Fig 1.
CUL4A-DDB1 E3 ligase promotes CRY1 protein degradation in cells and the mouse liver.
(A) Endogenous CRY1 interacts with DDB1-CUL4A E3 ligase complex. U2OS cells were first synchronized by serum shock to elevate the endogenous CRY1 protein expression. Cells were then treated with MG132 for 8 hr and harvested to detect CRY1 interaction with DDB1-CUL4A E3 ligase. About 5 mg of total nuclear protein were incubated with anti-CRY1 (1:100) for 16 hr at 4C. The presence of CRY1 and DDB1 complex were detected by using antibodies against CUL4A, DDB1, and CRY1. The representative one of three individual IP experiments is shown here. (B) Overexpression of DDB1 and CUL4A reduces the CRY1 abundance in 293T cells. 293T cells were co-transfected with Cry1-Flag along with either GFP control or a mixture of Ddb1 and Cul4A. Cells were collected 48 hr later to examine the expression of CRY1-FLAG protein by immunoblotting. (C) Depletion of Ddb1 elevates the expression of CRY1 in 293T cells. 293T cells were co-transfected with Cry1-Flag along with either control shRNA or Ddb1 shRNA. Cells were collected 48 hr later for examining CRY1-FLAG protein by immunoblotting. (D) Ddb1 knockdown increases CRY1 protein stability in 293T cells. 293T cells were co-transfected with Cry1-Flag along with either control shRNA or shDdb1 for 48 hr. Cells were then treated with cycloheximide (CHX, 100 μg/mL) for 0, 1, 3, and 8 hr before harvest. The expression levels of CRY1-FLAG were determined by immunoblotting. A representative of three individual experiments was shown here. The expression of CRY1-FLAG was quantified and normalized to loading control ß-tubulin. (E) Acute Ddb1 knockdown increases CRY1 protein in primary mouse hepatocytes. Primary mouse hepatocytes were transduced with either Ad-shLacZ or Ad-shDdb1 and then isolated after 48 h for CRY1 and DDB1 protein expression by immunoblotting. The expression of CRY1-FLAG was quantified, normalized to loading control ß-tubulin, and plotted as mean ± S.D. (n = 3). *p-value < 0.05 by the Student’s t-test. (F) Liver-specific acute knockdown of Ddb1 augments the CRY1 protein expression in the liver. Mice of 8–10 weeks were injected with either Ad-shLacZ or Ad-shDdb1 via the tail vein. Three weeks later, liver CRY1 protein levels were detected by immunoblotting with anti CRY1 (n = 3). The knockdown effect of Ad-shDdb1 was verified by the levels of DDB1 in the same samples. The expression of CRY1-FLAG was quantified, normalized to loading control ß-tubulin, and plotted as mean ± S.D. (n = 3). *p-value < 0.05 by the Student’s t-test.
Fig 2.
Cul4A-DDB1 E3 ligase promotes CRY1 ubiquitination both in vivo and in vitro.
(A) Enhanced CRY1 ubiquitination in the presence of CUL4A-DDB1 E3 ligase. 293T cells were co-transfected with Cry1-Flag and Myc-ubiquitin in the presence of GFP, HA-Cul4A plus T7-Ddb1, or HA-Cul4A-DN plus T7-Ddb1. 24 hr after transfection, cells were treated with proteasome inhibitor MG132 (5 μM) for 16 hr and then harvested for detection of CRY1 ubiquitination. The protein levels of CRY1, CUL4A, DDB1 and DN-CUL4A in the whole cell lysates were determined by immunoblotting with specific antibodies. All the experiments were repeated at least three times and a representative result was shown here. (B) In vitro CRY1 ubiquitination by CUL4A-DDB1 E3 ligase. The purified GST-CRY1 fusion protein was mixed with the DDB1-CUL4A complex isolated from adenovirus-transduced 293T cells in the presence of E2, E3, and ubiquitin at 37°C for 2 hr before immunoblotting with anti-ubiquitin. GST-CRY1 and GST control were shown by Coomassie blue staining.(C) Reduced CRY1 ubiquitination in Ddb1-LKO primary mouse hepatocytes. After isolation from either 8-wk old Ddb1flox/flox or Ddb1-LKO mice, primary mouse hepatocytes were transduced with Ad-Cry1-Flag for 24 hr and treated with MG132 (5 μM) for 16 hr before harvest. The protein lysates were subjected to denaturing IP to detect CRY1 ubiquitination. The protein levels of CRY1 and DDB1 in the whole cell lysates were measured by immunoblotting with either anti-FLAG or anti-DDB1. All the experiments were repeated three times and the representative result was shown here. (D) Adenoviral overexpression of Ddb1 rescues CRY1 ubiquitination in Ddb1-LKO primary mouse hepatocytes. Primary mouse hepatocytes were isolated from Ddb1-LKO mice and transduced by Ad-Cry1 along with either Ad-GFP or Ad-Ddb1. 24 hr post-transduction, cells were treated with MG132 for 8 hr and then harvested for detecting CRY1-ubiquitination.
