Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jul 20;293(29):11296-11309.
doi: 10.1074/jbc.RA117.001432. Epub 2018 Jun 5.

Ubiquitin-conjugating Enzyme UBE2O Regulates Cellular Clock Function by Promoting the Degradation of the Transcription Factor BMAL1

Affiliations
Free PMC article

Ubiquitin-conjugating Enzyme UBE2O Regulates Cellular Clock Function by Promoting the Degradation of the Transcription Factor BMAL1

Suping Chen et al. J Biol Chem. .
Free PMC article

Abstract

Dysregulation of the circadian rhythm is associated with many diseases, including diabetes, obesity, and cancer. Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Arntl or Bmal1) is the only clock gene whose loss disrupts circadian locomotor behavior in constant darkness. BMAL1 levels are affected by proteasomal inhibition and by several enzymes in the ubiquitin-proteasome system, but the exact molecular mechanism remains unclear. Here, using immunoprecipitation and MS analyses, we discovered an interaction between BMAL1 and ubiquitin-conjugating enzyme E2 O (UBE2O), an E3-independent E2 ubiquitin-conjugating enzyme (i.e. hybrid E2/E3 enzyme). Biochemical experiments with cell lines and animal tissues validated this specific interaction and uncovered that UBE2O expression reduces BMAL1 levels by promoting its ubiquitination and degradation. Moreover, UBE2O expression/knockdown diminished/increased, respectively, BMAL1-mediated transcriptional activity but did not affect BMAL1 gene expression. Bioluminescence experiments disclosed that UBE2O knockdown elevates the amplitude of the circadian clock in human osteosarcoma U2OS cells. Furthermore, mapping of the BMAL1-interacting domain in UBE2O and analyses of BMAL1 stability and ubiquitination revealed that the conserved region 2 (CR2) in UBE2O significantly enhances BMAL1 ubiquitination and decreases BMAL1 protein levels. A Cys-to-Ser substitution experiment identified the critical Cys residue in the CR2 domain responsible for BMAL1 ubiquitination. This work identifies UBE2O as a critical regulator in the ubiquitin-proteasome system, which modulates BMAL1 transcriptional activity and circadian function by promoting BMAL1 ubiquitination and degradation under normal physiological conditions.

Keywords: BMAL1; UBE2O; circadian function; clock gene; degradation; polyubiquitination; transcription factor; transcriptional activity; ubiquitin; ubiquitin-conjugating enzyme (E2 enzyme); ubiquitylation (ubiquitination).

