Influence of the -dependent circadian on diurnal, circadian, and aperiodic expression in

Yiing Lin*, Mei Han†, Brian Shimada‡, Lin Wang‡, Therese M. Gibler§, Aloka Amarakone†, Tarif A. Awad‡, Gary D. Stormo*, Russell N. Van Gelder§¶, and Paul H. Taghert†ʈ

Departments of *Genetics, †Anatomy and Neurobiology, §Ophthalmology and Visual Sciences, and ¶Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, MO, 63110; and ‡Affymetrix, Santa Clara, CA 95051

Communicated by Robert H. Waterston, Washington University School of Medicine, St. Louis, MO, May 6, 2002 (received for review February 2, 2002) We measured daily gene expression in heads of control and period Materials and Methods mutant Drosophila by using oligonucleotide microarrays. In control Details of fly stocks, fly collections, and microarray target flies, 72 showed diurnal rhythms in light-dark cycles; 22 of preparation are provided in Supporting Materials and Methods, these also oscillated in free-running conditions. The period gene which is published as supporting information on the PNAS web significantly influenced the expression levels of over 600 nonoscil- site, www.pnas.org. lating transcripts. Expression levels of several hundred genes also differed significantly between control flies kept in light-dark ver- Data Analysis. Average difference calls for each gene were sus constant darkness but differed minimally between per01 flies calculated by using Affymetrix MICROARRAY ANALYSIS SUITE kept in the same two conditions. Thus, the period-dependent software. The data on each chip were normalized to the mean circadian clock regulates only a limited set of rhythmically ex- expression of that chip. To classify a gene as having circadian pressed transcripts. Unexpectedly, period regulates basal and expression across two cycles of data, three criteria were applied. light-regulated gene expression to a very broad extent. First, the gene had to be called present by the analysis software in at least half the time points of each data set. Second, orward genetic screens in Drosophila melanogaster have expression was required to be highly reproducible between the Fidentified at least eight genes [period (per), (tim), data sets as assessed by correlation coefficient (12). Finally, if (cyc), clock (Clk), vrille (vri), doubletime, cryptochrome (cry), highly reproducible, the gene needed to show a circadian (as and shaggy] necessary for the normal functioning of the circadian opposed to ultradian) expression pattern by autocorrelation time-keeping system. Null in most of these genes analysis. Further details of the analysis methods are provided in render flies behaviorally arrhythmic in constant conditions, but Supporting Materials and Methods. they otherwise have minimal morphologic phenotype (1). A model for the mechanism by which specific gene products give Results rise to a stable clock mechanism has been formulated over the Analysis of Rhythmic Gene Expression in Control and Period Mutant past 10 years (2, 3). These clock genes appear to function in a Fly Heads. We studied gene expression in control and period null time-delayed -translation loop. A rhyth- mutant (per01) flies in four conditions: (i) control flies kept in LD mically expressed subset of the core clock genes (per, tim, and 12:12 (ii), control flies kept in dark-dark (DD), (iii) per01 flies Clk) and a nonrhythmically expressed core clock gene (cyc) are kept in LD 12:12, and (iv) per01 flies kept in DD (Fig. 1A). We thought to function as the state variables of the oscillator initially compared two replicate time series for each condition. mechanism (4). This model predicts that these core clock genes Control flies had 377 rhythmically expressed genes in LD and also should influence the rhythmic expression of ‘‘output’’ genes 447 rhythmically expressed genes in DD. To estimate the inci- important in regulating physiologic and biologic processes con- dence of false-positive results, we performed data-randomiza- trolled by the circadian clock (5). tion analysis (Fig. 6, which is published as supporting informa- Previous screens for such clock-controlled output genes have tion on the PNAS web site). Approximately 176 genes in LD and yielded varying estimates of their abundance and character in 298 genes in DD are expected to show rhythmic expression by different organisms. An insertional reporter screen in the pho- chance. Thus in control flies in LD conditions, about half tosynthetic prokaryote Synechococcus suggested that most genes (Ϸ176͞377) of the observed, oscillating genes are scored rhyth- in this organism are transcribed in circadian fashion (6). Using microarray analysis, Harmer et al. identified 453 genes under- mic by chance. In the absence of additional data, there are no going rhythmic expression under constant conditions in the plant means to discriminate between true and false-positive results. (7), representing Ϸ6% of the expressed We therefore increased the stringency of our analysis by adding genome. In Drosophila, analysis of 280 expressed sequence tags a third 24-h data set for LD 12:12 control flies. We performed from the fly head revealed 20 diurnally varying transcripts, the three two-set comparisons and found 72 genes that were rhyth- majority of which were extremely rare, long messages of unclear mic in all comparisons (Fig. 1B). Analysis of the randomized data physiologic function (8). The full extent of circadian gene sets demonstrated that, by chance, only one false-positive gene expression is not known in any organism. The recent availability is expected among these 72. The number 72 also provides a lower of an oligonucleotide-based microarray containing probes for limit to the number of detectable, oscillating genes in the fly nearly all known and predicted Drosophila genes allows estima- head. Because our initial estimate from two data sets suggested Ϸ tion of the number of clock-controlled genes in the fly. Here we there were 200 true positive, oscillating genes (i.e., the 377 describe results of measuring circadian gene expression in con- identified genes less the 180 genes that were expected to oscillate trol and period mutant flies in both light-dark (LD) and free- running conditions. While this article was in preparation, three Abbreviations: LD, light-dark; DD, dark-dark. other studies were published describing circadian gene expres- ʈTo whom reprint requests should be addressed at: Department of Anatomy and Neuro- sion in Drosophila heads (9–11). We discuss and reinterpret biology, Box 8108, Washington University Medical School, 660 South Euclid Avenue, St. some of these results in the context of the present analysis. Louis, MO 63110. E-mail: [email protected].

