A Novel Protein, CHRONO, Functions as a Core Component of the Mammalian Circadian Clock

Two independent studies, one of them using a computational approach, identified CHRONO, a gene shown to modulate the activity of circadian transcription factors and alter circadian behavior in mice.


Introduction
Circadian rhythms with a period of approximately 24 h endow organisms with the ability to adapt to changes of solar light following earth's rotation. The mammalian circadian clock system consists of inputs from light and feeding, a core pacemaker located in a paired nuclei, called suprachiasmatic nucleus (SCN), and outputs including, but not limited to, cycles of locomotor activity, sleep-awake, and hormonal secretion. Disturbance of the biological clock causes not only sleep rhythm disruptions but also various pathological conditions such as cancer, metabolic, and psychiatric disorders [1][2][3][4][5].
The clock gene, period, was first identified in fly [6][7][8] and later in various organisms [9]. The molecular mechanism of circadian transcription was then found to be based on interconnected transcription-translation feedback loops (TTFL), conserved from prokaryotes to humans [10][11][12]. In mammals, the complex of positive elements BMAL-CLOCK (NPAS2) activates PER and CRY that repress their own transcription to form a negative feedback loop. An accessory feedback loop involves ROR and REV-ERBa, which regulate BMAL1 transcription positively and negatively, respectively, whereas BMAL1 activates REV-ERBa expression. More members of the circadian clock components have been identified [13][14][15][16]. Because of the complexity of circadian timekeeping, mathematical modeling has emerged as an important tool to understand data and make novel predictions [17][18][19]. In particular, a recently published mathematical model reproduces much of the known data on circadian timekeeping (e.g., mutant phenotypes) and correctly predicts the pharmacological manipulation of circadian rhythms [20,21].
Although it is widely believed that the major components of the mammalian circadian clock have been identified, the search for additional clock components continues. In our previous study [22] and in others [23,24], the systematic screening by using ChIP (Chromatin immunoprecipitation)-Chip and ChIP-seq revealed several uncharacterized genome-wide BMAL1 targets. Among strong BMAL1 binding sites, only one novel gene, Gm129, was found besides known clock and clock-controlled genes such as Per1, Per2, Cry1, Cry2, Dbp, and Tef. Gm129 expression shows a robust circadian rhythm antiphasic to Bmal1. In light of its circadian expression, Gm129, now renamed Chrono (ChIP-derived Repressor of Network Oscillator), appeared to be a core clock gene.
Here, we study the functional role of Chrono in the circadian clock. CHRONO binds to the promoters of clock genes and functions as a negative regulatory component of the circadian clock. In vivo loss-of-function of Chrono including an Avp neuronspecific knockout (KO) mouse model displays a longer circadian period of locomotor activity. We demonstrate that Chrono is a coreclock component similar to Cry2, although its repression mechanism operates through an independent pathway. In silico study using a modified Kim-Forger model predicts that the recently identified residual rhythmicity in the Cry1, Cry2 double KO [25], is dependent on Chrono. Remarkably, Chrono is involved in glucocorticoid receptor (GR)-mediated metabolic physiology. We conclude that Chrono is part of the negative feedback loop of the mammalian circadian clock and a potential link between the clock and stress metabolism.

ChIP-Seq Identifies a Novel Clock Gene Regulated by BMAL1
Our previous ChIP-based genome-wide analyses using a core clock transcription factor BMAL1 identified hundreds of target molecules [22]. Among these targets, Gm129, now called Chrono, was one of the groups with the strongest binding including core clock proteins PER and CRY. Another ChIP-seq experiment using in vivo brain samples also identified Chrono as a BMAL1 target. Chrono exists only in mammals, is well conserved among mammals ( Figure S1), and consists of 375 amino acids with no functional domains. To examine whether Chrono encodes a polypeptide, we performed an in vitro translation experiment. Bands of approximately 45 kDa (CHRONO) and 46 kDa (CHRONO-FLAG) were observed as an in vitro translation product ( Figure 1A).

