Competing E3 ubiquitin ligases govern circadian periodicity by degradation of CRY in nucleus and cytoplasm

Abstract

Period determination in the mammalian circadian clock involves the turnover rate of the repressors CRY and PER. We show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss of function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.

Figure 1. Positional cloning of Past-time (Psttm) and identification of the Fbxl21 mutation

A. Histogram showing circadian period distribution of ENU mutagenized generation 3 (G3) mutant mice in a recessive screen. The shaded area represents the average period for WT C57BL/6J mice ± 3 standard deviations. The Past-time founder mouse is indicated by the arrow. B. Representative actogram of a WT C57BL/6J mouse (upper). The actogram is double plotted where each horizontal line represents 48 hrs of activity. The mice were kept on an LD12:12 cycle (represented in the bar above) for the first 7 days and then released into constant darkness for 21 days (indicated by the arrowhead on the right). Actogram of the Past-time founder G3 mouse (period = 22.91 hours) (lower). C. Period distribution of F2 intercross mice used for genetic mapping. The 3 panels from top to bottom represent WT, Psttm/+ and Psttm/Psttm mice, respectively. ANOVA of the period was performed on the 3 populations of F2 mice (grouped by genotype) (DF = 2; F= 78.80; p = 3.983 ×10⁻²⁹). D. Psttm was initially mapped to a 40 Mb region on chromosome 13 between rs13481788 and rs367922 (left). The chromosome 13 schematic lists the markers used to map Psttm to a smaller genetic interval. Haplotypes of the 78 Psttm/Psttm F2 intercross progeny (156 meioses) are shown on the right. Black boxes represent C57BL/6J WT alleles, and white boxes represent C3H/HeJ alleles. The number of recombinants per total meioses is indicated to the right of the haplotype map. Sequencing of Fbxl21 where a single-base change from G to A is indicated by the asterisk (right). E. Structural modeling of the FBXL21 LRR3 motif based on the Skp2 structure in WT and mutant proteins. See also Figure S1.

Figure 2. The Psttm mutation shortens period and antagonizes the period lengthening effect of the Ovtm mutation in FBXL3

A. Representative actogram of tetO::Psttm #11 single transgenic mouse (left). Representative actogram of tetO::Psttm #11 transgenic mouse with the Scg2::tTA driver (right). Arrowheads indicate LD to DD transition. Doxycycline-containing (10 μg/ml) water was administered during the interval indicated by yellow shading on the actogram. B. Free-running periods of single and double transgenic mice during constant darkness with water (black), Dox (yellow), and water after Dox treatment (grey). Error bars show SEM (tetO::Psttm #11 n = 3, Scg2::tTA/tetO::Psttm #11 n=5; *p < 0.05, Bonferroni corrected pair-wise comparison). C. Representative PER2::LUC bioluminescence recording of SCN and pituitary explants from WT and homozygous Psttm mice. Blue and red traces represent PER2 rhythm from WT and Psttm mice, respectively. WT SCN mean circadian period: 24.76 hr (n=6), Psttm SCN mean circadian period: 23.36 hr (n=6). t test: *p-value 0.0027. WT pituitary mean circadian period: 23.2hr, N=6; Psttm pituitary mean circadian period: 22.61hr, N=6. t test: **p-value 0.0004. D. Representative actograms of WT, Ovtm/Ovtm, Psttm/Psttm and Ovtm/Ovtm Psttm/Psttm mice subjected to the same experimental schedule as in Fig. 1B. E. Period distribution. The four panels from top to bottom represent WT, Ovtm/Ovtm, Psstm/Psstm and Ovtm/Ovtm Psttm/Psttm mice, respectively. ANOVA of the period was performed among the genotype groups. Results: DF = 3; F= 388.507; p = 2.295 × 10^−51. See also Figure S2 and Table S1.

Figure 3. Psttm alters circadian clock gene expression in mice

A. Real-time RT-PCR analysis of clock gene expression in WT and Psttm mice. Blue and red circles represent WT and Psttm mice, respectively. Error bars represent SEM for each time point from four independent replicates. 2-way ANOVA shows significant statistical differences between WT and Psttm mutants for Per1 (p < 0.001) and Per2 (p < 0.0001). Cry1 expression shows statistically significant difference at CT12 (p < 0.01) between WT and Psttm mice. Clock protein oscillation in cerebellum. (B) and liver (C). Western blotting was performed using total protein extracts with the indicated antibodies. Representative blots from 3 independent experiments are shown. Quantification of 3 independent experiments are shown to the right of each representative blot. Blue and red circles represent values from WT and Psttm mice. Error bars represent SEM for each time point from 3 independent repeats. In B, 2-way ANOVA shows significant statistical differences between WT and Psttm for PER2 (p < 0.0001), CRY2 (p < 0.0001), and FBXL21 (p < 0.0001). In C, 2-way ANOVA shows significant statistical differences between WT and Psttm for FBXL21 (p < 0.0001). D. Immunohistochemical staining of CRY1 in SCN sections from WT and Psttm mice. Representative images from ZT12 are shown. E. Number of CRY1-positive nuclei in the SCN sections collected from WT and Psttm mice at ZT0, 8, and 12. Student t-test shows statistically significant difference between WT and Psttm mice at ZT0 and ZT12 (*** p < 0.001) and ZT8 (* p < 0.01). See also Figure S3.

