CHRONO protein as a key part of the mammal's internal body clock

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).

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 (−195, −138, −87, −52, and −16 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 E-boxes 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 (). This oscillation was observed in both the mouse CHRONO antibody we generated and the human C1orf51 antibody (ab106120, Abcam) (). Figure S2A and C Figure S2B

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.

Next, we asked how Chrono is involved in circadian transcription. The luciferase activity of the Per2 promoter (∼−2,817+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 (−1,333 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 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).

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.

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 23.81±0.08 h (mean ± standard deviation, n = 13), whereas that of Chrono-deficient mice was significantly longer (23.96±0.11 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., CK1δ/ε) [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).

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) or Cry2 knockdown (Cry2sh) NIH3T3 cells (Figures 5B 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 (27.29±0.06 h; mean ± 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 (26.91±0.07 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 (26.66±0.15 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.

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 up-regulated 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 up-regulated 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 up-regulated by stress-dependent behavior and contributes to repressive function in the glucocorticoid response.

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 (24.04±0.12 h, n = 6) was significantly longer than that of Chrono^flx/flx littermates (23.84±0.06 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.

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.

NIH3T3, COS7 cells and MEFs were maintained in Dulbecco's Modified Eagle Medium (DMEM; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) and penicillin–streptomycin antibiotics at 37°C and 5% CO₂.

NIH3T3 and MEF cells were stimulated with 100 nM dexamethasone containing medium at each time point. Cells were fixed in 1× PBS containing 0.5% formaldehyde. Glycine was added to a final concentration of 0.125 M, and the incubation was continued for an additional 15 min. After washing the samples with ice-cold phosphate-buffered saline, the samples were homogenized in 1 mL of ice-cold homogenize buffer (5 mM PIPES [pH 8.0], 85 mM KCl, 0.5% NP-40, and protease inhibitors cocktail) and centrifuged (15,000× g, 4°C, 5 min). The pellets were suspended in nucleus lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS, protease inhibitors) and sonicated 20 times for 30 s each time at intervals of 60 s with a Bioruptor (Diagenode, Inc.) or sonicated 10 times for 10 s each time at intervals of 50 s with a MICROSON (Misonix, Inc.) for brain samples. The samples were centrifuged at 15,000 rpm at 4°C for 5 min. Supernatants were diluted 10-fold in ChIP dilution buffer (50 mM Tris-HCl [pH 8.0], 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate, protease inhibitor).

Whole brain samples from mice (C57BL/6J) were used for ChIP-seq. BMAL1-bound DNA was purified by SDS-PAGE to obtain 150–200 bp fragments and sequenced on an Illumina GA sequencer at the Research Center of Research Institute for Radiation Biology and Medicine (RiRBM), Hiroshima University. We generated 15,000–20,000 clusters per “tile,” and 26 cycles of the sequencing reactions were performed according to the manufacturer's instructions. The identification of each DNA fragments was performed using Genome Studio software (Illumina Inc.).

CHRONO, BMAL1, HDAC1 (ab7028, Abcam), Acetyl-Histone H3 (06-599, Millipore), and IgG-bound DNA were used for quantitative real-time reverse-transcription PCR (RT-PCR). The primers were designed for amplifying the E-box-like regions in Per2 and Dbp promoters.

NIH3T3 cultures at the concentration of 1×10⁵ 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].

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.

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 anti–c-Myc antibodies (Sigma). After washing three times, the precipitates were resuspended in the 2× SDS-PAGE sample buffer, boiled for 5 min, and run on a 10% SDS-PAGE gel followed by Western blot analysis. Immunoreactive bands were detected by ODYSSEY Infrared Imaging System (LI-COR). These experiments were independently performed three times. The intensity of the band (BMAL1 and CHRONO rpar; was calculated by using ODYSSEY Infrared Imaging System.

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 −80°C until processing.

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).

CHRONO protein was synthesized using the TNT T7 Coupled Reticulocyte Lysate System (Promega). We added 2 µg of template DNA (Chrono/pcDNA3 or Chrono–Flag/pcDNA3) to an aliquot of the TNT Quick Master Mix and incubated it in a 50 µl reaction volume for 60–90 min at 30°C. Synthesized proteins were detected by 10 mCi/ml (specific activity, 30 TBq/mmol) [³⁵S]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% β-mercaptoethanol, 4.5% SDS, 100 mM 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).

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 50°C for 2 min and then at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min.

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).

BLOCK-iT Lentiviral RNAi System (Invitrogen) was used for RNAi experiments. NIH3T3 cells at the density of 5×10⁴ 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.

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).

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–βgeo–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 β-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 (+/−). Ultimately, we obtained homozygous KO (−/−) 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).

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.

The Chrono conditional KO mice (Accession No. CDB0913K; http://www.cdb.riken.jp/arg/mutant%20mice%20list.html↗) were generated as described (http://www.cdb.riken.jp/arg/Methods.html↗). To generate a targeting vector, genomic fragments of the Chrono locus were obtained from the RP23-385O12 BAC clone (BACPAC Resources). A 950 bp region containing exons 2 and 3 of the Chrono gene was flanked by loxP sites (Figure 7A). Targeted ES clones were microinjected into ICR eight-cell stage embryos, and injected embryos were transferred into pseudopregnant ICR females. The resulting chimeras were bred with C57BL/6 mice, and heterozygous offspring were identified by PCR. Primers for 5′ loxP were used—forward E1F (5′-CAGACAGTGAAGAAGCTGCATA-3′) and reverse loxR2 (5′-CAGACTGCCTTGGGAAAAGC-3′)—yielding no product for WT allele and 603 bp products for the targeted allele, respectively. Primers for 3′ loxP were used—forward loxF1 (5′-GGCATGGGCTATTCTGTTTG-3′) and reverse loxR1 (5′-TTGAGGGAAACAGCAGAGGT-3′)—yielding 121 bp products for WT allele and 179 bp products for the targeted allele, respectively (Figure S10A and C).

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).