9562–9567 ͉ PNAS ͉ July 9, 2002 ͉ vol. 99 ͉ no. 14 www.pnas.org͞cgi͞doi͞10.1073͞pnas.132269699 Downloaded by guest on September 28, 2021 Fig. 1. (A) Overall experimental design. Numbers in boxes refer to hours of collection for control (yw) and per01 flies; gold numbers indicate times of lights on. (B) Venn-diagram summary of results from control flies. Based on two cycles, 377 circadianly expressed genes were identified in LD, and 447 were identified in DD. Based on three cycles in LD, a subset of 72 genes were identified as diurnally expressed. Twenty-two of these genes were found also to be rhythmic in DD flies.

by chance), we estimate that the number of true oscillating genes in control LD 12:12 fly heads is between 72 and Ϸ200. We also examined two replicate data sets from per01 flies (13) Fig. 2. (A) Phase-ordered expression patterns of the 72 genes identified as diurnally expressed in control flies. The positions of five known rhythmic in LD and DD conditions. We found 287 cycling transcripts in 01 genes of the circadian system are denoted on the Left. Tick marks show the per LD 12:12. By data randomization described above, we positions of those diurnally expressed genes also found to be rhythmic in estimate that 182 of these genes show oscillation in this paradigm control flies kept in DD (B), per01 flies kept in LD 12:12 (C), and per01 flies kept by chance. The probability of all 287 identified genes occurring in DD (D). by chance was less than 1 ϫ 10Ϫ4.Inper01 flies kept in DD conditions, we found 336 cycling transcripts; the probability of all 336 identified genes occurring by chance was greater than in Table 2, which is published as supporting information on the 1 ϫ 10Ϫ2. PNAS web site. The average expression levels of the 22 clock- We analyzed the 72 genes showing rhythmic expression in the driven genes, considering both LD and DD conditions, varied three LD 12:12 data sets in greater detail (Fig. 2). These genes over nearly a 100-fold range (Affymetrix average difference can be categorized further by their persistent oscillations in values from 383 to 25,198). Among these 22 genes, four are constant darkness (22 of the 72 are ‘‘clock-driven’’ genes) or their known components of the circadian clock (per, tim, vri, and Clk). persistent oscillations in the absence of the period gene in the Three previously described rhythmically expressed genes, Dreg-2 presence of an LD cycle (18 of the 72 are ‘‘masked’’ genes; ref. (8), cry (15), and takeout (16) were found in the set of 72 diurnally 14). Four of the 22 clock-driven genes were also expressed rhythmic genes. rhythmically in per01 flies kept in LD 12:12 (i.e., these were both clock-driven and masked). A distinct set of 4 of the 22 clock- Analysis of Basal Gene Expression Differences Between Control and driven genes also were expressed rhythmically in per01 flies kept Period Mutant Flies. We next determined whether the per- in DD. These potentially are clock-independent oscillations and dependent circadian clock or environmental LD conditions included the circadian gene vrille and a cytochrome P450 enzyme affected expression of nonrhythmically expressed genes (Table 3, Cyp4d21. Significantly, no gene was found to be expressed which is published as supporting information on the PNAS web rhythmically in all four conditions tested (control LD and DD site). Initially, we compared basal gene expression levels from 20 and per01 LD and DD). The remaining 32 of 72 oscillating genes control and 12 per01 points from flies kept in DD conditions. We required both the period gene and an LD cycle for their identified 650 genes that showed significant differences between oscillations (i.e., ‘‘period-dependent masking’’). genotypes (P Ͻ 0.0001, Mann–Whitney U test). Randomization Expression waveforms in LD and DD of a representative of the data sets indicated an expected mean false-positive subset of the 22 circadianly expressed genes are shown in Fig. 3. incidence of Ͻ1. A heat-plot rank profile of these genes is shown The range of phases seen in this set of circadian genes includes in Fig. 4A. peaks at all times of day. Interestingly, 15 of the 22 circadian To validate these findings, we analyzed CG3397, which is genes peaked Ϸ4 h earlier in DD versus LD conditions. The detected in per01 but not in control flies, and CG13406, which is identities, average expression levels, LD and DD peak phases, detected in control but not in per01. By reverse transcription– and proposed functions of the 22 clock-driven genes are shown PCR analysis, each gene was detected only in the genotype PHYSIOLOGY in Table 1, and the list of the 72 diurnally rhythmic genes is shown predicted by the microarray experiments (Fig. 5A). We next used

