralog. TRPL and TRP open in response to a G protein-coupled phosphoinositide cascade that is initiated by the isomerization of rhodopsin by light. Their opening produces the light-sensitive conductance in the photoreceptors. Amorphic mutants of each channel show visual defects, while the double null genotype results in a blind fly. A cycling trpl transcript could contribute to the visual sensitivity cycle: (1) the two phenomena are in the same phase with both sensitivity and trpl expression peaking around lights-off; (2) it is estimated that TRPL contributes about half of the wild-type conductance, allowing for a substantial range of modulation by reducing TRPL function. Another possible role for oscillating TRPL function is connected with circadian entrainment. In addition to being blind, trp/trpl double null mutants show reduced circadian behavioral resetting and less TIM degradation in response to light pulses. It is noted that the established entrainment factor CRY is known to cycle and that interactions of CRY and TIM are essential for light-dependent TIM degradation. The oscillating, clock-related protein VIVID has also been shown to regulate photo-entrainment in Neurospora (Claridge-Chang, 2001).

The microarray experiments show that two opsin genes are under circadian control: Rh5 and Rh4. The Rh5 mRNA rhythm peaks at ~ZT 21, while the Rh4 array data show a circadian pattern with a peak 4 hr later, at ZT1. Rh5 is a blue-absorbing rhodopsin expressed in a subset of R8 cells at the base of the retina, while Rh4 is a UV-absorbing pigment expressed in apical R7 cells. Rh5 is never expressed in an R8 cell underlying an Rh4-expressing R7 cell, so in this way all ommatidia would contain one cycling rhodopsin. In terms of regulating sensory receptiveness to light, it is unclear why these two opsins should be targets for clock control. The major blue rhodopsin Rh1 does not cycle so it seems unlikely that an oscillation in these two minor pigments would produce overall tuning of the sensitivity of the fly visual system (Claridge-Chang, 2001).

NinaA is a rhodopsin chaperone and is required to move Rh1 from the endoplasmic reticulum (ER) to the rhabdomeric membrane. ninaA mutants display aberrant accumulation of Rh1 protein in the ER. ninaA mRNA shows cycling expression in fly heads by both array and Northern blot, with peak expression around ZT2. It is tempting to hypothesize that early morning ninaA upregulation would have the effect of releasing a reservoir of Rh1 from the ER, making it available for use in visual transduction. However, a previous assay of NinaA levels in an LD regime revealed no oscillation in protein levels. If true, this would represent a surprising example of a robustly oscillating mRNA producing a constitutive cognate protein. Finally, although its function is still unknown, Photoreceptor dehydrogenase is a robustly oscillating transcript with an extremely high level of expression in the screening pigment cells of the eye (Claridge-Chang, 2001).

Rhythmic Proteases and Accessory Factors
Fifteen of the identified oscillatory genes are implicated in protein cleavage. CG7828 and BcDNA:GH02435 (peak phase ZT6 and ZT10, respectively) mediate ubiquitination of protein substrates, thus targeting them for degradation. Conversely, CG5798 and CG7288 cycle with a peak at respectively, ZT14 and ZT16, and each produces a ubiquitin specific protease (Ubp; cleaves ubiquitin from ubiquitin-protein conjugates) that may act to prevent the degradation of specific protein targets. Two metalloprotease genes, two aminopeptidase genes, and five serine peptidases show circadian oscillation. Four of the five serine peptidase genes cycle with a peak phase between ZT4-7. This profusion of oscillating proteases suggests that circadian proteolysis may represent a broad mechanism of clock control, both of clock components themselves, as well as output factors (Claridge-Chang, 2001).

While circadian transcriptional mechanisms are relatively well understood, less is known about posttranslational mechanisms of circadian regulation. Proteases are known to be involved in circadian control of the degradation of some central clock components, and the clock proteins PER, TIM, CLK, CRY, and VRI are all known to undergo daily cycles of protein accumulation. Degradation of TIM is responsible for photic resetting of the Drosophila clock. This is thought to be mediated by interaction with CRY, followed by ubiquitination and proteasome-dependent loss of TIM. Nothing is known about the factors mediating TIM degradation in the dark, yet patterns of CG5798 expression may be of interest in this regard as this gene encodes a cycling Ubp whose peak expression (ZT14) immediately precedes an interval of rapid TIM accumulation in pacemaker cells (Claridge-Chang, 2001).

A likely clock-related target of one or more proteases is the neuropeptide PDF, whose regulation may be crucial to linking the clock to behavior. While pdf RNA is expressed constitutively, the peptide accumulates rhythmically under indirect control of the clock gene vri. This mechanism has not been further explored, but a clear possibility is that a PDF propeptide is cleaved rhythmically, allowing cyclical release of active PDF. Of the 15 cycling proteases suggested by this study, CG4723 may be of special interest due to its inclusion in a class of proteases known to cleave neuropeptides. The phase of CG4723 expression, ZT4, also coincides with that of PDF immunoreactivity in the Drosophila head (Claridge-Chang, 2001).