Chrono Transcripts Display Robust Circadian Expression
Our previous study showed robust circadian oscillation of Chrono mRNA in the mouse SCN and liver [22]. We further examined its expression in five different mouse peripheral tissues (heart, lung, stomach, kidney, and testis) by quantitative RT-PCR. After entrainment of mice housed for 2 wk under a 12-12 h light-dark (LD) cycle, samples were collected every 4 h starting at circadian time (CT) 0 (n = 3 at each time point) in the third dark-dark (DD) cycle. The temporal expression of Chrono transcripts in all tested tissues except testis displayed robust circadian rhythms peaking at approximately CT 12 ( Figure 1B), which were antiphasic to Bmal1. This result supports that Chrono encodes a component of the circadian clock loops [26]. The ChIP-seq experiment using brain samples revealed that BMAL1 strongly binds to CpG islands on the Chrono promoter in vivo ( Figure 1C). Studying differently sized Chrono promoter constructs (2195, 2138, 287, 252, and 216 bp from the transcriptional start site (TSS) of Chrono/PGL3B) showed that the closest E-box to the TSS is necessary to generate circadian oscillation of Chrono in NIH3T3 cells and that all of the three Eboxes contribute to robust circadian rhythms ( Figure 1D and E). These results suggest that BMAL1 strongly binds to the E-boxes on the Chrono promoter and regulates circadian expression of Chrono, making Chrono a novel clock gene.
We next asked whether CHRONO is also expressed rhythmically at the protein level. We prepared liver samples at CT 2,8,14, and 20. We raised a specific antibody against the CHRONO protein. CHRONO showed circadian rhythm antiphasic to BMAL1 as in the mRNA expression ( Figure S2A and C). This oscillation was observed in both the mouse CHRONO antibody we generated and the human C1orf51 antibody (ab106120, Abcam) ( Figure S2B).

CHRONO Forms a Complex with Other Clock Components
Because CHRONO showed a similar rhythmic expression profile to other core clock proteins, we asked if CHRONO binds directly with clock proteins. Various clock proteins with tags were expressed in COS7 cells and the expression was assessed by immunoprecipitation (IP) and blotting with anti-tag antibodies. CHRONO bound to BMAL1, PER2, CRY2, and DEC2 but not to PER1, CRY1, and DEC1 ( Figure 2A and B and Figure S2D). Among these interactions, we asked if CHRONO-BMAL1 binding occurs endogenously in vivo. We observed a band of BMAL1 in the CHRONO antibody IP from mouse liver lysate that was absent in the IP from Chrono-deficient mouse liver ( Figure 2C). These results suggest that CHRONO endogenously binds to BMAL1 in vivo.

Chrono Is Involved in HDAC-Dependent Transcriptional Repression
Next, we asked how Chrono is involved in circadian transcription. The luciferase activity of the Per2 promoter (,22,817+110 bp from TSS/PGL3B) in NIH3T3 cells was repressed by co-expression with Cry2 and Dec2 ( Figures 2D and S3A). The basic transcription activity of Per2 was increased by Bmal1 and Clock co-expression, and this activation was repressed by Chrono as well as Cry2. This repression was also seen in the Chrono promoter (21,333 bp from TSS/ PGL3B) ( Figure S3B). Moreover, overexpression of Chrono reduced the transcriptional amplitude of expression on the Dbp promoter, just as Cry2 ( Figure S3C and D). These results suggest that Chrono

Author Summary
The circadian clock has a fundamental role in regulating biological temporal rhythms in organisms, and it is tightly controlled by a molecular circuit consisting of positive and negative regulatory feedback loops. Although many of the clock genes comprising this circuit have been identified, there are still some critical components missing. Here, we characterize a circadian gene renamed Chrono (Gm129) and show that it functions as a transcriptional repressor of the negative feedback loop in the mammalian clock. Chrono binds to the regulatory region of clock genes and its occupancy oscillates in a circadian manner. Chrono knockout and Avp-neuron-specific knockout mice display longer circadian periods and altered expression of core clock genes. We show that Chrono-mediated repression involves the suppression of BMAL1-CLOCK activity via an epigenetic mechanism and that it regulates metabolic pathways triggered by behavioral stress. Our study suggests that Chrono functions as a clock repressor and reveals the molecular mechanisms underlying its function.
functions as a negative element of circadian transcription, similar to Cry2.
Histone modification by histone deacetylase (HDAC) is one of the mechanisms of transcriptional regulation [27]. HDAC is often recruited during the transcriptional repression process. We hypothesized that CHRONO is in a repressor complex that includes CRY2. To investigate the potential role of HDAC in Chrono-mediated transcriptional repression, we treated cells with trichostatin A (TSA), an HDAC inhibitor, in the reporter assay. Chrono, as well as Cry2, repressed the enhanced luciferase activity of the Per2 promoter. However, in the presence of TSA, Chrono did not repress activity but rather enhanced activity, whereas Cry2 did not change its repression ( Figure 2E). To confirm the involvement of HDAC in Chrono-mediated transcriptional repression, we investigated the interaction of CHRONO with HDAC1. We expressed and showed co-IP of CHRONO-HA and HDAC1-FLAG in COS7 cells, indicating that CHRONO is bound with HDAC1 ( Figure 2F and G).

