Casein kinase 1 promotes synchrony of the circadian clock network.

Casein Kinase 1 Promotes Synchrony of the Circadian Clock Network Department of Neuroscience, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USAa; Howard Hughes Medical Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USAb

Casein kinase 1, known as DOUBLETIME (DBT) in Drosophila melanogaster, is a critical component of the circadian clock that phosphorylates and promotes degradation of the PERIOD (PER) protein. However, other functions of DBT in circadian regulation are not clear, in part because severe reduction of dbt causes preadult lethality. Here we report the molecular and behavioral phenotype of a viable dbtEY02910 loss-of-function mutant. We found that DBT protein levels are dramatically reduced in adult dbtEY02910 flies, and the majority of mutant flies display arrhythmic behavior, with a few showing weak, long-period (⬃32 h) rhythms. Peak phosphorylation of PER is delayed, and both hyper- and hypophosphorylated forms of the PER and CLOCK proteins are present throughout the day. In addition, molecular oscillations of the circadian clock are dampened. In the central brain, PER and TIM expression is heterogeneous and decoupled in the canonical clock neurons of the dbtEY02910 mutants. We also report an interaction between dbt and the signaling pathway involving pigment dispersing factor (PDF), a synchronizing peptide in the clock network. These data thus demonstrate that overall reduction of DBT causes long and arrhythmic behavior, and they reveal an unexpected role of DBT in promoting synchrony of the circadian clock network. n Drosophila melanogaster, daily rhythmic behavior is governed by a network of ⬃150 brain clock neurons (1, 2), each of which contains a molecular oscillator (3). Within this oscillator, the CLOCK (CLK) and CYCLE (CYC) proteins activate transcription of the repressor genes period (per) and timeless (tim) during the day, and accumulated PER and TIM proteins repress the transcriptional activity of CLK-CYC in the late night/early morning. Phosphorylation of clock proteins, which is mainly regulated by casein kinases (CK1 and CK2) (4–6), glycogen synthase kinase 3 beta (GSK3␤) (7), and specific protein phosphatases (8, 9), plays a pivotal role in circadian timing, at least in part by regulating the stability and nuclear localization of PER and TIM (3). Robust circadian behavior relies on the coupling of clock neurons within the network. The neuropeptide pigment dispersing factor (PDF) is required for synchrony of the circadian clock circuit and its output (10–12). A similar framework operates in the mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN), where a peptide, vasoactive intestinal polypeptide (VIP), released by some clock cells, synchronizes individual oscillators to generate a coherent output (13, 14). How PDF and VIP synchronize the circadian clock network and its output is unclear. Interestingly, in Ck1εtau hamsters, there is a tissue-specific misalignment of some peripheral clocks with the SCN, suggesting that the tau mutation in CK1 causes a defect in synchronization (15). However, tau appears to be a gain-of-function mutation (16), and the redundancy of CK1 isoforms in mammals makes it difficult to address consequences of CK1 loss. In Drosophila, Ck1ε is known as double-time (dbt), and mutations in this gene, such as dbtS, dbtL, and dbtar, produce different effects on circadian rhythms, specifically short periods, long periods, and arrhythmia, respectively. However, on a molecular level, they all produce relatively normal levels of protein with reduced overall kinase activity (4, 17–21). It is possible that allele-specific noncatalytic functions of DBT contribute to different circadian phenotypes. Overexpression of kinase-dead DBT results in cytoplasmic localization of PER in some cells but nuclear localization in others, raising the possibility that DBT plays a role in synchronizing PER expression (22). A severe hypomorphic mutation,

dbtP, causes pupal lethality (4, 23), thus precluding analysis of adult flies. Here, we report the adult circadian phenotype of a new dbt allele that dramatically reduces protein levels but is viable. In addition to robust behavioral and molecular phenotypes, dbt hypomorphs show heterogeneous expression of PER expression in the central brain, among different groups of canonical clock neurons, and also within each group. TIM expression is also heterogeneous and decoupled from PER. These data thus uncover an unexpected role of DBT in synchronizing the circadian clock network.

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Molecular and Cellular Biology

MATERIALS AND METHODS Drosophila genetics. A w1118 or y1, w1 genetic background was used in most experiments. The P[EPgy2] insertion allele of dbtEY02910 was obtained from the Bloomington stock center, outcrossed into an isogenic w1118 background for 4 generations, and balanced over TM6, Sb to establish a stock. perS, dbtS, dbtL, dbtAR, dbtP, tim01, Pdfr5304, cry-Gal4, and tim-Gal4 lines were described previously (1, 4, 20, 24–27). To generate the upstream activation sequence (UAS)-dbt transgenes, the corresponding coding sequences of dbt were inserted into a pUAS-V5 vector. We hypothesized that cytoplasmic DBT would more likely interact with PDF signaling, so we added a nuclear export sequence at the N terminus of dbt. However, we do not know if this transgene is expressed exclusively in the cytoplasm. P-element transformation was performed by Rainbow Transgenic Flies Inc. (Camarillo, CA). Behavioral assays. Flies were maintained on standard Drosophila food containing cornmeal, molasses, and yeast. Five-day-old male flies were entrained to a 12-h/12-h light/dark (LD) cycle at 25°C for 3 days and then loaded into locomotor assay tubes containing 5% sucrose and 2% agar. Activity records were collected in 1-min bins in the DAM system (TriKinetics, Waltham, MA) for more than 7 days and analyzed using the

Received 2 December 2013 Returned for modification 3 January 2014 Accepted 5 May 2014 Published ahead of print 12 May 2014 Address correspondence to Amita Sehgal, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/MCB.01571-13

July 2014 Volume 34 Number 14

Downloaded from http://mcb.asm.org/ on December 31, 2014 by UZH Hauptbibliothek / Zentralbibliothek Zuerich