Detoxification and Oxidative Stress
One hypothesis of sleep characterizes it as a cellular detoxification and repair process. Twelve genes were found that have a predicted role in detoxification. Three additional redox enzymes may also participate in this process. Toxins are initially modified into reactive species by reductases/dehydrogenases and cytochrome P450 molecules. Then glutathione-S-transferases (GSTs) and UDP-glycosyl transferases add polar groups to the substrates to render them hydrophilic for elimination by secretion. Four results are noteworthy: (1) following this pathway, the genes for three circadian dehydrogenases: Pdh, CG10593, and CG12116, were found.; (2) both morning and night cytochrome P450 genes (Cyp6a21 and Cyp305a1) were found to peak early in the day (ZT0 and ZT5), whereas Cyp18a1 and Cyp4d21 peak at approximately the same time late at night (ZT18 and ZT19); (3) while CG17524 is the only GST gene found in the core set, it was noticed that two other members of the GST type III gene cluster on chromosome 2R show 24 hr periodicity (CG17523; CG17527) and (4) UDP-glycosyl transferase genes are represented in the circadian set by Ugt35a, Ugt35b, and Ugt36Bc. Of these, Ugt35b encodes an antennal specific transcript with a potential role in olfaction, whereas Ugt35a is expressed more uniformly (Claridge-Chang, 2001).

CG13848 is an alpha-tocopherol transfer protein (alpha-TTP) that is strongly expressed in certain basal cells of the eye, showing peak expression around dawn. Humans with mutations in the homologous alpha-TTP show neurodegenerative ataxia that is associated with a deficiency in alpha-tocopherol (vitamin E) incorporation into lipoprotein particles. Vitamin E acts as an antioxidant, and it is thought that this activity allows it to protect neurons from damage by free radicals. Also from human studies, it is known that photoreceptor cells are particularly susceptible to oxidative damage due to high levels of polyunsaturated fatty acids in the photoreceptor membrane, and their exposure to visible light. Thus, it is proposed that the dawn phase of CG13848 represents an upregulation of alpha-TTP for increased daytime transfer of photoprotective vitamin E into the photoreceptor membrane. Also in the circadian set, Catalase (encoded by Cat) and a thioredoxin (encoded by CG8993) are both involved in neutralizing reactive oxygen species (Claridge-Chang, 2001).

Different aspects of metabolism are represented among the selected set of oscillating transcripts: lipid metabolism (five genes), amino acid metabolism (three), carbohydrate metabolism (three), and glycoprotein biosynthesis (two). Intriguingly, Zw encodes glucose-6-phosphate 1-dehydrogenase of the pentose-phosphate pathway (PPP), while CG10611 encodes fructose-bisphosphatase in gluconeogenesis. The two pathways have antagonizing roles in glucose metabolism; both genes are key control points in their respective pathway and are maximally expressed in opposite phases. Zw transcripts are at their zenith just before dusk (ZT11), whereas CG10611 transcripts peak at dawn (ZT0). Thus, the antiphase oscillation of these two genes may produce daily alternation between glucose anabolism and catabolism. Zw and CG10611 transcript levels also respond in opposite fashion to clock defects: Zw levels are significantly decreased in per0 and tim01 mutants, whereas, CG10611 is significantly upregulated in tim01 (Claridge-Chang, 2001).

In more direct relation to the clock itself, Zw is the first committed step in the PPP and generally thought to control flux through this pathway. The PPP is the major pathway of NAD (or NADP) conversion to NAD(P)H. It was recently shown that NAD(P)H can bind homologs of CLK and CYC, promoting their dimerization and DNA binding. Maximal Zw expression at ZT11 -- and therefore presumably NAD(P)H production via the PPP -- is coincident with maximal per and tim transcription by CLK/CYC. This information is consistent with Zw participating in a NAD(P)H-mediated autoregulatory loop of the clockworks (Claridge-Chang, 2001).

Nucleic Acid Metabolism
A subset of 15 genes involved in nucleic acid metabolism were found. This includes five genes encoding specific RNA polymerase II transcription factors. Four of these encode parts of the circadian clock itself (per, tim, vri, and Clk), whereas the fifth one, apterous (ap), generates a homeobox transcription factor with a role in neurogenesis and the expression of neuropeptides. Taf30alpha2 is a subunit of the general transcription factor TFIID, while moira (mor) is part of the SWI-SNF chromatin-remodeling complex. One splicing factor gene (DebB) and one DNA repair gene (CG4049) are also found among this set of oscillating transcripts (Claridge-Chang, 2001).

Circadian genes with a role in the cytoskeleton include those encoding two actin binding proteins (Chd64, CG11605), a troponin C (TpnC47D), and two septins (Sep-1 and CG9699). Chd64 (phase peak ZT4) and TpnC47D (phase peak ZT7) function specifically in muscle contraction. Another Drosophila Troponin C, TpnC73F, is found to peak at ZT6 (Claridge-Chang, 2001).

In conclusion, a set of 158 genes expressed with a robust circadian rhythm in the adult Drosophila head was found by microarray screening. These encompass a wide variety of molecular functions, and expression patterns represented essentially all circadian phases. A larger set of genes was identified (393 entries; 293 entries after secondary filters), and the statistical approach again indicated significant circadian rhythmicity for these, but they were characterized by somewhat less robust oscillations than those of the smaller set. Independent verifications indicated substantial enrichment for cycling gene expression in this larger set and beyond. 532 genes passed secondary filters for 24 hr autocorrelation, noise, and the range-to-noise measure. From the frequency of Northern-verified oscillations detected in this larger pool of candidate genes, it is believed that the total complement of circadian genes would include ~400-500 in the adult head (Claridge-Chang, 2001).