CHRONO Rhythmically Binds the E-Boxes on Circadian Gene Promoters
We then asked if endogenous CHRONO participates in clock function as a core clock component. A ChIP experiment with the CHRONO antibody in NIH3T3 cells after induction with dexamethasone showed endogenous binding of CHRONO to the E-boxes on Per2 ( Figure 3A) and Dbp ( Figure 3B) promoters. The levels of chromatin occupancy around the E-boxes on Per2 and Dbp were significantly different between 32 h and 44 h after dexamethasone stimulation ( Figure 3A and B, *p,0.05, **p, 0.01). Moreover, endogenous CHRONO occupancy showed circadian oscillation antiphasic to BMAL1 ( Figures 3C and  S3G). These results strongly suggest that Chrono behaves as an auto-regulated clock component.

Chrono KO Mice Have a Lengthened Circadian Period
To evaluate the physiological and circadian clock function of Chrono in vivo, we generated Chrono KO mice by using a gene trap method ( Figures 4A and S4). After entrainment in the 12-12 h LD condition, the locomotor activity rhythm under DD was recorded. In DD the average circadian period length of wild-type (WT) mice was 23.8160.08 h (mean 6 standard deviation, n = 13), whereas that of Chrono-deficient mice was significantly longer (23.9660.11 h, n = 12) (*p,0.001; Student's t test) ( Figure 4B-D). To logically confirm that the behavioral Chrono KO phenotype is an outcome of the observed biochemical and in vitro characteristics of Chrono (Figure 2), we adopt a mathematical modeling approach. We used a recently developed mathematical model of mammalian circadian clock (Kim-Forger model) because this model successfully reproduced and predicted the circadian period change in response to mutations of clock genes (e.g., Per1/2, Cry1/2, Bmal1, Clock, and etc.) and pharmacological inhibition of kinase (e.g., CK1d/e) [20,21]. The model is extended to include biochemical mechanisms of Chrono, such as binding with other clock components (Figure 2A and B), transcriptional repression by Chrono ( Figure 2D-G), and rhythms of Chrono ( Figure 1B) (see details in Text S1). Although CHRONO acts similarly to CRY2, we also incorporate key differences, including their very different mRNA time profiles ( Figure S5) and the fact that CHRONO does not bind PER1. When the transcription of Chrono is inhibited in the model, the model predicts that Chrono KO lengthens the period ( Figure 4D, right), which indicates that the biochemical mechanisms we have identified for Chrono-mediated repression of BMAL1-CLOCK ( Figure 2D) are expected to cause the Chrono KO phenotypes. There was no difference of basal locomotor activity between WT and Chrono KO mice during the light phase and dark phase after 1 wk in LD (p.0.5; Student's t test) ( Figure 4E). We also examined how Chrono KO mice responded to shifts in the LD cycle. When the lighting cycle was advanced 6 h, both WT and Chrono KO mice re-entrained progressively over 10 d, and there were no difference between the genotypes ( Figure S6A and B). Moreover, no induction of Chrono mRNA in the SCN was observed after light stimulation for 30 min ( Figure S6C).