Lin et al. PNAS ͉ July 9, 2002 ͉ vol. 99 ͉ no. 14 ͉ 9563 Downloaded by guest on September 28, 2021 between control flies kept in LD versus DD conditions (P Ͻ 0.0001, Mann–Whitney U test). A rank profile of these genes is shown in Fig. 4B. To determine whether per is necessary for this photic regulation, we examined the differences in basal expres- sion levels between per01 flies kept in LD 12:12 and DD conditions. The basal expression levels of only 15 genes were regulated by LD in the per01 flies. It thus seems that basal, aperiodic levels of expression of many genes in the fly are influenced by period either directly or by per-dependent modu- lation of photic masking effects. Discussion The rhythmic genes we have identified under LD and DD conditions display bone fide rhythmic expression. The inclusion of seven known rhythmic genes (per, tim, vri, Clk, Dreg-2, cry, and takeout) in the set of 72 diurnally oscillating genes and four (per, tim, vri, and Clk) in the set of 22 circadianly oscillating genes provides confidence that the autocorrelation method we applied identifies rhythmic gene expression with fidelity. The true number of circadianly regulated genes in Drosophila can only be estimated; the accuracy of any estimate depends on both methodological constraints and assumptions used in the data analysis. Our estimates of 72–200 robustly oscillating genes in LD 12:12 and a minimum of 22 genes in DD likely underes- timate the true number of oscillating genes in the fly head. First, by necessity the choices of A0 minima were determined empir- ically. In the absence of true positive controls (i.e., rhythmically spiked foreign, polyadenylated message), we relied on the re- producible waveforms of known cycling genes and visual inspec- tion of the records to establish useful minima. Second, current microarray technology does not detect very rare mRNAs. Of the 20 diurnally varying genes identified in the expressed sequence tag screen of Van Gelder et al. (8), at least four were expressed at levels that are undetectable on the present microarray. Similarly, we have used the oligonucleotide microarray to de- termine average expression levels in whole heads. Genes dis- playing rhythmic oscillation in only a small subset of head tissues, but statically expressed elsewhere, might escape detection. Third, although the current generation of oligonucleotide gene arrays includes probes for most known and predicted genes, the arrays remain incomplete. For example, several known rhythmic gene sequences including clock regulated gene-1 (17) and several of the Dregs (8) are not represented on the current microarray. Finally, some rhythmically expressed genes such as Dreg-5 (18) show oscillating expression levels only in certain caging and feeding conditions. Analysis of flies kept in other conditions may yield a different estimate for the number of rhythmically ex- pressed genes. Even considering these caveats, it seems that circadian control of gene expression in Drosophila heads is a limited phenomenon. Our set of 22 circadianly expressed genes includes four of the known clock component genes, per, tim, vri, and Clk. These genes Fig. 3. Representative circadian expression patterns found in control flies have among the highest amplitude oscillations in the fly head under three cycles in LD (Right) and two cycles in DD (Left). (Table 2), and very few other genes have similarly robust oscillations. This