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A Genome-wide RNAi Screen for Modiers of the Circadian Clock in Human Cells
Eric E. Zhang,1,2,5 Andrew C. Liu,1,3,5 Tsuyoshi Hirota,1,2,5 Loren J. Miraglia,1 Genevieve Welch,1 Pagkapol Y. Pongsawakul,2 Xianzhong Liu,1 Ann Atwood,2 Jon W. Huss III,1 Jeff Janes,1 Andrew I. Su,1 John B. Hogenesch,4,* and Steve A. Kay2,*
Institute of the Novartis Research Foundation, San Diego, CA 92121, USA of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0130, USA 3Department of Biology, The University of Memphis, Memphis, TN 38152, USA 4Department of Pharmacology, Institute for Translational Medicine and Therapeutics, Penn Genome Frontiers Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160, USA 5These authors contributed equally to this work *Correspondence: hogenesc@mail.med.upenn.edu (J.B.H.), skay@ucsd.edu (S.A.K.) DOI 10.1016/j.cell.2009.08.031
2Section 1Genomics

SUMMARY

Two decades of research identied more than a dozen clock genes and dened a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and conrmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.
INTRODUCTION Circadian rhythms in mammals are exemplied by the sleep/ wake cycle and regulated by an internal timing system. A central clock in the suprachiasmatic nuclei (SCN) of the hypothalamus coordinates input and, through a complex signaling cascade, synchronizes local clocks in the brain and throughout the body (Reppert and Weaver, 2002; Panda et al., 2002b; Liu et al., 2007a). Over the past two decades, extensive genetic, genomic, molecular, and cell biological approaches identied more than

a dozen clock genes that collectively comprise a biochemical feedback loop that drives circadian oscillations (Reppert and Weaver, 2002; Panda et al., 2002b). Recent models consider the clock a biochemical and cellular oscillator, and also a genetic network. A highly conserved negative feedback loop was discovered and elucidated at biochemical, cellular, and organismal levels in mammals and Drosophila; transcription factors play a prominent role in the clock mechanism. The bHLH-PAS transcriptional activators BMAL1 (ofcial name: ARNTL, an ortholog of the Drosophila cyc gene) and its heterodimeric partner CLOCK (an ortholog of the Drosophila Clk gene) interact to bind to E-box cis elements present in the promoter regions of their target genes. These targets include two families of repressor proteins, the PERIODs (PER1, PER2, and PER3) and the CRYPTOCHROMEs (CRY1 and CRY2), which interact in a protein complex that translocates from the cytoplasm to the nucleus. In the nucleus, this repressor complex physically associates with the BMAL1-CLOCK complex to inhibit E-box-mediated transcription. This process results in the cyclic transcription of these repressor genes, as well as thousands of transcriptional output genes elsewhere in the genome (Hughes et al., 2009; Panda et al., 2002a; Ueda et al., 2002). In addition to rhythmic transcription, PER and CRY protein levels and subcellular localization also oscillate. Protein level cycling is a consequence of the transcriptional regulation mentioned above, but also through posttranscriptional and posttranslational mechanisms that regulate the stability and degradation of messages and proteins. These processes are mediated by kinases (e.g., CSNK1D, CSNK1E, CSNK2, and GSK3B) (Vanselow et al., 2006; Maier et al., 2009; Hirota et al., 2008; Etchegaray et al., 2009) and the proteasomal machinery, including the E3 ligase, FBXL3 (Siepka et al., 2007; Busino et al., 2007; Godinho et al., 2007; Reddy et al., 2006). Thus, while transcriptional regulation generates rhythmic RNA levels, regulated posttranslational modications control protein abundance, subcellular localization, and repressor activity of PER and CRY.

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Importantly, these additional regulatory steps introduce a delay, critical for rhythm generation and period regulation, in the clock mechanism (Gallego and Virshup, 2007). The circadian oscillator is also a highly interconnected genetic network that uses other transcription factors and response elements. In addition to the biochemical feedback loop that regulates cycling at the E-box (termed the core loop), circadian gene expression is mediated by transcription at the ROR/ REV-ERB (RORE) and the DBP/E4BP4 (D-box) binding elements. Two subfamilies of nuclear hormone receptors, the NR1Ds (NR1D1 and NR1D2, or REV-ERBa and b) and RORs (a, b and g, or RORa, RORb and RORc), either repress or activate gene transcription from the ROR elements in several clock genes (Ukai-Tadenuma et al., 2008). The bZIP transcription factors, DBP, TEF, and HLF, perform similar functions on the D-box element (Gachon et al., 2006). The role of these genes was examined in vivo and in vitro. Knockout mice for Rev-erba, Rora, or Rorb, have lower amplitude rhythms, abnormal period lengths, and interindividual variability in both phenotypes (Andre et al., 1998; Preitner et al., 2002; Sato et al., 2004). A coactivator shown to interact with the ROR proteins, PGC1a, modulates Bmal1 expression, and Pgc1a knockout mice display long-period locomotor activity behavior (Liu et al., 2007c). Furthermore, dose-dependent knockdown of ROR and REV-ERB genes in vitro has a potent effect on the baseline and the amplitude of circadian gene expression (Baggs et al., 2009). Taken in sum, these data demonstrate the important role of the clock gene network in regulating circadian amplitude, resistance to perturbation, and, in several cases, modulation of period length. Although the clock can function in a cell-autonomous manner (for discussion purposes, we extend the denition of autonomy to include potential auto regulation in trans), in vivo rhythms in physiology and behavior result from more complex cellular and humoral interactions. Genetic dissection of the circadian system showed the important role of intercellular coupling in generating physiological and behavioral responses; these explain the apparent discordance among observations from cells, organotypic SCN cultures, and whole animals (Liu et al., 2007b). For example, although Cry1 deletion in mice causes short period length of locomotor activity rhythms, dissociated SCN neurons and peripheral cells derived from these mice are arrhythmic (Liu et al., 2007b). These results demonstrate that intercellular coupling in the SCN compensates for the loss of Cry1 function and, as a result, masks the more severe cellular clock decits. Similarly, while Clock gene knockout mice are nearly wild-type in their behavioral rhythms, lung and liver preparations from these mice display arrhythmic gene expression (DeBruyne et al., 2006, 2007). In fact, cellular clock defects are often more severe than behavioral in clock gene knockout mice (Brown et al., 2005; Liu et al., 2007b). Thus, although transcriptional rhythms are cell autonomous and reect the cellular nature of the circadian signaling, physiology and behavior result from more complex in vivo interactions. The circadian research community took nearly four decades to identify the above clock components. In metazoan models, successful approaches included forward mutagenesis in ies and mice and cellular and biochemical methods followed by