Xiangzhong Zheng,a Mallory Sowcik,b Dechun Chen,a Amita Sehgala,b

CK1 Synchronizes the Circadian Clock Network


pig or -mouse, and Cy5–anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1.5 h at room temperature, followed by extensive washes in PBS. Samples were mounted on slides with Vectashield mounting medium (Vector Laboratories, CA). Primary antibody dilutions were as follows: PER (GP1140), 1:1,500; PDF (C7; Developmental Studies Hybridoma Bank), 1:1,000; TIM (UPR41), 1:1,000. Secondary antibody dilutions were 1:1,000. Fluorescent images were taken using a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany). For cell counting, samples were visually examined under the fluorescence microscope, and PER- and TIM-positive neurons were arbitrarily scored (0 to 5) for fluorescence intensity. For the analysis of light-dependent degradation of TIM, flies were entrained to an LD cycle for 4 days, and at zeitgeber time 20 (ZT20) (ZT0 is light on and ZT12 is light off) of the fourth LD, one group of flies was exposed to a 10-min light pulse (LP) (2,000 lx) and then returned to the dark for 50 min. Both light-pulsed (LP) and unpulsed (ctl) groups were collected at ZT21 for immunohistochemistry procedures as described above.

RESULTS

Characterization of new dbt loss-of-function allele. We obtained a strain from the Bloomington Stock Center that contains a P[EPgy2] insertion in the 5= untranslated region (UTR) of dbt. The insertion dramatically reduces DBT expression (Fig. 1). However, the DBT protein is detectable when the immunoblots are overexposed (Fig. 1B), indicating that dbtEY02910 is a strong hypomorphic allele. We did not find gross defects of the adult brain, in that PER is expressed in canonical clock neurons in the central brain of this new dbt mutant (Fig. 1D; also see below), and neuronal processes from central clock cells, the ventral lateral neurons, are largely intact. We tested dbtEY02910 for circadian rhythms of locomotor activity and found that under free-running conditions of constant darkness (DD), more than half the mutant flies are arrhythmic, and about 40% display very weak rhythms with a period of 32 h (Fig. 2 and Table 1). A small proportion of mutant flies (6%) retain relatively strong rhythmicity, with a period of ⬃32 h. Since the orientation of the UAS element in the P[EPgy2] transposon allows it to drive transcription of dbt (Fig. 1A), we were able to rescue the behavioral phenotype by tim-Gal4-driven expression of endogenous dbt (Fig. 2A). When heterozygous over other dbt alleles or a wild-type chromosome, this new dbt allele (Table 1) behaves like the dbtP or deficiency alleles (4). Thus, it is a bona fide loss-of-function allele. These data demonstrate that a severe reduction of DBT causes long periods and disrupts behavioral rhythms. Importantly, we found that light-dependent degradation of TIM is largely intact in the mutant flies (Fig. 2B), suggesting that the primary response of the clock to light is unimpaired. Phosphorylation of the PER and CLK proteins is altered in the dbtEY02910 mutants. As noted above, DBT is a critical kinase that phosphorylates PER and CLK, but due to a lack of viable null mutants, most assays of this kinase have relied upon the expression of mutant proteins or deletion of the DBT binding domain in PER (4, 18, 19, 23, 29–31). This new loss-of-function allele allowed us to directly examine the effect of severe reduction of DBT on the PER phosphorylation profile over a 24-h day. We examined molecular oscillations under LD conditions, so the population of flies is maximally synchronized. In wild-type flies, PER is phosphorylated in a cyclic fashion, such that on Western blots of adult head extracts, it appears largely in a hyperphosphorylated, lowmobility form at ZT2 and a hypophosphorylated, high-mobility form at ZT14 (Fig. 3). In dbtEY02910 mutants, the phosphorylation

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Clocklab software program (Actimetrics, Wilmette, IL). For the Pdfr5304 experiments, data from day 4 to day 9 in constant darkness (DD) were used for analysis. Circadian periodicity was determined by ␹2 periodogram analysis, coupled with visual inspection of the actogram. A fly is considered rhythmic when activity is clearly rhythmic in the actogram and ␹2 periodogram analysis reveals a single peak above the 95% confidence line. For those individuals that had a weak rhythmic pattern and/or the periodogram displayed multiple peaks over the 95% confidence line, the strength of the rhythm corresponding to the major peak was determined via a fast Fourier transform (FFT) algorithm. An FFT value of ⬎0.01 was generally considered rhythmic, and an FFT value of 0.004 to 0.01 was scored as weakly rhythmic. Note that these rhythms with very low FFT values are unique to this dbt line, since typically the period of a rhythmic fly has an FFT value of ⬎0.01. Detection of very low strength rhythms may also have been facilitated by our use of smaller bin sizes (1 min) for activity data collection. Quantitative real-time PCR. To maximally synchronize the population of flies, 5-day-old adult flies were maintained in a 12-h/12-h LD cycle at 25°C for 3 days and then collected on dry ice at indicated time points on the third day of LD. Total RNA was isolated from heads by using TRIzol (Invitrogen, Carlsbad, CA), and cDNAs were synthesized by using a highcapacity cDNA Archive kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed using an ABI Prism 7000 sequence detection system using a SYBR green kit (Applied Biosystems), following the manufacturer’s suggestions. Values for clock gene mRNAs were normalized over those of Actin. The dbt primers were designed downstream of the p[EY02910] insertion site. Primer sequences are as follows: Actin-F, 5=-GCGCGGTTACTCTTTCACCA-3=; Actin-R, 5=-ATGTCACGGACG ATTTCACG-3=; per-1803F, 5=-CGTCAATCCATGGTCCCG-3=; per1861R, 5=-CCTGAAAGACGCGATGGTG-3=; Pdp1 RD1401F, 5=-TTTG AACAGCTTGAAAGCGC-3=; Pdp1RD1453R, 5=-GAGATTTCCTGCCT GAGCTGG-3=; Clk-1462F, 5=-TTCTCGATGGTGTTCTCGGTG-3=; Clk1512R, AGTTCGCAAAGCCAACGG; cry-1365F, 5=-GCAGTACGTCCC GGAGTTGA-3=; cry-1417R, 5=-AGGGCTCGTGAACAAATTCCT-3=; dbt-F, 5=-CTCCTCGGGTTCCTCGGAT-3=; dbt-R, 5=-GACGAAAGGAC AGTGCCAGAA-3=. Western blot analysis. To maximally synchronize the population of flies, 5-day-old flies were entrained to 12 h/12 h LD cycles for 3 days and collected at various time points. Fly heads were separated on dry ice for total protein extraction using a passive lysis buffer (Promega, Madison, WI), with the additions of protease inhibitors (Roche) and the phosphatase inhibitor okadaic acid (Sigma, St. Louis, MO). For the larval tissues, third-instar larvae were dissected in phosphate-buffered saline (PBS). SDS loading buffer was added to the whole homogenate, and samples were boiled. For the ␭-phosphatase treatment, okadaic acid was not included in the lysis buffer, and samples were equally divided into untreated and treated groups. ␭-Phosphatase (New England BioLabs, MA) treatment was carried out following the manufacturer’s suggestion. A higher volume was loaded into the gel to compensate for sample dilution. Total proteins were separated through SDS-PAGE, transferred onto a nitrocellulose membrane, and processed for antibody incubation. Primary antibodies rabbit anti-PER (PA1139), rat anti-TIM (UPR41), and guinea pig anti-CLK (GP50) (28) were used at a 1:1,000 dilution. Rat anti-DBT (17) was used at a 1:3,000 dilution. Mouse anti-Armadillo (Developmental Studies Hybridoma Bank, University of Iowa) was used to control the loading of total proteins. Following enhanced chemiluminescence, blots were exposed to X-ray film, and images were processed in the software program Adobe Photoshop (Adobe Systems Inc., San Jose, CA). Immunohistochemistry and microscopy. Brains were dissected in fresh 4% paraformaldehyde (in PBS) and fixed for 20 min at indicated time points, washed for 1 h in PBS buffer, and incubated with primary antibodies (in PBS buffer with 3% normal donkey serum and 0.3% Triton X-100) overnight at 4°C. Following 3 washes of 30 min each at room temperature, brain samples were incubated with Cy3– donkey anti-rat (or -guinea pig), fluorescein isothiocyanate (FITC)– donkey anti-guinea