finding suggests that circadian control of gene expression in the fly head does not represent an extension of the reverse transcription–PCR to measure the expression levels of autoregulatory feedback loop controlling expression of the core these two genes in three other clock gene mutants (Clk, tim, and clock genes, as has been proposed (1–4). How the circadian clock cyc). CG3397 was expressed strongly in all three mutant flies, controls output behaviors is not known. A neuropeptide, suggesting that this gene is activated by any loss of the circadian pigment-dispersing factor (pdf), is necessary for coupling the time-keeping mechanism. In contrast, CG13406 was expressed central clock mechanism to behavioral rhythmicity and is hy- minimally in tim mutant fly heads but expressed at higher levels pothesized to represent the transmitter output of the primary in Clk and cyc (Fig. 5B). This gene seems to require both per and pacemaker neurons (19, 20). The pdf gene, however, is not tim function for expression but is expressed normally in flies expressed rhythmically (21). We speculate that rhythmic gene lacking a circadian clock because of Clk or cyc . expression in the fly head is used primarily as part of the Finally, we performed an analysis for genes with basal expres- time-keeping mechanism, and that circadian output relies pri- sion levels that were influenced by LD conditions. A total of 178 marily on posttranscriptional or other physiologic mechanisms genes had significant differences in basal expression levels (22). Indeed, the necessity of the transcription-translation feed-

9564 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.132269699 Lin et al. Downloaded by guest on September 28, 2021 Table 1. Identities of the 22 genes displaying strong circadian oscillations in control flies in both LD 12:12 and DD

Gene A0 Phase LD (ZT) Phase DD (CT) Expression mean (min:max) Function

tim 0.96 12 8 3,374.9 (876.6:8,390.2) per 0.94 16 8 957.7 (97.9:2,531.9) Circadian rhythm vri 0.84 12 8 3,350.5 (1,564.5:7,146.2) Circadian rhythm; RNA polymerase II transcription factor Clk 0.92 20 20 477.2 (44.2:1,098.7) Circadian rhythm Dreg-2 0.86 4 4 1,573.4 (1,112.0:2,107.6) Haloacid dehalogenase͞epoxide hydrolase family puc 0.94 12 4 1,845.6 (1,157.9:2,814.9) JUN phosphatase Ugt35b 0.9 0 0 5,342.6 (2,615.3:8,887.5) UDP-glucuronosyltransferase Cyp4d21 0.91 20 16 2,081.3 (969.8:4,728.8) Cytochrome P450 Amp 0.77 20 0 3,323.5 (2,134.5:5,517.0) Synaptic vesicle endocytosis Pdh 0.98 0 20 18,375.0 (5,817.3:32,515.9) Oxidoreductase, acting on the CH-OH group of donors, NAD or NADP as acceptor Slob 0.99 16 8 6,260.6 (3,054.3:9,768.0) Potassium channel-binding ; signal transduction CG5798 0.94 12 8 2,732.0 (958.2:6,526.6) Ubiquitin-specific protease CG7149 0.9 20 16 1,949.5 (1,221.4:3,395.0) Diacylglycerol cholinephosphotransferase CG10091 0.88 8 4 1,895.7 (930.5:3,467.3) Glutathione transferase CG10237 0.8 20 16 6,465.7 (3,994.5:12,993.8) ␣-Tocopherol transfer protein-like; retinaldehyde binding CG11407 0.91 20 20 3,058.6 (1,661.2:6,640.5) Long-chain fatty acid transporter CG11796 0.83 4 4 8,371.2 (4,750.0:11,891.0) 4-Hydroxyphenylpyruvate dioxygenase CG17386 0.98 20 16 533.3 (Ϫ47.1:2,319.8) RNA binding CG9497 0.9 0 16 2,642.4 (949.3:5,657.0) Unknown CG4962 0.89 12 16 2,355.6 (431.1:4,998.2) Unknown CG10553 0.85 0 16 1,725.0 (824.9:3,937.9) Unknown CG11891 0.86 0 16 1,425.9 (351.1:3,682.3) Unknown