conrmatory reverse genetics. Although these efforts were fruitful, they remain incomplete because evidence, including quantitative trait loci (QTL) studies, suggests that additional clock components and modulators exist (Takahashi, 2004). Remarkably, circadian clocks exist in cell lines (Balsalobre et al., 1998). Some of these lines are amenable to complementary DNA (cDNA) overexpression and RNA interference (RNAi) techniques and have been used to identify new components of many signal transduction pathways (for example, see Conkright et al., 2003; Iourgenko et al., 2003; Aza-Blanc et al., 2003). To conduct a whole-genome screen for circadian clock modiers, we used the U2OS (human osteosarcoma) cell line that was successfully employed in chemical screens (Hirota et al., 2008), in dissection of network properties of the oscillator (Baggs et al., 2009), and in a limited-scale RNAi screen (Maier et al., 2009). We screened $90,000 individual small interfering RNAs (siRNAs) and measured luciferase reporter gene expression every 2 hr for 4 days. We applied statistical algorithms to nd modiers of rhythm amplitude and period length. Knockdown of nearly 1000 genes reduced rhythm amplitude. On the other hand, knockdown of hundreds of genes by multiple independent siRNAs generated strong circadian phenotypes. Characterization of a subset of these genes demonstrated a dosage-dependent effect on clock function. Protein interaction network analysis indicated that some candidates directly or indirectly interact with known clock components. Gene expression studies showed that some affect the clock by regulating clock component expression levels. Finally, to disseminate this data and integrate with existing public resources, we constructed a database (http://biogps.gnf.org/circadian/). This resource will serve the research community in genetic and biochemical investigations of circadian regulation of physiology and behavior. RESULTS Assay Development Because circadian rhythms are dynamic, we employed kinetic, rather than steady-state, bioluminescence detection to assess the persistence and characteristics of rhythms after perturbation. To conduct such a screen, we required a cell line amenable to efcient reverse transfection, where siRNAs are deposited on plates, dried, mixed with transfection reagents and, nally, cells are added to complete the transfection process. We chose U2OS cells for assay development as this model was extensively characterized by functional studies examining knockdown effects of all known clock components with cellular and behavioral phenotypes in knockout mice (Baggs et al., 2009; Liu et al., 2007b). In addition, this model was used in small molecule screening and limited-scale RNAi screening to nd modiers of amplitude and periodicity (Hirota et al., 2008; Maier et al., 2009; Baggs et al., 2009). We established cell lines harboring a rapidly degradable form of luciferase, dLuc, driven by the mouse Bmal1 or Per2 gene promoter (Liu et al., 2008) (Figure S1 available online). Knockdown of CRY1, CRY2, and BMAL1 in both lines resulted in phenotypes consistent with previous knockout mouse and cellular knockdown studies (Liu et al., 2007b, 2008; Maier et al., 2009; Baggs et al., 2009) (Figure S1). For instance, CRY1

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knockdown shortens period length and compromises rhythm persistence, CRY2 knockdown lengthens period length, and knockdown of both results in arrhythmicity. We next adapted these assays for high-throughput screening (HTS) in 96- and 384-well plates. By optimizing growth conditions and transfection efciency, we obtained consistent bioluminescence rhythms in a HTS format (e.g., 0.5 hr of SD in control wells of 384-well plates, n = 7680 wells) with knockdown effects for period and amplitude (see below). Knockdown of known clock components such as CRY1, CRY2, and BMAL1 in this format produced similar cellular clock phenotypes to those in LumiCycle assays using 35 mm dishes (Figure S1). These conditions enabled large-scale siRNA screens. Primary Screen The screening protocol and logic are outlined in Figures 1A and S2 and describe the primary screen, data mining, hit selection, secondary screen, and validation studies. The Bmal1-dLuc line was more robust than our Per2-dLuc line and therefore was used for primary screens in a 384-well format. CRY2 siRNAs were used as positive controls in each plate. We screened an siRNA library (QIAGEN) targeting 17,631 known and 4837 predicted human genes. In these libraries, four siRNA constructs were designed for each gene with a pool of two siRNAs per well (two siRNAs/well, two wells/gene) for a total of 89,872 siRNAs. These siRNAs were prespotted on 384-well plates. After addition of transfection reagent, we seeded approximately 2000 cells into each well to complete the transfection. We measured bioluminescence every 2 hr for 4 days for a total of 48 time points for each well; this temporal resolution was chosen based on simulation studies using data collected from known clock gene perturbation. The primary screen produced a total of >4.3 3 106 data points, which are also deposited in the PubChem Bioassay database (accession number AID 1904; see the Experimental Procedures for details). Data Mining We used the MultiCycle Analysis program (Actimetrics) to analyze period length and rhythm amplitude for each well. Plate-to-plate variation in period length and amplitude was modest in control wells (period length or t = 25.20 hr 0.55 [mean SD], n = 1176; amplitude or A = 4380 1010, n = 1176) (Figures 1B and S3). Knockdown of CRY2, a period length control, resulted in robust lengthening (29.43 hr 0.65; n = 1176), while knockdown of BMAL1, an amplitude control, potently reduced amplitude (692 626 arbitrary luminescence units; n = 1176). Gratifyingly, many known clock components had phenotypes in this screen consistent with those seen in knockout animals (Liu et al., 2007b; Liu et al., 2008) or dose-dependent siRNA knockdown (Baggs et al., 2009) (Figure 1C). For example, CRY2 knockdown lengthened period, and CRY1 knockdown led to rapid loss of rhythmicity. Knockdown of BMAL1 or CLOCK genes resulted in low amplitude/arrhythmicity, and knockdown of PER1 or PER2 also caused almost immediate arrhythmicity. Consistent with its role as a repressor for RORE-mediated transcription, knockdown of NR1D1 raised overall reporter expression levels and lengthened the period (see below), consistent with a previous

report (Baggs et al., 2009). We also saw knockdown effects for CSNK1D and CSNK1E (Xu et al., 2005; Meng et al., 2008; Gallego and Virshup, 2007; Etchegaray et al., 2009), GSK3B (Hirota et al., 2008), FBXL3 (Siepka et al., 2007; Busino et al., 2007; Godinho et al., 2007), FBXW11 (or b-TRCP2), and CSNK2 (Maier et al., 2009). Collectively, these results support the suitability of our primary screen. Primary Hit Selection To identify genes whose knockdown modulates the circadian clock, rather than the overall health of the cells, we focused on period length decits and increased amplitude of rhythmicity. Low-amplitude traces usually exhibit poor curve tting and generate inconsistent period length data between replicate wells; they constitute less than 4.5% of all wells and were excluded from the analysis (Figure S4). We found 1028 shortperiod hits, including 76 genes that were hit by two independent siRNA pairs (double hits), 4230 long-period hits (274 double hits), and 493 high-amplitude hits (18 double hits) (Figure 1B). Consistent with data from small molecule screens (Hirota et al., 2008) (T.H and S.A.K., unpublished data) and previous model organism screens (Takahashi, 2004), many more genes generated long period length than short. As siRNAs have well described potential off target effects, we focused on genes where two or more independent siRNAs generated a consistent phenotype (Echeverri et al., 2006). The plotted traces for these genes were individually inspected to remove false positives caused by poor curve tting. We selected 254 genes whose knockdown resulted in strong circadian phenotypes, dened by three or more standard deviations from the mean, in period length and rhythm amplitude (Figure S2). We also selected 89 single siRNA pair hits that showed strong circadian phenotypes in duplicate wells (Figure S2). Thus, our primary hit selection list contains a total of 343 genes including known clock components (Table S1 and Figure 1). Secondary Conrmation Screen Next, we performed a secondary screen to conrm the knockdown phenotypes identied in the primary screen (Figure S2). We tested at least four siRNA constructs (one siRNA/well, at least four wells/gene) for each gene in both the Bmal1-dLuc and Per2-dLuc reporter cell lines (Figure S1). We hypothesized that consistent changes in both assays indicate that resultant phenotype is not reporter or response element specic, but rather reports an impact on clock function in cells. As expected, the secondary screen using a Bmal1-dLuc reporter assay generated highly concordant results with the primary screen (Figures 2A and S2): 222 of 238 genes that were identied in the primary screen with two siRNA pairs and 47 of 83 with a single siRNA pair were conrmed (Table S1). In Per2-dLuc reporter assay, 219 were also independently conrmed (Figures 2B2D and S2). The remaining unconrmed genes either failed to have more than one independent siRNA conferring a consistent phenotype or conrmed in the Bmal1dLuc but not the Per2-dLuc assay. In addition, a few genes altered both period length and amplitude in these assays (Figure S2). In summary, we observed a high level of concordance between our primary and secondary screens, and we

