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. 2013 Nov 12;110(46):18537-42.
doi: 10.1073/pnas.1317608110. Epub 2013 Oct 28.

Epstein-Barr virus nuclear antigen leader protein localizes to promoters and enhancers with cell transcription factors and EBNA2

Daniel Portal  1 , Hufeng Zhou, Bo Zhao, Peter V Kharchenko, Elizabeth Lowry, Limsoon Wong, John Quackenbush, Dustin Holloway, Sizun Jiang, Yong Lu, Elliott Kieff
Affiliations

Epstein-Barr virus nuclear antigen leader protein localizes to promoters and enhancers with cell transcription factors and EBNA2

Daniel Portal et al. Proc Natl Acad Sci U S A. .

Abstract

Epstein-Barr virus (EBV) nuclear antigens EBNALP (LP) and EBNA2 (E2) are coexpressed in EBV-infected B lymphocytes and are critical for lymphoblastoid cell line outgrowth. LP removes NCOR and RBPJ repressive complexes from promoters, enhancers, and matrix-associated deacetylase bodies, whereas E2 activates transcription from distal enhancers. LP ChIP-seq analyses identified 19,224 LP sites of which ~50% were ± 2 kb of a transcriptional start site. LP sites were enriched for B-cell transcription factors (TFs), YY1, SP1, PAX5, BATF, IRF4, ETS1, RAD21, PU.1, CTCF, RBPJ, ZNF143, SMC3, NFκB, TBLR, and EBF. E2 sites were also highly enriched for LP-associated cell TFs and were more highly occupied by RBPJ and EBF. LP sites were highly marked by H3K4me3, H3K27ac, H2Az, H3K9ac, RNAPII, and P300, indicative of activated transcription. LP sites were 29% colocalized with E2 (LP/E2). LP/E2 sites were more similar to LP than to E2 sites in associated cell TFs, RNAPII, P300, and histone H3K4me3, H3K9ac, H3K27ac, and H2Az occupancy, and were more highly transcribed than LP or E2 sites. Gene affected by CTCF and LP cooccupancy were more highly expressed than genes affected by CTCF alone. LP was at myc enhancers and promoters and of MYC regulated ccnd2, 23 med complex components, and MYC regulated cell survival genes, igf2r and bcl2. These data implicate LP and associated TFs and DNA looping factors CTCF, RAD21, SMC3, and YY1/INO80 chromatin-remodeling complexes in repressor depletion and gene activation necessary for lymphoblastoid cell line growth and survival.

Keywords: gene expression; genome-wide ChIP-seq analysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Genomic distribution and TF DNA binding sequences enriched at LP, E2, and LP/E2 sites. (A) Pie charts showing the genome-wide distribution of all LP or E2 sites, as well as the genome-wide distribution of LP-only, E2-only, and LP/E2 sites. (B) List of most highly enriched cell transcription factor DNA binding sequences at LP, E2, and LP/E2 sites (±100 b) (P < 0.01).
Fig. 2.
Fig. 2.
Anchor plots of cell TF coverages at LP, LP/E2, and E2 sites. The upper 12 panels show higher cell TF coverage at LP/E2 (red line) than at LP (green line), or E2 (blue line) sites (±1.5 kb). EBF differs in having higher coverage at the E2 site than at LP or the LP/E2 site. The lower three panels show higher cell TF coverage for DNA looping factors SMC3, RAD21, and CTCF at LP sites than at E2 or LP/E2 sites.
Fig. 3.
Fig. 3.
Anchor plots for cell epigenetic marks and affected gene expression data. (A) Coverage of RNAPII, P300, H3K9ac, H3K27ac, H3K4me1, and H3K4me3 ±1.5 kb of LP (green line), E2 (blue line), and LP/E2 (red line) sites. (B) Box plots of RNA-seq at LP, E2, or LP/E2 annotated peaks for the nearest promoter versus a control (Ctrl) random gene set. LP, E2, and LP/E2 genes were significantly more highly expressed than control genes (P < 2 × 10−16). LP had lower expression than E2 (P < 0.00056) or LP/E2 (P < 2.7 × 10−12), and E2 was lower than LP/E2 (P < 0.05). The box plots indicate the data distribution in percentiles, with the horizontal line being the median. The top of the box represents upper 25% quartile (25% of the data over that value), and the bottom of the box, the lower quartile (25% of the data below that value). The horizontal lines at the ends of the dotted lines are the maximum observed values.
Fig. 4.
Fig. 4.
LP site-associated cell TFs separated into 10 K-means clusters and gene expression. (A) Cell TFs YY1, PAX5, BATF, IRF4, and PU.1 were prominent components of most clusters, consistent with their 30–59% LP site association. Notably, E2, RBPJ, and TBLR1 clustered separately from looping factors CTCF, SMC3, and RAD21. (B) Box plots of RNA-seq gene expression from LP-affected promoters. All LP-affected genes were more highly expressed than random control genes (P < 2 × 10−16). The box plots indicate the data distribution in percentiles, with the horizontal line being the median. The top of the box represents upper 25% quartile (25% of the data over that value), and the bottom of the box, the lower quartile (25% of the data below that value). The horizontal lines at the ends of the dotted lines are the maximum observed values, excluding the outliers.
Fig. 5.
Fig. 5.
Model. LP occupies mostly promoters, whereas E2 is found at enhancers (17). Enhancers regulate transcription via long-distance DNA interactions mediated by CTCF, RAD21, and SMC3. Several transcription factors are associated with LP sites and E2 sites and likely help mediate the interactions between LP and E2 (blue box). The genes associated with LP sites are highly expressed and rich in activation histone marks (Fig. 3). LP and HA95 regulate the dismissal of the NCOR repressive complex (9, 59).
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