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profile of PER is delayed, and PER is never present in an exclusively, or largely, hyperphosphorylated form. Even when hyperphosphorylated forms are seen (at ZT6 and ZT10), a large amount of the PER protein still appears in a hypophosphorylated state. These observations indicate that a severe reduction of DBT mainly affects the synchronized phosphorylation of the PER proteins over the course of the day. Levels of PER are also higher in dbtEY02910 mutants, which is consistent with the role of DBT in promoting the degradation of PER (4). However, overall levels of PER, as well as its phosphorylation status, still cycle in mutant flies, albeit with a much lower amplitude than they do in wild-type flies. Since a decrease in DBT stabilizes PER, enhancing PER degradation in a dbt mutant background might be expected to rescue the phenotype. Indeed, it was previously reported that short period alleles of per can rescue the arrhythmic behavior of the dbtAR allele (20). Accelerated degradation of nuclear PERT (or PERS) proteins (32–34) apparently restores PER turnover in the dbtAR background, which would explain the rescue of rhythmicity. Interestingly, the perS allele did not rescue the arrhythmicity of dbtEY02910, even though it shortened the period of a few weakly rhythmic flies (Table 1). These data indicate that severe reduction of DBT does not just stabilize PER but causes additional defects in the clock network (see below). In wild-type flies, levels of CLK do not cycle over the course of a day, but its phosphorylation status displays a rhythmic pattern,

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such that maximal phosphorylation occurs toward the end of the night/beginning of the day (31, 35) (Fig. 3A). In the dbtEY02910 mutants, maximal phosphorylation of CLK is reduced (compare ZT22 between wild type and mutant in Fig. 3A and ZT2 and -24 in 3B). However, considerable hyperphosphorylation of CLK is still observed at these time points, and at other time points CLK phosphorylation in the dbtEY02910 mutants is slightly higher than or equivalent to that in wild-type flies (Fig. 3A). Thus, severe reduction of DBT does not significantly reduce total CLK phosphorylation; rather, it dampens and delays the circadian oscillation of CLK phosphorylation. Such a dampening of molecular rhythms may be due to a weaker oscillator, but it may also reflect desynchrony of individual clocks (see below). Role of DBT in circadian clock gene transcription. The role of DBT in the repression of CLK-mediated transcription is still unclear. While some studies support the idea that DBT regulates transcription indirectly through its effect on PER localization (although the effect of DBT on PER localization is also controversial) (21, 29, 36), others propose that DBT participates directly in transcriptional repression (18, 30). To investigate the effect of severe reduction of DBT on clock gene transcription, we assayed mRNA oscillations of clock genes in adult heads of dbtEY02910 mutants (Fig. 4A and B). We found that peak expression of per and Pdp1ε is reduced and delayed, whereas trough levels are increased, in dbtEY02910 mutants. Daily cycling of Clk and cry expression is abol-



FIG 1 Identification of a new dbt mutant allele. (A) A P[EPgy2] element insertion in the 5= UTR of all isoforms (RA, RB, RC, and RD) of dbt disrupts their expression. A pair of blue triangles denotes the location of quantitative PCR (qPCR) primers. (B) DBT is dramatically reduced in dbtEY02910 mutants. Levels of DBT protein in adult heads are quantified based on 3 Western blots (6 samples in each blot) (right panel). The bottom panel is overexposed to visualize the weak band of DBT in the mutants. The Armadillo (ARM) protein is shown as a loading control. wt, wild type; EY, dbtEY02910. (C) Levels of DBT protein are barely detectable in the larval tissues of dbtEY02910 and dbtP mutants. (D) No gross defect of the circadian clock network is observed in the central brain of dbtEY02910 mutants. PDF projections from the s-LNvs and l-LNvs are intact and dorsal neuron groups are present in dbtEY02910 mutants. Green, PDF; red, TIM (ZT21).


