The NRON complex controls circadian clock function through regulated PER and CRY nuclear translocation

Post-translational regulation plays a central role in the circadian clock mechanism. However, nucleocytoplasmic translocation of core clock proteins, a key step in circadian timekeeping, is not fully understood. Earlier we found that the NRON scaffolding complex regulates nuclear translocation of NFAT and its signaling. Here, we show that components of the NRON complex also regulate the circadian clock. In peripheral cell clock models, genetic perturbation of the NRON complex affects PER and CRY protein nuclear translocation, dampens amplitude, and alters period length. Further, we show small molecules targeting the NFAT pathway alter nuclear translocation of PER and CRY proteins and impact circadian rhythms in peripheral cells and tissue explants of the master clock in the suprachiasmatic nucleus. Taken together, these studies highlight a key role for the NRON complex in regulating PER/CRY subcellular localization and circadian timekeeping.

Intriguingly, we found that the same complex that regulates NFAT also regulates translocation of clock proteins PER and CRY 20 . More specifically, the kinases in the complex (CSNK1E, GSK3B, DYRK1A) were shown to regulate NFAT function 10,27,28 . These kinases also regulate phosphorylation-related proteolysis and nuclear entry of PER and CRY proteins 13,14,20,22 . Further, two other components of the NRON complex, nuclear importin beta (KPNB1) and nuclear import carrier Transportin 1 (TNPO1) were recently shown to play a key role in post-translational regulation of the PER/CRY complex 20,29 . Thus, the NFAT and circadian clock pathways share functional components.
Here, we extend these studies to other components of the NRON complex and characterize their role in nucleocytoplasmic shuttling of the PER/CRY complex. We demonstrate that these regulations are functional using both peripheral clock models and ex vivo suprachiasmatic nucleus (SCN) explants. Collectively, these studies point to the role of the NRON complex as a flexible module regulating nucleocytoplasmic shuttling of at least two critical signal transduction pathways, NFAT, and the circadian clock.

Knockdown of the NRON complex components alters circadian rhythms in human and mouse cellular clock models.
Previous studies including ours show that several components of the NRON complex, including CSNK1E, GSK3B, and DYRK1A, regulate clock function in mammals 13,21,22 . Several components and their analogs have knockdown phenotypes in our previous siRNA genomic screen, including PSMD11, PSMD13, DDX3X, DDX56, KPNB1, and KPNA4 30 . We showed that importin-beta (KPNB1) directly interacts with PER2 and mediates PER/CRY nuclear translocation, and in so doing, regulates circadian rhythms in human cells and in flies 20 . As these NRON complex components perform independent but interrelated functions in regulating nucleocytoplasmic protein abundance and activities, we hypothesized that other members of the complex also play roles in regulating clock function. To test this, we used RNA interference (RNAi) to knock down the complex components individually in human U2OS cells stably expressing the Bmal1 promoter-driven luciferase reporter and performed kinetic bioluminescence assays to assess clock function. We show that knockdown of 12 of 14 genes altered circadian rhythms in these cells (Fig. 1B-E), without adversely impacting cell viability (Fig. S1A). For example, knockdown of nuclear import and export components, KPNB1 and CSE1L, caused arrhythmicity, consistent with our previous findings 20 . Three genes involved in protein synthesis and proteolysis (EIF3E, PSMD11, HUWE1) led to arrhythmicity upon knockdown. Similar results were obtained from an independent mouse MMH-D3 hepatocyte clock model 31 (Fig. S1B). The majority of these genes that are involved in phosphorylation and signal transduction caused long period phenotypes upon knockdown, including DYRK1A. It is noted that the DYRK1A knockdown phenotype is consistent with our previous genome-wide screen in the U2OS cell model 30 , but different from a previous report that showed a short period length phenotype in a fibroblast cell model 13 . We confirmed the long period phenotype in U2OS cells using Harmine, a specific DYRK1A inhibitor 32 (Fig. S1C), which is also consistent with a previous study showing a similar phenotype in Per2::Luc embryonic fibroblasts and the SCN 33 . This phenotypic difference may reflect cell line or type-specific clock function. Taken together, these results suggest that the NRON complex, known for its role in regulating NFAT signaling, also regulates the circadian clock.
Components of the NRON complex interact with core clock proteins. CSNK1E, GSK3B, and DYRK1A, three kinase components of the NRON complex, are known to be involved in phosphorylation-dependent stability and nuclear localization of PER and CRY in both flies and mammals 21,22 . To further investigate the direct role of the NRON complex, we examined the interactions of the complex components with core clock proteins. Our recent study showed that KPNB1, which directs protein nuclear trafficking, directly interacts with PER and CRY. Perturbation of KPNB1 has strong effects on PER2 nuclear translocation and PER-CRY complex formation 20 . Previous studies also characterized the interaction between CSNK1E and PER2 11,34 . Using RNA-protein immunoprecipitation (RIP) and quantitative PCR analyses, we show that Venus-tagged PER2 (PER2-V) associated with NRON in U2OS cells (Figs 2A and S2). Co-immunoprecipitation assays detected PER2-V interactions in transfected U2OS cells with endogenous clock regulatory kinases DYRK1A and GSK3B, as well as other complex components including IQGAP1 (CaM-binding scaffolding protein), TNPO1 (nuclear import protein), PPP2R1A (protein phosphatase regulatory subunit), and PSMD11 (a 19S proteasome lid component) (Fig. 2B). Importantly, we also detected endogenous PER2 interactions with DYRK1A and GSK3B in the mouse liver (Fig. 2C,D).
If PER2 interacts with the NRON complex, they must co-localize in the same subcellular compartment. Using immunofluorescence imaging combined with RNA fluorescence in situ hybridization (FISH), we show that PER2 colocalizes with NRON and the NRON complex components (DYRK1A, CSNK1E, GSK3B, IQGAP1, PPP2R1A, and PSMD11) at the perinuclear membrane in U2OS cells stably expressing NRON (Figs 2E,F and S3). Notably,  www.nature.com/scientificreports www.nature.com/scientificreports/ NRON reduced the abundance of PER2 in the nucleus and the other complex components. These data suggest that PER2 associates with the NRON complex components and colocalizes at the perinuclear region.
NRON knockdown alters the status of core clock proteins and clock function. Stable expression of NRON in U2OS cells affected the subcellular localization of PER2, as well as other NRON complex components. Next, we examined the effects of NRON loss of function on core clock proteins and clock function. In contrast to the effect of NRON overexpression, RNAi knockdown significantly increased nuclear accumulation of endogenous PER2, CRY1 and CRY2 ( Fig. 3A-C). NRON knockdown also substantially increased PER2 phosphorylation without an obvious effect on its overall abundance, whereas CRY2 levels, but not CRY1, were increased (Fig. 3D). Further, compared to a non-specific control siRNA, NRON knockdown caused significantly longer period length and reduced rhythm amplitude in a dose-dependent manner (Fig. 3E,F). This clock phenotype is consistent with the increased abundance of nuclear PER and CRY repressors.

