ERK/c-Jun Recruits Tet1 to Induce Zta Expression and Epstein-Barr Virus Reactivation through DNA Demethylation

Abstract

DNA demethylation plays an essential role in the reactivation of Epstein-Barr virus (EBV) from latency infection. However, it is unclear how epigenetic modification is initiated in responding to stimuli. Here, we demonstrate that ERK/c-Jun signaling is involved in DNA demethylation of EBV immediate early (IE) gene Zta in response to 12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulation. Remarkably, Ser73 phosphorylation of c-Jun facilitates Zta promoter demethylation and EBV reactivation, whereas knockdown of c-Jun attenuates Zta demethylation and viral reactivation. More importantly, we reveal for the first time that c-Jun interacts with DNA dioxygenase Tet1 and facilitates Tet1 to bind to Zta promoter. The binding of c-Jun and Tet1 to Zta enhances promoter demethylation, resulting in the activation of Zta, the stimulation of BHRF1 (a lytic early gene) and gp350/220 (a lytic late gene), and ultimately the reactivation of EBV. Knockdown of Tet1 attenuates TPA-induced Zta demethylation and EBV reactivation. Thus, TPA activates ERK/c-Jun signaling, which subsequently facilitates Tet1 to bind to Zta promoter, leading to DNA demethylation, gene expression, and EBV reactivation. This study reveals important roles of ERK/c-Jun signaling and Tet1 dioxygenase in epigenetic modification, and provides new insights into the mechanism underlying the regulation of virus latent and lytic infection.

Figure 1. ERK/c-Jun pathway is involved in the demethylation of Zta promoter and the expression of Zta gene.

(a–c) B95-8 cells were pretreated with ERK signal pathway specific inhibitor U0126 (20 μM) for 2 h and then treated with phorbol ester 12-O-Tetradecanoylphorbol 13-acetate (TPA) (60 ng/ml) for 24 h. The Zta promoter was analyzed by sodium bisulphite sequencing. The white and black circles indicate unmethylated and methylated CpGs, respectively (a). The level of EBV IE gene Zta mRNA was determined by real-time PCR (b). The level of Zta protein was detected by Western blot analyses (c). Relative protein levels, indicated by numbers below the blots, were determined by densitometry, with internal normalization to β–actin and external normalization to the negative control sample processed in parallel. (d) B95-8 cells were pretreated with U0126 (20 μM) for 2 h and then treated with TPA (60 ng/ml) for 24 h. The levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were determined by dot blot analyses. Bulk genomic DNA was isolated, sonicated, cross-linked to nitrocellulose membrane, and probed with the monoclonal antibody specific for 5hmC or 5mC. The loading control is showing by the methylene blue stain in the bottom panel. The blot was quantitated by densitometry, and a representative experiment is shown from three independent experiments. All data were collected from 3 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2. c-Jun Ser73 phosphorylation is essential for ERK-dependent demethylation and activation of Zta promoter.

(a) B95-8 cells were pretreated with U0126 (20 μM) for 2 h and then treated with TPA for 1, 3, 6, 12, and 24 h, respectively. Whole-cell lysates were isolated and the levels of phosphorylated ERK (p-ERK), phosphorylated c-Jun (p-c-Jun), phosphorylated c-Fos (p-c-Fos), ERK, c-Jun, c-Fos, Zta, and GAPDH proteins were determined by Western blotting using corresponding antibodies. (b) B95-8 cells were pretreated with U0126 (20 μM) for 2 h and then treated with TPA for 15 and 90 min. Whole-cell lysates were isolated and the levels of phosphorylated c-Jun (p-c-Jun) and GAPDH proteins were determined by Western blotting. (c) B95-8 cells were treated with TPA for 0.5, 1, 3, 6 and 9 h. Total RNAs were isolated from the cells and the level of Zta mRNA was analyzed by qPCR. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 3. c-Jun is required for ERK-dependent DNA demethylation of Zta promoter.

(a) B95-8 cells were lentiviruses expressing control shRNA (shCtrl) or shRNA against c-Jun (shc-Jun) for 2 weeks and then selected with puromycin. The total RNAs were isolated and the c-Jun mRNA was analyzed by qPCR (a). (b–d) B95-8 cells were transduced with lentiviruses expressing shCtrl or shc-Jun for 2 weeks, selected with puromycin, and then treated with TPA for 18 h. The total RNAs were isolated form the cells and the level of Zta mRNA was determined by qPCR (b). The cell extracts were prepared and the level of Zta, c-Jun, and β-actin proteins were detected by Western blot analyses (c). The Zta promoter was analyzed by sodium bisulphite sequencing. The white and black circles indicate unmethylated and methylated CpGs, respectively (d). All data were collected from 3 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 4. Tet1 binds to Zta promoter through interacting with c-Jun.