ished, with intermediate levels of both transcripts throughout the day. As in the case of the Western blot analysis described above, these RNA assays of adult heads largely report clock gene expression in the compound eye (37, 38), which constitutes a peripheral oscillator. We infer that the peripheral clock in the eye is dampened in dbtEY02910 mutants. It was previously reported that PER is a strong repressor of CLK-mediated transcriptional activity in the dbtAR mutants in the absence of TIM (21). Those experiments could not address the role of DBT in PER-mediated transcriptional repression, since DBTAR is expressed at levels similar to those of wild-type DBT and


so may have contributed to this repression. To evaluate the role of DBT in repression by PER, we conducted experiments similar to those reported by Cyran et al. (2005) (21) but using the dbtEY02910 mutant. Thus, we crossed dbtEY02910 into a tim01 null (25, 39) background. Because PER levels are very low in tim01 flies, there is little repression of CLK transcriptional activity. Thus, the CLK target Pdp1ε is expressed at close to peak levels in tim01 flies (Fig. 4D). We found that Pdp1ε and vri mRNA levels are only slightly reduced in the tim01; dbtEY02910 double mutants. Thus, stabilization of PER in the double mutants is not sufficient to repress Pdp1ε and vri expression, which suggests a role for DBT. Yet an-

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FIG 2 Behavioral rhythms are disrupted in dbtEY02910 mutants. (A) 5-day-old adult males were entrained to 3 light/dark (LD) cycles and released into constant darkness (DD) for 7 days. The majority of dbtEY02910 mutants (lower panel) display arrhythmic behavior in DD; about 40% of the mutants exhibit weak rhythms with a period of ⬃32 h. This behavioral phenotype can be rescued by expression of endogenous dbt (trapped by the EY insertion) driven by tim-Gal4 (TG); R, rhythmic; WR, weakly rhythmic; AR, arrhythmic. (B) Light-dependent degradation of TIM protein is intact in dbtEY02910 mutants. (Left panels) compared to unpulsed controls (ctl), TIM levels are dramatically reduced by a 10-min light pulse (LP). (Right panels) TIM is dramatically reduced in both wild types and mutants at ZT02 compared to results at CT02.

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TABLE 1 Behavioral analysis for dbt mutants

w1118/Y (iso31) w1118/Y;; dbtEY02910/⫹ w1118/Y;; dbtEY02910 w1118/Y; tim-Gal4/⫹; dbtEY02910 w1118/Y;; dbtEY02910/dbtL w1118/Y;; dbtEY02910/dbtS w1118/Y;; dbtAR/⫹ w1118/Y;; dbtEY02910/dbtAR y1, w1, perS/Y;; dbtEY02910/⫹ y1, w1, perS/Y;; dbtEY02910 Pdfr5304/Y; ⫹; ⫹ Pdfr5304, cry-Gal4/Y; UAS-dbtwt/⫹ Pdfr5304, cry-Gal4/Y; UAS-dbtAR/⫹d

28 23 62 52 13 6 21 37 21 37 30 26 24

AR flies (%)

55 8

46 86 23 23 13

R fliesb

WR fliesc

% flies

Period

FFT value

100 100 6 90 92 100 100 16 100

23.8 ⫾ 0.06 23.9 ⫾ 0.04 31.8 ⫾ 0.45 24.6 ⫾ 0.06 25.6 ⫾ 0.13 19.3 ⫾ 0.09 27.6 ⫾ 0.07 37.3 ⫾ 0.92 19.5 ⫾ 0.06

0.059 ⫾ 0.009 0.051 ⫾ 0.004 0.015 ⫾ 0.001 0.019 ⫾ 0.002 0.037 ⫾ 0.005 0.078 ⫾ 0.013 0.051 ⫾ 0.009 0.017 ⫾ 0.003 0.054 ⫾ 0.005

37 38 58

23.7 ⫾ 0.18 23.8 ⫾ 0.17 23.9 ⫾ 0.24

0.022 ⫾ 0.003 0.045 ⫾ 0.009 0.057 ⫾ 0.016

% flies

Period

FFT value

39 10

32.2 ⫾ 0.51 24.9 ⫾ 0.36

0.007 ⫾ 0.001 0.007 ⫾ 0.001

38

36.9 ⫾ 0.65

0.013 ⫾ 0.002

14 40 38 29

27.9 ⫾ 0.35 23.6 ⫾ 0.11 24.2 ⫾ 0.29 23.5 ⫾ 0.55

0.004 ⫾ 0.001 0.007 ⫾ 0.001 0.007 ⫾ 0.001 0.006 ⫾ 0.001

a

Total number of male flies tested (excluding the ones that did not survive for more than 7 days in DD). Percentage of rhythmic flies (R) that have a single significant peak (P ⬍ 0.05) based on the ␹2 periodogram analysis; their respective period and FFT (fast Fourier transformation) values are shown. c Percentage of weakly rhythmic flies (WR) (see Materials and Methods); their respective period and FFT values are also indicated. d Significant difference (P ⬍ 0.001) by ␹2 test when compared to results for Pdfr5304 mutants. b

other CLK target, per, is even slightly increased in tim01; dbtEY02910 double mutants relative to levels in tim01 single mutants (Fig. 4D), again supporting the idea that DBT participates in the feedback repression of per transcription. As discussed later (see Discussion), we believe the effects on Pdp1ε and per are compatible with a role of DBT in repression of CLK-mediated transcription. Heterogeneous expression of PER in clock neurons of dbtEY02910 mutants. In a normal daily cycle, hyperphosphorylation of PER is correlated with maximal nuclear localization in the late night and early morning (39–42). Although phosphorylation of PER at specific sites may promote its nuclear localization, the accumulation

FIG 3 Cyclic phosphorylation of the circadian clock proteins PER and CLK is altered in dbt mutants. (A) Hyperphosphorylation of PER, which occurs in the late night and early morning in wild-type flies, is delayed in dbtEY02910 mutants. Hypophosphorylated forms of both the PER and CLK proteins are present at most times of day, even at times of peak phosphorylation, thus resulting in dampened oscillation of phosphorylation levels. Asterisks indicate nonspecific bands also present in Clkjrk mutants (note that the top nonspecific band is variable from blot to blot). Similar results were obtained from more than 3 experiments. (B) ␭-Phosphatase treatment eliminates the slow-moving bands, demonstrating that the majority of CLK proteins are phosphorylated in both the wild type and dbtEY02910 mutants. Similar results were obtained in two independent experiments.

