Genome-Wide DNA Methylation as an Epigenetic Consequence of Epstein-Barr Virus Infection of Immortalized Keratinocytes

Characterization of EBV-infected and transiently infected EBV-negative NOK. (A) Reverse transcriptase PCR for detection of latent and lytic viral transcripts in an uninfected clone (U), EBV-infected cell lines 1 and 2 (I1 and I2, respectively), and an EBV-positive clone from I1 (clone 4, cl4). B958 (+) was used as a positive control. W indicates water as the template control. (B) EBNA1 immunofluorescence analysis. Cells shown in the left panels were stained with the nuclear stain DAPI; EBNA1-positive cells are shown in red in the right panels. NOK E+ bulk, EBV-positive NOK cell line. (C) Southern blotting for detection of fused and linear EBV terminal repeats probed with the BamHI NJhet fragment. The asterisk denotes end fragments from linear EBV DNA. (D) Cell proliferation using an MTS [3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt] assay was measured at 24-h intervals for 96 h. Shown are the average absorbance values and standard errors of the means from three independent biological experiments. (E) Transepithelial electrical resistance following calcium-induced differentiation. Data are the averages and standard errors of the means from three independent biological experiments. Lower TEER measurements for EBV-positive and EBV-negative transiently infected clones were statistically significant relative to uninfected and vector controls starting at days 4 to 9. (F) Involucrin (IVL) expression following methylcellulose treatment. Cells were either seeded as a monolayer (mono) or suspended in methylcellulose for 24 h (MC) to induce differentiation. qRT-PCR was used to quantify the amount of IVL transcripts relative to cyclophilin A (PPIA) transcripts. Data are the averages and standard errors of the means of two independent experiments. U, uninfected cells; V, vector control; EBV+, EBV-positive clone; EBV-cl 1, 3, and 4, three EBV-negative transiently infected clones.

Methylome analysis of NOK by RRBS. (A) Distribution of mapped CpG residues in NOK. (B) Box-and-whisker plots comparing the distribution of methylated CpG residues between uninfected (U) cells, a vector control (V), an EBV-positive clone (EBV+), and three transiently infected EBV-negative clones (EBV-cl1, -cl3, and -cl4) at CpG islands, promoters, and within gene bodies. The yellow line across the bar represents the median level of methylation, the top whisker represents the maximum level of methylation, and the bottom whisker, which goes to 0, represents the minimum level of methylation.

Distribution of differentially methylated CpG residues relative to the transcription start site (TSS). CpGs that either gained methylation (hypermethylated) or lost methylation (hypomethylated) in the EBV-positive and three EBV-negative transiently infected clones in relation to the uninfected and vector control were mapped relative to the TSS.

CpG island hypermethylation as an epigenetic consequence of EBV infection of oral keratinocytes. (A) Number of hypermethylation events per locus. (B) Number of hypomethylation events per locus. Differentially methylated CpG residues that were located 500 bases from the previous CpG methylation event were counted. Blue bars indicate the number genes that acquired a certain number of methylation events, while red bars indicate CpG methylation at intergenic locations. (C to E) Methylation tracks at CpG islands in the NEDD4 binding protein 2-like 1 (N4BP2L1) gene, retinoic acid receptor responder 1 (RARRES1) gene, and protocadherin 19 (PCDH19) gene, respectively. Red bars represent unmethylated CpGs, and yellow bars represent methylated CpGs. Solid blue bars represent exons, and green bars represent CpG islands. Direction of transcription is indicated with blue or white arrows. Methylation tracks were visualized using the UCSC (University of California, Santa Cruz) Genome Browser. Samples shown are the uninfected parental control, vector control, an EBV-positive clone (EBV pos), and three EBV-negative transiently infected clones (EBV neg cl1, cl3, and cl4). hg19, human genome build 19.

Methylation of the EBV genome in stably infected NOK. The outer circle displays the EBV genome position. The inner gray circle annotates select EBV genes, with purple bars showing rightward reading open reading frames and gold bars indicating the leftward reading open reading frames. Repetitive elements and origins of DNA replication are indicated as gray bars. The light gray circles map the positions of methylated (50 to 100% meCpG) and unmethylated (0 to 20% meCpG) CpG residues.

Altered gene expression patterns as a consequence of EBV-induced epigenetic alterations. (A) Principle component analysis (PCA) of gene expression using a subset of 5,500 genes of uninfected (UN) cells, a vector control (vector), an EBV-positive clone (Pos), and three EBV-negative transiently infected clones (Neg cl1, -cl3, and -cl4). (B) Dendrogram based on PCA showing clustering of samples according to EBV exposure.

Positional effect of DNA methylation on gene expression. (A) Differentially expressed genes were grouped according to whether the position of DNA methylation was near the TSS (<2,000 kb) or far from the TSS (>2,000 kb). (B) Distribution of methylation from the transcription start site (TSS) on differentially expressed genes (upregulated or downregulated) in an EBV-positive clone and the three EBV-negative transiently infected clones compared to uninfected cells and vector controls. Black horizontal bars indicated the mean distances of methylation from the TSS.