Chrono Acts as a Transcriptional Repression Independent of Cry2
Because Chrono is a putative repressor in the circadian clock mechanism, we next asked whether the expression of clock genes is altered after the deletion of Chrono in mice. In mouse embryonic fibroblasts (MEFs) derived from Chrono KO mice ( Figure S7A), Per2 expression was increased at 48 h and 52 h after dexamethasone stimulation ( Figure S7B), compared with WT MEFs. In the liver derived from Chrono KO mice ( Figure  S7C), Per3, Cry1, Dbp, and Rev-erbs were increased at CT12 (*p,0.05, **p,0.01, Student's t test), consistent with the idea that Chrono is involved with negative feedback regulation of the core clock component via E-box. The similar trend was observed in the Chrono-deficient SCN ( Figure S7D). Moreover, Acetyl-Histone H3 occupancy on Per2 and Dbp promoters was enhanced in Chrono KO MEFs compared to WT ( Figure S7E and F; **p,0.01, Student's t test). This result suggests that Chrono changed the epigenetic modification of cells with the expression status.
Chrono's role in circadian timekeeping seems to mimic that of Cry2 both in vivo and in cells. Because CHRONO interacts with CRY2 and DEC2, we hypothesized that CHRONO represses BMAL1-CLOCK activity only when partnered with CRY2 or DEC2 to form a complex. To confirm this hypothesis, we performed a luciferase reporter assay using Cry2 KO MEFs ( Figure 5A  and S8) and Dec2 KO MEFs ( Figure S3E). The BMAL1-CLOCK complex induced Per2 transcription in all cells and Chrono repressed the BMAL1-CLOCK activity in all cells, including those without Cry2 and Dec2, indicating that CHRONO acts as a transcriptional repressor via an independent pathway from CRY2 and DEC2. Moreover, we established a stable NIH3T3 cell line with Bmal1 promoter luciferase and Cry2 knockdown. The rhythm of fibroblasts with overexpression of Flag as a control showed a robust circadian oscillation with a lengthened period (27.2960.06 h; mean 6 S.E.M.; n = 4) due to Cry2 knockdown. As expected, constitutive overexpression of Cry2 driven by the CMV promoter partially restored the period to a shorter value (26.9160.07 h; n = 4; *p,0.01, Welch's t test) in the Cry2-sh/ NIH3T3 cells. Consistent with the idea that Chrono mimics the Cry2 phenotype, overexpression of Chrono similarly shortened the period (26.6660.15 h; n = 5; *p,0.01, Welch's t test) of oscillation in the Cry2-sh/NIH3T3 cells ( Figure 5C and D). These relative modulations in periods are predicted in parallel by the extended Kim-Forger model ( Figure 5E), under the simulated experimental conditions of Cry2 knockdown to 30% of its original value and/or constitutive overexpression of either Cry2 or Chrono. These results indicate that the circadian phenotypes of Chrono are similar to Cry2, although the repression mechanisms operate via different pathways.
Given the phenotypic similarities between Chrono and Cry2, we postulated that Chrono could overtake the role of Cry2 under KO conditions. To test this idea, we generated Chrono, Cry1 double KO mice by mating Chrono KO with Cry1 KO mice. The Cry1, Cry2 double KO mouse shows a circadian arrhythmicity in DD [28], and we expected a Chrono, Cry1 double KO would exhibit similar arrhythmicity. However, the circadian locomotor activity persisted under DD. In DD condition, Cry1 KO mice showed a shorter period than WT, similar to Cry1, Chrono double KO mice ( Figure 5F). There was no significant difference of period between Cry1 KO and Cry1, Chrono double KO mice ( Figure 5G). These results are predicted by our mathematical model, which shows short period rhythms under the double Chrono, Cry1 KO (22.95 h) and Cry1 single KO (22.89 h), respectively ( Figure 5G, right). The model also predicted that the Chrono KO does not compromise the amplitude of Per2 mRNA rhythms ( Figure 5H). Interestingly, the model predicts initially oscillating but damping Per2 rhythms in Cry1, Cry2 double KO ( Figure 5H, blue). However, the triple, Chrono, Cry1, Cry2 KO completely removed any residual oscillations ( Figure 5H, red). These results led us to conclude that, although CHRONO acts as a repressor of the BMAL1-CLOCK complex just as CRY2, the mechanism of action of CHRONO is independent of CRY2 and that CHRONO may be required for rhythmicity in certain mutant backgrounds.

Chrono Mediates Clock Repression in the Glucocorticoid Response
The Cryptochromes (Cry1 and Cry2) have been reported to mediate rhythmic repression of the GR [29]. Because Chrono behaves as a transcriptional repressor with an effect similar to Cry2, we next verified the physiological role of the Chrono KO against stress responses. We first examined the expression of serum/glucocorticoid-regulated kinase 1 (Sgk1) after dexamethasone stimulation. We found that Sgk1 mRNA was upregulated in Chrono KO MEFs when compared to WT MEFs after 1-h and 4-h exposures to dexamethasone ( Figure 6A, *p, 0.05, **p,0.01, Student's t test). The relative expression of Sgk1 was also activated by 4-h exposure to dexamethasone ( Figure 6B; *p,0.05, Student's t test). Serum corticosterone levels in vivo were also robustly increased in Chrono KO when compared with WT mice after 0.5 h and 1 h of restraint stress ( Figure 6C; *p,0.05; one-way ANOVA followed by Tukey-Kramer's multiple comparisons test, as follows, *p,0.05 versus 0.5 h time after restraint stress; **p,0.01 versus 1 h time after restraint stress). In a WT background, Chrono mRNA was upregulated in MEFs ( Figure 6D; **p,0.01, Student's t test) and hypothalamus ( Figure 6E; *p,0.05, Student's t test) after 1 h of exposure to dexamethasone and restraint stress, respectively, whereas Cry2 mRNA was not significantly increased under restraint stress in both WT and Chrono KO hypothalamus as well as MEFs ( Figure S9). Furthermore, the IP-Western experiment indicated CHRONO endogenously interacted with GR in vivo ( Figure 6F). These results suggest that Chrono is upregulated by stress-dependent behavior and contributes to repressive function in the glucocorticoid response.