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of hyperphosphorylated, low-mobility forms of PER appears to be secondary to its nuclear localization (43). The presence of hypoand hyperphosphorylated PER at many time points in dbtEY02910 mutants raised the possibility that nuclear expression of PER is desynchronized in these mutants. To test this idea, we examined PER expression in the central clock neurons at various time points in a daily cycle. In wild-type flies, PER expression peaks and is nuclear in all cell types at the end of the night/beginning of the day, and it is low at the end of the day. In dbtEY02910 mutants, PER expression is variable among clock neurons at most time points (Fig. 5A). Within each group of the canonical clock neurons, namely, ventral lateral neurons (LNvs), dorsolateral neurons (LNds), and dorsal neuron group1 (DN1s), at almost all time points we noticed high levels of PER in some cells but low levels in others. Because of the high variability in expression across cells, we quantified the number of cells that strongly express PER at each time point rather than the intensity of expression. Even with this measure, wild-type neurons show robust daily rhythms of PER expression. In the dbtEY02910 mutant, the large ventral lateral neurons (l-LNvs) and LNds show a trough at ZT18 but mixed expression at other time points. The subcellular localization of PER is likewise variable; indeed, high levels of PER usually correspond to nuclear expression. Strong nuclear PER is present in some small ventral lateral neurons (s-LNvs) and DN1s throughout the day (Fig. 5B). Heterogeneous expression of PER in the s-LNvs is more obvious at ZT16 to ZT17 than at other time points (Fig. 6B). Such prolonged nuclear PER and heterogeneity among clock neurons may account for the weak and long-period behavioral rhythms of some dbtEY02910 flies. PER and TIM expression is decoupled in clock neurons of the dbtEY02910 mutants. To evaluate the role of DBT in promoting synchrony of the clock network in further detail, we examined the temporal expression of PER and TIM in the adult lateral neurons. In general, TIM is low during the daytime, and it undergoes cytoplasm-to-nucleus translocation at night in dbtEY02910 mutants, as it does in wild-type flies. As noted above, PER does not show this cycle in the dbtEY02910 mutants, so the expression of PER and TIM appears to be decoupled in the dbtEY02910 mutant. Interestingly, in



Genotype

No. of fliesa


contrast to the wild type, where TIM accumulates shortly after lights-off (ZT14) in all clock neurons, we found fewer TIM-positive cells in the mutants, suggesting that the underlying phase of TIM accumulation is altered among these cells. In addition, although the general cycling of TIM is maintained in LNvs, we found that at ZT22 (Fig. 6A) and ZT18 (Fig. 6B and D), TIM is expressed in the nucleus in some cells but in the cytoplasm in others. Decoupling between TIM and PER is evident at times when PER is stable and nuclear and TIM is absent (e.g., at ZT8) and also after TIM accumulates. Figure 6 shows that at ZT14, PER is expressed strongly in the nucleus of 4 l-LNvs, whereas TIM is expressed weakly in the cytoplasm; in 2 s-LNvs, PER is expressed in the nucleus, whereas in the other 2 s-LNvs, TIM is cytoplasmic, but there is no detectable PER (Fig. 6A). Although PER is nuclear for the most part in dbtEY02910 mutants, there is also some variability in its localization: it is in the nucleus of some LNvs is but cytoplasmic in other LNvs at ZT16 to ZT17 (Fig. 6B and D). These data suggest that PER and TIM expression in the central clock cells is desynchronized when DBT is severely reduced.


To independently confirm that a severe reduction of DBT is the underlying cause of the phenotype, we used the dbtP mutation. This causes severe reduction of dbt mRNA (23) and results in pupal lethality but allows for clock gene expression analysis in larvae. In wild-type larvae, TIM starts accumulating before PER and is visible at ZT14 in all clock neurons, and both PER and TIM are present in all clock neurons from ZT16 to ZT23. However, as in the adult lateral neurons of dbtEY02910 mutants, we found that PER and TIM expression is heterogeneous and decoupled in the clock neurons of dbtP larval brains (Fig. 7). At most time points, some cells show high expression of PER and low expression of TIM while other cells express low PER and high TIM. Importantly, PER is present in the nucleus in some cells but in the cytoplasm in others from ZT16 to ZT23, and we also found similar coexistence of neurons with cytoplasmic and nuclear expression of TIM from ZT7 to ZT23. Thus, overall reduction of DBT results in heterogeneous expression of PER and in decoupling of PER and TIM in the central pacemaker cells. To further investigate the temporal dynamics of PER and TIM

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FIG 4 Role of DBT in transcriptional regulation. (A) Dampened oscillations of circadian clock gene mRNAs in dbtEY02910 mutants. Peak levels of per and Pdp1ε mRNA levels are reduced and delayed, whereas trough levels are increased. Clk and cry mRNAs are expressed at intermediate levels throughout the day. Data are shown as means ⫾ SEM (n ⫽ 5). The area under the curve (but above the trough point) is significantly smaller for the mutants, as shown in panel B (ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; Student’s t test, n ⫽ 5). (C) While PER is very low in tim01 mutants, it is present in the nucleus of lateral neurons (upper panel) and fat body cells (lower panel) in the tim01; dbtEY02910 double mutants (ZT21). PDF staining (green) labels LNvs. Nuclei are counterstained with 4=,6-diamidino-2-phenylindole (DAPI) (blue). (D) Compared to results for the tim01 single mutant, the per mRNA level is slightly increased in tim01; dbtEY02910 double mutants; on the other hand, Pdp1ε and vri mRNA levels are slightly reduced in the double mutants. For the clockless mutants in which mRNA levels do not cycle, data from ZT2 and ZT14 were pooled. Asterisks indicate significant differences (P ⬍ 0. 05) by Student’s t test (n ⬎ 4).