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
UBE2O interacts with BMAL1. A, flow chart for the identification of BMAL1-interacting proteins by IP-MS. B, list of IP-MS–identified BMAL1-interacting proteins that are associated with the UPS. The accession number, protein name, and peptide spectral matches are provided. Note that PSM of the listed proteins is 0 for the control immunoprecipitate. C, a representative MS/MS spectrum of a UBE2O tryptic peptide identified from MS analysis of the affinity-purified BMAL1-interacting proteins. The b- and y-ions are annotated, and the peptide sequence, MH+, charge state (z), and Δmass are shown in the spectrum. D, BMAL1 co-immunoprecipitates UBE2O. Myc-UBE2O was cotransfected with pcDNA3.1 or FLAG-BMAL1 plasmid into HEK293T cells using PEI transfection reagent. Cell lysates were prepared 48 h after transfection, and FLAG-BMAL1 was purified with FLAG M2 affinity gel. The immunoprecipitates and whole-cell lysates were immunoblotted for the indicated antibodies. E, BMAL1 was detected in the UBE2O pulldown sample. Strep-FLAG-UBE2O (SF-UBE2O) was cotransfected with pcDNA3.1 or FLAG-BMAL1 plasmid into HEK293T cells using PEI transfection reagent. Cell lysates were prepared 48 h after transfection, and UBE2O was pulled down with Strep-tactin agarose beads. The purified samples and whole-cell lysates were immunoblotted. F and G, BMAL1 interacts with UBE2O endogenously. Endogenous BMAL1 was immunoprecipitated with a BMAL1 antibody from N2a cell lysates (F) or mouse brain cell lysates (G). Immunoprecipitates and cell lysates were immunoblotted for UBE2O, BMAL1, and/or GAPDH.
Figure 2.
Figure 2.
UBE2O regulates BMAL1 protein level. A, UBE2O reduces the protein level of endogenous BMAL1 in a dose-dependent manner. HEK293T cells in 6-well plates were transfected with the indicated amount of SF-UBE2O and/or pcDNA3.1 plasmid using PEI transfection reagent for 48 h. Cell lysates were blotted for BMAL1, FLAG, and β-tubulin. Three biological replicates were carried out, and densitometric quantification was performed with ImageJ. Student's t test was used to calculate the p value against the control sample transfected with the pcDNA3.1 vector. Error bars, S.D. ns, not significant; *, p < 0.05; **, p < 0.01. B, UBE2O does not affect the CLOCK protein level. HEK293T cells were transfected with HA-CLOCK plasmid and equally split into three 6-cm plates 12 h after transfection. Cells were further transfected with either pcDNA3.1 (balance plasmid) or SF-UBE2O plasmid. Cell lysates were prepared 48 h after the second transfection and immunoblotted for HA (CLOCK), FLAG (UBE2O), and α-tubulin. C, UBE2O knockdown increases BMAL1 protein level in HEK293T cells. Control or three UBE2O-specific siRNAs were transfected into HEK293T cells using Lipofectamine 2000 for 48 h. Cell lysates were immunoblotted for the indicated proteins. D, Ube2o knockdown increases BMAL1 protein level in N2a cells. Control or Ube2o-specific siRNAs were transfected into N2a cells using Lipofectamine 2000. Cell lysates were prepared 48 h after transfection and immunoblotted for the indicated proteins. The protein levels were quantified for data from three biological replicates, and data are expressed as mean ± S.D. Statistical analyses were performed using Student's t test. *, p < 0.05; **, p < 0.01.
Figure 3.
Figure 3.
UBE2O reduces BMAL1 stability by promoting its ubiquitination. A, UBE2O reduces BMAL1 stability. pcDNA3.1 or SF-UBE2O plasmid was cotransfected with the FLAG-BMAL1 plasmid into HEK293T cells using PEI transfection reagent and split into four plates 12 h after transfection. At 48 h post-transfection, cells were treated with CHX (200 μg/ml) for the indicated time. Cell lysates were immunoblotted for FLAG and β-tubulin. S.E., short exposure; L.E., long exposure for FLAG-BMAL1. B, densitometric quantification of Western blotting images from three biological replicates of A. Two-way ANOVA was used to calculate the p value, and mean ± S.D. (error bars) values are shown. ***, p < 0.001. C, proteasomal inhibition rescues the BMAL1 protein level reduced by UBE2O expression. pcDNA3.1 or Myc-UBE2O plasmid was transfected into HEK293T cells using PEI transfection reagent. At 48 h post-transfection, cells were treated with DMSO or MG132 (10 μm for 12 h), respectively, and lysed. Cell lysates were immunoblotted with the indicated antibodies. Student's t test was used to calculate the p values for data from three biological replicates, and mean ± S.D. values are depicted in the bar graph. *, p < 0.05; **, p < 0.01 compared with the first bar (transfected with pcDNA3.1 empty vector and treated with DMSO); ns, not significant. D, UBE2O promotes BMAL1 ubiquitination. pcDNA3.1 or Myc-UBE2O plasmid was cotransfected with the FLAG-BMAL1 plasmid into HEK293T cells using PEI transfection reagent. At 48 h post-transfection, cells were treated with MG132 (10 μm for 12 h) and lysed. FLAG-BMAL1 was immunoprecipitated with FLAG M2 affinity gel. The immunoprecipitates and cell lysates were immunoblotted for the indicated antibodies.
Figure 4.
Figure 4.
UBE2O regulates the BMAL1 transcriptional activity. A, UBE2O attenuates the mRNA level of PER1, CRY1, RORA, and NR1D1 but not BMAL1. pcDNA3.1 or Myc-UBE2O plasmid was cotransfected with HA-CLOCK plasmid into HEK293T cells using PEI transfection reagent for 48 h. Total RNA was isolated from the first half of the samples with TRIzol reagents. The first-strand cDNA was synthesized, and qPCR was performed. The second half of the samples were lysed for immunoblotting of BMAL1, Myc (UBE2O), HA (CLOCK), and β-tubulin. Student's t test was used to perform statistical analysis of the three technical and three biological replicates, and mean ± S.D. (error bars) values are plotted in the bar graph. ns, not significant; *, p < 0.05; **, p < 0.01 compared with the sample transfected with pcDNA3.1 empty vector. B, knockdown of Ube2o with siRNA in N2a cells increases the expression of BMAL1 downstream target genes. N2a cells were transfected with siNC (negative control) or siUbe2o with Lipofectamine 2000. Samples were prepared as described in A, and qPCR was performed with primers specific for mouse mRNA. Student's t test was used for statistical analysis of data from three technical and three biological replicates, and mean ± S.D. values are shown in the bar graph. ns, not significant; *, p < 0.05; **, p < 0.01 compared with the sample transfected with the pcDNA3.1 empty vector.