Chrono in the SCN Plays a Central Role in Behavioral Rhythms
To identify the role of Chrono in the SCN, we generated mice carrying a conditional Chrono KO allele using the Cre-loxP system ( Figures 7A and S10). Avp-Cre mice express Cre specifically in arginine vasopressin (AVP) neurons (GENSAT project) [30]. AVP is the most abundant neuropeptide in the hypothalamus and specifically in the SCN [31,32]; therefore, Avp-Cre Chrono flx/flx mice can be considered as SCN-targeted Chrono KO mice. The average locomotor activity rhythm of Avp-Cre Chrono flx/flx male mice (24.0460.12 h, n = 6) was significantly longer than that of Chrono flx/flx littermates (23.8460.06 h, n = 8) (*p,0.01, Student's t test) ( Figure 7B-D). The basal locomotor activity was measured during the light phase and the dark phase after 1 wk in LD by the infrared beam breaking. The activity amount of the Avp-Cre Chrono flx/flx mouse was not altered compared with Chrono flx/flx mouse ( Figure 7E). These results demonstrate that Chrono expression in the Avp neurons plays a central role in behavioral rhythms.

Discussion
In this study, we characterized a gene called Chrono that serves as a component in the negative arm of the core mammalian circadian clock. Indeed, based on our genomic analysis of circadian promoters, CHRONO appears to be one of the last remaining components of the clock. The most interesting feature of CHRONO is its regulation by epigenetic control and behavioral stress. These findings place CHRONO in a central position to couple stress metabolism to clock regulation.
Our results suggest that CHRONO operates as a repressor of the core circadian feedback loop through the recruitment of HDAC. The repressive effects of CHRONO were also seen in Cry2 KO MEFs and Dec2 KO MEFs, which shows that CHRONO repression does not require cooperative interactions with CRY2 or DEC2 [33,34]. It has been reported that the recruitment of histone-modifying enzymes is regulated in a circadian manner [35][36][37][38][39] and some complexes are observed in transcriptional repression as in PER and SIN3-HDAC [14]. HDAC1 rhythmically bound to the Per2 promoter ( Figure S3F), and this result was consistent with a recent report [40]. Acetyl-Histone H3 occupancy around E-boxes was enhanced in Chrono KO MEFs compared to WT ( Figure S7E and F), suggesting that Chrono also has the potential to form complexes with other histonemodifying enzymes. However, HAT (histone acetyltransferase) activity by CLOCK was not affected by CHRONO in vitro experiments ( Figure S11). Further mechanistic study is required. CHRONO endogenously binds to the E-boxes of circadian genes with circadian rhythmicity, suggesting that Chrono is a core circadian repressor that operates as an auto-regulated component of the clock.
The Chrono KO mouse showed a lengthened circadian period similar to the Cry2 KO mouse [28]. Overexpression of Chrono restored the period of Bmal1-luc oscillations in Cry2 knockdown cells in a manner comparable to Cry2 overexpression. On the other hand, the Chrono, Cry1 double KO mouse did not show arrhythmic behavior as seen in the Cry1, Cry2 double KO mouse [28]. Together, these results suggest that Chrono plays a similar but independent role from Cry2. A conditional Chrono KO mouse driven by the Avp promoter (AvpCre Chrono flx/flx ) also showed a lengthened circadian period. This points to Chrono's central role in the core clock and that its expression in Avp neurons is critical for the determination of behavioral circadian period. It also demonstrates that the AvpCre system (GENSAT line number QZ20_CRE) can potentially prove useful in dissecting the output pathway of the SCN that may perform coding by internally distributed periods [41].
The role of Chrono was also tested in silico by modeling mechanisms of Chrono-mediated repression of BMAL1-CLOCK ( Figure 2D) within the most comprehensive and realistic mathematical model of the mammalian circadian clock [20]. The extended model successfully predicted that the reduced (or enhanced) Chrono expression results in a lengthened (or shortened) period ( Figures 4D and 5E), matching the phenotypes of Chrono KO or overexpression (Figures 4D and  5D). This indicates that the proposed biochemical mechanisms of Chrono-mediated repression of BMAL1-CLOCK ( Figure 2D) are consistent with the overall phenotypes observed when Chrono levels are changed.
Chrono is likely to be a physiological response-dependent regulator. In response to dexamethasone application, Sgk1 was up-regulated in Chrono KO MEFs when compared with WT MEFs. In vivo serum corticosterone levels were also increased in the Chrono KO mouse when compared with the WT mouse under restraint stress and CHRONO itself interacted with the GR. Along with the observation that Chrono mRNA expression was induced by the stress response in MEFs and hypothalamus, we conclude that Chrono is a potential repressor activated by behavioral stress and can couple the clock with the hypothalamic-pituitary-adrenal (HPA) axis. Our previous results showed acute physical stress also elevated Per1 mRNA through a glucocorticoid-responsive element [42]. Further experimental work is needed to reveal the detailed molecular interactions between the circadian clock and the HPA axis.
Our discovery of the role of Chrono opens up many possibilities for future work. Our mathematical modeling raises the interesting possibility that the Per2 oscillations from Cry1, Cry2 double KO neonatal SCN [25] is mediated by Chrono. In further studies, it would be interesting to see if the Cry1, Cry2, Chrono triple KO eliminates the ability to oscillate in neonatal SCNs as predicted by our model ( Figure 5H). Further work should also examine the role of Chrono in linking the HPA to the circadian clock. Taken together, we conclude that Chrono is a novel circadian clock gene that acts as a repressor in the circadian system and modulates physiology.