‘‘Phase LD’’ and ‘‘Phase DD’’ refer to collection times of peak expression. ZT, Zeitgeber time; CT, circadian time. ‘‘Expression’’ lists mean, peak, and trough expression levels across the three LD cycles in microarray fluorescence units. Functional annotations based on Affymetrix annotations and the Gene Ontology database (36).

back loop in Drosophila for generation of circadian rhythms has We unexpectedly found that many genes had significantly been questioned recently (23). different levels of basal expression between per and control flies During submission of these data, three parallel studies of kept in DD. The flies used in those experiments were derived circadian gene expression in Drosophila heads were published from a ywgenetic background, and thus this feature is unlikely (9–11). The present study and that of ref. 11 analyzed data to represent a nonspecific strain difference. In previous microar- representing LD and DD conditions separately. The other two ray studies (9–11), similar large effects on basal gene expression studies combined LD and DD data to analyze circadian gene were noted in the cases of flies mutant for tim, per, and Clk. The expression. Each study used unique criteria for identification of mechanism for this broad regulation of gene expression is rhythmic gene expression. Table 4, which is published as sup- unknown, but several hypotheses may be considered. First, the porting information on the PNAS web site, presents the union of per-dependent circadian clock could control the rhythmic tran- all four lists describing ‘‘circadianly rhythmic genes’’: 134 genes scription of these genes, but mRNA half-lives could be greater (9); 158 genes (10); 115 genes (11); 22 genes (this study). The than 24 h, thereby damping circadian rhythmicity. Drosophila concordance among the three longer lists is very low: 19 genes mRNAs vary considerably in half-life, with some messages (24). However, of the 22 circadian genes identified in this study, having half-lives over 90 h (25). Second, changes in basal, 18 are scored circadian on at least one of the other three lists. aperiodic levels of gene expression may represent a widespread Thus, despite methodological differences, our analysis identified compensation to the absence of a circadian clock or the absence genes with independently reproducible rhythmic expressions. of the period gene in particular. Several studies have suggested Of the 340 unique genes scored rhythmic by at least one of the pleiotropic phenotypes in per mutants that are not attributable four groups, 84% were listed by only one group. We infer that directly to the circadian clock (26–30). Finally, these broad-scale large subsets of the three other lists represent false-positive effects could result from per01-induced dysregulation of one or results. Most of the genes that were present on any of the other more widely acting regulators of gene expression such as lists but were not scored rhythmic in the present analysis poly(A)-binding protein (31). Loss of the per-dependent regu- produced weak rhythmicity in our data set; they mostly displayed lation of this gene leads to a change in its static levels, which then a random distribution of correlation values (Table 4). Eleven could lead to significant changes in message half-life for a large genes were scored rhythmic by all other groups but not by us. number of nonoscillating transcripts. Among these genes, five were weakly rhythmic in our data set or The lighting condition of the flies also altered basal gene were eliminated because of less than 50% ‘‘present’’ calls. In sum, expression. Light affects fly behavior through two routes: the we find that the published data do not yet provide a consistent circadian clock and direct masking effects. The circadian clock description of circadian gene expression in Drosophila heads. We is entrained to external LD cycles, and light can transiently affect propose that the present analysis provides a useful and minimal gene expression through the clock (32, 33). Even in the absence set of bona fide circadian genes in Drosophila with which to of a functioning clock, however, flies still show rhythmic behav- initiate future studies. We offer access to all the raw data used ior when kept in an LD cycle (34). We found that 18 genes of the for the analysis of rhythmic gene expression (http:͞͞ 72 showing robust oscillations in control flies in LD 12:12 also PHYSIOLOGY circadian.wustl.edu). showed oscillations in a per01 flies in LD 12:12, presumably