Perturbation of Ca 2+ signaling alters clock function in U2OS cells.
Our data here and previous findings suggest that the NRON complex regulates both NFAT signaling and the circadian clock 20,23,27 . NFAT is regulated by the Ca 2+ sensor calmodulin (CaM) and the phosphatase calcineurin (CaN). CaN is a central regulator of the NFAT pathway; CaN dephosphorylates NFAT, leading to its nuclear translocation and transactivation. Therefore, we reason that this pathway might also impact the clock and that perturbation of Ca 2+ signaling would affect cellular circadian rhythms. To test this hypothesis, we leveraged well-established pharmacological agents and cellular clock models and tested their effects on circadian rhythms in U2OS cells. Cyclosporine A (CsA) and FK506 (Tacrolimus) are classic CaN inhibitors that block NFAT dephosphorylation, trapping NFAT in the cytoplasm 35 . We show that, while phorbol 12-myristate 13-acetate (PMA), an activator of NFAT signaling, shortened the period length, both CsA and FK506 lengthened period in these cells (Figs 4A and S5A).
We asked whether genetic perturbation of CaN via RNAi knockdown also affects clock function. CaN is composed of a catalytic subunit and a regulatory subunit. There are three catalytic subunits encoded by PPP3CA, PPP3CB and PPP3CC. We show that, among the three subunit genes, PPP3CC knockdown in human U2OS cells significantly lengthened period (Fig. S4A). A similar phenotype was also observed in mouse MMH-D3 cells when Ppp3cc was knocked down by lentiviral shRNAs (Fig. S4B). Taken together, our data from both genetic and pharmacological perturbation suggest that the CaM-CaN axis regulates circadian clock function.

Perturbation of Ca 2+ signaling alters PER/CRY nuclear translocation in U2OS cells. The CsA and
FK506 effect on clock function in U2OS cells raised the possibility that they affected the nuclear translocation of core clock proteins, particularly PER and CRY, as in their effect on NFAT. To test this, we performed immunofluorescence imaging to determine the effect of perturbation of Ca 2+ signaling on subcellular localization of PER1, PER2, CRY1 and CRY2 in these cells 25 . Compared to DMSO control, PMA significantly increased the abundance of all PER and CRY repressor proteins in the nucleus (Figs 4B and S5B,C), consistent with the shortened period length in PMA treated cells (Figs 4A and S5A). However, we did not observe the opposite effects from the CsA and FK506 inhibitors. Overall, CsA and FK506 lowered the relative nuclear/cytoplasmic levels of PER and CRY repressors, but did not considerably alter nuclear PER/CRY levels. On the other hand, PMA did not increase the relative nuclear/cytoplasmic ratio, as would be expected from the CsA and FK506 effects. The PMA did increase the protein levels in the cytosol in most cells. This increase may be attributable to transcriptional induction and reduced protein degradation [36][37][38] . Thus, our data support a role of Ca 2+ signaling and the CaM-CaN axis in regulating clock function, but the mechanistic details are not clear and require future studies.

