Teemu Andersin1,2, Francis Vilbois3 and Ueli Schibler1,2
1Department of Molecular Biology, Sciences III, University of Geneva 30, Quai Ernest Ansermet, CH-1211 Geneva-4 Switzerland
2National Center of Competence in Research (NCCR) Frontiers in Genetics
3 Merck Serono S.A. 9, chemin des Mines Case postale 54 CH-1211 Geneva 20 Switzerland
Abstract
Mammalian circadian oscillators are believed to depend on a transcription/translation feedback loop where Cryptochrome (CRY) 1 and 2 play important role as negative regulators. In this study we purified and identified CRY associated proteins from NIH
3T3 cells. These included two proteins involved in ubiquitination pathways, the SCF ubiquitin E3-ligase component SKP1 and OTUD4. Depletion of SKP1 by RNA interference (RNAi) resulted in period lengthening in NIH-3T3 cells. OTUD4 is an otu
domain containing putative de-ubiquitinase that interacted with both CRY1 and CRY2.
Depletion of endogenous OTUD4 resulted in the stabilization of CRY proteins, but had no effect on PER2 accumulation. OTUD4 depletion also led to the loss of circadian expression of genes driven by the BMAL1/CLOCK heterodimer. Our results suggest that OTUD4 plays an important role in the generation of circadian rhythms, by regulating the stability of CRY proteins.
Introduction
Most light-sensitive organisms from cyanobacteria to humans possess circadian clocks.
Many behavioral and physiological aspects of life are governed by circadian timekeepers, including sleep wake cycles, heartbeat frequency, arterial blood pressure, renal plasma flow, urine production, intestinal peristaltic motility, and metabolism.
In mammals the circadian timing system is build in a hierarchical fashion and consists of a master clock and subsidiary peripheral oscillators. The master clock resides in the suprachiasmatic nucleus (SCN) in the ventral hypothalamus just above the optic chiasm and receives direct input from the retina through the retino-hypothalamic tract.
Whereas light seems to be the major timing cue for the master clock, cyclic feeding behavior is the most dominant timing cue for peripheral clocks (Damiola et al., 2000).
Therefore, it seems that the SCN synchronizes peripheral oscillators through imposing rest-activity cycles and thus rhythmic feeding behavior.
In both the SCN and the periphery molecular oscillators are thought to consist of negative transcriptional and translational feedback loops. The PAS domain basic helix-loop-helix transcription factors BMAL1 and CLOCK bind to the promoters of two cryptochrome (Cry1 and Cry2) and two period (Per1 and Per2) genes and transactivate
their transcription. Once CRY and PER proteins reach a critical threshold concentration and/or activity, they translocate into the nucleus and inhibit the activity of the BMAL1
CLOCK heterodimer, presumably by attenuating their DNA-binding activity. This leads to an autorepression of Cry and Per genes (Hastings and Herzog, 2004; Reppert, 2006).
In addition, Bmal1 is rhythmically activated and repressed by orphan nuclear receptors of the ROR and REV-ERB families, respectively. As the transcription of Rev-erbα(and probably Rev-erbβ) is regulated by the same mechanisms as that accounting for the rhythmic expression of Cry and Per genes, the transcription of Bmal1 is coupled to the PER/CRY feedback loop (Guillaumond et al., 2005; Preitner et al., 2002).
Post-translational modifications, such as phosphorylation, acetylation,
sumoylation and ubiquitination, affect the nucleo-cytoplasmic shuttling, stability and degradation of several core clock components and thereby make an essential contribution to the generation of circadian rhythms.
Three recent studies have shown that FBXL3, a member of the SCF (SKP1-CUL1-F-BOX protein) family of E3 ubiquitin ligases, mediates the poly-ubiquitination and subsequent proteasomal degradation of CRY proteins. The FBXL3 mutant mice display a free running period length that is two to three hours longer than that of wild-type animals (Busino et al., 2007; Godinho et al., 2007; Siepka et al., 2007). The lengthened period phenotype shows that the periodic degradation of CRYs is a key feature of the exact time-keeping molecular circuitry.
The activity of enzymes adding post-translational modifications to their target proteins is often counteracted by the activity of enzymes that remove these modifications.
