PMC

Coordinate regulation of the EBV epigenome and TET2 gene transcript by TET2 and EBNA2. Model depicting TET2 interacting with EBNA2 at RBP-jκ sites to regulate the demethylation of the EBV epigenome and the TET2 gene locus in type III latency.

TET2 depletion alters the EBV epigenome. (A to F) LCLs transduced with shTET2-1 (red) or control lentivirus (black) were assayed by DIP for 5hmC (A) or 5mC (B) or by ChIP for EBF1 (C), RBP-jκ (D), H3K4me3 (E), or total H3 (F) at EBV regions for EBER, FR, LMP2Ap, LMP1p, Cp, Qp, or Zp. *, P < 0.05.

TET2 depletion alters EBNA2-responsive genes in the cellular genome. The process was as described for , except at cellular genes. (A to F) LCLs transduced with shTET2-1 (red) or control lentivirus (black) were assayed by DIP for 5hmC (A) or 5mC (B) or by ChIP for EBF1 (C), RBP-jκ (D), H3K4me3 (E), or total H3 (F) cellular genes for IL-7, HES1, FCER2, or actin. (G) RT-qPCR for shTET2-1 (red) or control (black) lentivirus-transduced LCLs assayed for RNA expression of IL-7, HES1, or FCER2 relative to GAPDH, as indicated. *, P < 0.05.

TET2 expression in B lymphocytes with EBV type III latency. (A) RT-qPCR for TET2 mRNA assayed in EBV-negative DG75 and Akata, in EBV type I latency Akata EBV+, Mutu I, Kem 1, and in EBV type III latency Kem III, Raji, LCL187, and LCL352 cells. (B) Western blot with antibodies to TET2 or GAPDH for cells as described for panel A. (C) RT-qPCR for TET2, TET1, or TET3 (left panel) or DNMT1, DNMT3a, or DNMT3b (right panel) in Mutu I (blue) or LCL (red). (D) Western blot with antibodies to TET3 or GAPDH for cells as described for panels A and B.

DNA methylation status and TET2 expression in EBV latency types. (A) MeDIP assay on Mutu I (blue), Mutu III (yellow), or LCL (red) was performed by qPCR for EBV regulatory regions for EBERs, family of repeats (FR), LMP2A promoter, LMP1 promoter, Cp, Qp, Zp, and Rp, and the percentage of input DNA was quantified. (B) RT-qPCR for TET2 mRNA analysis in Mutu I, Mutu III, or LCL, as indicated, relative to GAPDH. (C) Western blot with antibodies to TET2, IDAX, EBNA2, or GAPDH in cell extracts from Mutu I, Mutu III, or LCL.

shRNA depletion of TET2 alters EBV gene expression. (A) Western blot analysis of LCLs transduced with either shTET2-1, shTET2-2, or control lentivirus and assayed with antibody to TET2, EBNA2, LMP1, EBNA3C, ZTA, EA-D, TET3, or actin. The depletion percentage of each shTET2 was determined by the signal intensity of TET2 relative to that of actin and then compared to that of shCtrl. (B) RT-qPCR for shTET2-1 (red), shTET2-2 (blue), or control (black) lentivirus-transduced LCLs assayed for RNA expression of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, LMP2A, ZTA, or RTA relative to GAPDH, as indicated. (C) qPCR of intracellular DNA for EBV using primers for either Cp relative to cellular actin. *, P < 0.05.

TET2 colocalization and coimmunoprecipitation with EBNA2. (A) ChIP-qPCR for TET2 in Mutu I (blue) or LCL (red) at EBV genome sites for EBER, FR, LMP2Ap, LMP1p, Cp, Qp, Zp, or Rp. (B) Same as described for panel A, except assayed as cellular RBP-jκ binding sites at IL-7, HES1, FCER2, or actin. (C) LCL or Mutu III cell lysate was subjected to IP with TET2 or IgG control and assayed by Western blotting with EBNA2 or TET2. Input is 5% of total. (D) 293T cells transfected with FLAG-EBNA2 were subjected to IP with TET2 or IgG (left panel) or with FLAG or IgG (right panel) followed by Western blotting with FLAG or TET2 antibody. (E) 293T cells transfected with Myc-EBNA2 were subjected to IP with TET2 or IgG (left panel) or with Myc or IgG (right panel) and then assayed by Western blotting with Myc or TET2 antibody. *, P < 0.05.

TET2 expression depends on EBNA2 and RBP-jκ in type III latency. (A) EREB cells withdrawn from estradiol (E2) for 24, 48, or 72 h were assayed by Western blotting for TET2, EBNA2, or actin. (B) RT-qPCR results for TET2 mRNA for EREB cells treated as described for panel A. (C) ChIP-qPCR results for EBNA2 at EBNA2_A, EBNA2_B, RBP_L, or RBP_R sites at TET2 loci or at control actin in EREB cells in the presence (+E2) or absence (−E2) of estradiol for 48 h. (D) ChIP-qPCR results for RBP-jκ at RBP_L or RBP_R sites or at control actin in EREB cells in the presence (+E2) or absence (−E2) of estradiol for 48 h. (E) LCLs transduced with three different shRNAs for RBP-jκ or control (shCtrl) and assayed by Western blotting with antibody to TET2, RBP-jκ, or actin. (F) Cells were treated as described for panel E and then assayed by RT-qPCR for TET2 mRNA relative to actin. *, P < 0.05.

Epigenetic features of TET2 gene locus in type I and III cell lines. (A) ChIP-seq tracks for EBNA2, EBF1, RPB-jκ, H3K4me3, or H3K36me3 in LCL (red) or Mutu I (blue) cells over the regions spanning TET2 and CXXC4/IDAX gene loci. Binding sites for EBNA2 are designated EBNA2_A and EBNA2_B and indicated by asterisks. (B) Zoom-in of ChIP-seq track for RBP-jκ for EBNA2 and RBP-jκ in Mutu I (blue) or LCL (red) cells over the TET2 promoter locus. Binding sites for RBP-jκ are designated RBP_L and RBP_R and indicated by asterisks. (C) ChIP-qPCR for validation of RBP-jκ binding at sites RBP_L and RBP_R. (D) MeDIP for 5mC in Mutu I (blue), Mutu III (yellow), or LCL (red) cells at TET2 loci for TSS_1, TSS_2, RBP_L, RBP_R, or at actin. (E) RT-qPCR for TET2 mRNA in Mutu I or LCL cells treated with 10 μM 5′ azacytidine (orange) or control (black) for 72 h. (F) ChIP-qPCR for H3K4me3 with primers for the TET2 TSS_1/2 in Mutu I (left) or LCL (right) cells treated with 5′ azacytidine (orange) or control (black) for 72 h.