There are important factors that might lead to an underestimation of the total complement of circadian genes. The approach that was used would favor genes that are homogeneously expressed in the head. If the same gene is expressed with varied phases in different head tissues, this will lessen the robustness of the apparent oscillation and phase. Similarly, if only a restricted portion of the head generates the cycling gene pattern, but constitutive expression is found elsewhere in the head, amplitude of the signal will be diminished. Differences of this sort might be expected in cases where a cycling gene product produces a limited physiological effect. Regulation of this type might be expected in the antennae, where, for example, electrophysiological responses to odorants vary with a circadian rhythm. It should also be stressed that only the fully differentiated adult head has been sampled. If transient patterns of circadian expression occur during development, these would go unnoticed in the experiment. Given the plethora of tissues housing autonomous circadian clocks, an expanded list of rhythmic genes would probably be derived from any related sampling of the body (e.g., wings, legs, excretory, and digestive tissues), especially as this tissue autonomy may reflect a requirement for tissue-specific pathways of circadian control that lie downstream from a largely uniform clock mechanism (Claridge-Chang, 2001).

How will the many patterns of cycling gene expression be further explored? Molecular tools that reveal the importance of oscillating gene activity have already been applied to a study of several clock genes in Drosophila. In these studies, oscillating patterns of a target gene's expression have been replaced with constitutive activity. Central questions related to vri, per, and tim function have each been explored in this manner. The present study allows an expansion of such work to address the molecular connections between individual behaviors and circadian clocks (Claridge-Chang, 2001).

Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA

Transcriptional feedback loops are central to the generation and maintenance of circadian rhythms. In animal systems as well as Neurospora, transcriptional repression is believed to occur by catalytic post-translational events. This study reports in the Drosophila model two different mechanisms by which the circadian repressor PERIOD (PER) inhibits CLOCK/CYCLE (CLK/CYC)-mediated transcription. First, PER is recruited to circadian promoters, which leads to the nighttime decrease of CLK/CYC activity. This decrease is proportional to PER levels on DNA, and PER recruitment probably occurs via CLK. Then CLK is released from DNA and sequestered in a strong, approximately 1:1 PER-CLK off-DNA complex. The data indicate that the PER levels bound to CLK change dynamically and are important for repression, first on-DNA and then off-DNA. They also suggest that these mechanisms occur upstream of post-translational events, and that elements of this two-step mechanism likely apply to mammals (Menet, 2010).

Circadian transcriptional repression is believed to occur by catalytic post-translational events in animal systems as well as Neurospora. In the Drosophila model, two different mechanisms occur sequentially. First, the beginning of the repression phase is associated with PER binding to circadian promoters, probably via a PER-CLK interaction. The PER-DNA interaction likely inhibits CLK-mediated transcription despite persistent CLK DNA binding. This 'on-DNA' phase is followed by the release of CLK from DNA and concomitant formation of a strong, close to 1:1 'off-DNA' PER-CLK complex with low affinity for DNA and in which most of CLK is sequestered (Menet, 2010).

The interaction of PER with DNA is prominent, as CLK-mediated transcription starts to decrease at ZT14 and is then maximal at ZT18 when the decrease in transcription (slope) is approximately maximal. The increase in on-DNA PER between ZT10 and ZT18 parallels the substantial, well-established rise in PER levels during these 8 h, and indicates that mass action may be sufficient to account for this increase. The data therefore suggest that this increase in on-DNA PER affects the rate of transcription, and that possible effects of post-translational mechanisms on DNA-bound CLK or of chromatin modification on transcription are downstream from this PER-CLK ratio. Although the mechanism of transcriptional inhibition is not known, PER presumably recruits or potentiates corepressors, or it inhibits the recruitment or activity of coactivators (Menet, 2010).

The inability to assay a soluble PER-CLK interaction until ZT19 suggests that formaldehyde cross-linking captures earlier interactions of PER with DNA-bound CLK that are too weak to survive a standard soluble IP assay. It is speculated that the in vivo stability of these early PER-CLK interactions may be enhanced by a high local concentration of CLK due to several adjacent DNA-binding sites, as indicated by the broad (~4-kb) CLK-interacting region of DNA. The mixed cytoplasmic/nuclear localization of PER compared with the predominantly nuclear localization of CLK also suggest a labile PER-CLK interaction before ZT18 (Menet, 2010).

Between ZT18 and ZT22, there is a decrease in the association of CLK with DNA as well as a striking increase in the levels of an ~1:1 soluble PER-CLK complex, suggesting that these two phenomena are mechanistically related. This 1:1 PER-CLK complex presumably has a low DNA affinity, which largely accounts for the low transcription rates after ZT19. The formation of a stable stochiometric repression complex contrasts with the transient, phosphorylation-based repression mechanism in Neurospora. In this system, the repressor FRQ is present in nuclei at a much lower molar ratio than the activator WC complex (Menet, 2010).

As there are no striking increases in PER levels after ZT18, key qualitative changes may occur after this time; for example, the addition of other components and/or post-translational modifications. These changes presumably contribute to removing CLK from DNA and to creating the strong 1:1 PER-CLK complex in the late night-early morning with a greatly reduced affinity for circadian promoters. It is of note that ZT18 is precisely when TIM goes from being predominantly cytoplasmic to being predominantly nuclear within l-LNvs. This event may therefore contribute to the removal of CLK and PER from DNA. The post-translational modification possibility is supported by the mobility change of CLK from ZT17 to ZT19, as well the lower mobility of both PER and CLK within the 1:1 stochiometric complex. It is suggested, however, that the prior on-DNA PER-CLK complex is the substrate for these modifications, and is therefore upstream of phosphorylation events that might increase the stability of the off-DNA (Menet, 2010).