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of clock neurons in wild-type flies, PER is present in some clock neurons most times of the day in dbt mutants, resulting in an apparent lack of cycling of PER in the central brain. However, at any given time point, PER is strong in some clock neurons but weak in others. This heterogeneity of PER expression is summarized in panel B, which depicts the number of cells in each clock cell group that strongly expresses PER at different times of the day. PER-positive clock neurons were arbitrarily scored (0 to 5) for fluorescence intensity under the microscope, and cells with a score higher than 2.5 were counted as strongly PER positive. About 8 brains were examined for each time point.

in individual clock neurons, we examined the larval dorsal neurons. We found that PER is constantly present in the nucleus in DN1 cells of dbtP larvae in LD cycles (Fig. 8). On the other hand, TIM expression undergoes normal cycling in DN1 cells of mutant larvae (Fig. 8). These data further support the idea that PER and TIM are decoupled in mutants that have a severe reduction of DBT. DBT interacts with the PDF signaling pathway. The strong effect of dbt mutants on the synchrony of the circadian clock net-

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work is reminiscent of the phenotype produced by loss of pigment dispersing factor (PDF), the only known factor that is released from the s-LNvs to synchronize the circadian clock network. In Pdf 01 null flies (44), oscillation of clock genes gradually loses synchrony in the s-LNvs and is also affected in some other clock neurons (10–12, 45). To test if DBT interacts with the PDF signaling pathway, we expressed wild-type and mutant forms of DBT in the Pdfr5304 mutant background. Consistent with previous reports, we found that most Pdfr5304 mutants are arrhythmic or have weak



FIG 5 Heterogeneous expression of PER in adult clock neurons of dbtEY02910 mutants. (A) In contrast to the robust rhythm of PER expression in all three groups


rhythms (26). Interestingly, when UAS-dbtAR expression was driven by cry-Gal4 in most clock cells, it partially restored rhythmicity in the mutant background (Table 1). We note that this genetic interaction does not necessarily indicate a direct interaction between DBT and PDF signaling. Nonetheless, these data suggest that DBT and PDF signaling act on some common pathways to regulate the circadian clock network. DISCUSSION

Despite the well-known function of CK1 in the phosphorylation and subsequent degradation of PER, the molecular mechanism by which CK1 phosphorylates a specific target site is just beginning to be unraveled. In Drosophila, substrate recognition of DBT seems to be temporally and spatially regulated (46, 47). A reduced rate of PER degradation can explain the behavioral phenotypes of dbtAR and dbtL (48) but not of dbtS, although all produce DBT proteins that have overall reduced kinase activity (4, 17, 19, 22, 23, 48). In addition, DBT may play a noncatalytic role in influencing CLKdependent transcription (18). Thus, studies of known dbt mutants have uncovered multiple allele-specific effects on the circadian clock (4, 18, 20, 21, 49). Developmental lethality associated with the dbtP strain, perhaps due to the dbt mutation itself, limited its use in investigating the effect of reduction of DBT on the circadian


clock (4, 23). The bona fide loss-of-function allele, dbtEY02910, that we describe here provides a valuable reagent to address basic questions regarding the circadian function of DBT. Severe reduction of DBT causes long periods and disrupts circadian rhythms. We found that more than half the dbtEY02910 flies are arrhythmic, and about 40% display very weak rhythms with a period of 32 h (Table 1). Since other allelic combinations of dbt show dissociations of period length and arrhythmicity (4, 20, 23) and some ultra-long-period mutants (timUL, 33 h, and perL; timUL, 41 h) are largely rhythmic (50), a long period itself is not necessarily the cause of disrupted rhythms. We suggest that DBT has separate roles in regulating period length and rhythmicity. In dbtEY02910 flies, both effects are probably caused by a general reduction of catalytic activity, because overexpression of a kinasedead DBT protein (K38R mutation) produces similar phenotypes (22). The expression of the DBT protein is reduced by ⬃95% in the dbtEY02910 mutant flies, reminiscent of the dbtP mutant larvae, which show a similar reduction of dbt mRNA levels (23). We were not able to reliably compare DBT protein levels between dbtEY02910 and dbtP mutant larvae due to high background noise. Behaviorally, dbtP seems to be a more severe mutation (20) than dbtEY02910 (Table 1) when tested in trans over the dbtAR allele. Interestingly, the

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FIG 6 Expression of PER and TIM is decoupled in the adult LNvs of dbtEY02910 mutants. (A) In contrast to the synchronized expression of PER and TIM in the lateral neurons of wild-type flies, PER and TIM expression is heterogeneous in the lateral neurons of dbtEY02910 mutants. TIM is low during the light phase, and it still undergoes cytoplasm-to-nucleus translocation in the dbtEY02910 mutants, while PER is largely nuclear at all time points. At ZT14, strong PER and TIM localize to different clock neurons. (B) Similar decoupling is observed at ZT16. Note that PER in the majority of mutant brains at ZT17 is similar to that in ZT16. In a few mutant brains (⬃20%), PER is strongly nuclear in some cells but cytoplasmic in others at ZT17, as shown. More than 8 brains were examined for each time point (A and B), and similar results were obtained from two experiments. This heterogeneity is summarized in panel C, which depicts the number of clock neurons (means ⫾ SEM; n ⬎ 7) with high PER and high TIM (Hper Htim), high PER but low TIM (Hper Ltim), low PER but high TIM (Lper Htim), or low PER and low TIM (Lper Ltim), based on relative levels of intensity. (D) Cellular localization of PER and TIM is less synchronized in dbtEY02910 mutants. Compared to uniform cytoplasmic (ZT16) and nuclear (ZT24) localization of PER and TIM in wild types, PER and TIM are present in the nucleus in some cells but are cytoplasmic in others in dbtEY02910 mutants.