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Figure 1. A Cell-Based Genome-wide siRNA Screen for Circadian Clock Modiers

(A) A schematic diagram of the genome-wide siRNA screen including the primary screen, data mining, hit selection, secondary screen, and validation of several selected targets. In the primary screen, reporter cells were transfected with siRNA in 384-well plates followed by kinetic bioluminescence recording (see the Experimental Procedures for details). Luminescence data were analyzed to obtain circadian parameters and select primary hits. Secondary screen and validation studies were performed to conrm circadian phenotypes of hits and to demonstrate the validity of the primary screen. To catalyze the use of this dataset by the research community, we constructed a comprehensive circadian genomic screen database in BioGPS (see Figure S6 for details). (B) Distribution of circadian parameters of the entire primary screen. Dots represent normalized period (upper) and amplitude values (lower). For period length, the average of duplicate wells was divided by the mean of the entire screen and indicated in Log2 space. The cutoff was 0.1 and +0.1 (corresponding to raw data 23.55 hr for short- and 26.85 hr for long-period hits). Traces that lack apparent $24 hr bioluminescence oscillation usually returned as a period length of 48 hr and are considered as arrhythmic. In addition, Log2 values above 0.4 (corresponding to 38 hr), for example, display poor curve tting and are also considered as arrhythmic. For rhythm amplitude, average of duplicate wells was divided by the mean of the entire screen, and the cutoff was 2.20 (corresponding to raw data 7390) for high-amplitude hits. The knockdown phenotypes of several representative clock genes are shown in colored dots. (C) Cellular clock phenotypes of siRNA knockdown of known clock genes. Plots of cellular oscillations upon knockdown of BMAL1, CLOCK, PER1, PER2, CRY1, or CRY2 by two independent pairs of siRNAs in the primary screen are presented. The spikes of initial 10 hr bioluminescence readings resulted from media change and were removed from the plot.

identied hundreds of genes whose knockdown generates strong circadian phenotypes. Dose-Dependent Phenotypic Validation To determine those genes that most sensitively impacted period length or increased amplitude (cellular rheostats of clock func-

tion), we conducted a dosage-dependent knockdown analysis as per our previous work (Baggs et al., 2009). In this study, we selected 17 genes from the primary screen with extreme phenotypes and generated an eight-point dose response. All but one showed dose-dependent effects on circadian rhythms (Figure 3A). Gene knockdown was conrmed by quantitative PCR

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25.38 26.29 26.89 26.89 25.08 26.59 26.59 26.89 25.98 27.20 25.68 26.59 26.59 28.41 26.59 27.80 26.89 25.38 27.20 25.08 25.98 27.20 27.20 23.56 25.08 25.68 28.71 29.02

24.67 25.27 24.67 25.88 24.67 27.39 27.09 26.79 23.15 26.18 24.36 25.88 27.70 32.24 25.58 48.00 26.18 24.67 23.45 24.36 25.88 25.58 24.67 48.00 24.06 24.36 28.00 28.00

25.58 25.27 26.48 26.18 24.67 28.00 27.09 27.09 48.00 26.18 24.06 26.48 27.09 22.24 24.97 48.00 25.88 24.67 24.36 24.36 26.48 26.18 24.67 25.27 24.06 24.06 28.30 28.30

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25.08 23.56 22.05 25.08 24.17 23.26 23.86 25.38 24.47 23.26 22.65 25.08 25.08 23.56 23.26 24.47 24.17 24.17 23.86 24.47 23.86 22.65 22.65 24.77

21.33 21.94 22.55 24.36 23.76 22.24 21.94 22.55 23.45 22.85 21.94 24.36 22.55 24.67 22.24 23.15 22.55 22.24 24.06 22.24 23.45 22.85 21.64 23.76

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Figure 2. Secondary Conrmation Assay of Cellular Clock Phenotypes

(A) A heat map of the secondary screen using Bmal1-dLuc cells. Eight hundred and seventy-two independent siRNAs were tested against 154 genes in duplicate, along with 20 wells for each of the controls (GL2, CRY2, BMAL1, and GL3 siRNAs) for a total of 1824 wells. Bioluminescence intensity for each well was plotted against time (hr), with each horizontal line representing luminescence recordings from a single well. The circadian proles from each well were classied by hierarchical clustering (clustering method: maximum complete linkage; similarity measure: correlation; ordering function: average value). (BD) Circadian parameters for 17 genes generating long-period (B), short-period (C), and high-amplitude phenotypes (D) in both Bmal1-dLuc and Per2-dLuc cell lines. CRY2 siRNA was a positive control. For the Bmal1-dLuc cell line, the period length of the control wells was 25.07 hr 0.59 and amplitude was 4120 1285 (mean SD; n = 768). For the Per2-dLuc cell line, the period length was 24.18 hr 0.55 and amplitude was 6438 1140 (n = 768). Cellular clock phenotypes are color coded, with darker colors in each category representing stronger alteration of circadian parameters (mean 3 3 SD) than lighter colors (mean 2 3 SD). Four different siRNAs (y axis) were tested for each gene, and the assay was conducted in duplicates (x axis) in each screen using either Bmal1-dLuc or Per2-dLuc cells.