In conclusion, these data provide a new mechanistic view of PER-mediated transcriptional repression and emphasize the importance of the PER levels and the PER:CLK ratio. This includes the increases that occur on-DNA during the early night as circadian transcription is decreasing, as well as the ~1:1 PER-CLK ratio that is found off-DNA in the late night-early morning when CLK DNA-binding affinity is at its nadir. Indeed, these two phases may be connected by the increasing ratio of PER-CLK on-DNA: It may dictate the circadian timing of the decrease in CLK DNA affinity, ultimately resulting in the departure from DNA of the stable PER-CLK complex. It is notable that a recent study in the mammalian system has described a prominent PER-CLK interaction that appears very important to repression of the CLK-BMAL1 complex. These new insights support the notion that PER acts as a stable complex component rather than catalytically to effect transcriptional repression in flies and, perhaps, also in mammals (Menet, 2010).

The ion channel Narrow abdomen is critical for neural output of the Drosophila circadian pacemaker

Circadian clocks consist of transcriptional feedback loops housed in interdependent pacemaker neurons. Yet little is known about the neuronal output components essential for rhythmic behavior. Drosophila mutants of a putative ion channel, narrow abdomen (na), exhibit poor circadian rhythms and suppressed daylight activity. NA is expressed in pacemaker neurons and induced expression within circadian neurons is sufficient to rescue these mutant phenotypes. Selective na rescue in distinct pacemaker neurons influences rhythmicity and timing of behavior. Oscillations of the clock protein Period are intact in na mutants, indicating an output role. Pore residues are required for robust rescue consistent with NA action as an ion channel. In na mutants, expression of potassium currents and the key neuropeptide PDF are elevated, the latter consistent with reduced release. These data implicate NA and the pacemaker neural network in controlling phase and rhythmicity (Lear, 2005).

Repression by Period and Cryptochrome

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

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

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

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

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

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

Cryptochrome antagonizes synchronization of Drosophila's circadian clock to temperature cycles

In nature, both daily light:dark cycles and temperature fluctuations are used by organisms to synchronize their endogenous time with the daily cycles of light and temperature. Proper synchronization is important for the overall fitness and wellbeing of animals and humans, and although a lot is known about light synchronization, this is not the case for temperature inputs to the circadian clock. In Drosophila, light and temperature cues can act as synchronization signals (Zeitgeber), but it is not known how they are integrated. This study investigated whether different groups of the Drosophila clock neurons that regulate behavioral rhythmicity contribute to temperature synchronization at different absolute temperatures. Using spatially restricted expression of the clock gene period, this study shows that dorsally located clock neurons mainly mediate synchronization to higher (20°C:29°C) and ventral clock neurons to lower (16°C:25°C) temperature cycles. Molecularly, the blue-light photoreceptor Cryptochrome (Cry) dampens temperature-induced Period (Per)-Luciferase oscillations in dorsal clock neurons. Consistent with this finding, this study shows that in the absence of Cry very limited expression of Per in a few dorsal clock neurons is able to mediate behavioral temperature synchronization to high and low temperature cycles independent of light. This study shows that different subsets of clock neurons operate at high and low temperatures to mediate clock synchronization to temperature cycles, suggesting that temperature entrainment is not restricted to measuring the amplitude of such cycles. Cry dampens temperature input to the clock and thereby contributes to the integration of different Zeitgebers (Gentile, 2013).

This study has shown that different sets of clock neurons play a role for synchronization to low and high temperature cycles with identical amplitude. This shows that temperature entrainment does not solely rely on measurement of temperature differences but rather on measurement of absolute temperatures. This task is divided between different neuronal groups, opening the possibility that multiple temperature receptors -- expressed either in different clock neurons, other neurons in the brain, or in the PNS -- contribute to temperature entrainment. Cry seems to actively block the entrainment strength of temperature both at a molecular and behavioral level, which most likely contributes to Zeitgeber integration (Gentile, 2013).

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

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

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

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

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

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

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

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

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

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

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

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

Postranscriptional Regulation

To investigate quantitative features of per and tim transcription, in vivo transcription rate was analyzed in fly-head nuclei with a nuclear run-on assay. The results show a robust transcriptional regulation, which is similar but not identical for the two genes. In addition, PER mRNA levels are regulated at a post-transcriptional level. This regulatory mode makes a major contribution to the PER mRNA oscillations from a previously described per transgenic strain as well as to the mRNA oscillations of a recently identified Drosophila circadianly regulated gene (Crg-1). The data show that circadian mRNA oscillations can take place without evident transcriptional regulation (So, 1997).

Circadian features of two period-lacZ (per-lacZ) fusion genes were examined in transgenic strains of Drosophila. Both genes manifest circadian fluctuations of mRNA levels, but fluctuations of only the larger chimeric protein are apparent. Fusion protein cycling is indistinguishable from the behavior of wild-type Per protein, including apparent temporal regulation of phosphorylation state. Several arguments indicate that the difference in the two constructs is proper regulation at the level of protein turnover: the smaller protein has much higher levels; a beta-galactosidase degradation products is visible in both strains but fails to manifest cycling, presumably due to a long half-life; and only the noncycling proteins accumulate as a function of adult age. The large cycling fusion protein also undergoes modest cycling in an arrhythmic per01 background. This is light dependent, resembles the regulation of the Timeless protein by light, and reflects a documented fusion protein-Tim interaction. The results are discussed with respect to the posttranscriptional regulation that is necessary for proper cycling of both Per and Tim as well as for clock function (Dembinska, 1997).

