Epstein–Barr Virus Nuclear Antigen Leader Protein Localizes to Promoters and Enhancers with Cell Transcription Factors and EBNA2

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

Daniel Portala,b,1, Hufeng Zhoua,b,c,1, Bo Zhaoa,b, Peter V. Kharchenkod, Elizabeth Lowrya,b, Limsoon Wongc, John Quackenbushe, Dustin Hollowaye, Sizun Jianga,b,d, Yong Luf, and Elliott Kieffa,b,2

aDepartment of Medicine, Brigham and Women’s Hospital, and bDepartment of Microbiology and Immunobiology, Harvard University, Boston, MA 02115; cSchool of Computing, National University of Singapore, Republic of Singapore 117417; dCenter for Biomedical Informatics, Harvard Medical School, Harvard University, Boston, MA 02115; eDepartment of Biostatistics and Computational Biology, Dana Farber Cancer Institute, Boston, MA 02115; and fLaboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

Contributed by Elliott Kieff, October 1, 2013 (sent for review August 16, 2013) Epstein–Barr virus (EBV) nuclear antigens EBNALP (LP) and EBNA2 (17). E2 increases H3K4me1 signals allowing E2 and cell TF (E2) are coexpressed in EBV-infected B lymphocytes and are critical occupancy and transcription activation. The E2 activation do- for lymphoblastoid cell line outgrowth. LP removes NCOR and main recruits basal and activation-related cell TFs, including RBPJ repressive complexes from promoters, enhancers, and ma- TAF40, TFIIH, TFIIE, and histone acetylases P300, CBP, and trix-associated deacetylase bodies, whereas E2 activates transcrip- PCAF (17–21). Focusing on 88 dynamically E2-regulated genes, tion from distal enhancers. LP ChIP-seq analyses identiﬁed 19,224 using ENCODE chromosome conformation capture (3C) data, LP sites of which ∼50% were ±2 kb of a transcriptional start site. the transcriptional start site (TSS) of 50 E2 dynamically regu- LP sites were enriched for B-cell transcription factors (TFs), YY1, lated genes are in proximity to approximately three E2 enhancers SP1, PAX5, BATF, IRF4, ETS1, RAD21, PU.1, CTCF, RBPJ, ZNF143, per gene. These enhancers are 61% on the same chromosome and κ SMC3, NF B, TBLR, and EBF. E2 sites were also highly enriched at a median distance of ∼330 kb from their affected genes. The for LP-associated cell TFs and were more highly occupied by RBPJ combined effect of three E2 enhancers accounts for E2’sstrongup- and EBF. LP sites were highly marked by H3K4me3, H3K27ac, regulatory effects (17). E2 induces MYC expression within 24 h of H2Az, H3K9ac, RNAPII, and P300, indicative of activated transcrip- RBL infection. MYC then drives RBL cell cycle entry and pro- tion. LP sites were 29% colocalized with E2 (LP/E2). LP/E2 sites liferation (14, 22). However, the nearest RBPJ and E2 sites are were more similar to LP than to E2 sites in associated cell TFs, >100 kb from myc (17). FISH and 3C assays connect the myc RNAPII, P300, and histone H3K4me3, H3K9ac, H3K27ac, and H2Az − occupancy, and were more highly transcribed than LP or E2 sites. TSS to an E2 site at 428 kb from myc (17). Gene affected by CTCF and LP cooccupancy were more highly LP and E2 cooperatively activate virus and cell gene tran- expressed than genes affected by CTCF alone. LP was at myc scription following transient or stable B-lymphocyte transfection enhancers and promoters and of MYC regulated ccnd2,23med (9, 23, 24). The experiments described here were undertaken to complex components, and MYC regulated cell survival genes, igf2r identify the mechanisms through which LP and E2 affect cell and bcl2. These data implicate LP and associated TFs and DNA gene transcription in LCLs. looping factors CTCF, RAD21, SMC3, and YY1/INO80 chromatin- Results and Discussion remodeling complexes in repressor depletion and gene activation LP, LP/E2, and E2 Genome Distributions. necessary for lymphoblastoid cell line growth and survival. Duplicate LCL LP and an E2 ChIP-seq dataset (17) were analyzed using HOMER, with genome-wide ChIP-seq analysis | gene expression a false discovery rate of P < 0.001. LP localized to 19,224 sites