Figure 5.
Figure 5.
The CR2 domain in UBE2O is the major domain responsible for the reduction of BMAL1 protein level. A, schematic representation of UBE2O domains and its truncations. B, CR2 domain reduces BMAL1 protein level. pcDNA3.1, Myc-UBE2O, or six UBE2O truncation plasmids were transfected into HEK293T cells using PEI transfection reagents. Cell lysates were prepared 48 h after transfection and immunoblotted for endogenous BMAL1, Myc (for UBE2O and its truncations), and β-tubulin. The asterisks at the left of the bands indicate the expected bands for UBE2O and its truncations. Student's t test was used to calculate the p value against the first bar (transfected with the pcDNA3.1 empty vector), and mean ± S.D. (error bars) values are presented in the bar graph. *, p < 0.05; **, p < 0.01. C, gradient expression of UBE2O CR2 truncation progressively reduces BMAL1 protein level. An increased amount of Myc-CR2 plasmid was transfected into HEK293T cells in 6-well plates using PEI transfection reagent. pcDNA3.1 empty vector was used to balance the total amount of transfected plasmid. Cell lysates were obtained 48 h after transfection and immunoblotted for endogenous BMAL1, Myc (CR2), and β-tubulin. Densitometric quantification was carried out for data from three biological replicates, and Student's t test was used for statistical analyses (compared with the sample transfected with the pcDNA3.1 empty vector). Data are presented as mean ± S.D. ns, not significant; *, p < 0.05; **, p < 0.01.
Figure 6.
Figure 6.
UBE2O CR2 domain interacts with, colocalizes with, and ubiquitinates BMAL1. A, CR2 colocalizes with BMAL1 in the nucleus. Myc-UBE2O or Myc-CR2 plasmid was cotransfected with pcDNA3.1 or FLAG-BMAL1 plasmid into HEK293 cells in a 24-well plate using PEI transfection reagent. At 24 h post-transfection, cells were fixed, permeabilized, and stained for Myc (UBE2O or CR2; green), FLAG (BMAL1; red), and DAPI (blue). B, UBE2O CR2 domain interacts with BMAL1. Myc-CR2 plasmid was cotransfected with pcDNA3.1 or FLAG-BMAL1 plasmid into HEK293T cells using PEI transfection reagent. Cell lysates were obtained 48 h after transfection, and BMAL1 was purified with FLAG M2 affinity gel. The immunoprecipitates and whole-cell lysates were immunoblotted. C, a UBE2O truncation lacking the CR2 domain does not interact with BMAL1. HEK293T cells were transfected with FLAG-BMAL1 and three UBE2O truncation plasmids (551–794, CR2; 795–1292, CC+UBC+NLS2; 551–1292, CR2+CC+UBC+NLS2). Cells were lysed 48 h after transfection, and FLAG-BMAL1 was immunoprecipitated with FLAG M2 affinity gel. The immunoprecipitates and cell lysates were immunoblotted for the indicated antibodies. D, the UBE2O CR2 domain enhances BMAL1 ubiquitination. pcDNA3.1 or Myc-CR2 plasmid was cotransfected with FLAG-BMAL1 plasmid into HEK293T cells using PEI transfection reagent. At 48 h post-transfection, cells were treated with MG132 (10 μm) for 12 h and lysed, and FLAG-BMAL1 was immunoprecipitated with FLAG M2 affinity gel. The immunoprecipitates and cell lysates were immunoblotted with the indicated antibodies. E, the location of four cysteines (Cys-566, -585, -598, and -617) in the CR2 domain of UBE2O. F, C617S mutation abolishes the CR2-mediated BMAL1 ubiquitination. HEK293T cells in two 10-cm plates were transfected with HA-Ub and FLAG-BMAL1 plasmids and were equally split 6 h after transfection. At 24 h post-transfection, cells were further transfected with either pcDNA3.1, Myc-CR2, or each of its Cys-to-Ser mutants for 36 h and treated with MG132 (10 μm) for 12 h. Cells were lysed, and BMAL1 was immunoprecipitated with FLAG M2 affinity gel. Both the cell lysates and immunoprecipitates were immunoblotted for the indicated antibodies. The experiment was repeated twice, and similar results were obtained.
Figure 7.
Figure 7.
Full-length UBE2O and the CR2 truncation rescue the effect of UBE2O knockdown on BMAL1 protein level. HEK293T cells (A) or N2a cells (B) were first transfected with the control or UBE2O (Ube2o)-specific siRNAs using RNAiMAX. At 24 h post-transfection, cells were further transfected with pcDNA3.1, Myc-UBE2O, or Myc-CR2 plasmid. Cells were lysed 48 h after the second transfection, and cell lysates were immunoblotted with the indicated antibodies. Asterisks on the right indicate the full-length UBE2O or the CR2 truncation. Quantification was performed for data from three biological replicates, and data are presented as mean ± S.D. (error bars). Student's t test was used for statistical analyses. ns, not significant; *, p < 0.05; **, p < 0.01.
Figure 8.
Figure 8.
UBE2O alters the amplitude of the circadian clock in U2OS cells. A, UBE2O knockdown alters the circadian clock. PER2-luciferase U2OS cells were transfected with control or UBE2O-specific siRNAs for 24 h, synchronized with recording DMEM containing 100 nm dexamethasone. Bioluminescence signals were recorded in a luminometer. The raw and detrended data are depicted. B, quantification of the relative amplitude and period obtained from bioluminescence signals of three biological replicates. Statistics were performed with Student's t test, and mean ± S.D. (error bars) values are depicted in the bar graph. *, p < 0.05; ns, not significant.

Similar articles

See all similar articles

Cited by 4 articles

Publication types

LinkOut - more resources

Feedback