(a) Yeast strain AH109 was co-transformed with the combination of one BD and one AD plasmid, as indicated. Transformed yeast cells containing both plasmids were grown on SD-minus Trp/Leu plates (DDO), and colonies were then replica-plated onto SD-minus Trp/Leu/Ade/His plates (QDO) containing X-α-gal to check for the expression of reporter gene (blue colonies). (b) GST pull-down assay with GST-c-Jun or GST alone incubated with HA-tagged Tet1CD. (c) Co-immunoprecipitation of HA-tagged Tet1 CD and GFP-tagged c-Jun or GFP-tagged c-Fos, followed by western blot analysis with anti-GFP antibody. (d) HA-tagged Tet1 CD and Flag-tagged c-Jun were transfected to HEK293 cells and immunoprecipitated using anti-Flag/HA antibody from cell extracts. Co-immunoprecipitation of HA-Tet1/Flag-c-Jun was detected by western blotting using anti-HA/Flag antibody. (e) HEK293 cells were transfected with pHA-tagged Tet1 CD and immunoprecipitated using anti-HA antibody from cell extracts. Co-immunoprecipitation of HA-Tet1/ c-Jun was detected by western blotting using anti-HA/ c-Jun antibody. (f) HEK293 cells were transfected with HA-Tet1 CD for 24 h. After fixation, the cells were immunostained with anti-HA and anti-c-Jun antibody. The nucleus stained by DAPI and visualized by confocal laser microscopy. (g) For ChIP, B95-8 cells were pretreated with U0126 (20 μM) for 2 h and treated with TPA (60 ng/ml) for 4 h. Cross-linked chromatin was immunoprecipitated with the specific antibodies. (h) For ChIP, c-Jun knockdown and control B95-8 cells were treated with TPA (60 ng/ml) for 4 h. Cross-linked chromatin was immunoprecipitated with the specific antibodies. Error bars indicate standard error of the mean. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5. Tet1 is essential for TPA-mediated Zta promoter demethylation and gene expression.

(a) B95-8 cells were transduced with lentiviruses expressing control shRNA (shCtrl) or shRNA against Tet1 (shTet1) for 2 weeks, and selected with puromycin. The total RNAs were isolated form the cells and the level of Tet1 mRNA was determined by qRT-PCR (upper panel). The cell extracts were prepared and the level of Tet1 and β-actin proteins were detected by Western blot analyses (lower panel). (b,c) B95-8 cells were transduced with lentiviruses expressing shCtrl or shTet1 for 2 weeks, selected with puromycin, and then treated with TPA for 18 h. The total RNAs were isolated form the cells and the level of Zta mRNA was determined by qRT-PCR (b, upper panel). The cell extracts were prepared and the level of Zta and β-actin proteins were detected by Western blot analyses (b, lower panel). The Zta gene promoter was analyzed by sodium bisulphite sequencing. White and black circles indicate unmethylated and methylated CpGs, respectively (c). All data were collected from 3 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 6. c-Jun and Tet1 are essential for ERK-dependent reactivation of EBV.

(a) B95-8 cells were pretreated with U0126 (20 μM) for 2 h and then treated with TPA (60 ng/ml) for different times. RNA was extracted and the BHRF1 gene mRNA was analyzed by qPCR. (b) B95-8 cells were pretreated with U0126 (20 μM) for 2 h and then treated with TPA (60 ng/ml) for 18 h. DNA was prepared from treated cells and the level of EBV genomic DNA was determined by qPCR. Values of viral genome copies are normalized to the control DMSO. (c–e) B95-8 cells were transduced with lentiviruses expressing shCtrl or shTet1 for 2 weeks, selected with puromycin, and then treated with TPA for 18 h. The total RNAs were isolated form the cells and the levels of BHRF1 mRNA (c), pg350/220 mRNA (d), and EBV genomic DNA (e) were determined by qPCR. Values are normalized to the control shCtrl. Error bars represent SD from the mean. All data were collected from 3–6 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.