ﬁ pstein–Barr virus (EBV) nuclear antigens EBNALP (LP) and Signi cance EEBNA2 (E2) are EBV-encoded transcription factors (TFs) that are coordinately expressed within hours after EBV infection Epstein–Barr virus nuclear antigen (EBNA) leader protein (LP) of resting B lymphocytes (RBLs) and are important for B-lym- and EBNA2 (E2) up-regulation of virus and cell gene expression phocyte conversion to lymphoblastoid cell lines (LCLs) (1–7). is important for human B-lymphocyte conversion to continu- However, the biochemical mechanisms by which LP and E2 co- ous, potentially malignant, lymphoblast cell lines. Although the ordinately affect RBL transformation to LCLs are largely un- molecular mechanism(s) underlying LP and E2 regulation of cell known. LP coactivates transcription by heterodimerizing with gene expression have been partially elucidated, LP ChIP- HA95 and Hsp70/72 to relocate HDAC4 from the nucleus to the sequencing studies have now revealed that LP and LP/E2 interact, cytoplasm, displaces Sp100 and Hp1α from ND10 bodies, and genome-wide, with human B-cell transcription factors, mostly at disrupts matrix-associated deacetylase (MAD) bodies, broadly or near prepatterned promoter sites, to increase cell transcription affecting repressor localization in cell nuclei (8–16). LP also factor occupancies, increase activation-associated histone decreases repressive NCOR and RBPJ occupancy at E2 sites, marks, and positively affect cell gene transcription. without altering E2 occupancy (9). Author contributions: D.P. and B.Z. designed research; D.P. and E.L. performed research; E2 enhances gene expression by localizing to cell TF sites P.V.K., L.W., J.Q., D.H., S.J., and Y.L. contributed new reagents/analytic tools; D.P., H.Z., through RBPJ or ZNF143 (17–19). E2/RBPJ sites localize in six S.J., and E.K. analyzed data; and D.P. and E.K. wrote the paper. clusters of EBF, ETS1, ZNF143, PU.1, NFκB, and RUNX1 sites. The authors declare no conﬂict of interest. Encyclopedia of DNA elements (ENCODE) ChIP-sequencing Data deposition: All information regarding access to data is included in SI Materials and experiments (ChIP-seqs) indicated high-level cell TF cooccu- Methods. pancy at E2 sites, consistent with these sites being open to cell or 1D.P. and H.Z. contributed equally to this work. virus TF occupancy. Indeed, E2 chromatin sites in LCLs are open 2To whom correspondence should be addressed. E-mail: [email protected]. chromatin sites in RBLs, before EBV infection, consistent with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CELL BIOLOGY EBF and RBPJ as pioneering factors that displace nucleosomes 1073/pnas.1317608110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1317608110 PNAS | November 12, 2013 | vol. 110 | no. 46 | 18537–18542 Downloaded by guest on September 23, 2021 A LP all E2 all up-regulate transcription through RBL genome-wide sites that (19,224) (19,845) are prepatterned for up-regulation of B-cell growth and survival. HOMER did not recognize a de novo LP DNA sequence, 16 fi 28 33 which may indicate that LP is not a DNA sequence-speci c 31 binding protein. 32 42 Cell TF Cooccupancy Levels at LP, E2, and LP/E2 Sites. ENCODE LP only E2 only LP/E2 ChIP-seq data were used to determine cell TF occupancies at (13,602) (14,195) (5,605) LP, LP/E2, and E2 sites. Cell TFs with statistically significant enrichment at LP, E2, or LP/E2 sites were YY1, SP1, PAX5, BATF, IRF4, ETS1, RAD21, PU.1, CTCF, RBPJ, ZNF143, 10 26 34 45 31 31 SMC3, NFκB, and TBLR1. LP sites were more occupied with most (9 of 15) of these factors than E2 sites, except for RBPJ and 31 40 32 EBF, which were more highly occupied at E2 sites. E2 stabilizes RBPJ interaction with DNA (Table 1) (17). However, 11 of 15 promoter intron intergenic exon other LP/E2 sites were more highly occupied by cell TFs than LP or E2 only sites (Table 1). These data are consistent with LP’s der- B Enriched DNA binding sequences epressive and cooperative effects with E2 being important in LP E2 LP/E2 transcription activation. CTCF RBPJ ETS1 Although LP’s frequent localization to promoters and E2’s ETS1 EBF PU.1 frequent localization to enhancers might limit their cooperation PU.1 ETS1 AP-1 in transcription activation, their high occupancies with many of IRF4 PU.1 ZNF143 the same cell TFs, including YY1, SP1, PAX5, BATF, IRF4, κ SP1 RUNX1 