(Q-PCR) (Figure 3B). Genes whose knockdown lengthened period included HCFC1, POLR3F, PRPF4, SEC13, UNC119, and ZMAT3, similar to CRY2 or FBXL3. Knockdown of ACSF3, B4GALT2, CEACAM21, TBCB, or MPG led to dose-dependent

period shortening, similar to CRY1. Finally, knockdown of COX4NB, FHIT, HIST1H1B, NMNAT1, or PDE1B caused dosedependent increases in rhythm amplitude. These data conrm primary screening results and show that many genes from our

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Figure 3. Dose-Dependent Phenotypic Validation

(A) Dose-dependent effects on circadian phenotypes. Bmal1-dLuc cells were transfected with the indicated amounts of siRNAs (eight-point, 2-fold dilution series; 81000 fmol/well) against 17 genes, and bioluminescence oscillations were recorded. Representative bioluminescence proles (left) and circadian parameters (right) are indicated. Data represent the mean SD (n = 3). (B) Knockdown of target genes. Bmal1-dLuc cells were transfected with 3000 fmol/well siRNAs (corresponding to 1000 fmol/well on 384-well plate) and subjected to Q-PCR analysis under unsynchronized conditions. mRNA levels of target genes relative to GAPDH are indicated. Data represent the mean SD (n = 2). In parallel experiments, bioluminescence rhythms were recorded and circadian phenotypes were conrmed.

screen impact clock function equivalently to or better than known clock components. Dose-Dependent Effects on Clock Gene Expression Because of the complex regulatory architecture of the clock gene network, perturbation of one clock component can lead to dynamic changes in the levels of others (we describe these as network effects). To determine whether genes from our screen had these effects, we selected a subset of genes that were conrmed in dose response (Figure 3): POLR3F, PRPF4, and SEC13 (long-period hits), ACSF3 and MPG (short-period hits), and COX4NB (high-amplitude hit) to analyze their potential for network effects. (The technical demands of this experiment prevented us from extending this analysis to all genes from

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Figure 3.) Knockdown of known clock genes had potent effects on circadian phenotypes, as well as clock gene expression (Figure 4), consistent with a previous study (Baggs et al., 2009). Interestingly, knockdown of most screen hits led to dose-dependent reduction of NR1D1 and DBP transcript levels (Figure 4), consistent with E-box-driven regulation (Ueda et al., 2005; Liu et al., 2008; Baggs et al., 2009). These observations provide additional support for the notion that regulation of E-box-mediated transcription represents a topological vulnerability in mammalian circadian clocks (Ueda et al., 2005). Similar to NR1D1 knockdown, SEC13 knockdown upregulated BMAL1 transcription; like NR1D1, SEC13 may impact clock function through network effects, rather than direct physical association with clock components. Intriguingly, knockdown

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Target si

Target si

0 12 24 36 48 60 72 84 96

0 8 16 31 63 125 250 500 1000

0 12 24 36 48 60 72 84 96

0 8 16 31 63 125 250 500 1000

24

40

0.0

Target si

0 12 24 36 48 60 72 84 96

0 12 24 36 48 60 72 84 96

40

of ACSF3 and POLR3F did not impact expression of core clock genes despite dose-dependent functional consequences on the clock (Figure 4). We note that CRY2 knockdown also does not alter the messenger RNA (mRNA) levels of known clock genes. These genes may regulate posttranslational modication of clock components and impact protein abundance, localization, or function to perturb the oscillator. Protein Interaction Network Analysis We hypothesized that some hits from our screen directly or indirectly associate with known clock components. Using the Entrez Gene and Prolexys protein-protein interaction databases (Prolexys Pharmaceuticals, UT), we identied a comprehensive set of proteins that interacted with gene products of screen hits or known clock components (Table S2). To visualize the global topography of these interactions, we constructed an expanded clock gene interaction network (Figure 5). Most genes identied in our screen are present in a highly connected cluster of proteins centered on the core clock components: BMAL1, CLOCK, PERs, and CRYs. While ZMAT3, BLNK, and RRP12 directly interact with core clock components, most others associate indirectly through bridging molecules. (Many of these bridging molecules have phenotypic effects in our screen, but fell short of our reporting threshold.) For example, P53 (ofcial name: TP53, tumor protein 53) physically interacts with CDK9, MAPK8, NCL, ABL1, BCR, DHFR, and BRCA2, as well as PCAF and CSNK1E; knockdown of these genes generate short-period, long-period, and high-amplitude phenotypes in our screen. Knockdown of P53 itself generated low-amplitude rhythms in our screen; U2OS cells are wild-type for P53 expression (Florenes et al., 1994). Interestingly, a recent RNAi screen showed that BMAL1 potently regulates P53 pathway function (Mullenders et al., 2009). Taken together, these observations provide evidence of the interconnectedness between the circadian clock and many other cellular pathways. Pathway Analysis of Primary Screen Hits Although the protein interaction network analysis gave some sense of pathway regulation, we sought to directly test for functional interconnectedness between the clock and other biological processes using the NIH DAVID pathway analysis tool (Huang et al., 2009; Dennis et al., 2003). We found that hits from our screen were members of dozens of cellular pathways. In addition, several pathways were overrepresented in this analysis, including insulin signaling (p < 0.013), hedgehog signaling (p < 0.0088), cell cycle (p < 0.054), and folate metabolism (p < 0.014) (Figures 6A and S5). While inhibition of multiple components of the insulin, hedgehog, or cell-cycle pathways resulted in long period length of circadian oscillations, knockdown of components of folate metabolism resulted in short- and longperiod, as well as high-amplitude, phenotypes. (We note that components of these pathways often play important roles in other pathways; for example, several components in insulin signaling are shared in B cell receptor, IL-4, IL-8, PDGF, and IGF-1 signaling.) Our previous work has demonstrated that many components in these pathways are themselves under clock control and expressed in a circadian fashion in various peripheral tissues such as the liver (Panda et al., 2002a;

Hughes et al., 2009). Collectively, these data show functional interconnectedness between the circadian clock and many other biological pathways. One dramatic example of this regulation is the insulin signaling pathway (Figure 6A). (Insulin signaling and many other pathways have inherent feedback mechanisms; in this regard, these pathways may not function in a wholly cell-autonomous manner, and instead may autoregulate in trans.) Downregulation of multiple components of the insulin pathway resulted in period changes, including JNK (MAPK8, long period), IKK (IKBKB, long period), PI3K (PIK3R5, long period), MTOR (FRAP1, long period), APKC (PRKCI, long period), PFK (PFKP, short period), and PYK (PKLR, long period) (Figure 6B). In addition, at least 19 components of the insulin pathway are regulated at the transcriptional level by the circadian clock (Figure 6A). These include IKK, PI3K, and MTOR, whose perturbation also impacts clock function. To provide additional validation, we employed small molecules targeting multiple components of the insulin pathway in the U2OS model. SP600125 (a JNK inhibitor) and the Dequalinium analog C14 linker (a PKC inhibitor) led to long period length of circadian oscillations, PMA (a PKC activator) resulted in short period length, and wortmannin and LY294002 (PI3K inhibitors) resulted in a phase delay (Figure 6C). These results are all consistent with genetic perturbation of their intended targets. These results highlight the functional intersection between two important biological pathways, insulin signaling and the circadian clock. Data Presentation in BioGPS To facilitate use of this resource, we constructed a database to enable visualization and exploration. Our screening data are displayed as a plug-in in BioGPS, an open-access, searchable database for aggregating gene annotation from multiple online sources (http://biogps.gnf.org/). We created a custom BioGPS circadian layout focused on the siRNA screen described in this manuscript (Figure S6) and accessible at http://biogps.gnf.org/ circadian/. This BioGPS layout displays plug-ins from our siRNA circadian screen and a recently published 1 hr resolution circadian gene expression databases from mouse liver and pituitary tissues (Hughes et al., 2009). Additional online resources are also available including the UCSC Genome Browser (http://genome.ucsc.edu/), our previously published reference gene expression data obtained from various tissues and cell lines (Su et al., 2002; Su et al., 2004), and Wikipedia, which describes the expanded gene annotations (e.g., PER2 as shown here). Furthermore, BioGPS provides a exible platform for building customized layouts that can be modied to include a selection of the more than 100 other online data sets and/or resources in the BioGPS plug-in library. DISCUSSION To identify additional clock genes or modulators, we carried out a genome-wide siRNA screen using a robust reporter gene assay in human U2OS cells. We found nearly 1000 genes whose knockdown resulted in low-amplitude circadian oscillations, which may indicate their potential in regulating the general health of cells. In addition, we found hundreds of genes whose