Rhythmic oscillations of the Period protein in brain neurons of the adult fly are strongly involved in the control of circadian rhythms. Temporal and spatial expression patterns were examined for three per-reporter fusion genes, which share the same 4 kb regulatory upstream region but contain increasing amounts of per's coding region fused in frame to the bacterial lacZ gene. The fusion proteins contain either the N-terminal half (SG), the N-terminal-two-thirds (BG), or nearly all (XLG) of the PER protein. All constructs lead to reporter signals only in the known per-expressing cell types within the anterior CNS and PNS. Whereas the staining intensity of SG flies is constantly high at different Zeitgeber times, the in situ signals cycle in BG and XLG flies, with approximately 24 hr periodicity in the Per-expressing brain cells in wild-type and per01 loss of function flies. Despite the rhythmic fusion-gene expression within the relevant neurons of per01 BG flies, their locomotor activity in light/dark cycling conditions and in constant darkness is identical to that of per01 controls, uncoupling protein cycling from rhythmic behavior. The XLG construct restores weak behavioral rhythmicity to (otherwise) per01 flies, indicating that the C-terminal third of PER (missing in BG) is necessary to fulfill the biological function of this clock protein (Stanewsky, 1997b).

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

takeout (to) is a Drosophila circadian clock-regulated output gene, a transcriptional target of the central clock. The Takeout amino acid sequence shows similarity to two ligand binding proteins, including juvenile hormone binding protein. Takeout mRNA is expressed in the head and the cardia, crop, and >antennae —- structures related to feeding. to expression is induced by starvation, which is blocked in all arrhythmic central clock mutants, suggesting a direct molecular link between the circadian clock and the feeding/starvation response. A takeout mutant has aberrant locomotor activity and dies rapidly in response to starvation, indicating a link between locomotor activity, survival, and food status. It is proposed that takeout participates in a novel circadian pathway target that conveys temporal and food status information to feeding-relevant metabolisms and activities (Sarov-Blat, 2000).

TO mRNA expression is down-regulated in cyc01 flies and in all other circadian mutants tested. Its level is undetectable in cyc01 and Clkjrk mutants, as measured by RNase protection and Northern blotting. In contrast, there is detectable TO mRNA in all other genotypes tested, though it is substantially lower than that in wild-type flies. Since there is little or no functional CLK-CYC heterodimer in the cyc01 and Clkjrk backgrounds, the simplest way to explain this observation is that to is directly regulated by CLK and CYC. The higher to transcription in per01, tim01, and per01;tim01 double-mutant flies is presumably due to residual functional CLK-CYC heterodimer in these backgrounds. per01 flies reproducibly show a higher level of to expression than tim01, indicating that Per and Tim may differentially regulate to expression. However, the mechanism underlying this difference is still unknown. When mRNA levels at different time points are measured, to does not show a significant cycling pattern in the clock mutants tested (So, 2000).

The to promoter sequence reveals a remarkable sequence identity with the E-box region of the per and tim promoters. In particular, there is a 9-bp sequence identity around this E-box sequence. The other E-box sequences known in circadian genes usually share the 6-bp core sequence or the core sequence with an additional A (CACGTGA), which has been shown to be strongly preferred by the mammalian BMAL1-MOP4 bHLH-PAS transcription factor heterodimer. In fact, the to and per promoters share 13 out of the 18 bp shown to be sufficient to drive transcriptional activation in S2 cells. This is also consistent with the fact that TO mRNA is undetectable in cyc01 and Clkjrk mutants, suggesting that Clk-Cyc regulates to transcription directly. This would be similar to per and tim transcriptional regulation, despite the phase difference. Neverless, extensive investigatio of the to promoter suggests that to transcription requires factors other than Clk and Cyc (So, 2000).

The Drosophila double-time (dbt) gene, which encodes a protein similar to vertebrate epsilon and delta isoforms of casein kinase I, is essential for circadian rhythmicity because it regulates the phosphorylation and stability of period (per) protein. In this study, the circadian phenotype of a short-period dbt mutant allele (dbtS) was examined. The present study shows that dbt affects posttranscriptional regulation of Per at the level of nuclear accumulation, in addition to the previously demonstrated effects on cytoplasmic stability. dbt therefore affects multiple aspects of the Per temporal program, and it is possible that further analysis will reveal additional aspects of clock biochemistry that are regulated by dbt. The circadian period of the dbtS locomotor activity rhythm varies little when tested at constant temperatures ranging from 20° to 29°C. However, perL;dbtS flies exhibit a lack of temperature compensation like that of the long-period mutant (perL) flies. Light-pulse phase-response curves were obtained for wild-type, the short-period (perS), and dbtS genotypes. For the perS and dbtS genotypes, phase changes are larger than those for wild-type flies, the transition period from delays to advances is shorter, and the light-insensitive period is shorter. Immunohistochemical analysis of per protein levels has demonstrated that per protein accumulates in photoreceptor nuclei later in dbtS than in wild-type and perS flies, and that it declines to lower levels in nuclei of dbtS flies than in nuclei of wild-type flies. Immunoblot analysis of per protein levels has demonstrated that total per protein accumulation in dbtS heads is neither delayed nor reduced, whereas RNase protection analysis has demonstrated that per mRNA accumulates later and declines sooner in dbtS heads than in wild-type heads. These results suggest that dbt can regulate the feedback of per protein on its mRNA by delaying the time at which it is translocated to nuclei and altering the level of nuclear Per during the declining phase of the cycle (Bao, 2001).