IRF4 ETS1, PU1, CTCF, RBPJ, RAD21, SMC3, NF B, and TBLR1, YY1 NF B NFkB enable multiple dynamic interactions among E2, LP, and their ZNF143 ZNF143 EBF associated cell TFs (9, 23, 24). E2 interactions at enhancer sites and LP at promoter sites, with the similar interacting cell TFs, NF B AP-1 CTCF positions LP and E2 in proximity to each other to mediate DNA Fig. 1. Genomic distribution and TF DNA binding sequences enriched at LP, looping and transcription activation (17). E2, and LP/E2 sites. (A) Pie charts showing the genome-wide distribution of LP, E2, and LP/E2 sites are differentially occupied by cell TFs. To assess all LP or E2 sites, as well as the genome-wide distribution of LP-only, E2-only, cell TF occupancies at LP, LP/E2, and E2 sites, ENCODE LCL and LP/E2 sites. (B) List of most highly enriched cell transcription factor DNA ChIP-seq data were used to quantify average occupancy or binding sequences at LP, E2, and LP/E2 sites (±100 b) (P < 0.01). coverage of cell TFs at LP, E2, and LP/E2 sites (Fig. 2). The most frequent pattern applied to YY1, SP1, PAX5, BATF, IRF4, ETS1, PU.1, ZNF143, NFκB, and TBLR1 sites, where LP/E2 and E2 localized to 19,845 sites (Fig. 1A) (17). In contrast to E2 coverages (red lines) were significantly higher than LP coverages sites without LP, which are mostly at enhancers (17) and only fi − + (green lines) or E2 coverages (Fig. 2, blue lines) indicating that 10% at promoters (de ned as 1to 0.1 kb from a TSS), LP LP/E2 sites are more highly occupied by these factors than LP or sites without E2 were 34% at promoters and more than 50% of E2 sites (Fig. 2). However, EBF occupancy was higher at E2 than LP sites without E2 were within 2 kb of a TSS (Fig. S1), in- at LP or LP/E2 sites, consistent with E2 and RBPJ or ZNF143 dicating that LP is much more promoter localized than E2 (Fig. stabilization of EBF coverage at promoter sites, as previously S1). The 5,605 LP/E2 sites were also 31% promoter associated, observed for E2. A third pattern was represented by CTCF, similar to the 13,602 LP-only sites, which were 34% promoter SMC3, and RAD21, whose coverages were highest at LP sites, associated (Fig. 1A). These data indicate that LP is substantially consistent with their important role in promoter derepression, promoter localized and dominantly maintains a similar level of enhancer-mediated chromatin looping, and INO80/YY1 tran- promoter localization with E2 cooccupancy. scription boundary effects at CTCF sites (31–33, 36–38, 44). At Cell TF Sites Associated with LP or E2. LP sites (±100 bp) were −958 significantly enriched (from P < 0.01 to <1 × 10 ) for cell TF Table 1. Cell TF cooccupancy at LP, LP/E2, or E2 sites binding sites important in lymphocyte development, including LP (13,602) LP/E2 (5,605) E2 (14,195) CTCF, ETS1, PU.1, IRF4, SP1, YY1, ZNF143, NFκB, and RUNX1 sites (Fig. 1B). LP-associated cell TF sites were unaffected TF No. sites % Total No. sites % Total No. sites % Total by increasing the LP site search window to ±250 bp. The LP site-associated TFs are remarkable for their impor- YY1 8,059 59 3,965 71 4,812 34 tance in B-cell development and mature B-cell responses to SP1 7,506 55 4,423 79 5,305 37 antigen. CTCF is a transcription insulator, which associates with PAX5 7,055 52 4,199 75 5,220 37 YY1, RAD21, and SMC3 to mediate long-range chromatin BATF 5,618 41 3,987 71 5,607 39 interactions (25–38). YY1-associated INO80 chromatin-remod- IRF4 5,526 41 3,870 69 4,753 33 eling complexes and PU.1 have prominent roles in development, ETS1 4,822 35 2,662 47 2,172 15 immune responses (39), and chromatin domain transcription RAD21 4,529 33 1,822 33 2,495 18 (40). ZNF143 and RBPJ mediate Notch or E2 interaction with PU1 4,064 30 2,999 54 4,276 30 cognate DNA (41, 42) in tissue development and in EBV RBL CTCF 3,800 28 686 12 822 6 conversion to LCLs (42). BATF/JUN/FOS/ETS family proteins RBPJ 3,737 27 4,434 79 11,529 81 heterodimerize with IRF4 or IRF8 and are essential for mature ZNF143 3,417 25 1,113 20 916 6 SMC3 3,127 23 955 17 923 7 B-lymphocyte immune responses (43). κ Most cell TF binding sites at LP sites were also at E2 and LP/ NF B 3,074 23 2,828 50 3,614 25 E2 sites (Fig. 1B, compare LP, E2, and LP/E2 columns), consis- TBLR1 2,594 19 2,472 44 2,130 15 tent with the hypothesis that LP and E2 evolved to cooperatively EBF 1,285 9 1,920 34 5,030 35