Figure 4. FBXL21 forms an SCF E3 ligase complex that slowly ubiquitinates CRY1 and antagonizes the activity of SCF^Fbxl3

A. Interaction of FBXL proteins with Cullin1 and Skp1. NIH3T3 cells were transfected with Flag-Fbxl3, Flag-Fbxl21, Flag-Psttm and V5-βTrcp1 expression constructs. Co-immunoprecipitated proteins were analyzed by western blotting with anti-CRY1, anti-CUL1, and anti-SKP1 antibodies. Representative blots from 3 independent experiments are shown. B and C. FBXL21 interacts with CRY1 (B) and CRY2 (C) in native extracts in a circadian manner. Liver extracts from CT0 to CT24 were immunoprecipitated (IP) with an FBXL21 antibody, and Western blotting was performed with CRY1 and CRY2 antibodies. Lower: quantification of co-immunoprecipitated CRY1 and CRY2 amounts. Data are taken from 2 independent experiments. Error bars show ± range (n = 2). 2-way ANOVA shows statistically significant differences between WT and Psttm for the amount of CRY1 co-precipitated with FBXL21 throughout the circadian cycle (p < 0.0001). D. Differential effects of FBXL21 and PSTTM on CRY1 stability. 293A cells were co-transfected with indicated constructs. 32 hrs after transfection, cells were treated with 20 μg/ml cycloheximide and incubated for the indicated time before harvest. Western blotting was performed to monitor CRY1 levels using an anti-HA antibody. Lower: Quantification of the effects of FBXL3, FBXL21, PSTTM on CRY1 stability. Data are taken from 3 independent experiments. Error bars show ± SEM (n = 3). Half life was determined by using nonlinear, one phase exponential decay analysis (the half-life parameter, K, is significantly different in all four conditions: p<0.0001) E. The Psttm mutation destabilizes FBXL21. 293A cells were transfected with Fbxl21-Flag or Psttm-Flag constructs. Cycloheximide treatment was performed as in D. Data are taken from 3 independent experiments. Error bars show ± SEM (n = 3). The half-life parameter, K, is significantly different: p=0.0436. F. Competition between FBXL3 and FBXL21/PSTTM modulates CRY1 degradation. Cycloheximide treatment and CRY1 western blotting were performed as in D. Data for quantification represent 3 independent experiments (Figure S4F). The half-life parameter, K, is significantly different: p<0.0001. Data are taken from 3 independent experiments. Error bars show ± SEM (n = 3). Lower: Competition between Ovtm and Fbxl21/Psttm in CRY1 degradation. Cycloheximide treatment and CRY1 western blotting were performed as in D. Data for quantification represent 3 independent experiments (representative western blots are shown in Figure S4G). The half-life parameter, K, is significantly different: p<0.0001. Error bars show ± SEM (n = 3). G. In vitro ubiquitination assay. Sf9 cells were infected with the indicated baculovirus constructs. Samples were analyzed by western blotting with an anti-Myc antibody. Upper shows a short exposure image, and the bracket to the right marks polyubiquitinated CRY1. Lower shows a long exposure image, and the bracket to the right marks highly polyubiquitinated CRY1. Results are representative of more than 3 replicates. H. In vitro ubiquitination was performed as indicated in G. Sf9 cells were infected with the indicated baculovirus. Co-infection of Fbxl baculovirus (Fbxl3+Fbxl21, Fbxl3+Psttm) attenuated the E3 ligase activity of FBXL3. Results are representative of 3 replicates. I. FBXL3/FBXL21 mediated CRY1 ubiquitination requires Ubc5 as an E2 ligase. Top: Ubc5-mediated robust ubiquitination by FBXL3-SCF complexes and multi ubiquitination by FBXL21/PSTTM SCF complex. Lower: lack of CRY1 ubiquitination in the presence of Ubc13/Uev1A as the E2 ligase. Results are representative of 3 replicates. J. 293A cells were co-transfected with Cry1-HA, ubiquitin (hUb-HA) and the indicated F-box constructs. Cells were treated with MG132 (10μg/ml) 6 hr before harvest. Whole-cell lysates were analyzed by western blotting with an anti-CRY1 antibody. Results are representative of 3 replicates. See also Figure S4.