Lin et al. PNAS ͉ July 9, 2002 ͉ vol. 99 ͉ no. 14 ͉ 9565 Downloaded by guest on September 28, 2021 Fig. 4. Basal gene expression is modulated by period and light. ‘‘Heat-plot’’ rank profiles of expression levels of genes exhibiting significant change in basal expression differences between experimental conditions (P Ͻ 0.0001, Mann–Whitney U test) are shown. From left to right, entire cycles (6 or 8 time points, in LD or DD) are grouped. (A) Expression levels of 650 genes from DD conditions on each of a total of 32 microarrays (yw, 20; per01, 12). (Upper) Those 330 genes with expression levels significantly higher in control flies (yw-Hi). (Lower) Those 320 genes with expression levels significantly higher in per01 (per-Hi). Pseudocolor indicates relative rank order in expression (yellow, highest; blue, lowest). (B) Comparison of control flies kept in LD versus DD. Those 178 genes that exhibit significant differences in expression between LD (17 measurements) and DD (20 measurements) are shown. LD-Hi, higher average expression in LD; DD-Hi, higher average expression in DD. (C) Comparison of per01 flies between LD versus DD (12 measurements each). Other markings are as described for B.(B and C) Data for the same 178 genes, displayed in register. (D) Representative expression levels of individual genes from B and C, as denoted by numbers in parenthesis. Microarray intensity values of control LD (17 points), control DD (20 points), per01 flies in LD (12 points), and per01 flies in DD (12 points) conditions are shown as means (ϩSE).

Fig. 5. Confirming the influence of period on basal gene expression. (A) Reverse transcription–PCR was performed on total RNA extracted from control and per01 flies pooled from multiple time points in DD. RP49, control; MW, molecular weight markers. (B) Reverse transcription–PCR performed on total RNA from pooled time points of other clock mutant flies (tim, Clk, and cyc) kept in DD conditions.

9566 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.132269699 Lin et al. Downloaded by guest on September 28, 2021 through direct light-masking effects. Interestingly, 32 genes parison between day length and internal circadian clock time. required both per function and an LD cycle for rhythmicity. We Drosophila exhibits photoperiodic control of ovarian diapause propose the term period-dependent masking to describe this (35). Our data indicate that day length and clock function may class of genes. Remarkably, nearly 200 genes showed substantial be integrated to determine the basal expression levels of nu- differences in basal expression depending on whether the flies merous genes. were housed in DD or LD conditions. These effects could be caused either by light acting through the per-dependent clock or We thank the Berkeley Drosophila Genome Project for providing the 01 genomic sequences used in this analysis. We also thank Erik Herzog and its direct masking effect on basal gene expression. Because per Louis Muglia for their constructive criticism of the manuscript. Y.L. flies had very few genes showing basal expression differences and G.D.S. are supported by National Institutes of Health (NIH) between LD- and DD-housed conditions, we propose that light’s Grant GM28755. R.N.V.G. is supported by a Research to Prevent effects on basal expression is mediated primarily through a Blindness Career Development Award, the Becker͞AUPO͞RPB circadian clock-dependent mechanism. Indeed, these broad- Clinician-Scientist Award, and NIH Grant EYK08-00403. P.H.T. is supported by NIH Grant NS-21749 and a grant from the Human Frontier scale changes in light-dependent static gene expression may Science Program Organization. The raw data from all oligonucleotide reflect the mechanism and output of the photoperiodic response. microarrays used in this study may be obtained from http:͞͞ The seasonal photoperiodic response is determined by a com- circadian.wustl.edu.

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