18538 | www.pnas.org/cgi/doi/10.1073/pnas.1317608110 Portal et al. Downloaded by guest on September 23, 2021 40 25 16 YY1 SP1 PAX5 30 20 12 15 20 8 10 10 4 coverage coverage 5 coverage

-1.5 -1 -0.5 0.50 1 1.5 -1.5 -1 -0.5 0.50 11.5 -1.5 -1 -0.5 0.50 1 1.5 Kb Kb Kb Kb Kb Kb 25 8 BATF 14 IRF4 ETS1 20 12 6 10 15 8 4 6 10 4 2 coverage coverage 2 coverage

-1.5 -1 -0.5 0.50 1 1.5 -1.5 -1 -0.5 0.50 11.5 -1.5 -1 -0.5 0.50 1 1.5 Kb Kb Kb Kb Kb Kb

50 40 30 PU.1 RBPJ ZNF143 40 30 20 30 20 20 10 10 coverage coverage 10 coverage

-1.5 -1 -0.5 0.50 11.5 -1.5 -1 -0.5 0.50 11.5 -1.5 -1 -0.5 0.50 1 1.5 Kb Kb Kb Kb Kb Kb 8 40 NF B TBLR1 EBF 12 6 30 8 4 20 coverage coverage 4 coverage 2 10

-1.5 -1 -0.5 0.50 11.5 -1.5 -1 -0.5 0.50 11.5 -1.5 -1 -0.5 0.50 1 1.5 Kb Kb Kb Kb Kb Kb

10 SMC3 16 RAD21 30 CTCF 8 25 12 20 6 8 15 4 10

coverage 2 coverage 4 coverage 5

-1.5 -1 -0.5 0.50 11.5 -1.5 -1 -0.5 0.50 11.5 -1.5 -1 -0.5 0.50 1 1.5 Kb Kb Kb Kb Kb Kb

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.