Figure 5. The Psttm mutation is mimicked by Fbxl21 knockdown and that promotes accelerated CRY1 degradation in the nucleus and stabilization in the cytoplasm

A. Psttm shortens circadian period in MEFs. Representative PER2::LUC bioluminescence recording from WT (blue trace) and Psttm (red trace) MEFs. B. Fbxl21 knockdown shortens circadian period in MEFs. Representative PER2::LUC bioluminescence recording from Lenti-GFP infected (green trace) and Lenti-Fbxl21sh infected (orange trace) PER2::LUC MEFs. C. Western blotting of WT, Psttm, Lenti-GFP and Lenti-Fbxl21sh cell lysates showing that FBXL21 is low in Psttm MEFs and depleted in Fbxl21 knockdown MEFs. Filled and open arrows indicate FBXL21 and a nonspecific band respectively. D. Average period values of the experimental groups in A and B. Statistically significant difference in the mean period was detected between WT (24.4h) and Psttm (23.1hr) MEFs (t test: * p < 0.001,). Likewise, statistically significant difference in the mean period was detected between Lenti-GFP (24.06 h) and Lenti-Fbxl21si (22.68 hr) MEFs (t test: * p < 0.001). E. The Psttm mutation decreases nuclear CRY1 levels and oscillation amplitude. Western blotting for CRY1 was performed using nuclear and cytosolic fractions. Right: Quantification from triplicates including examples shown here. CRY1 levels from Psttm nuclei (red) were significantly reduced (upper, 2-way ANOVA, p < 0.001) relative to CRY1 from WT nuclei (blue). In contrast, cytoplasmic CRY1 levels from Psttm (red) were significantly elevated throughout the circadian cycle (lower, 2-way ANOVA, p < 0.001). Error bars show ± SEM (n = 3). F. Fbxl21 depletion reveals that Psttm behaves as a loss-of-function mutation relative to WT Fbxl21. Lenti-GFP and Lenti-Fbxl21sh infected mPER2::LUC MEFs were treated the same as in E. Right: Quantification from triplicates including example shown in F. Error bars show ± SEM (n = 3). CRY1 levels in Lenti-Fbxl21sh nuclei were significantly reduced (orange, upper, p < 0.001), whereas cytoplasmic CRY1 levels were significantly increased throughout the circadian cycle (orange, lower, p < 0.001). G. Accelerated nuclear CRY degradation and decelerated cytoplasmic CRY degradation in Psttm and Fbxl21 knockdown MEFs. Western blotting was performed using anti-CRY1, -CRY2, -FBXL21 antibodies for nuclear and cytoplasmic fractions. Representative blots from 3 independent experiments are shown. H. Quantification of nuclear and cytoplasmic CRY1 and CRY2 degradation in Psttm and Fbxl21sh MEFs from triplicate experiments including the representative blots shown in G. Error bars show ± SEM (n = 3). See also Figure S5.

Figure 6. Differential localization of FBXL3 and FBXL21 SCF complexes in the nucleus and cytoplasm

A. Differential subcellular localization of FBXL3, FBXL21 and PSTTM. Full-length Venus tagged FBXL3, FBXL21, PSTTM, and CRY1 were expressed in 293A cells, and percentages of cells with fluorescence signals in nuclei only, cytoplasm only, or both were measured. Whereas FBXL3 (96%) and CRY1 (92%) were predominantly localized to the nucleus, FBXL21 and PATTM were found in the both nucleus and cytoplasm with 51% and 54% of cells in the cytoplasm, respectively. Smaller percentages of cells showed nuclei only localization (FBXL21: 18%, PSTTM: 12%) or both (FBXL21: 30%, PSTTM: 36%). Green: Venus; Blue: DAPI. Bar graphs show the mean ± SD of 3 replicate experiments. 2-way ANOVA shows the localization is significantly different, p < 0.0001. B. Differential subcellular localization of CRY-FBXL complexes. VenN tagged FBXL3, FBXL21 and PSTTM formed BiFC complexes with VenC tagged CRY1 in 293A cells. FBXL3-CRY1 complexes were primarily localized to the nuclei (88%); however, FBXL21-CRY1 (62%) and PSTTM-CRY1 (70%) were distributed in both nuclei and cytoplasm. Green: Venus; Blue: DAPI. Bar graphs show the mean ± SD of 3 replicate experiments. 2-way ANOVA shows the localization is significantly different, p < 0.0001. C. Reciprocal 2-color, 3-way BiFC competition assays in 293A cells using CRY1-CerC complementation with CerN-FBXL3 + VenN-FBXL21 or VenN-PSTTM (upper), VenN-FBXL3 + CerN-FBXL21 or CerN-PSTTM (lower). FBXL21, and to a lesser degree PSTTM, interact with CRY1 more strongly than FBXL3 (Bottom: quantification). Green: Venus; Blue: Cerulean. Bar graphs show the mean ± SEM of 3 replicate experiments; * p < 0.01, ** p < 0.001, *** p < 0.0001. D. FBXL3 and SKP1 form complexes predominantly in the nuclei of 293A cells, whereas FBXL21 and PSTTM interact with SKP1 in the cytoplasm. Bar graphs show the mean ± SD of 3 replicate experiments. 2-way ANOVA shows the localization is significantly different, p < 0.0001. E. FBXL3 and CULLIN form complexes mainly in the nuclei of U2OS cells (63%), whereas FBXL21 (75%) and FBXL21^Psttm (74%) bind to CULLIN mainly in the cytoplasm. All scale bars are 30 μm. Bar graphs show the mean ± SD of 3 replicate experiments. 2-way ANOVA shows the localization is significantly different, p < 0.0001.