Cell 139, 199210, October 2, 2009 2009 Elsevier Inc. 205

Bioluminescence Rhythms
31
200 100 0 -100 -200 0 12 24 36 48 60 72 84 96

Knockdown Efficiency
CRY2 ACTB

Effects on Gene Expression

Long period
1.5 1.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 0.5 0.0 0.5 0.0 0.5 0.0

BMAL1

1.5 1.0

CLOCK

1.5 1.0

PER1

2.0 1.5 1.0 0.5 0.0

PER2

2.0 1.5 1.0 0.5 0.0

CRY1

1.5 1.0 0.5 0.0

CRY2

1.5 1.0 0.5 0.0

NR1D1

2.0 1.5 1.0 0.5 0.0

DBP

1.5 1.0 0.5 0.0

FBXL3

29 27 25

CRY2 si

CRY2 si

Normalized Luminescence

200 100 0 -100 -200 0 12 24 36 48 60 72 84 96

31 29

1.5 1.0

FBXL3

1.5 1.0 0.5 0.0

ACTB

1.5 1.0 0.5 0.0

BMAL1

1.5 1.0 0.5 0.0

CLOCK

1.5 1.0 0.5 0.0

PER1

1.5 1.0 0.5 0.0

PER2

1.5 1.0 0.5 0.0

CRY1

1.5 1.0 0.5 0.0

CRY2

1.5 1.0 0.5 0.0

NR1D1

1.5 1.0 0.5 0.0

DBP

1.5 1.0 0.5 0.0

FBXL3

Period (hr)

27 25

FBXL3 si

0.5 0.0

FBXL3 si

31 29 27 25

200 100 0 -100 -200 0 12 24 36 48 60 72 84 96

1.5 1.0

POLR3F

1.5 1.0 0.5 0.0

ACTB

2.0 1.5 1.0

BMAL1

1.5 1.0 0.5 0.0

CLOCK

1.5 1.0 0.5 0.0

PER1

1.5 1.0 0.5 0.0

PER2

2.0 1.5 1.0 0.5 0.0

CRY1

2.0 1.5 1.0 0.5 0.0

CRY2

1.5 1.0 0.5 0.0

NR1D1

1.5 1.0 0.5 0.0

DBP

1.5 1.0 0.5 0.0

FBXL3

POLR3F si

0.5 0.0

POLR3F si

0.5 0.0

200 100 0 -100 -200 0 12 24 36 48 60 72 84 96

35 33 31 29 27 25

1.5 1.0

PRPF4

1.5 1.0 0.5 0.0

ACTB

2.0 1.5 1.0

BMAL1

1.5 1.0 0.5 0.0

CLOCK

1.5 1.0 0.5 0.0

PER1

1.5 1.0 0.5 0.0

PER2

2.0 1.5 1.0 0.5 0.0

CRY1

2.0 1.5 1.0 0.5 0.0

CRY2

1.5 1.0 0.5 0.0

NR1D1

1.5 1.0 0.5 0.0

DBP

1.5 1.0 0.5 0.0

FBXL3

PRPF4 si

0.5 0.0

PRPF4 si

0.5 0.0

33

200 100 0 -100 -200 0 12 24 36 48 60 72 84 96

1.5 1.0

SEC13

1.5 1.0 0.5

ACTB

2.0 1.5 1.0

BMAL1

1.5 1.0 0.5

CLOCK

1.5 1.0 0.5

PER1

2.0 1.5 1.0 0.5

PER2

2.0 1.5 1.0 0.5

CRY1

2.0 1.5 1.0 0.5

CRY2

1.5 1.0 0.5

NR1D1

1.5 1.0 0.5

DBP

1.5 1.0 0.5

FBXL3

31 29 27
0 8 16 31 63 125 250 500 1000

SEC13 si

0.5
CTRL 8 16 31 63 125 250 500 1000

SEC13 si
CTRL 16 31 63 125 250 500 1000 8

0.5
16 31 63 125 250 500 1000 16 31 63 125 250 500 1000 CTRL CTRL

16 31 63 125 250 500 1000

16 31 63 125 250 500 1000

16 31 63 125 250 500 1000

16 31 63 125 250 500 1000

16 31 63 125 250 500 1000

16 31 63 125 250 500 1000

Short period
Relative mRNA Levels (relative to GAPDH) Relative mRNA Levels (relative to GAPDH)
27 200 100 0 -100 -200 0 12 24 36 48 60 72 84 96
27 26
1.5 1.0 0.5 0.0

CRY1

1.5 1.0 0.5 0.0

ACTB

1.5 1.0 0.5 0.0

BMAL1

1.5 1.0 0.5 0.0

CLOCK

2.0 1.5 1.0 0.5 0.0

PER1

2.0 1.5 1.0 0.5 0.0

PER2

1.5 1.0 0.5 0.0

CRY1
2.5 2.0 1.5 1.0 0.5 0.0

CRY2

1.5 1.0 0.5 0.0

NR1D1

CTRL 8 16 31 63 125 250 500 1000

CTRL

CTRL

CTRL

CTRL

CTRL

CTRL

25

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

2.0 1.5 1.0 0.5 0.0

DBP

2.0 1.5 1.0 0.5 0.0

FBXL3

26 25

CRY1 si

CRY1 si

Normalized Luminescence

200 100 0 -100 -200 0 12 24 36 48 60 72 84 96

Period (hr)

1.5 1.0 0.5 0.0

ACSF3

1.5 1.0 0.5 0.0

ACTB

1.5 1.0 0.5 0.0

BMAL1

1.5 1.0 0.5 0.0

CLOCK

1.5 1.0 0.5 0.0

PER1

1.5 1.0 0.5 0.0

PER2

1.5 1.0 0.5 0.0

CRY1

1.5 1.0 0.5 0.0

CRY2

1.5 1.0 0.5 0.0

NR1D1

1.5 1.0 0.5 0.0

DBP

1.5 1.0 0.5 0.0

FBXL3

ACSF3 si
25

ACSF3 si

27
200 100 0 -100 -200 0 12 24 36 48 60 72 84 96
0 8 16 31 63 125 250 500 1000

1.5 1.0 0.5

MPG

1.5 1.0 0.5

ACTB

2.0 1.5 1.0 0.5

BMAL1

1.5 1.0 0.5

CLOCK

1.5 1.0 0.5

PER1

1.5 1.0 0.5

PER2

2.0 1.5 1.0 0.5

CRY1

2.0 1.5 1.0 0.5

CRY2

1.5 1.0 0.5

NR1D1

1.5 1.0 0.5

DBP

1.5 1.0 0.5

FBXL3

26

MPG si
25

MPG si
CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

Amplitude change
1000 800 600 400 200 0 0 12 24 36 48 60 72 84 96 50 40 30 20 10 0
1.5 1.0