the myc promoter, high CTCF and LP cooccupancies likely lower H3K27ac, H2Az, H3K9ac, RNAPII, and P300 levels. Over- prime these sites for distal E2/RBPJ enhancer looping to myc all, LP, LP/E2, and E2 sites had active promoter or enhancer epi- (17) (Fig. S2). genetic marks and occupancies by RNAPII and P300 (Table 2), LP, LP/E2, and E2 sites are associated with RNAPII, P300, and activation- indicative of active transcription. related promoter or enhancer chromatin marks. Because LP, LP/E2, and E2 sites were highly occupied by cell TFs, the relationship of cell TF occupancies to canonical epigenetic chromatin activation Table 2. Basal TF cooccupancy and epigenetic marks at LP, marks at LP, LP/E2, and E2 sites were evaluated using high- LP/E2, or E2 sites quality ENCODE histone mark datasets to impute relative ac- tivities (Table 2 and Fig. 3 A and B). LP (13,602) LP/E2 (5,605) E2 (14,195) Histone The 13,602 LP-only sites were highly associated with chromatin mark/TF No. sites % Total No. sites % Total No. sites % Total marks characteristic of promoter-associated, activated transcription effects, including high-level H3K4me3, H3K27ac, H2Az, H3K4me3 7,539 55 3,404 61 4,176 29 H3K9ac, as well as RNAPII and P300 signals. The 5,605 LP/E2 H3K27ac 7,513 55 4,162 74 5,687 40 sites had even higher promoter-associated H3K4me3, H3K27ac, H2AZ 7,412 54 3,172 57 4,083 29 H2Az, H3K9ac, RNAPII, and P300 coverage, albeit lower H3K9ac 6,905 51 3,441 61 4,049 29 H3K4me1, consistent with their promoter localization, whereas the RNA Pol II 4,798 35 2,361 42 2,148 15 H3K4me1 2,300 17 1,960 35 4,154 29 14,195 E2, mostly enhancer sites, had substantially lower H3K4me3 CELL BIOLOGY P300 1,986 15 2,233 40 2,121 15 promoter marks, higher H3K4me1 enhancer activation marks, and

Portal et al. PNAS | November 12, 2013 | vol. 110 | no. 46 | 18539 Downloaded by guest on September 23, 2021 25 < H3K27ac However, LP-annotated genes were less expressed than E2 (P A H3K9ac −4 −12 20 5.6 × 10 ) or LP/E2 (P < 2.7 × 10 ) annotated genes (Fig. 3B), 15 15 and LP/E2-annotated genes were more highly expressed than E2 10 < 10 annotated genes (P 0.05), despite substantial differences in cell 5 5 TF occupancies, as well as RNAPII, P300, and activating histone coverage coverage epigenetic marks (Fig. 3B). These data highlight a complexity of -1.5 -1 -0.5 0.50 1 1.5 -1.5 -1 -0.5 0.50 1 1.5 LP and E2 activation of proliferation and survival pathways that Kb Kb Kb Kb remains to be deconstructed using shRNAs for individual LP-, 6 14 H3K4me1 H3K4me3 E2-, and LP/E2-associated cell TFs. 5 12 10 4 8 LP Sites Clusters Differed in Cell TF Composition and Transcription 3 6 Effects. To better understand the range of LP site cell TF occu- 2 4 coverage coverage pancies, a K-means clustering segregation analysis of LP sites was 1 2 undertaken (Fig. 4). Clusters 1–7 were highly occupied by B-cell -1.5 -1 -0.5 0.50 1 1.5 -1.5 -1 -0.5 0.50 1 1.5 Kb Kb Kb Kb developmentalTFs,YY1,PAX5,BATF,IRF4,andPU.1.Cluster 25 2 had more YY1, PAX5, BATF, and IRF4, less PU.1, and high RNAPII 6 P300 20 RAD21, CTCF, SMC3, E2, RBPJ, and ZNF143 levels, TFs char- 15 4 acteristic of B-cell enhancers and promoter looping factors. Cluster 3 was uniformly PAX5, an EBF-induced early B-cell transcription 10 ∼ coverage coverage 2 activator, and 50% YY1. Cluster 4 was uniformly RBPJ and al- 5 most 50% E2. Cluster 5 was solidly PU.1, an activating B-cell developmental TF, and TBLR1, an NCOR/SMRT repressive -1.5 -1 -0.5 0.50 1 1.5 -1.5 -1 -0.5 0.50 1 1.5 Kb Kb Kb Kb complex component that may be an LP target. Cluster 5 was B enriched for E2 and RBPJ, which enhance MYC and MYC-driven