Figure 7. Identification of CRY1 ubiquitination sites for FBXL3 and FBXL21

A. Left: CRY1:R co-immunoprecipited with FBXL3, FBXL21 and PER2. Upper: Co-immunoprecipitation of CRY1WT and CRY1:R with FBXL proteins. Lower: Reciprocal co-IP of WT and CRY1:R with PER2. WT or Cry1:R expression constructs were transfected into 293A cells as indicated (+). Right: 293A cells were co-transfected with indicated constructs. Whole-cell lysates were analyzed by western blotting with an anti-CRY1 antibody. Representative blots from 3 independent experiments are shown. B. CRY1:R repressed CLOCK/BMAL1-mediated transcriptional activation of the Per2 promoter. pGL6 was transfected into 293A cells with the indicated constructs (+). Luciferase reporter assay showed similar repression efficiency for CRY1:R and CRY1 WT. Results are mean ± SEM for 3 independent experiments in duplicate. C. Representative western blots of HA-tagged CRY1 (WT, R, R-10Ks, R-6Ks, or R-4Ks). The Cry1 construct was transfected alone or with Fbxl3 or Fbxl21 into 293A cells. Cells were treated with CHX (100 μg/ml) and collected after 0, 3, 6, or 9 hrs. D. Quantification of CRY1 and CRY1 mutant degradation by Fbxl3 or Fbxl21 from 2 independent experiments in duplicate (n = 4 western blots; representative blots shown in C). Data represent mean ± SEM for expression of Cry1 alone (blue), Cry1 + Fbxl3 (green), or Cry1 + Fbxl21 (orange). Half life was determined by nonlinear, one phase exponential decay analysis. CRY1 half life: alone 5 hr; + Fbxl3 0.7 hr; + Fbxl21 9 hr. CRY1:R half life: alone >24 hr; + Fbxl3 21 hr; + Fbxl21 >24 hr. CRY1:R-10Ks half life: alone 6 hr; + Fbxl3 1.8 hr; + Fbxl21 21 hr. CRY1:R-6Ks half life: alone 8.9 hr; + Fbxl3 1.3 hr; + Fbxl21 >24 hr. CRY1:4Ks half life: alone 10.6 hr; + Fbxl3 4.6 hr; + Fbxl21 >24 hr. The half-life parameter, K, is significantly different in all conditions, p<0.0001, with the exception of the CRY1:R group. E. FBXL21-mediated CRY1 degradation via the K11 residue. Representative western blots of Cry1:R-K11-HA, Cry1:R-HA are shown. The experiment was performed as in C. The K11 revertant underwent moderate degradation by FBXL21. K11 alone half life: >24 hr, + Fbxl3 18.4 hr, + Fbxl21 6.7 hr. The half-life parameter, K, for Fbxl21 is significantly different: p<0.0001). F. 11 candidate CRY1 lysines subject to ubiquitination. All 31 lysine residues of CRY1 are shown, and candidate lysines for FBXL3- and FBXL21-mediated degradation are indicated with green and orange boxes, respectively. G. Differential roles of FBXL21 in nuclear and cytoplasmic CRY turnover. FBXL21 appears to form SCF complexes only in the cytoplasm and functions as a cytoplasmic-specific, weak E3 ligase for CRY degradation. In contrast, nuclear FBXL21 antagonizes FBXL3-mediated CRY degradation, thus conferring a CRY-protective function. In the absence of FBXL21, cytoplasmic CRY is stabilized, whereas in the nucleus CRY is destabilized because FBXL21 cannot antagonize the action of FBXL3. See also Figure S6, S7.