Ethics Statement
All protocols of animal experiments followed in the present study were approved by the Animal Research Committee of Hiroshima University and Animal Care and Use Committees of the RIKEN Brain Science Institute.

In Vitro Real-Time Oscillation Monitoring System (IV-ROMS)
NIH3T3 cultures at the concentration of 1610 5 cells in Opti-MEM (Gibco) supplemented with 10% FBS in a 35 mm dish were transfected with the desired plasmids by using the Lipofectamine reagent (Invitrogen). The medium was exchanged 24 h after transfection with 100 nM dexamethasone containing medium, and 2 h later this medium was replaced with Opti-MEM supplemented with 1% FBS and 0.1 mM luciferin-10 mM HEPES (pH 7.2). Bioluminescence was measured by using the IV-ROMS (Hamamatsu Photonics) as described previously [26,43].

Luciferase Assay
NIH3T3 cells and MEFs were cultured and transfected with the desired plasmids by using Lipofectamine 2000 (Invitrogen) or Nucleofection (Lonza). Cells were harvested 24 h after transfection, and cell lysates were prepared and then used in the dual luciferase assay system (Promega). For exogenous expression, we transfected cells with pcDNA3 driven by ubiquitous cytomegalovirus (CMV) promoter.

Western Blotting and IP
Rabbit antibody against HA-tag and mouse antibodies against Flag-tag and Myc-tag were subjected to Western blot according to the manufacturer's protocol. For IP, COS7 cells transfected with the desired plasmids by using Lipofectamine 2000 (Invitrogen) were lysed in TNE buffer with protease inhibitor. Following the standard protocol, lysates were precleared with Dynabeads Protein G (Invitrogen) and then immunoprecipitated with rabbit anti-HA antibodies (Cell signaling), mouse anti-Flag antibodies (Sigma), or mouse antic-Myc antibodies (Sigma). After washing three times, the precipitates were resuspended in the 26 SDS-PAGE sample buffer, boiled for 5 min, and run on a 10% SDS-PAGE gel followed by Western blot analysis. Immunoreactive bands were

Animal Care and Behavioral Analysis
Circadian rhythms of locomotor activity were analyzed as previous described [43]. Each mouse was individually housed for 2 wk in 12-12 h LD cycles, and then for 4 wk in constant darkness (DD). Locomotor activity was monitored with a locomotor activity recording apparatus (Biotex, Kyoto, Japan) that measures events of infrared beam breaking in 1-min bins. The data from the first week under DD were used to estimate the period of circadian locomotor activities of WT and Chrono KO mice. For RNA sampling, dissected tissues were immediately frozen in liquid nitrogen and stored at 280uC until processing.