BMAL1

1.5 1.0 0.5 0.0

ACTB

1.5 1.0 0.5 0.0

BMAL1

1.5 1.0 0.5 0.0

CLOCK

4
3 2

PER1

2.0 1.5 1.0 0.5 0.0

PER2

4
3 2 1 0

CRY1

4
3 2 1 0

CRY2

CTRL 8 16 31 63 125 250 500 1000

1.5 1.0 0.5 0.0

NR1D1

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

1.5 1.0 0.5 0.0

DBP

1.5 1.0 0.5 0.0

FBXL3

BMAL1 si

0.5 0.0

BMAL1 si

1 0

1000 800 600

80 70 60

1.5 1.0

NR1D1

1.5 1.0 0.5 0.0

ACTB

2.0 1.5 1.0

BMAL1

1.5 1.0 0.5 0.0

CLOCK

1.5 1.0 0.5 0.0

PER1

1.5 1.0 0.5 0.0

PER2

1.5 1.0 0.5 0.0

CRY1

2.5 2.0 1.5 1.0 0.5 0.0

CRY2

1.5 1.0 0.5 0.0

NR1D1

1.5 1.0 0.5 0.0

DBP

1.5 1.0 0.5 0.0

FBXL3

Luminescence

400 0

NR1D1 si

Amplitude

200 0 12 24 36 48 60 72 84 96

0.5 0.0

NR1D1 si

50 40

0.5 0.0

1000 800 600 400 200 0 0 12 24 36 48 60 72 84 96

80 70 60 50 40

1.5 1.0

COX4NB

1.5 1.0 0.5 0.0

ACTB

2.0 1.5 1.0

BMAL1

1.5 1.0 0.5 0.0

CLOCK

1.5 1.0 0.5 0.0

PER1

1.5 1.0 0.5 0.0

PER2

1.5 1.0 0.5 0.0

CRY1

1.5 1.0 0.5 0.0

CRY2

1.5 1.0 0.5 0.0

NR1D1

1.5 1.0 0.5 0.0

DBP

1.5 1.0 0.5 0.0

FBXL3

COX4NB si

0.5 0.0

COX4NB si

0.5 0.0

600 400 200 0 0 12 24 36 48 60 72 84 96

0 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

Time (hr)

siRNA amount (fmol in 384-well format)

siRNA amount (fmol in 384-well format)

siRNA amount (fmol in 384-well format)

BMAL1 CLOCK PER1 CRY2 FBXL3 POLR3F PRPF4 up down SEC13 up CRY1 down ACSF3 MPG BMAL1 down NR1D1 up COX4NB down down up

siRNA

mRNA levels PER2 CRY1 CRY2 NR1D1 down down down down down down up up down up down down down down

DBP down down down down

FBXL3 down

up down

up up

down down down

Figure 4. Dose-Dependent Effects of siRNAs on Clock Gene Expression

(A) Dose-dependent effects on circadian phenotype. Bmal1-dLuc cells were transfected with siRNAs (eight-point, 2-fold dilution series, 81000 fmol/well) designed against 11 genes including clock gene controls, and bioluminescence rhythms were recorded. Representative bioluminescence proles (left) and circadian parameters (right) are indicated. Data represent the mean SD (n = 3). (B and C) Dose-dependent knockdown of target genes (B) and effects on known clock gene expression (C). Bmal1-dLuc cells were transfected with siRNAs (eight-point, 2-fold dilution series, 243000 fmol/well) and analyzed by Q-PCR in unsynchronized conditions. mRNA levels of target gene (left) and ACTB (right,

206 Cell 139, 199210, October 2, 2009 2009 Elsevier Inc.

CTRL 8 16 31 63 125 250 500 1000

CTRL 8 16 31 63 125 250 500 1000

50 40 30 20 10 0

1.5 1.0

ACTB

1.5 1.0 0.5 0.0

BMAL1

1.5 1.0 0.5 0.0

CLOCK

1.5 1.0 0.5 0.0

PER1

1.5 1.0 0.5 0.0

PER2

1.5 1.0 0.5 0.0

CRY1

1.5 1.0 0.5 0.0

CRY2

1.5 1.0 0.5 0.0

NR1D1

1.5 1.0 0.5 0.0

DBP

1.5 1.0 0.5 0.0

FBXL3

Control si

0.5 0.0

Control si

CDK9 DHFR

MAPK8 SCAMP2 BCR EMP2 NUFIP2 CSE1L HNRPR PIAS1 SYCP2 TMEM2 NCL FLJ36812 PPP5C EPN1 TGFB1I1 TRRAP PRO0149 PTPN12 UBC RXRA PCAF TES CYLN2 LOC402110 MYD88 NOLA2 C13ORF24 MAP3K2 BLNK DVL3 PEG3 RORB ARNTL CLOCK CRY2 ARNTL2 NPAS2 CSNK1E FBXL3 PER3 AXIN1 PIK3R1 CSNK1A1 PER2 DVL2 HCFC1 CRY1 PER1 DVL1 CSNK1D CSNK2A1 MT2A C14ORF78 TBCB BRCA2 MPG

Figure 5. The Network

Expanded

Clock

Gene

ABL1

TP53

SFN

PLSCR1

NCOA6 RORA ICAM5

PPARGC1

IRF4

TRAF2

RORC

Clock components in the core feedback loop (blue), as well as other clock components known to regulate clock mechanism (light blue), were used to identify a list of interactors from the hits identied in the primary siRNA screen (green, short period; red, long period; purple, high amplitude) (Table S2). Protein-protein interactions were collated from Entrez Gene and the Prolexys proteinprotein interaction databases. Common interacting proteins (pink) are depicted as nodes (circles). Edges are depicted as protein-protein interactions (black), phosphorylation reactions (blue), transactivation (green), and transrepression (red). The graph was generated in Cytoscape (http://www. cytoscape.org/).