Perturbation of calcineurin and Ca 2+ signaling alters the SCN clock function. Our findings that
CaN and the NRON complex regulate clock function in multiple cell models (U2OS, MMH-D3), and the well recognized role of Ca 2+ signaling in regulating SCN circadian timekeeping in the SCN 39 , suggested a ubiquitous modifier role and raised the possibility that the pathway also regulates the SCN clock. We dissected SCN slices from the PER2::LUC fusion knockin (Per2 Luc ) mice and cultured the tissue explants ex vivo 40,41 . Compared to DMSO control treatment (Fig. 5A), both CsA and FK506 significantly lengthened period in SCN explants (Fig. 5B,C). Taken together, our data suggest that the NRON complex regulates clock function not only in peripheral oscillators but also in the master SCN clock.

Discussion
Circadian oscillations are cell-autonomous and self-sustaining. These properties distinguish the clock from many other cellular processes. One of the key steps in establishing circadian oscillation is the cytoplasmic to nuclear translocation of the PER/CRY repressor complex. This process occurs via PER/CRY protein degradation and gradual accumulation in the cytoplasm, followed by nuclear transport and subsequent repression of BMAL1/ multiple-comparisons test. Data are presented as mean ± SD (n = 27~30). Fluorescence microscopy filter sets: FITC (green, PER2), TRITC (red, NRON), and DAPI (blue, nuclei). (F) Immunofluorescence imaging analysis to detect subcellular colocalization of PER2 with the NRON complex proteins. U2OS cells stably expressing NRON (NRON Stable ) were fixed and immunostained with antibodies against PER2 and other proteins as indicated. The nuclear/cytoplasmic ratio of the NRON complex proteins in control (Ctrl) and NRON Stable cells (NRON stable ) is shown (right). ****p < 0.00001, ***p < 0.001, **p < 0.01 by one way ANOVA followed by Dunnett's multiple-comparisons test. Data are presented as mean ± SD (n = 10~38). Fluorescence microscopy filter sets: FITC (green, PER2), TRITC (red, individual NRON components), and DAPI (blue; nuclei). DAPI merged images show colocalization.
www.nature.com/scientificreports www.nature.com/scientificreports/ CLOCK activity. Several players known to modify PER/CRY play critical roles in setting the clock speed 21,22 . In particular, to maintain circadian gene transcription, clock protein abundance must be coupled with nuclear translocation. In this context, the NRON complex, which is positioned to couple protein synthesis with degradation, and cytoplasmic protein abundance with nuclear transport, provides the missing link. Our studies show that the NRON complex regulates nucleocytoplasmic partitioning of PER/CRY, and most complex members display strong knockdown phenotypes in circadian assays.
The NRON complex links enzymes that catalyze protein modifications with those that target them either for degradation or for nuclear transport. More specifically, NRON mediates the assembly of Ca 2+ signaling scaffolding protein (IQGAP1), kinases and phosphatases (CSNK1, DYRK1A, GSK3B), proteasome components and stability factors (PSMD11, CUL4B), and nuclear import and export factors (KPNB1, TNPO1, CSE1L). This complex appears to localize at the perinuclear regions, where the nuclear transport machinery is located. This perinuclear scaffolding architecture confers regulatory specificity, efficiency, and strength 42,43 . While we have a reasonable understanding of the steady-state nuclear localization of PER/CRY, we do not have a detailed description of how their cytoplasmic degradation and nuclear translocation are linked dynamically throughout the day. In this context, it is plausible that the interrelated functions of the NRON complex coordinate to enable circadian oscillations.
As most members of the NRON complex are required not only for NFAT signaling but also for clock function, these two processes are mechanistically linked. The NRON complex regulates the NFAT pathway via the CaM-CaN axis and consequently NFAT nuclear translocation. However, it is not clear whether the NRON complex affects  www.nature.com/scientificreports www.nature.com/scientificreports/ differences provoked by activators (PMA) and inhibitors (FK506, CsA). The NRON complex may also indirectly affect the clock via NFAT's transcriptional activity, e.g. by affecting core clock gene expression. Thus, the mechanisms of NRON function likely involve PER/CRY protein post-translational modification, stability and degradations, nucleocytoplasmic partitioning, and ultimately their transcriptional activities in the nucleus. It is conceivable that these dynamic interactions and regulatory functions work to maintain a robust clock.