For example, phosphorylation of Drosophila PER by doubletime (DBT), which is required for the nuclear translocation and degradation of PER, is balanced by the action of protein phosphatase 2A (PP2A) (Gallego and Virshup, 2007). Likewise, acetylated murine PER2 (mPER2) is de-acetylated and de-stabilized by Sirtuin1 (SIRT1) (Asher et al., 2008).
In order to identify CRY-associated proteins that might modulate CRY expression or function, we purified CRY complexes from NIH-3T3 cells stably expressing affinity tagged CRYs. The identified proteins included two RNA binding proteins, RBM5 and PSF, proteins involved in transcription regulation, TRAP150, BTF and KRIP1, and
proteins that are involved in ubiquitination and possibly de-ubiquitination, SKP1 and OTUD4, respectively. Furthermore, the depletion of SKP1 by RNAi resulted in a long period phenotype, similar to the FBXL3 mutant phenotype. OTUD4, an otu-domain containing protein is a putative de-ubiquitinase that interacted with both CRY1 and CRY2. We further showed that depletion of OTUD4 by RNAi led to a stabilization of CRY proteins, thereby attenuating circadian transcription in NIH-3T3 cells.
Results
Identification of cryptochrome-associated proteins
Both CRY1 and CRY2, together with PER1 and PER2 are the major players in the negative limb of the mammalian circadian oscillator. To identify new factors associated with the negative limb, we generated NIH-3T3 cell lines stably expressing CRY1 and CRY2 proteins containing an N-terminal 6-histidine tag and a C-terminal Flag-tag. The expression levels of His-CRY1-Flag in unsynchronized cells were comparable to the endogenous CRY1 expression in the parental line (Fig.1A). The endogenous CRY1 levels in this transgenic cell line appeared to be dramatically suppressed (Fig.1A). This result was expected and probably reflected the repression of endogenous Cry genes by the constitutively expressed Cry transgenes. The His-CRY2-Flag protein was strongly overexpressed as compared to endogenous CRY2 in the parental cell line (Fig.1A).
Similar to the observation made with the CRY1 cell line, the endogenous Cry2 allele was downregulated in the presence of constitutively expressed His-CRY2-Flag (Fig.1A).
Since overexpression of CRY1 has been shown to impair circadian rhythmicity (Ueda et al., 2005), we wished to examine whether our cell lines still exhibited circadian
oscillations. Therefore, we transiently transfected the above cell lines with luciferase reporter genes driven by either the Bmal1 or the Rev-Erbα promoter (Bmal1-luc and RevErbα-luc) and recorded the bioluminescence of transfected cells in real-time. In contrast to the circadian oscillation of the luciferase reporters observed in the parental cell line, the constitutive expression of both His-CRY1-Flag and His-CRY2-Flag abolished the circadian oscillations of Bmal1-luc and Rev-Erbα-luc (Fig. 1B).
Whole cell extracts from His-CRY1-Flag and His-CRY2-Flag cells were harvested four hours after the cells had been synchronized by a short treatment with
dexamethasone (Balsalobre et al., 2000). These extracts were then used for NiNTA affinity chromatography, eluted by increasing concentration of imidazole, and subjected to M2-agarose chromatography. Proteins were eluted from the M2 column with an excess of Flag peptide, size-fractionated by SDS-PAGE, and visualized by silver-staining (Fig.1C and D). In order to assess the specificity of the affinity enrichments, mock-purifications were also performed with extracts from the parental cell line (see
Supplementary Figure 1). Individual bands were excised and tryptic peptides from the associated proteins were identified by tandem mass spectrometry. In addition to peptides from PER1, which is already know to interact with CRYs (Lee et al., 2001), many peptides from other proteins were identified (Table 1). Among them, we identified two RNA binding proteins, RBM5 (associated with both CRY1 and CRY2), and PSF (only observed in the CRY1 complex). Three additional proteins, involved in transcription regulation also copurified with CRY proteins: BTF and TRAP150, which were associated with both CRYs, and KRIP1 which was associated with CRY1 only. Finally, we
identified two proteins that are involved in ubiquitination and protein degradation pathways namely SKP1, which was found in both CRY1 and CRY2 complexes, and OTUD4, which was only detected in the CRY1 complex. SKP1 is a component of the SCF (SKP-CULLIN-F-box) class ubiquitin E3-ligase complex.