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visible in all LNvs from ZT14 to ZT20. At ZT23, TIM and PER localize to the nucleus in all lateral neurons, and PER expression persists in nuclei to ⬃ZT7. In dbtP mutants, PER is visible in the nucleus of most clock neurons at all time points. However, expression levels are more variable, and cytoplasmic PER is detected in some clock neurons at ZT16 to ZT23. TIM levels are generally lower and also variable in the dbtP mutants. In most cells (ZT16 to ZT23), intensities of PER and TIM are generally decoupled from each other (summarized in panels B and C). Note that TIM is mostly undetectable at ZT01 and ZT07. More than 8 brains were examined for each time point, and similar results were obtained from two independent experiments.

perS mutation does not rescue the arrhythmicity of dbtEY02910mutants (Table 1), although it rescued the arrhythmicity of dbtAR mutants (20). Perhaps DBTAR prevents phosphorylation at specific degradation-promoting sites on PER, and this can be offset by the destabilizing effect of the perS mutation (20, 33). Lack of rescue by perS

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supports the idea that severe reduction of DBT in dbtEY02910 mutants has some effects that are different from those of other dbt alleles, e.g., desynchrony of the clock network (see below). DBT kinase activity regulates nuclear localization of PER in vivo. We found that nuclear localization of PER and TIM in the



FIG 7 Expression of PER and TIM is decoupled in the larval lateral neurons of dbtP mutants. (A) In the wild-type larval brain, cytoplasmic TIM expression is


late night is fairly well coupled in wild-type flies. Although we did not find clear sequential nuclear localization of PER and TIM as previously reported (51), PER was more nuclear than TIM in some cells from ZT16 to ZT18 (Fig. 6). It should be noted that although Shafer et al. (51) reported a higher abundance of TIM in the cytoplasm at ZT16 to ZT20, they also detected TIM in the nucleus at this time (51). The absolute amount of TIM in the nucleus is difficult to quantify, because different antibodies are used to label PER and TIM. In contrast to the situation in the wild type, nuclear localization


of PER and TIM is decoupled in the dbtEY02910 and dbtP mutants. These data suggest that DBT plays an important role in regulating the nuclear localization of PER and TIM. Severe reduction of DBT in dbtEY02910 flies results in predominantly nuclear expression of PER, which is consistent with previous reports showing that PER is constitutively nuclear in clock neurons when its phosphorylation is blocked by phosphatase expression (9) or by reduction of DBT activity (4, 21, 22). In addition, the PER⌬ protein, which lacks the DBT binding domain, is mainly nuclear in clock cells (30). All these data suggest that reduction of DBT activity in-

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FIG 8 TIM cycling persists in DN1 neurons of dbtP larvae. (A) In the dbtP mutants, PER is constantly present in the nucleus, whereas TIM expression is similar to that in wild-type larval brains. This decoupling is summarized in panels B and C. More than 8 brains were examined for each time point, and similar results were obtained from two independent experiments.

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CLK. On the other hand, phosphorylation events critical for repression may not occur in the mutant. DBT promotes coherence of the circadian clock network. We uncovered an unexpected role of DBT in synchronizing the circadian clock network in the central brain. dbtEY02910 mutants show variable numbers of neurons expressing high levels of PER from time point to time point, indicating a loss of coherence in timing. PER is mostly nuclear, perhaps because DBT activity or levels set the gate for nuclear translocation of PER, so in cells with severely reduced DBT, the default state of PER is nuclear. However, we do find coexistence of neurons that express cytoplasmic and nuclear PER at certain time points, particularly at ZT16 to ZT18, the time of PER nuclear translocation in wild-type flies. These data, together with the finding that about 40% of the dbtEY02910 flies display weak behavioral rhythms, indicate that the residual DBT is capable of maintaining a weak molecular clock in some cells. However, the coherence of the network is lost. We note that heterogeneous expression of PER in the early night has also been reported for flies overexpressing the kinase-dead DBTK38R (22). While prolonged nuclear expression of PER likely accounts for the long period, it probably does not account for the weak behavioral rhythms of dbtEY02910. As mentioned above, some extremely long-period mutants maintain stable rhythms without arrhythmicity (20, 50). Furthermore, the dosage effect of DBT on period length and rhythmicity does not seem to be correlated (23) (Table 1). We suggest that the long period of dbtEY02910 mutants derives from effects on clock proteins, while the arrhythmia is attributable, at least in part, to disruption of the circadian circuit. A role of CK1 in maintaining coherence of the circadian timing system has also been proposed in mammals. In Ck1εtau mutants, some peripheral clocks are misaligned with the SCN, suggesting that this gain-offunction mutation affects synchrony of the circadian timing system (15). In addition, loss of the VIP receptor in the SCN desynchronizes clock cells and abolishes oscillations of Per2-luciferase, which can be restored by transiently inhibiting CK1␦ (53). As noted earlier, the Drosophila equivalent of VIP is PDF, which has also been associated with synchrony of the clock network. Indeed, we showed that overexpression of dbtAR by the cryGal4 driver can partially restore rhythmicity to the Pdfr5304 mutants. We hypothesize that dbt is epistatic to Pdfr, such that a severe reduction in DBT levels causes a strong phenotype even in LD, while the effect of Pdfr mutation manifests a weak phenotype over time in DD. We hypothesize that DBT and PDF share some common mechanisms that synchronize the circadian clock network. On the other hand, given that the dbt desynchrony phenotype is manifest in the presence of light/dark cycles, it is possible that dbt mutations affect entrainment of the clock network to light. TIM degradation by light is normal in these mutants, but we cannot exclude the possibility that events downstream of the TIM response are affected and prevent light-mediated synchrony of the circuit. Regardless, the data indicate that changes in CK1 activity, perhaps in response to peptidergic signals including PDF or VIP, mediate communication across the circadian network. Intriguingly, a recent study using the unicellular marine alga Ostreococcus tauri indicated that CK1 activity is circadian gated (54), which could also be the case in higher organisms. ACKNOWLEDGMENTS We thank Jeffery Price, Paul Hardin, Michael Young, and Michael Rosbash for fly stocks and antibodies.