Splicing of the period gene 3'-terminal intron is regulated by light, circadian clock factors, and phospholipase C

The daily timing of circadian (congruent with 24-h) controlled activity in many animals exhibits seasonal adjustments, responding to changes in photoperiod (day length) and temperature. In Drosophila, splicing of an intron in the 3' untranslated region of the period (per) mRNA is enhanced at cold temperatures, leading to more rapid daily increases in per transcript levels and earlier 'evening' activity. Daily fluctuations in the splicing of this intron (herein referred to as dmpi8) are regulated by the clock in a manner that depends on the photoperiod (day length) and temperature. Shortening the photoperiod enhances dmpi8 splicing and advances its cycle, whereas the amplitude of the clock-regulated daytime decline in splicing increases as temperatures rise. This suggests that at elevated temperatures the clock has a more pronounced role in maintaining low splicing during the day, a mechanism that likely minimizes the deleterious effects of daytime heat on the flies by favoring nocturnal activity during warm days. Light also has acute inhibitory effects, rapidly decreasing the proportion of dmpi8-spliced per transcript, a response that does not require a functional clock. The results identify a novel nonphotic role for phospholipase C (no-receptor-potential-A) in the temperature regulation of dmpi8 splicing (Majercak, 2004).

Seasonal behavior in Drosophila melanogaster requires the photoreceptors, the circadian clock, and phospholipase C

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Pigment dispersing factor-dependent and -independent circadian locomotor behavioral rhythms

Circadian pacemaker circuits consist of ensembles of neurons, each expressing molecular oscillations, but how circuit-wide coordination of multiple oscillators regulates rhythmic physiological and behavioral outputs remains an open question. To investigate the relationship between the pattern of oscillator phase throughout the circadian pacemaker circuit and locomotor activity rhythms in Drosophila, the electrical activity and pigment dispersing factor (PDF) levels of the lateral ventral neurons (LNv) were perturbed, and their combinatorial effect on molecular oscillations was assayed in different parts of the circuit and on locomotor activity behavior. Altered electrical activity of PDF-expressing LNv causes initial behavioral arrhythmicity followed by gradual long-term emergence of two concurrent short- and long-period circadian behavioral activity bouts in ~60% of flies. Initial desynchrony of circuit-wide molecular oscillations is followed by the emergence of a novel pattern of period (PER) synchrony whereby two subgroups of dorsal neurons (DN1 and DN2) exhibit PER oscillation peaks coinciding with two activity bouts, whereas other neuronal subgroups exhibit a single PER peak coinciding with one of the two activity bouts. The emergence of this novel pattern of circuit-wide oscillator synchrony is not accompanied by concurrent change in the electrical activity of the LNv. In PDF-null flies, altered electrical activity of LNv drives a short-period circadian activity bout only, indicating that PDF-independent factors underlie the short-period circadian activity component and that the long-period circadian component is PDF-dependent. Thus, polyrhythmic behavioral patterns in electrically manipulated flies are regulated by circuit-wide coordination of molecular oscillations and electrical activity of LNv via PDF-dependent and -independent factors (Sheeba, 2008).

The period values of the short and long-period activity bouts (~22.5 and ~25 h) observed in these studies closely match those reported earlier for several clock mutants of Drosophila. The fact that robust multiple behavioral rhythms are observed in numerous mutant backgrounds strongly suggests that homeostatic behavioral resynchrony to period values of ~22.5 and ~25 h is a circuit-level constraint rather than a property of any single subgroup of clock neurons. Nevertheless, it is noted that the two periods that emerge in the mutant cryb under dim LL undergo continuous and opposite changes in period length with increasing light intensity. Thus, it is likely that inputs perceived by individual components of the pacemaker circuit may differentially influence components of the circuit during the intermediary or transition states, whereas the steady state period is determined by the entire network. Although many studies have proposed the dominance of the sLNv in determining the free-running rhythm in DD, previous reports suggest that the molecular oscillations in DN1 neurons play an important role in determining rhythmic activity/rest behavior both under DD and LL conditions. The results of a study which manipulated molecular oscillations in circadian pacemaker circuit by targeted expression of Shaggy (Sgg; a clock component whose overexpression speeds up the oscillations in mRNA of circadian genes) suggests that whereas sLNv, LNd, DN1, and DN3 cells are part of a circuit that regulates locomotor activity rhythm, the lLNv and DN2 cells form a separate and independent circuit that apparently does not influence locomotor activity rhythm and that the DN2 cells are the dominant component of this second circuit and regulate the oscillations in the lLNv (Sheeba, 2008).