Methods for Light Treatment
The experiment was performed as described previously [44]. Male ICR mice (SLC, Japan) were exposed to an incandescent light stimulus (1,000 lux, 30 min) at CT16 in the second DD cycle. Animals were sacrificed 60 min after the initiation of the light exposure. Total RNA was prepared from six pooled pairs of SCN at each time point using PicoPure RNA Isolation kit (Applied Biosystems).

In Vitro Translation
CHRONO protein was synthesized using the TNT T7 Coupled Reticulocyte Lysate System (Promega). We added 2 mg of template DNA (Chrono/pcDNA3 or Chrono-Flag/pcDNA3) to an aliquot of the TNT Quick Master Mix and incubated it in a 50 ml reaction volume for 60-90 min at 30uC. Synthesized proteins were detected by 10 mCi/ml (specific activity, 30 TBq/mmol) [ 35 S]methionine, and resolved on SDS-PAGE (10%) gels using one-fifth of each translation reaction product mixed with an equal volume of sample buffer (15% glycerol, 5% b-mercaptoethanol, 4.5% SDS, 100 mM expression vectors. All detrended traces (6-7 samples) from each set of experiments (Flag, Cry2, and Chrono overexpression) are superposed. The baselines are intentionally separated for each case for ease of view. The abscissa indicates the day in culture, and the ordinate the relative bioluminescence intensity (kcpm, 1,000 photon counts per minute). Cry2sh/NIH3T3 cells prolonged the period, and Chrono recovered the period length similar to Cry2. *p,0.005. (E) Model prediction of Bmal1 transcript expression in the Cry2 knockdown (Flag, black), under additional constitutive overexpression of Cry2 (blue), and Chrono (red). The baselines are separated intentionally for ease of view. (F) Raw actograms of WT, Chrono, Cry1 double KO and Cry1 KO mice and comparison of free-running period estimates. A significantly shorter free-running period was observed in Chrono, Cry1 double KO and Cry1 KO mice compared to WT in DD (G, middle), in line with the prediction through in silico simulation (G, right). Chrono, Cry1 double KO mice did not show arrhythmicity in DD. *p,0.05, Student's t test. (H) Model prediction of Per2 mRNA time courses in Cry1, Cry2 double KO (blue) and Cry1, Cry2, Chrono triple KO (red) compared to WT (black). The amounts of KO transcripts are set to 0 from the beginning of the simulated day 0, and dynamics before reaching steady state is illustrated. doi:10.1371/journal.pbio.1001839.g005 Tris-Cl, pH 6.8, 0.03% bromophenol blue). Gels were fixed, dried, and exposed to Hyper-film (Amersham) for 16 h to 2 d. The molecular masses (in kDa) of the translated proteins were derived using standard curves generated from protein size standards (Bio-Rad).

Quantitative RT-PCR
Each quantitative real-time RT-PCR was performed using the ABI Prism 7900HT sequence detection system as described previously [42,45]. The PCR primers were designed with the Primer Express software (Applied Biosystems). The reaction was first incubated at 50uC for 2 min and then at 95uC for 10 min, followed by 40 cycles at 95uC for 15 s and 60uC for 1 min.

Corticosterone Level Under the Restraint Stress
Blood samples were collected by tail bleeding at time points of 0, 0.5, 1, 2, and 4 h after restraint stress. Corticosterone in mouse serum was measured using YK240 corticosterone enzyme immunoassay (EIA) kit (Yanaihara Institute).

RNAi Experiment
BLOCK-iT Lentiviral RNAi System (Invitrogen) was used for RNAi experiments. NIH3T3 cells at the density of 5610 4 were infected with a lentiviral vector and cultured. We selected stably transfected cells with zeocin. The shRNA/NIH3T3 cells were infected with Bmal1 promoter-driven luciferase lentiviral vector and selected for stable expression with blasticidin.

Antibodies
Antibody against BMAL1 was generated as described previously [22]. Purified glutathione S-transferase (GST) -Gm129 N-terminal (amino acids 1 to 187) protein was produced as a recombinant protein in the competent cells BL21 (DE3) (Stratagene). After removing GST by using GSTrap FF and PreScision Protease (GE Healthcare), the produced antigen was used to immunize rabbits. The antiserum was subjected to affinity purification using Affi gel 10 (Bio-Rad) conjugated with the antigen. The anti-CHRONO antibody recognized its target protein in immunochemical analysis ( Figure S4D).