PNRC2

IKBKB

ZMAT3

NR1D1 YT521

FXR1 UBAP2

NCOA3

NR1D2 PPP1R9A PUM1

olism, are overrepresented. Previous data has shown that these pathways are HIF1A CAMK2G URG4 MPHOSPH10 RBAF600 MCM3AP clock regulated. Collectively, we conPKP2 RRP12 RAB3A clude the clock is massively interconCXORF15 HIF1AN NUP88 KALPHA1 nected and functionally intertwined with TRPC4AP UBE1C EPAS1 CNOT2 MLH1 many biological pathways. As a cauTNK2 tionary note, observations from this cellular model may not be directly applicable to clock function in the whole organism as Core clock component Protein-protein interaction Short-period hit many genes identied in our screen are Other clock component Phosphorylation reaction Long-period hit tissue specic. Trans-activation High-amplitude hit Why is this interconnectedness important? Research in other elds has Common interacting protein Trans-repression shown perturbation of one pathway often has deleterious and unintended conseknockdown led to long or short period length of oscillation or quences on another. For example, COX2 inhibitors such as Vioxx increased amplitude. Many of these factors had phenotypic were designed to inhibit pain and inammation; however, intereffects on the oscillator similar to known clock components mediates produced by COX2 (and inhibited by this class of small including dose-dependent perturbation. These genes are excel- molecules) also afford cardio protection (FitzGerald, 2004). Alterlent points of intervention for perturbation of autonomous clock natively, this interconnectedness may provide advantages properties and may later prove important in regulation of physi- many of the above pathways have specic inhibitors that may ology and behavior in the whole organism. Molecular analysis prove useful in altering circadian phenotypes. Knowledge of of these dose-dependent genes suggests that most of these this interconnectedness will be valuable in exploiting existing genes function by regulating clock gene expression levels. tools while avoiding the potentially deleterious consequences These observations point the way to new aspects of genetic of doing so blindly. Finally, we provide a publicly accessible resource to explore network architecture and possibly new feedback loops in the and visualize this data. It is our hope that this resource will serve clock. Protein interaction network analysis showed that some factors as a useful launching point for colleagues studying the circadian physically interact with core clock components, while others clock in interpretation of their biochemical and genetic results to interact with bridging proteins that physically interact with the accelerate understanding of how the clock regulates physiology clock (one degree of separation). This analysis also showed and behavior. that the P53 pathway is functionally interconnected with the EXPERIMENTAL PROCEDURES clock. Focused pathway analysis of screen hits dramatically extend this observationgenes from dozens of pathways are Materials represented on our hit list, and several pathways, including U2OS cells were obtained from the American Type Culture Collection (ATCC). insulin and hedgehog signaling, the cell cycle, and folate metab- We generated Bmal1-dLuc and Per2-dLuc reporter cell lines. The siRNA
HSP90AA1 USP9X EPB41L1 MAP7D1 PRPF4

as control) relative to GAPDH are indicated in (B), and mRNA levels of nine clock genes relative to GAPDH are indicated in (C). Data represent the mean SD (n = 2). In parallel experiments, bioluminescence rhythms were recorded and circadian phenotypes were conrmed. (D) Summary of dose-dependent effects on known clock gene expression.

Cell 139, 199210, October 2, 2009 2009 Elsevier Inc. 207

A
INSULIN SIGNALING PATHWAY
Glucose Homeostasis Flotiliin APS
+p

CAP Cbl
+p

CrkII GRF2

TC10

Exo70 CIP4/2

GLUT4 vesicle

translocation to PM GLUT4
+p

O Glucose

Glucose uptake

AMPK SHIP SKIP SOCS PTP1B JNK IKK

-p -py +py +ps

ACC FAS PFK PYK GK

Lipogenesis Fatty acid biosynthesis (path 1)

PRKCI
aPKC
+p SREBP-1c

PFKP
O DNA PGC-1a

PKLR
Glycolysis Glycolysis / Gluconeogenesis

MAPK8 IKBKB
O PIP3
+p

G6PC
+p

FOXO1

O DNA
+p

FBP PEPCK Glycogen O

+p

+p

INS
-p

INSR

IRS
-py

PI3K

PDK1/2

Akt
+p

GSK-3b
-p

GYS
-p

Glycogenesis Starch and sucrose metabolism

PIK3R5

Phosphatidyl inositol signaling system +ps : serine phosphorylation +py : tyrosine phosphorylation -py : tyrosine dephosphorylation

TSC2 TSC1 Rheb

+p +p +p +p

PP1 PDE3 BAD S6 eIF4E

+p +p

PHK PKA

PYG
+p

O cAMP

HSL Antiapoptosis Apoptosis

Antilipolysis

+p +p

LAR

FRAP1
+p

Lipid homeostasis

mTOR Raptor
+p

p70S6K 4EBP1
+p

MAPK signaling pathway GRB2 SOS Ras

+p

Protein synthesis

MNK Elk1 O DNA Proliferation, differentiation

SHC

Raf

MEK1/2

ERK1/2
+p

Period Change

Cycles in Liver

Period Change & Cycles in Liver

Implicated in Clock

Phase delay (hr)

29 27 25 23
Control si MAPK8 si1 MAPK8 si2 IKBKB si1 IKBKB si2 PIK3R5 si1 PIK3R5 si2 FRAP1 si1 FRAP1 si2 PRKCI si1 PRKCI si2 PFKP si1 PFKP si2 PKLR si1 PKLR si2

C
32

Period change

Phase change
3 2 1 0 -1 -8 -7 -6 -5

Period (hr)

Period (hr)

30 28 26 24 22 -8 -7 -6 -5

21

log[conc] (M)
DMSO SP600125 (JNK inhibitor) Dequalinium analog (PKC inhibitor) PMA (PKC activator)

log[conc] (M)
DMSO Wortmannin LY294002 (PI3K inhibitors)

Figure 6. Regulation of Cellular Circadian Clock by Components in the Insulin Signaling Pathway
(A) Insulin signaling pathway genes that impact circadian function. Using the NIH DAVID Pathway Analysis tool, we identied many components of the insulin signaling pathway represented in our screen hits. Genes that impact clock function and genes regulated by the clock are indicated with colored boxes. (B) Effects of siRNAs against genes involved in insulin signaling. The representative results from the secondary screen using individual siRNA are presented. Data represent the mean SD (n = 2). (C) Effects of chemical inhibitors against protein kinases involved in insulin signaling. Bioluminescence rhythms of Bmal1-dLuc cells were monitored in the presence of various concentrations of compounds (eight points of 3-fold dilution series; nal 3 nM7 mM). Period or phase parameter was plotted against nal concentrations of the compound. Data represent the mean SD (n = 4). The results for SP600215 and PMA are consistent with our previous observations (Hirota et al., 2008).

libraries used in the primary and secondary screens were purchased from QIAGEN. Additional siRNAs were purchased from Invitrogen and Dharmacon and used as controls in target validation experiments. GL2 and GL3 siRNAs designed against the Luc in pGL2 and pGL3 vectors, respectively, were purchased from QIAGEN. The GL2 siRNA was used as a negative control. Screen controls purchased from Invitrogen included CRY1-HSS102308, CRY2-HSS102311, and BMAL1 or ARNTL-HSS100703. CRY1 and NR1D1

siRNAs used in dose-dependent experiment were previously described (Baggs et al., 2009). Other siRNAs used in the dose experiment are listed in Table S3 and were used as pools. Cell Line Establishment U2OS cells were grown in regular Dulbeccos modied Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics.