Is there a clear association with the phase of molecular oscillation in any of the circadian pacemaker neurons with the phase of behavioral locomotor activity? Under LD, wild-type flies show two peaks in locomotor activity, yet, all known pacemakers exhibit a synchronized phase of Per oscillation that peaks coincident with the morning peak in activity. Wild-type flies when subjected to DD show a single peak in activity (apparently a derivative of the evening peak), and the peak in Per levels of all cells remain synchronized for the first few days, occurring at the trough of behavioral activity. After ~5 d in DD, the phase of peak Per levels appears to drift apart among the members of the circuit, with DN2 cells being out-of-phase, as determined by sampling at 12 h or 6 h intervals. When Per is overexpressed in the pacemaker circuit using the tim-GAL4 driver, it is possible to induce a robust single-period rhythm under LL in >90% of flies. In this case, molecular oscillation appears to persist in DN1 up to the fourth day in LL as quantified by cell counts based on PDP-1 staining. The peak in PDP-1-immunopositive cell numbers coincides with falling levels of locomotor activity (although not the trough). Two simultaneously occurring behavioral rhythms are seen in LL in cryb mutants, with increasing fraction of flies showing polyrhythmic locomotor behavior with increasing light intensity. Per levels in the PDF positive sLNv and the PDF negative fifth sLNv are antiphasic as determined by sampling at time points (12 h intervals) corresponding to the peak activity of each of the two polyrhythmic bouts in LL in cryb mutants and a subset of LNd appears to be in phase with the PDF negative fifth sLNv and has high Per levels during one of two activity peaks (the short-period bout apparently derived from the LD morning peak). For flies expressing voltage-gated sodium channel (NaChBac) in the LNv, the phase distribution of Per cycling between the different pacemaker neuronal subgroups shows an antiphasic relationship between the PDF-positive and PDF-negative subsets of sLNv, but is opposite to that seen in cryb flies under LL (sampled at ~6 h intervals). Furthermore, two peaks in PER levels are seen in DN1 and DN2 and also a clear oscillation in LNd that is not seen in controls or any other study in prolonged DD. Thus, the association between the short-period and long-period activity bouts with the PDF positive and negative sLNv is labile and is highly dependent on environmental or intercellular signals. Furthermore, the polyrhythmicity in behavior and novel pattern of synchrony among the different members of the circadian pacemaker circuit in NaChBac flies appears to be a result of the alteration of electrophysiological properties in either the sLNv, lLNv, or both types of LNv (Sheeba, 2008).

To summarize what has been observed for the temporal relationship between clock cycling in the pacemaker neurons and overt locomotor behavior, from these results and those from previous studies, it is concluded that there is no absolute relationship between activity level and the phase of Per cycling in sLNv. This is particularly clear when comparing the experimental conditions which lead to two behavioral activity peaks, including the morning and evening bouts for wild type flies in LD; and polyrhythmic bouts seen for LL-cryb; DD-NaChBac expressed in the LNv. Furthermore, it is proposed that the relationship between the pattern of molecular oscillations among the different neuronal subgroups and rhythmic activity rest behavior in the absence of external time cues under DD and LL is likely to be a result of continuous recalibration of signals being perceived by members of the pacemaker circuit. When these signals are below a certain threshold the circuit remains fairly tightly synchronized in terms of the molecular oscillation of clock proteins. Whereas when membrane electrical properties of parts of the circuit are altered to produce higher-than-threshold signals, molecular oscillations in the circuit are rendered asynchronous along with a loss of temporally regulated behavioral activity (Sheeba, 2008).

The circuit gradually recovers from perturbations applied to parts of the network via circuit-wide plasticity as demonstrated by the emergence of novel behavior pattern with novel consolidated activity patterns and synchrony in molecular oscillation in different subgroups of the circuit. The electrically perturbed LNv do not recover their normal pattern of spontaneous action potential firing. Previous work shows that current injection evokes firing in otherwise silent large LNv. The absence of spontaneous firing in this previous study is probably caused by technical differences in the recording procedures. The results of the current study provide empirical evidence for the idea that gradual resynchrony among a large number of constituent oscillators could account for initial arrhythmicity and subsequent emergence of rhythmic behavior seen in animals when exposed to LL. More recent studies in mammals indicate a mechanism for plasticity in neural networks and outputs involving emergence of a coherent oscillation from previously asynchronous oscillator cells in sub areas of SCN when animals are transferred to DD from LL. In summary, the adult circadian pacemaker circuit exhibits electrical-activity dependent circuit-level plasticity of oscillators as shown by the reorganization of molecular oscillation and rhythmic activity/rest behavior. Electrophysiological studies of pacemaker cells in the accessory medulla of cockroaches has led to the hypothesis that pacemaker cells are organized into assemblies with distinct phases which are synchronized by neurotransmitters such as PDF and GABA. In Drosophila this study shows clear evidence for the existence of PDF-independent factors that contribute toward the regulation of circadian activity/rest rhythm also suggesting the existence of compensatory/redundant mechanisms in the pacemaker neuronal circuit. It is hypothesized that the emergence of rhythmic activity due to NaChBac expression using pdfGAL4 driver in pdf01 flies may cause the rhythmic release of yet unknown neurotransmitters that are otherwise released at lower levels than would be necessary to elicit robust rhythmic behavior. Electrophysiological measurements of NaChBac expressing flies have shown that lLNv show giant action potentials, which may likely trigger downstream neurons to release, signals that can compensate for the lack of PDF. Together, the results of the current experiments along with those of others that have examined the relationship between activity and the pattern of phase distribution among the different pacemaker components suggests that different genetic manipulations and environmental conditions place the architecture of the pacemaker circuit to unique steady states, each of which is able to use different components of the circadian pacemaker circuit to generate distinct circadian patterns in behavior (Sheeba, 2008).

The novel gene twenty-four defines a critical translational step in the Drosophila clock

Daily oscillations of gene expression underlie circadian behaviours in multicellular organisms. While attention has been focused on transcriptional and post-translational mechanisms, other post-transcriptional modes have been less clearly delineated. This study reports mutants of a novel Drosophila gene twenty-four (tyf; CG4857) that show weak behavioural rhythms. Weak rhythms are accompanied by marked reductions in the levels of the clock protein Period (Per) as well as more modest effects on Timeless (Tim). Nonetheless, Per induction in pacemaker neurons can rescue tyf mutant rhythms. Tyf associates with a 5'-cap-binding complex, poly(A)-binding protein (PABP), as well as per and tim transcripts. Furthermore, Tyf activates reporter expression when tethered to reporter messenger RNA even in vitro. Taken together, these data indicate that Tyf potently activates Per translation in pacemaker neurons to sustain robust rhythms, revealing a new and important role for translational control in the Drosophila circadian clock (Lim, 2011).