creases nuclear expression of PER in flies. However, in S2 cells, PER is expressed predominantly in the cytoplasm, even when endogenous dbt expression is knocked down by RNA interference (RNAi) (29). It is likely that the lack of circadian oscillation and dynamic interaction of the PER-TIM-DBT proteins (52) in S2 cells accounts for some of the differences. For instance, DBT displays PER-associated rhythmic redistribution between the nucleus and the cytoplasm in wild-type clock neurons, while it is mostly nuclear in per01 (no PER) and tim01 (very low PER) mutants (52). Role of DBT in transcriptional repression of CLK target genes. The relationship between DBT-mediated hyperphosphorylation and the repressor potency of PER is not clear, although one model posits that PER brings DBT to phosphorylate and repress CLK activity (18, 31, 35). Most data for the DBT effect in this model are based upon experiments using S2 cells. As a viable mutant with very little DBT expression, dbtEY02910 provided us with a valuable tool to address the role of DBT in the transcriptional repression of CLK by PER in vivo. In the DBT bridge model (18), DBT recruits some unknown kinase to phosphorylate CLK and suppress its activity. We predicted a dramatic reduction of CLK hyperphosphorylation in the dbtEY02910 mutants. Surprisingly, we found that CLK is still abundantly phosphorylated in the dbtEY02910 mutants (Fig. 3), which suggests that a 1:1 stoichiometric ratio of DBT to CLK is not necessary for CLK hyperphosphorylation. Alternatively, DBT may not be the primary kinase that phosphorylates CLK. Importantly, CLK in the mutant is always expressed as hypo- and hyperphosphorylated forms, and it still has significant transcriptional activity (see below). We found that rhythms of mRNA levels of CLK target genes per and Pdp1ε are dampened, such that peak levels are reduced, whereas trough levels are increased, in the dbtEY02910 mutants. This dampening of clock gene transcription can be simply explained by dampened oscillation of PER and CLK protein levels and phosphorylation states. In order to examine the steady state of clock gene transcription, we analyzed CLK-mediated transcription in the tim01 mutant background. We observed a small increase in per mRNA levels in clockless tim01; dbtEY02910 mutants (compared to those in tim01 single mutants (Fig. 4D). Although we did not detect an increase in other CLK target genes, Pdp1ε and vri, between tim01; dbtEY02910 double and tim01 single mutants (Fig. 4D), these data do not negate a role for DBT in repression (see below). A previous report showed that Pdp1ε and vri are actually dramatically reduced in tim01; dbtAR mutants (compared to levels in tim01 single mutants) (21). Presumably the very stable and nuclear PER protein in the tim01; dbtAR double mutant (in tim0 flies, PER is very unstable) effectively represses transcription of Pdp1ε. However, even though PER is stabilized in the tim01; dbtEY02910 mutant, it does not efficiently repress transcription of per, Pdp1ε, and vri, most likely because the DBT protein itself is required for this effect. Thus, a noncatalytic function of DBT (18), present in DBTAR, is sufficient to repress CLK activity. The role for DBT indicated by the dbtEY02910 mutant is consistent with a previous finding that RNAi knockdown of dbt decreases PER repressor activity (29). The mechanism by which DBT modulates repression is unclear. Given that considerable phosphorylation of CLK still occurs in dbtEY02910 flies, it is possible that phosphorylation is not the trigger and DBT recruits other factors that repress


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This work was supported by a grant, 5R01NS048471, from the NIH to A.S. A.S. is an Investigator of the Howard Hughes Medical Institute. X.Z. was supported by a 2010 NARSAD Young Investigator Award. We declare that we have no competing financial interests.

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The Circadian Clock Mutation Promotes Intestinal Dysbiosis.

Casein kinase 1 promotes initiation of clathrin-mediated endocytosis.

Constant light enhances synchrony among circadian clock cells and promotes behavioral rhythms in VPAC2-signaling deficient mice.

Timing of neuropeptide coupling determines synchrony and entrainment in the mammalian circadian clock.

Tyrosine kinase regulation of a molluscan circadian clock.

Neurodegeneration and the Circadian Clock.

Casein kinase 1ε promotes cell proliferation by regulating mRNA translation.

Physiology. Partitioning the circadian clock.

Pharmacological control of circadian clock. Pharmacological manipulation of the circadian clock: from animal to human studies.

Site-specific phosphorylation of casein kinase 1 δ (CK1δ) regulates its activity towards the circadian regulator PER2.

Light evokes rapid circadian network oscillator desynchrony followed by gradual phase retuning of synchrony.

Assembly of a comprehensive regulatory network for the mammalian circadian clock: a bioinformatics approach.

The Drosophila circadian clock is a variably coupled network of multiple peptidergic units.

A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock.

Lack of Casein Kinase 1 Delta Promotes Genomic Instability - The Accumulation of DNA Damage and Down-Regulation of Checkpoint Kinase 1.

Physiological links of circadian clock and biological clock of aging.

Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1.

ε rapidly delays peripheral clock gene rhythms.

The MAP kinase p38 is part of Drosophila melanogaster's circadian clock.

Mining for novel candidate clock genes in the circadian regulatory network.

Circadian rhythms. Resetting the human clock.

Circadian clock, cancer, and chemotherapy.

Nuclear envelope regulates the circadian clock.

Strategies for resetting the human circadian clock.