Fig 3.
CUL4A-DDB1 E3 ligase targets the N-terminal lysine 585 of CRY1 for ubiquitination and degradation.
(A) Comparison of the CRY1-585KA-Flag vs. CRY1-WT-Flag protein stability. 48 hr later after transfection with either Cry1-WT-Flag or Cry1-585KA-Flag, 293T cells were treated with CHX for 0, 2, and 8 hr before harvest. The expression levels of FLAG-tagged CRY1 were determined by immunoblotting by anti-FLAG, quantified and normalized by loading control control RAN. The percentage of the remaining CRY1 at each time point was calculated by comparing to the CRY1 abundance at time 0 hr. (B) CRY1-585KA-FLAG mutant is resistant to degradation triggered by DDB1-CUL4A E3 ligase. After transfection with Cry1-WT-Flag vs. Cry1-585KA-Flag along with GFP control or T7-Ddb1 plus HA-Cul4a, 293T cells were harvested 48 hr later for immunoblotting with anti-FLAG. (C) Cry1-585KA-FLAG mutant is resistant to ubiquitination mediated by DDB1-CUL4A E3 ligase. 293T cells were transfected with expression vectors encoding either Cry1-WT-Flag or Cry1-585KA-Flag in the presence of GFP control or T7-Ddb1 plus HA-Cul4a. Cells were then treated with MG132 for 16 hr and harvested for ubiquitination IP. The expression levels of CRY1-FLAG, DDB1 and CUL4A in the whole cell lysates were determined by immunoblotting. (D) CRY1-585KA mutant interacts with the CUL4A-DDB1 complex. 293T cells were co-transfected with Cry1-585KA-Flag vs. Cry1-WT-Flag in the presence or absence of HA-Cul4a plus T7-Ddb1. 24 hr post-transfection, cells were treated with MG132 for 16 hr and collected for co-IP with anti-FLAG. The presence of CRY1, CUL4A, and DDB1 in the immunocomplex was detected by antibodies against CUL4A, DDB1, and FLAG.
Fig 4.
CDT2 functions as a substrate-binding protein for DDB1-CUL4A-mediated regulation of CRY1 protein.
(A) CRY1 interacts with both the endogenous and overexpressed CDT2. U2OS cells were transfected with Cry1-Flag in the presence or absence of Cdt2 expression vector. Cells were treated with MG132 for 16 hr prior to IP with anti-CRY1. The presence of CDT2 was detected by anti-CDT2. (B) CRY1 and CDT2 co-localize in the nucleus. U2OS cells were transfected with both mCherry-Cry1 and GFP-Cdt2 and treated with MG132 at 10 μM for 3 hr prior to methanol fixing, Triton X-100 permeabilization, and DAPI counterstaining. mCherry-CRY1 and GFP-CDT2 fluorescent protein signals were captured by confocal imaging and overlaid by Image J software. (C) Cdt2 knockdown increases CRY1 protein stability in 293T cells. 293T cells were co-transfected with Cry1-Flag along with either control shRNA or Cdt2 shRNA for 48 hr. Cells were then treated with cycloheximide (CHX) for 0, 2, 4, and 8 hr before harvest. The expression levels of CRY1-FLAG were determined by immunoblotting and quantified. The percentage of the remaining CRY1 at each time point was calculated by comparing to the CRY1 abundance at time 0 hr. The experiments were repeated three times and a representative one is shown here. (D) Effect of knockdown of CDT2 co-factor Pcna on the CRY1 protein degradation. 293T cells were co-transfected with CRY1-Flag along with either shControl or shPcna vector. 48 h later, Cells were treated with CHX at 10 μg/mL for the indicated times prior to immunoblotting. The percentage of the remaining CRY1 at each time point was calculated by comparing to the CRY1 abundance at time 0 hr. (E) Depletion of Cdt2 reduces CRY1 interaction with DDB1-CUL4A complex. U2OS cells were transfected with CRY1-Flag together with either shControl or shCdt2 vectors. 