It remains unclear how TYF controls translation of its target RNAs. Specific effects on Per and Tim were observed but not on other clock components, and Tyf was found to interact with translation components such as the eIF4E-containing cap-binding complex and PABP. It is proposed that RNA-binding translational repressors associate with newly transcribed per RNA, temporarily postpone translation and thus delay Per feedback repression on its own transcription. Such a delay could contribute to the observed lag between protein and RNA particularly in pacemaker neurons, although post-translational mechanisms may also contribute, at least in the eyes. Tyf, which does not have a known RNA-recognition motif, could then be recruited to target transcripts by these translational repressors, releasing them to stimulate initiation of per translation. It has not been possible to biochemically or genetically link Tyf to RNA-binding proteins FMR, LARK, or the translation regulator Thor/4E-BP, which have been shown to contribute to circadian clock function. Nonetheless, TYF association with eIF4E and their similar polysome profiles implicate TYF as a novel translation initiation factor. In addition, the effects of TYF may be more evident on poorly adenylated transcripts on the basis of in vitro data. Of note, the Drosophila homologue of the clock-regulated deadenylase nocturnin has been shown to be important in dorsal neurons for circadian light responses but neither a LN function nor an RNA target has been described. Nevertheless, unique features of Tyf-regulated transcripts may mediate the highly selective TYF effects on clock components in vivo (Lim, 2011).

Post-transcriptional regulation on per RNA has been considered to be modulatory to clock function. The identification of a critical role for Tyf highlights an important role for Per translation in the Drosophila neural clockwork. It will be of interest to determine if proteins functionally analogous to Tyf serve similarly important and specific functions in the mammalian clock (Lim, 2011).

The circadian clock gates the intestinal stem cell regenerative state

The intestine has evolved under constant environmental stresses, because an animal may ingest harmful pathogens or chemicals at any time during its lifespan. Following damage, intestinal stem cells (ISCs) regenerate the intestine by proliferating to replace dying cells. ISCs from diverse animals are remarkably similar, and the Wnt, Notch, and Hippo signaling pathways, important regulators of mammalian ISCs, are conserved from flies to humans. Unexpectedly, this study identified the transcription factor Period, a component of the circadian clock, as critical for regeneration, which itself follows a circadian rhythm. Hundreds of transcripts were found that are regulated by the clock during intestinal regeneration, including components of stress response and regeneration pathways. Disruption of clock components leads to arrhythmic ISC divisions, revealing their underappreciated role in the healing process (Karpowicz, 2013).

Circadian pathway mutants are viable and their cells readily proliferate during development. Unlike other tissues, cell-cycle regulators do not seem to be clock targets in the intestine. Although they are readily detected, neither cyclins nor regulators such as Wee1 exhibit circadian rhythms in this tissue. In the absence of acute damage, clock mutant ISCs divide normally and have no ISC-autonomous phenotypes. So it is quite surprising that PER and CYC are critical for adult ISC division during regeneration (Karpowicz, 2013).

The ISC-autonomous phenotypes that occur during regeneration are modest compared with those that arise when the clock is disrupted systemically or in all ISCs/ECs by RNAi. This suggests that the clock predominantly regulates nonautonomous functions and may be involved in the synchronization of cell states across this tissue during the damage response. Indeed, because esg-Gal4 is expressed in both ISCs and their immediate progeny (enteroblasts or EBs) for some time while they differentiate, it is possible that the clock regulates EB-to-ISC signaling. Intriguingly, disruption of the circadian clock in different cells leads to the accumulation of ISCs in different cell states; for instance, the cyc0 mutant stalls during mitosis when CYC is absent systemically, whereas it stalls during G1 if CYC is depleted in all ISCs. This G1 lag explains why cyc RNAi ISCs show reduced mitoses compared with the cyc0 mutant; however, given that the mechanisms underlying these processes are unresolved, it is possible that these differences are due to genetic background. At present, it is thus concluded that rhythmic cell proliferation normally occurs in the damaged intestine and that this is dependent on the clock. it is also noted that forced expression of per or cyc in ISCs is able to partially restore rhythmic divisions in their respective mutant backgrounds, whereas disruption of these genes in only ECs perturbs ISC rhythmic division. This highlights the complexity of clock-regulated processes and suggests that desynchrony between ISCs and their surrounding cells can have different outcomes (Karpowicz, 2013).

Circadian rhythms occur in many intertwined processes, including metabolism, posttranscriptional regulation, and oxidationreduction cycles. The rhythmic expression of Connector of kinase to AP-1 (Cka), which brings together kinases and transcription factors to transduce JNK signal, and Ipk2, an Inositol polyphosphate kinase that may boost the activity of cytokines involved in regeneration, suggests that the clock sensitizes the intestine to engage the regenerative response at specific times. For instance, several of the genes that exhibit circadian rhythms during regeneration also show these rhythms prior to damage. An emergent function of the clock could be to coordinate stem cell states according to either local niche signals or systemic signals, each of which would be under autonomous circadian control (Karpowicz, 2013).

Although per mutation increases cancer incidence and cancer cell proliferation, the current work suggests it is not simply a tumor suppressor. Recently, the circadian clock was shown to influence mammalian blood and hair stem cell biology. In particular, hair stem cells are strikingly heterogenous in their circadian rhythm activity, for unknown reasons. The coordination of proliferation, by synchronizing internal with external rhythms, may thus represent an important difference between normal stem cells and neoplastic cells (Karpowicz, 2013).

period: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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