The SCF ubiquitin-E3-ligase associated adapter protein SKP modulates circadian period length
To gain more insight into the function of SKP1, we generated short hairpin RNAs (shRNAs) against Skp1 and tested their ability to reduce the levels of co-transfected HA-tagged SKP1 (HA-SKP1). shRNA2 and shRNA3 efficiently knocked down HA-SKP1, whereas shRNA1 proved to be inefficient (Fig 2B). Next we investigated the effect of SKP1 loss-of-function on circadian oscillation. To this end, we co-transfected the Skp1 shRNA constructs together with the Bmal1-luc reporter and measured bioluminescence in real time for 72 hours. Depletion of SKP1 with shRNA2 and shRNA3 lengthened the period of Bmal-luc oscillations by 3 hours from 24 hours to 27 hours (Fig 2A). As expected, transfection of the ineffective shRNA1 did not change the period length.
Furthermore, we did not observe any changes in the expression of a CMV
promoter/enhancer-driven luciferase reporter gene, ruling out the possibility that the
SKP1 depletion had an effect on luciferase stability. We would like to point out that we did not detect peptides of the F-box protein FBXL3 (Busino et al., 2007; Godinho et al., 2007; Siepka et al., 2007) in the purified CRY complexes, whereas SKP appears to be present in nearly stoichiometric quantities in these complexes. A possible explanation for this is that during the cell lysis, FBXL3 may have dissociated from the CRY protein complexes.
The putative deubiquinase OTUD4 is an ubiquitously expressed component of CRY complexes
We next focused on OTUD4, a large protein encompassing 1107 amino acid. This protein contains only one predicted domain, the OTU-domain spanning amino acids 42 to 149.
OTU domain-containing proteins have previously been shown to function as de
ubiquitinating enzymes in a number of cases (Balakirev et al., 2003; Evans et al., 2003;
Wertz et al., 2004). To verify the interaction we cloned the full length mouse Otud4 cDNA from mouse liver into hemagglutinin (HA)-tag containing expression vector and expressed the HA-OTUD4 protein in the His-CRY1-Flag and His-CRY2-Flag cell lines.
Immunoprecipitation experiments with anti-Flag antibody showed that OTUD4 co-precipitates with both CRY1 and CRY2 (Fig. 3), whereas a control immunoprecipitation with an irrelevant antibody (anti-myc) yielded little if any co-precipitated HA-OTUD4. In the mass-spectrometric analysis we found OTUD4 only associated with CRY1. It was thus possible that the OTUD4 associated more avidly with CRY1 than with CRY2, and that we did therefore not detect OTUD4 peptides in CRY2 complexes. Since little is known about OTUD4, we decided to investigate its temporal and spatial expression. To this end we measured mRNA accumulation of Otud4 by quantitative real-time PCR and northern blot hybridization. Otud4 mRNA levels did not show circadian oscillation in mouse liver, in contrast to the high-amplitude circadian expression of Bmal1 transcripts (Fig. 4A). As judged from the northern blot experiments, the size of the Otud4 transcript was approximately 7000 nucleotides, which closely fits the expected size of 6825
nucleotides indicated by the NCBI database. Of note, we detected another Otud4 transcript, supposedly generated by alternative splicing. Since nearly all cells possess circadian clocks, one would expect that a gene involved in the generation or modulation
of circadian rhythms is expressed ubiquitously. We therefore monitored the expression of Otud4 mRNA in different tissues by quantitative real time PCR. Otud4 transcripts were found in all tissues examined, including brain, heart, intestine kidney and liver (Figure 4).
The mRNA levels were similar in brain, intestine, kidney and liver, but about 5 fold higher in heart.
OTUD4 is required for circadian rhythms
Next, we investigated whether Otud4 loss-of-function had an effect on the core clock. We generated short hairpin RNA (shRNA) sequences against the Otud4 transcripts and tested their ability to reduce the levels of co-transfected HA-OTUD4 (Fig. 5A). As a control we used the empty pSUPER-neo-gfp vector. Efficient knock-down was achieved with Otud4 shRNA1, whereas shRNA2 did not elicit a depletion of the co-transfected HA-OTUD4.