Chrono (Gm129) Gene Trap Mice
C57BL/6 gene trap ES cell clone IST11761C7 was an embryonic stem cell provided by the gene trap method [46]. Long terminal repeat (LTR) -splice acceptor-bgeo-polyA-LTR sequence was inserted between exons 1 and 2 in one allele of the Chrono (Gm129) gene ( Figure 4A) (TIGM, Texas A&M Institute for Genomic Medicine). In substitution for mRNA producing CHRONO protein, mRNA producing fusion protein of the neomycin-resistant gene product and b-galactosidase (B-geo) was transcribed from this variation allele. We generated chimeric mice from C57BL/6 gene trap ES cell clone IST11761C7, mated the chimeric mice with WT C57BL/6 (+/+), and subsequently obtained heterozygous KO mice (+/2). Ultimately, we obtained homozygous KO (2/2) mice by mating heterozygous KO mice. Absence of Chrono mRNA and CHRONO protein was confirmed by PCR, RT-PCR, and Western blotting ( Figure S4B-D).

mRNA Expression of Core Circadian Genes in Chrono KO MEFs and Liver
WT and Chrono KO MEFs were stimulated with 100 nM dexamethasone containing medium. After 32 h, mRNA were extracted by TRI reagent (Molecular Research Center, Inc.) every 4 h. Quantitative real-time RT-PCR was described above. Adult WT and Chrono KO mice were exposed to 2 wk of LD cycles and then kept in complete darkness as a continuation of the dark phase of the last LD cycle. mRNA expression was examined CT12 and next CT0 from the third DD cycle.

In Silico Evaluation of Chrono in the Oscillator Network
We incorporated Chrono into a recently developed mathematical model of the mammalian circadian clock [20] based on our biochemical findings. We assumed that the mRNA degradation rate of Chrono is the same as Per2, as the Chrono mRNA time course is similar to Per2 mRNA [42]. We further assumed the time course of PER2-CHRONO nuclear entry is similar to the PER-CRY complexes. However, as found in our experiments, the CHRONO protein binds to the PER2 protein but not to PER1 protein ( Figure 2A) and that the CHRONO protein interacts with BMAL1-CLOCK to inhibit its E-box activation on Per1, Per2, Cry1, Cry2, and Rev-erbs promoters. In the model, we did not consider binding between CHRONO and CRY2, as BMAL1-CLOCK repression by CHRONO did not depend on the presence of CRY2 ( Figure 5). In total, 38 variables were newly added to the Kim-Forger model, which account for all the possible complexes involving CHRONO (details of computer simulation are described in Text S1, Tables S1, S2, S3, and Appendix S1). All the simulations were performed with Mathematica 8.0 (Wolfram Research). Figure S1 Sequence conservation of Chrono. The protein sequence alignment of Chrono across species was performed with Homologene database (NCBI). Chrono is highly conserved in mammals. (TIF) Figure S2 Characterization of CHRONO. (A-C) CHRONO protein expression showed circadian rhythm antiphasic to BMAL1 in the liver. We prepared liver samples at CT 2,8,14,and 20. Each time point has four or five samples, which were dissolved with RIPA buffer. The x-axis represents time in CT and y-axis protein amounts. The relative levels of protein were normalized to the b-ACTIN protein levels. The maximum protein amount was set to 100. (D) A schematic model of CHRONO interaction. CHRONO interacts with BMAL1, PER2, CRY2, and DEC2 (red line), but not PER1, CRY1, and DEC1 (see Figure 2A [22,47]. That is, the amplitude of the Chrono mRNA rhythm is much larger than that of Cry2 mRNA, and the phase of the Chrono mRNA rhythm is more advanced than that of Cry2 mRNA. (TIF)  Figure 7A). The WT (6.7 kb) and Chrono flx (8.7 kb) alleles were detected. (C) Mouse tail DNA from mice carrying WT and/or the Chronoflx allele were genotyped by PCR using primer sets E1F/loxR2 or loxF1/loxR1, as shown in (A). E1F/loxR2 amplifies a fragment of 603 bp only from the Chrono flx allele. loxF1/loxR1 amplifies fragments of 121 bp from the WT allele and 179 bp from the Chrono flx allele. (TIF) Figure S11 HAT activity in NIH3T3. HAT activity in NIH3T3 cells, which were transfected with the desired plasmids by using Lipofectamine 2000 (Invitrogen). After 24 h from transfection, the nuclear protein was extracted and the Histone acetyltransferase activity was measured by using HAT assay kits (ab65352, Abcam). (TIF)   Appendix S1 Newly added and modified equations. (DOCX)