Experimental evidence supports rhythmic Ca 2+ /CaM-CaN-NFAT activity in the SCN and that perturbation of the NRON complex alters both circadian Per2 Luc and NFAT-RE::dLuc rhythms. In particular, CaN subunits (including its catalytic and regulatory subunits, e.g. Ppp3ca, Ppp3r1) are highly rhythmic in the liver and modestly rhythmic in the SCN 7 . ChIP-seq analysis revealed that CaN subunits are targeted by BMAL1, PER1/2 and CRY1/2 9 . Conversely, our data suggest that the NRON-NFAT pathway impacts clock function. As NFAT plays roles in diverse processes, including immune and inflammatory responses, its circadian regulation in SCN has implications for these processes as well. Hundreds of NFAT target genes are rhythmically expressed in the SCN and liver. These genes peak immediately before and after dawn in the liver and the SCN, respectively, raising the possibility that NFAT plays a role in regulating entrainment to light and food.
Previous studies show that SCN explants of Bmal1 −/− and Cry1 −/− ;Cry2 −/− mice display residual quasi circadian Per2 Luc oscillations 44,45 . The molecular origins for these oscillations remain elusive. In light of our findings, we speculate that Ca 2+ homeostasis, signaling through the NRON pathway, might contribute to PER/CRY nuclear transport regulation; this process impinges on the E-box to generate stochastic gene expression, which underlies weak but quasi circadian oscillations. This notion is supported by findings that GPCR signaling and intracellular Ca 2+ activities play an essential role in enabling circadian oscillations in the SCN and in fly free-running behavior 39,46,47 . Intracellular Ca 2+ concentrations in SCN neurons display a circadian rhythm, which is cell autonomous and dependent on the core clock mechanism, but also is reinforced by a synchronized SCN neuronal network 48,49 . Further, Ca 2+ oscillations may also arise from rhythmic behavioral and physiological functions, including locomotor activity and feeding. It is interesting to note that the CaN-NFAT pathway, as an input to the clock, controls activity-dependent circadian gene expression in skeletal muscle 50 . This crosstalk between Ca 2+ signaling and the circadian clock provides a mechanism where each process can reinforce or influence the other during normal or pathological conditions. Cell culture and reagents. U2OS or HEK 293 T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 1% L-Glutamine, and 1% penicillin-streptomycin (Invitrogen) at 37 °C under 5% CO2. The cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen) and DNA plasmids using Fugene HD reagents (Promega). The combined transfection of DNA plasmids with siRNA into the cells were performed using Lipofectamine 2000 (Invitrogen). Cell culture and growth conditions for MMH-D3 hepatocyte were performed as previously described 31 . Plasmids. For NRON overexpression and generation of stable cell lines, the full-length genomic DNA fragments of NRON from U2OS cells was amplified by PCR with primers containing the flanking restriction sites (NotI, XhoI) and inserted into pcDNA3.1 mammalian expression vector (Invitrogen). For plasmids expressing Venus-tagged mouse Per2 (Per2-Venus), full-length DNA fragment of each gene was subcloned into pCMV-Venus, pCMV-VN, or pCMV-VC using a restriction-free (RF) cloning method as described previously 51-53 . Antibodies. The following antibodies were used for immunoprecipitation, immunoblotting, and immu- Immunofluorescence analysis. After 24 h post-transfection, cells were fixed with 4% paraformaldehyde in PBS and visualized under a fluorescence microscope. For IP analysis, cells were incubated with various antibodies and then secondary antibodies, and visualized using FITC/TRITC/DAPI filter sets. DAPI merged images indicate colocalization. For nuclear and cytoplasmic protein quantification, original red fluorescent images were first converted to white and black mode, nuclear and cytoplasmic areas in each stained cell manually demarcated, and signal intensities measured using Image J software to obtain the ratio.

RNA-fluorescence in situ hybridization (RNA-FISH) analysis.
A set of 48 Quasar 570 labeled probes (Stellaris) targeting NRON mRNA were designed using the Stellaris probe designer. RNA-FISH procedure was performed according to the manufacturer's protocol (Biosearch Technologies). For hybridization, the probes were incubated with control and NRON stable expression cells at 37 °C overnight, followed by immunostaining. Images were acquired using FITC/TRITC/DAPI filter set in fluorescence microscopy.
RNA-binding protein immunoprecipitation (RIP) assay. Cell lysates were cross-linked by formaldehyde for 15 min and the protein/RNA complex were immunoprecipitated with antibodies. The eluted NRON RNA samples were analyzed RT-PCR to determine the association between RNA and protein of interest.

Transfection efficiency validation for RNAi knockdown screen.
To validate the siRNA knockdown efficiency, cells were synchronized and transferred to luminescence recording medium as described above in