48 hr later, cells were exposed to MG132 for 8 hr before harvest. The interaction between CRY1-FLAG and the endogenous DDB1-CUL4A complex was examined by immunoprecipitation with anti-FLAG. (F) CDT2 promotes CRY1 ubiquitination via a functional CUL4A-DDB1 complex. Myc-Cry1 was co-transfected with or without Cdt2 expression vector. To block the endogenous CUL4A-DDB1 complex, HA-Cul4a-DN was co-transfected along with Cry1 and Cdt2 vector. Cells were treated with MG132 for 16 hr before detection of CRY1 ubiquitination, as described above. (G) Cdt2 depletion blocks on DDB1-CUL4A-mediated CRY1 degradation. U2OS cells were transfected with Cry1-Flag or Cry1-Flag/T7-Ddb1/HA-Cul4a in the presence of either shControl or shCdt2 vector. Cells were then harvested 48 hr later for examining levels of CRY1-FLAG, DDB1, CUL4A, CDT2, and β-tubulin.
Fig 5.
Knockdown of Ddb1 enhances both circadian oscillations of CRY1 protein and Bmal1 promoter-driven clock activity.
(A) Depletion of Ddb1 by adenoviral shRNA elevates CRY1 protein levels in Hepa1 cells. Mouse Hepa1 cells were transduced with Ad-shLacZ or Ad-shDdb1. 24 hr post-transduction, cells were synchronized by serum shock to reset circadian cycles. Protein samples were collected every 4 hr between 24 and 48 hr post-synchronization. The endogenous CRY1 and DDB1 levels were determined by immunoblotting. The protein level of RAN was measured as loading control. The levels of CRY1-FLAG were measured by immunoblotting, quantified, and normalized by the levels of loading control RAN (bottom panel). (B) Effect of acute Ddb1 depletion on circadian activities of Bmal1-luc in U2OS cells. The Bmal1-luc U2OS stable cells were transfected with either shLacZ or shDdb1 prior to synchronization. Lumicycle monitored the oscillations of luciferase activities for five circadian cycles. Knockdown efficiency of Ddb1 by shRNA was confirmed by anti-DDB1 immunoblotting in the top panel. Circadian period length and amplitude values were compared between shLacZ and shDdb1 groups (n = 4). *p < 0.05 by the Student's t-test.
Fig 6.
Impact of 585KA mutation on circadian oscillations of CRY1 protein, Bmal1 promoter activity as well as clock output gene.
(A) Circadian oscillations of CRY1-WT vs. CRY1-585KA in Hepa1 cells. 24 hr after transfection with Cry1 expression vectors (WT vs. 585KA), Hepa1 cells were synchronized by serum shock and harvested every 8 hr between 16 and 64 hr. The protein levels of CRY1 protein (WT vs. 585KA), BMAL1, and CLOCK were determined by immunoblotting with anti-FLAG or with protein-specific antibodies. (B) Impact of CRY1 WT or CRY585KA on oscillations of Bmal1-luc in U2OS cells. The Bmal1-luc U2OS stable cells were transfected with GFP, Cry1-WT, or Cry1-585KA and then subjected to Lumicycle analysis. The period length and amplitude values were calculated and compared. *p-value < 0.05 between CRY1-WT and Cry1-585KA group by the student’s t-test (n = 6). (C) Increased amplitude of the endogenous clock gene oscillations in cells overexpressing CRY1-585KA. U2OS cells were transfected with expression vectors encoding either Cry1-WT or Cry1-585KA. 24 hr post-transfection, cells were synchronized by serum shock and collected at the indicated time points. The endogenous Dbp mRNA levels were measured by RT-qPCR. The data were plotted as Mean ± S.D. (n = 4). The AUC (area under curve) analysis for Dbp mRNA oscillations was presented as well. * p-value < 0.05 by Student’s t-test.