Previous studies have shown that CRYs are ubiquitinated, a step mediated by F-box protein FBXL3, and subsequently degraded by the proteasome (Busino et al., 2007;
Godinho et al., 2007; Siepka et al., 2007). Since OTUD4 is a putative de-ubiquitinase, we wished to examine if the depletion of OTUD4 affected the stability of CRYs. Therefore, we cotransfected NIH-3T3 cells with the Otud4 shRNA1 expression vector or control shRNA (shRNA2) together with either Flag-tagged CRY1, CRY2, or PER2. Depletion of OTUD4 had no discernable effect on the protein levels of PER2 (Fig. 5B). In sharp contrast, the protein levels of CRY1 were markedly elevated upon OTUD4 depletion. We also observed a similar increase in CRY2 protein in cells cotransfected with Otud4 shRNA1. To assess the effect of OTUD4 depletion on circadian transcription, we cotransfected Otud4 shRNAs together with circadian luciferase reporters and measured the bioluminescence in real time for 92 hours after the synchronization of circadian gene expression with dexamethasone. In control NIH-3T3 cells, the expression of Bmal1
driven luciferase (Bmal1-luc) displayed robust circadian cycles with a period length of approximately 24 hours (Fig 6A), the first peak value being reached approximately 16 hours after the synchronization. Cotransfection of the control shRNA had no effect on this oscillation. However, depletion of OTUD4 by shRNA1 disrupted the circadian expression of Bmal1-luc. As circadian Bmal1 transcription is controlled by nuclear orphan receptors of the ROR and REV-ERB families, and we hence also wished to examine the consequence of OTUD4 depletion on a gene whose cyclic expression is
directly governed by the CLOCK-BMAL1 and PER/CRY proteins. The rhythmic expression of RevErbα is controlled by CLOCK/BMAL1 heterodimer (Preitner et al., 2002; Triqueneaux et al., 2004; Ueda et al., 2002). The real-time monitoring of cells transfected with a RevErbα-driven luciferase reporter gene (Rev-erbα-luc) revealed bioluminescence cycles with periodicity of about 24 hours (Fig.6B). As expected (Asher et al., 2008), the expression of Rev-erbα-luc was almost completely anti-phasic to the expression of Bmal1-luc, with the first peak value observed at 30 hours following the dexamethasone synchronization. Cells co-transfected with the control shRNA displayed a nearly identical circadian oscillation of Rev-erbα-luc expression. In sharp contrast, the circadian expression of Rev-erbα-luc was lost when we depleted OTUD4 with shRNA1.
We further monitored the expression of two additional luciferase reporter genes under the control of the CLOCK-BMAL1 heterodimer, Per2-luc and Dbp-luc, upon OTUD4 depletion. As reported previously (Asher et al., 2008; Brown et al., 2005), cells
transfected with Per2-luc or Dbp-luc showed robust circadian cycles of bioluminescence, with an approximately 24 hour period length (Fig. 6C and 6D), and control shRNA did not affect these expression cycles. However, depletion of OTUD4 by shRNA1
cotransfection resulted in the abolishment of circadian Per2-luc and Dbp- luc oscillation.
Furthermore, the depletion of OTUD4 markedly decreased the expression of both Per2-luc and Dbp-Per2-luc, but not that of CMV-Per2-luc (Supplementary Fig. 2). The most parsimonious interpretation of these results is that the higher accumulation of CRY proteins in the absence of OTUD4 repressed the E-box driven reporter genes. We therefore also wished to examine the effect of CRY gain of function on the expression of the circadian
expression of Bmal1-luc, Rev-erbα−luc, Per2-luc, and Dbp-luc. To this end, we cotransfected NIH-3T3 cells with CRY1 or CRY2 expression plasmids together with various circadian luciferase reporter plasmids and recorded bioluminescence in real-time for 92 hours. Overexpression of CRY1 disrupted the circadian oscillations of Bmal1-luc, Rev-erbα−luc. and Dbp-luc (Fig 6A, B, D). Per2-luc expression was strongly attenuated in cells overexpressing CRY1, but still displayed residual circadian cycles with a phase advance of approximately 4 hours (Fig. 6C).