208 Cell 139, 199210, October 2, 2009 2009 Elsevier Inc.

Lentiviral Per2-dLuc and Bmal1-dLuc reporters (Liu et al., 2008) were introduced into U2OS cells via lentivirus-mediated infection as described (Liu et al., 2008). We selected stable cell lines with blasticidin and clonal lines by uorescence-activated cell sorting (FACS)-based single-cell sorting in 96-well plates; we tested these lines as described in the LumiCycle (Liu et al., 2008). The clonal lines are genetically and morphologically indistinguishable from parental cells and represent the average period length of the infected cell population. Transfection and ViewLux Recording For primary siRNA screens, U2OS circadian reporter cells were reverse transfected with siRNAs in 384-well plates. In brief, we trypsinized rapidly growing cells and resuspended them in DMEM containing 20% FBS without antibiotics at 0.1 3 106 cells/ml. We next added 20 ml of transfection reagent mixture (3.3 ml/ml or 0.066 ml/well Lipofectamine 2000 in Opti-MEM) to each well containing prespotted siRNA constructs (1 pmol; 0.5 pmol for each siRNA; nal concentration of 12.5 nM), incubated them at room temperature for 20 min, and added 20 ml of cells (2000 cells/well) with our robotic system. Approximately 18 hr after transfection, we replaced this media with 60 ml prewarmed fresh DMEM containing 10% FBS and antibiotics and allowed the cells to grow for an additional 2 days. Three days after transfection, we replaced this media with 60 ml HEPESbuffered explant medium supplemented with luciferin (1 mM) (Promega) and B-27 supplements (Invitrogen), and the plates were sealed with an optically clear lm (USA Scientic). We next loaded these plates in a 36 C incubator and recorded bioluminescence expression with a ViewLux (Perkin Elmer). We measured bioluminescence for 30 s every 2 hr for 4 days. Secondary screens were performed in the same manner. For technical reasons, 20 out of the 292 plates in the primary screen were recorded for 3 days. Data Mining The primary screen produced a total of >4.3 3 106 data points (Tables S4 and S5). We used the MultiCycle circadian data analysis program (Actimetrics) to analyze recorded bioluminescence data. In brief, data was detrended by subtraction of a best t line (rst order polynomial) and, subsequently, were t to a sine wave to obtain circadian parameters such as rhythm period length and amplitude. Mechanistic Validation The Bmal1-dLuc cells were transfected as described (Hirota et al., 2008) with a small modication, 0.4 ml/well of Lipofectamine 2000. Parallel transfection experiments were performed for Q-PCR and functional analyses. For Q-PCR analysis, the cells were harvested prior to medium change for rhythm recording and therefore unsynchronized. Total RNA preparation and Q-PCR were performed as described (Hirota et al., 2008). SYBR Green PCR Master Mix (Applied Biosystems) or QuantiTect SYBR Green PCR kit (QIAGEN) was used for Q-PCR. The primers used in Q-PCR analysis are listed in Table S4. ACCESSION NUMBER The primary screening data reported in this paper has been deposited in the PubChem Bioassay database of the National Center for Biotechnology Information with the accession number AID 1904. SUPPLEMENTAL DATA Supplemental Data include six gures and six tables and can be found with this article online at http://www.cell.com/supplemental/S0092-8674(09)01099-X. ACKNOWLEDGMENTS We thank Buu Tu for technical support in the screens, Jia Zhang and Tony Orth for help with Qiagen siRNA libraries, Chunlei Wu and Ghislain Bonamy for help with BioGPS, and Richard Glynne and Peter Schultz for their institutional support and encouragement. We also thank Jose Pruneda-Paz, Elizabeth Hamilton, Dmitri A. Nusinow, Michael Hughes, Julie Baggs, and Jason

DeBruyne for critically reading the manuscript. This work is supported by National Institutes of Health grants to S.A.K. (MH51573 and GM74868). J.B.H. is supported by the Pennsylvania Commonwealth Health Research Formula Funds, the National Institute of Neurological Disease and Stroke (1R01NS054794), and the National Institute of Mental Health (P50 MH074924-01, awarded to Joseph S. Takahashi, Northwestern University). A.C.L. is supported by The University of Memphis startup fund and the National Science Foundation (IOS-0920417). S.A.K. is a founder of ReSet Therapeutics and is a member of its Scientic Advisory Board. BioGPS development is supported by NIGMS (1R01GM083924 to A.I.S.). This is manuscript number 090513 of the Genomics Institute of the Novartis Research Foundation. Received: May 30, 2009 Revised: July 3, 2009 Accepted: August 20, 2009 Published online: September 17, 2009 REFERENCES Andre, E., Conquet, F., Steinmayr, M., Stratton, S.C., Porciatti, V., and BeckerAndre, M. (1998). Disruption of retinoid-related orphan receptor beta changes circadian behavior, causes retinal degeneration and leads to vacillans phenotype in mice. EMBO J. 17, 38673877. Aza-Blanc, P., Cooper, C.L., Wagner, K., Batalov, S., Deveraux, Q.L., and Cooke, M.P. (2003). Identication of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol. Cell 12, 627637. Baggs, J.E., Price, T.S., DiTacchio, L., Panda, S., FitzGerald, G.A., and Hogenesch, J.B. (2009). Network features of the mammalian circadian clock. PLoS Biol. 7, e52. Balsalobre, A., Damiola, F., and Schibler, U. (1998). A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93, 929937. Brown, S.A., Fleury-Olela, F., Nagoshi, E., Hauser, C., Juge, C., Meier, C.A., Chicheportiche, R., Dayer, J.M., Albrecht, U., and Schibler, U. (2005). The period length of broblast circadian gene expression varies widely among human individuals. PLoS Biol. 3, e338. Busino, L., Bassermann, F., Maiolica, A., Lee, C., Nolan, P.M., Godinho, S.I.H., Draetta, G.F., and Pagano, M. (2007). SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins. Science 316, 900904. Conkright, M.D., Guzman, E., Flechner, L., Su, A.I., Hogenesch, J.B., and Montminy, M. (2003). Genome-wide analysis of CREB target genes reveals a core promoter requirement for cAMP responsiveness. Mol. Cell 11, 1101 1108. DeBruyne, J.P., Noton, E., Lambert, C.M., Maywood, E.S., Weaver, D.R., and Reppert, S.M. (2006). A clock shock: mouse CLOCK is not required for circadian oscillator function. Neuron 50, 465477. DeBruyne, J.P., Weaver, D.R., and Reppert, S.M. (2007). Peripheral circadian oscillators require CLOCK. Curr. Biol. 17, R538R539. Dennis, G., Jr., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., and Lempicki, R.A. (2003). DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 4, 3. Echeverri, C.J., Beachy, P.A., Baum, B., Boutros, M., Buchholz, F., Chanda, S.K., Downward, J., Ellenberg, J., Fraser, A.G., Hacohen, N., et al. (2006). Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat. Methods 3, 777779. Etchegaray, J.P., Machida, K.K., Noton, E., Constance, C.M., Dallmann, R., Di Napoli, M.N., DeBruyne, J.P., Lambert, C.M., Yu, E.A., Reppert, S.M., and Weaver, D.R. (2009). Casein kinase 1 delta regulates the pace of the mammalian circadian clock. Mol. Cell. Biol. 29, 38533866. FitzGerald, G.A. (2004). Coxibs and cardiovascular disease. N. Engl. J. Med. 351, 17091711. Florenes, V.A., Maelandsmo, G.M., Forus, A., Andreassen, A., Myklebost, O., and Fodstad, O. (1994). MDM2 gene amplication and transcript levels in

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