PMC

In EBV latency type I cells, hypermethylated DNA at the miR-155 CpG island represses miR-155 expression. In EBV latency type III cells, hypomethylation of the CpG island allows for miR-155 expression. What is more, EBV latency genes such as LMPs induce expression of AP1 genes, which bind to the AP1 site on the miR-155 promoter to activate miR-155 expression.

A–C. Latency membrane proteins induced pri-miR-155 expression. Stable expression of EBV genes, LMP2A (LMP2A-1 & LMP2A-2, L2A) and/or LMP1 (LMP1-1, LMP1-2 & LMP1-3, L1) was performed in the EBV-negative cell line BL30, and RNA was extracted for real-time RT-PCR to detect endogenous pri-miR-155 expression. D. Assessment of the effect of EBV latent genes on pri-miR-155 expression. Transient transfection was conducted to co-express EBNA3C, LMP2A and EBNA3C (E3C-L2A), LMP2A, EBNA3C, EBNA2 and LMP1 (E3C-L2A-E2-L1), or LMP2A, EBNA3A/3B/3C, EBNA2 and LMP1 (E3A-B-C-L2A-E2-L1) in EBV-negative cells (DG75), and followed by RNA extraction for RT-PCR 72 hours post transfection. The relative expression fold change of pri-miR-155 was calculated based on the 2^−ΔΔCT value.

A. An electrophoretic motility shift analysis (EMSA) was performed using the miR-155 AP1 elements as the probe; with or without competitor oligos, the wild type miR-155 AP1 (AP1wt) and the mutant miR-155 AP1 (AP1mut); and supershift was conducted with AP1 antibodies, JunB and FosB; in EBV latency type I cells (MutuI or Akata) or type III (X50-7 or JY) cells. − indicates without competitor oligonucleotide (oligo). B. A ChIP assay was conducted with antibodies against Histone 3 (H3), acetyl-Histone 4 (Ac-H4) and AP1 protein, JunB, FosB, ATF3 and JunD, and PCR with specific primers spanning the miR-155 promoter (155p) region and non-specific primers spanning the first intron of the miR-155 gene (Intron 1) in EBV latency type III cells (X50-7 and IB4). Input was 1/10 of each ChIP reaction. ImageJ software was used for quantitation.

A. LMP2A and EBNA3C activate miR-155 promoter activity in a dose dependent manner. Co-transfection of wild type miR-155 promoter (pwt) with LMP2A and/or EBNA3C at the indicated concentrations into EBV-negative cells (MutuE1dn) was performed and a luciferase assay was conducted after 48 hours. The RLU fold change was calculated on the basis of the RLU value without LMP2A and/or EBNA3C. B. The miR-155 promoter AP1 mutant (pAP1mut) was activated by 5ug of EBNA3C but not by 0.5ug of LMP2A or 0.5ug of LMP2A plus 5ug of EBNA3C (LMP2A-EBNA3C). LMP2A and/or EBNA3C were co-transfected with miR-155 promoter wild type (pwt) or miR-155 promoter mutants (pAP1mut, pEtsmut, pNF-κBmut and pPU.1muts) into EBV-negative MutuE1dn cells and cells were harvested for a luciferase assay after 48 hours. The RLU fold change was calculated on the basis of the RLU value of control (Cntl), pGL3-basic. C. MEK kinase inhibitors inhibit LMP2A-mediated miR-155 promoter activity but not EBNA3C-mediated miR-155 promoter activity. Wild type miR-155 promoter (pwt) was co-transfected with LMP2A or/and EBNA3C, then cells were treated with the MEK inhibitors U126 or PD98095, or with the NF-κB inhibitor Bay11, and a luciferase assay was carried out after 48 hours. The RLU fold change was calculated on the basis of the RLU value of control (Cntl), no LMP2A and/or EBNA3C.

DNA methylation was sequenced across part of the promoter and first exon of miR-155 from −11 to 443 relative to the TTS. A. The sequence of the miR-155 CpG island and the sequenced CpG sites are labeled in red. B. MiR-155 CpG island methylation status in EBV-positive and EBV-negative cells. DNA methylation levels at indicated regions were shown as a percentage value in the various cell lines. The analyzed cells include EBV latency type I cells (Mutu, Akata and Rael), EBV latency type III cells (JC5, Dana, Boston, Alwife, JY and P3HR1) and EBV negative cell (DG75). Mutu Bulk Cntl, an EBV-positive latency type I cell line, was treated with DNA demethylating agents Zebularine (Mutu Bulk Zeb) or 5-aza-deoxycytidine (Mutu Bulk 5A).

A–C. X50-7 (A) and IB4 (B & C) cells were treated with an NF-κB inhibitor and MEK kinase inhibitors for 48 hours and cells were harvested for RT-PCR to detect pri-miR-155 expression (top panel) and for western blot to detect AP1 protein expression (bottom panel). Bay-U indicates treatment with Bay 11 plus UO126. Relative expression in percentage was compared to the DMSO 2^−ΔΔCT value. D. A luciferase assay was performed to analyze the activity of miR-155 promoters; wild type (155pwt) vs. AP1 mutant (155pAP1mut), in response to treatment with signaling pathway inhibitor Bay11 in IB4 cells. The relative luminescence units (RLU) fold change value is compared with the RLU measured in control (Cntl), pGL3-basic.