Fig 1.
EBV modulates expression of E2F6 and other E2F family members.
A) 10 million human peripheral blood mononuclear cells (PBMC) were infected with BAC-GFP-EBV for 4 hours. Cells were harvested at indicated time periods, and total RNA was isolated and reverse transcribed to cDNA according to the manufacturer’s instructions. Quantitative Real-time PCR was performed to detect E2Fs transcription levels. B) Bar graph shows log2 fold changes of E2F1 and E2F6 mRNA expression in detail that were analyzed previously. C-D) 10 million HEK-293 or Saos-2 cells were co-transfected with Myc-EBNA3C and control plasmid or Flag-tagged E2F1, E2F2, E2F3a, E2F4, E2F6 expression plasmids. Transfected cells were harvested and lysed at 36 hours post-transfection. About 5% of total lysates were reserved as the input. After incubating with 1 μg of mouse anti-Flag (M2) antibody, Immunoprecipitations were prepared for detection. Input and immunoprecipitated proteins were identified with 10% SDS-PAGE and analyzed by western blot using specific antibodies.
Fig 2.
EBNA3C is an important contributor to the enhanced expression of E2F6.
A-C) 10 million EBV-negative BJAB, Ramos, EBNA3C stable expressing BJAB7, BJAB10, EBV-transformed LCL1, LCL2, and sh-ctrl (stable control knockdown), sh-EBNA3C (stable EBNA3C knockdown) LCL1 cells were harvested and lysed. Western blot analysis of E2F6 protein expression levels was shown with indicated antibodies. D-E) 10 million Ramos or HEK-293 cells were co-transfected with increasing doses of EBNA3C expressing constructs (0, 5, 10, 15, 20 μg) and E2F6, EBNA3C, GAPDH proteins were detected with western blot analysis. GAPDH was used as an internal loading control.
Fig 3.
E2F6 binds to both amino and carboxy terminal domains of EBNA3C.
A) Myc-tagged EBNA3C full length and different truncated mutants were translated in vitro using a T7-TNT kit. The S35-radiolabeled in vitro-translated proteins were pre-cleared with GST-beads, then incubated with GST control or GST-E2F6 protein and GST beads. The mixture were resolved by 10% SDS-PAGE, exposed to phosphorImager plates and scanned with Typhoon Scanner. B-C) Cell lysates form 50 million different B-cells (BJAB, BJAB7, BJAB10, LCL1, LCL2) were collected and incubated with either GST control or GST-E2F6 protein and GST beads. The mixture samples were identified by western blot analysis using specific mouse antibody for EBNA3C (A10). D) Purified GST control and GST-E2F6 proteins were separated by 10% SDS-PAGE and stained with Coomassie Blue. E) The schematic diagram shows various interactive domains of EBNA3C with E2F6.
Fig 4.
EBNA3C forms a complex with E2F6 in EBV-transformed B-cells.
A-D) Cell lysates from different B-cell lines (BJAB, BJAB7, BJAB10, LCL1, and LCL2) were immunoprecipitated with E2F6 or EBNA3C specific antibody (A10). Then immunoprecipitated samples were analyzed with western blot and endogenous EBNA3C, E2F6 proteins were detected by the indicated antibodies.
Fig 5.
EBNA3C co-localizes with E2F6 in nuclear compartments in transformed LCLs.
A-B) 0.3 million U2OS or Saos-2 cells were seeded on coverslips and transiently transfected with control plasmid, GFP-EBNA3C or Flag-E2F6 expression constructs using Polyplus jetPRIME. Exogenous E2F6 protein was incubated with mouse anti-Flag (M2) antibody and Alexa Fluor 594-conjugated secondary antibody, while GFP-tagged EBNA3C was detected by GFP-fluorescence directly. C) BJAB, BJAB10, LCL1 cells were semi-air-dried on slides. Endogenous EBNA3C and E2F6 proteins were detected with their specific primary antibodies, followed by the corresponding secondary antibodies. And the nuclei was stained with DAPI. The images were captured using Olympus Fluoview confocal microscope and analyzed with FLUOVIEW software.
Fig 6.