Discussion
In mammals, the CRY1 and CRY2 proteins have been proposed to be the most potent repressors of BMAL1/CLOCK mediated transactivation (Ueda et al., 2005). Here we present a biochemical purification of CRY1 and CRY2 protein complexes from mouse NIH-3T3 fibroblasts and the identification of new CRY associated factors. As expected, we found the known CRY-interacting protein PER1 associated with both CRY1 and CRY2. In contrast, PER2 was not detected in these complexes. However, this was not unexpected, since we purified CRY-complexes after dexamethasone-mediated
synchronization. Since owing to its two nearly perfect GRE sequences, PER1 is strongly induced by the glucocorticoid receptor agonist dexamethasone (Balsalobre et al., 2000) and may have saturated the PER docking sites on CRY proteins. In addition to PER1, we identified RBM5 and PSF, two RNA-binding proteins, TRAP150, BTF and KRIP1, three proteins involved in transcription regulation, and SKP1 and OTUD4, two proteins
possibly participating in protein degradation pathways. In this study, we focused on the putative functions of the latter two proteins.
We found SKP1 associated with both CRY1 and CRY2, and the depletion of SKP1 by RNAi resulted in a lengthened period in NIH-3T3 cells. SKP1 is a member of the SKP-Cullin-F-box (SCF) class of E3-ubiquitin ligase complex. Recently, three independent groups identified FBXL3 as an F-box protein responsible for ubiquitination of both CRY1 and CRY2. Since it is generally believed that F-box proteins form the substrate recognition part of the SCF-complex, it remains possible that the interaction of CRYs with SKP1 we observed was mediated by FBXL3. However, we failed to identify peptides of this protein in the complexes we purified and hence suspect that SKP1 does not require F-box proteins to assemble with CRY-PER complexes. The degradation of PER1 is also mediated by a SKP1 containing SCF complex, in which the F-box protein βTRCP recognizes casein kinase 1ε (CK1ε) phosphorylated PER1 (Eide et al., 2005).
While the initial mass-spectrometry analysis revealed OTUD4 associated with only CRY1, we showed by co-immunoprecipitation that OTUD4 also interacted with CRY2. Otud4 is expressed ubiquitously in all examined mouse tissues and the expression was found to be constant throughout the day, at least in liver. While many core clock components are circadianly expressed, most enzymes involved in the generation of circadian rhythms, such as CK1ε, display temporally invariable mRNA levels (Lowrey et
al., 2000). It would be important to determine whether OTUD4 protein levels display circadian oscillations in spite of constant mRNA levels, as has been reported for the protein deacetylase SIRT1 (Asher et al., 2008) and heat shock transcription factor HSF1 (Reinke et al., 2008). Unfortunately, OTUD4 antibodies yielding specific signals in immunoblot experiments are not yet available for this purpose.
The depletion of endogenous OTUD4 from NIH-3T3 cells by shRNA-mediated RNA interference led to marked elevation of co-transfected CRY protein levels. More importantly, the knock-down of OTUD4 resulted in the loss of circadian accumulation of firefly luciferase specified by co-transfected reporter genes Bmal1-luc, Rev-erbα-luc, Dbp-luc, and Per2-luc, and the suppression of OTUD4 thus closely phenocopied the effects of CRY1 or CRY2 overexpression.
Domain predictions from the OTUD4 amino-acid sequence showed that the otu
domain, encompassing amino acids 42 to 149, is the only recognizable peptide motif of this large protein. Otu-domain containing proteins constitute a class of cysteine proteases with specificity for ubiquitin moieties. Intuitively, one would thus expect that a reduced activity of a de-ubiquitinating enzyme would lead to a faster turnover rate of its target.
domain, encompassing amino acids 42 to 149, is the only recognizable peptide motif of this large protein. Otu-domain containing proteins constitute a class of cysteine proteases with specificity for ubiquitin moieties. Intuitively, one would thus expect that a reduced activity of a de-ubiquitinating enzyme would lead to a faster turnover rate of its target.