A-B) 10 million MEF or Saos-2 cells were co-transfected with control plasmids, Myc-E3C, and Flag-EBNA3C expression plasmids. At 36 hours post-transfection, cells were treated with 40 μg/ml cycloheximide (CHX) for 0, 4, 8, 12 hours, and then lysed and analyzed by immunoblotting with specific antibodies. C-D) Different B-cells (BJAB, BJAB7, LCL1, and sh-ctrl, sh-EBNA3C LCL1) were obtained after treating with 40 μg/ml cycloheximide (CHX) for indicated time periods, and analyzed by western blot with specific antibodies. GAPDH was used as an internal loading control.
Fig 7.
EBNA3C and E2F6 can repress the transcriptional activity of E2F1.
A-F) 10 million HEK-293 or Saos-2 cells were transfected with pGL2 control vector, full-length E2F1 promoter construct, Flag-E2F6, either increasing doses of EBNA3C or E2F6 expression plasmids. Thirty-six hours post-transfection, cells were harvested and carried out for luciferase reporter assays. Expression of transfected constructs were detected by western blot. G-H) 10 million HEK-293 or Saos-2 cells were transfected with pGL2 control vector, full-length E2F1 promoter construct, Flag-E2F1, Flag-E2F6, and EBNA3C expression plasmids. Luciferase reporter assays were carried out at 36 hours post-transfection as above. Expression levels of transfected constructs were also examined by western blot.
Fig 8.
EBNA3C competes with E2F1 for E2F6 binding.
A-B) Cell lysates from indicated B-cells (BJAB, BJAB7, BJAB10, LCL1, and LCL2) were immunoprecipitated with E2F1 specific antibody. Then immunoprecipitations were analyzed by western blot to identify expressions of endogenous EBNA3C, E2F6 proteins. C-D) 10 million HEK-293 or Saos-2 cells were electroporated with indicated combinations of Myc-E2F6, Flag-E2F1, EBNA3C expression constructs. The cell lysates were immunoprecipitated using anti-Myc antibody and the immunoprecipitations were examined with western blot.
Fig 9.
EBNA3C promotes E2F6 binding to E2F1 promoter.
A-B) HEK-293 cells were co-transfected with appropriate combinations of Myc-EBNA3C, Flag-E2F6, and pGL2-E2F1 promoter reporter plasmid or pGL2 control plasmid. Then the ChIP assay was performed with E2F6 antibody and recovered DNA was quantitated by Real-time PCR using primers specific for transfected E2F1 promoter (A) or endogenous E2F1 promoter (B). NC, negative control. C) BJAB, BJAB7, LCL2 cells were harvested directly and performed ChIP assay as previously mentioned. The recovered DNA was detected with primers specific for endogenous E2F1 promoter. Shown are the results from independent experiments. Error bars indicate standard deviations.
Fig 10.
E2F6 is critical for EBNA3C-mediated cell proliferation.
A-B) 1 million BJAB cells were infected with the indicated lentivirus in combination 20 μg/ml polybrene. After 72 hours incubation, puromycin antibiotic was added as a 0.5 μg/ml concentration for selection. The selected cells were observed the GFP immunofluorescence (A) and the target protein expressions were detected by western blot (B). C-D) 1 million BJAB7 cells were infected and selected for 2 weeks using 0.5 μg/ml puromycin. GFP immunofluorescence (C) was checked and the target proteins were also analyzed by western blot (D). E) 3 million BJAB or BJAB7 stable cell lines were treated with 5μM CFSE solution for 10 minutes at room temperature. Then cells were washed, cultured, and harvested at indicated time points (0, 24, 48, and 72h). Flow cytometry was used to analyze CFSE-labeled cells.
Fig 11.
A schematic that illustrates the role of E2F6 in EBNA3C-mediated E2F1 regulation.
EBNA3C interacts with E2F6 specifically and enhances the stability of E2F6. EBNA3C can also compete with E2F1 for E2F6 binding. E2F6 recruited together with EBNA3C binds E2F1 promoter and inhibits its activity, which contributes to B-cell proliferation by reducing the expression of E2F1. This mechanism describes that the contribution of E2F6 in EBNA3C-related oncogenic activity important for EBV-transformed B-cell proliferation.