100 cell survival gene expression. Cluster 6 was uniformly TBLR1, which associates with NCOR, YY1, PAX5, BATF, IRF4, RBPJ, and E2 (17). Cluster 7 was uniformly YY1, PAX5, BATF, and IRF4 occupied, consistent with B-cell developmental transcription activation.Cluster7wasalsohighinRAD21,CTCF,SMC3,and ZNF143; ZNF143 is a potential alternative mediators of E2 or Notch interaction with DNA. Cluster 8 was uniformly PAX5, an activating B-cell TF, and RAD21, CTCF, and SMC3, looping factors that mediate enhancer interactions with promoters. Cluster 9 was YY1, CTCF, RAD21, SMC3, and ZNF143 occupied, 020406080 Gene expression (RNA-seq) LP E2 LP/E2 Ctrl Fig. 3. Anchor plots for cell epigenetic marks and affected gene expression AB data. (A) Coverage of RNAPII, P300, H3K9ac, H3K27ac, H3K4me1, and H3K4me3 YY1 PAX5 BATF IRF4 PU.1 RAD21 CTCF SMC3 ±1.5 kb of LP (green line), E2 (blue line), and LP/E2 (red line) sites. (B)Boxplots EBNA2 RBPJ ZNF143 TBLR1 of RNA-seq at LP, E2, or LP/E2 annotated peaks for the nearest promoter versus 1 a control (Ctrl) random gene set. LP, E2, and LP/E2 genes were signiﬁcantly − 2 more highly expressed than control genes (P < 2 × 10 16). LP had lower ex- − pression than E2 (P < 0.00056) or LP/E2 (P < 2.7 × 10 12), and E2 was lower than 3

LP/E2 (P < 0.05). The box plots indicate the data distribution in percentiles, with 50 100 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, 4 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. 5

6 0 7 Consistent with LP’s association with active promoters, LP Gene expression (RNA-seq) 12345678910 ctrl 8 sites were highly occupied by RNAPII (Fig. 3A, green line) P300, 9 H3K9ac, and H3K27ac, whereas, consistent with E2’s enhancer localization, E2 and LP/E2 sites had higher H3K4me1 levels 10 than LP. LP/E2 sites were overall most coincident with activation-associated histone marks and highest RNAPII and P300 levels (Fig. 3A, red lines). The strong differences in cell TF and Fig. 4. LP site-associated cell TFs separated into 10 K-means clusters and gene epigenetic mark anchor plots at LP, LP/E2, and E2 sites were expression. (A) Cell TFs YY1, PAX5, BATF, IRF4, and PU.1 were prominent com- less evident when LP, E2, and LP/E2 sites in promoter regions ponents of most clusters, consistent with their 30–59% LP site association. Notably, were assessed (Fig. S3). E2, RBPJ, and TBLR1 clustered separately from looping factors CTCF, SMC3, and To correlate LP-, E2-, and LP/E2-associated cell TF occu- RAD21. (B) Box plots of RNA-seq gene expression from LP-affected promoters. pancies and chromatin activation marks with activated tran- All LP-affected genes were more highly expressed than random control genes (P < × −16 scription, high-quality ENCODE LCL RNA-seq data for genes 2 10 ). The box plots indicate the data distribution in percentiles, with the − + horizontal line being the median. The top of the box represents upper 25% with LP, LP/E2, or E2 sites ( 1/ 0.1 kb from a TSS) indicated quartile (25% of the data over that value), and the bottom of the box, the lower that LP, LP/E2, and E2 annotated genes are more highly quartile (25% of the data below that value). The horizontal lines at the ends of −16 expressed than random genes (P < 1 × 10 ) (Fig. 3B). the dotted lines are the maximum observed values, excluding the outliers.

18540 | www.pnas.org/cgi/doi/10.1073/pnas.1317608110 Portal et al. Downloaded by guest on September 23, 2021 −16 H3K27ac NCOR having CTCF sites without LP (P < 2 × 10 ). These data indicate H3K4me1 LP HA95 that LP localized with CTCF at promoter sites increases tran- H3K9ac H2Az NCOR scription. LP-associated transcription increases are most likely RBPJ EBF mediated by LP dismissal of CTCF-, SMC3-, or RAD21-associ- CTCF E2 ated NCOR or HDACs and may also be affected by long-distance RAD21 SMC3 enhancer interaction with CTCF, RAD21, and SMC3 at CTCF/LP

INO80-YY1/PAX5/SP1/ETS sites. Overall, LP, localized with CTCF at or near promoters had IRF4/BATF/ZNF143 derepressive effects comparable to E2’s activating effects (9, 10, 24, 48, 49). RAD21 SMC3 To illustrate LP and associated TFs roles in up-regulating bi- LP HA95 RNAPII ++++ CTCF ologically important genes for LCL proliferation and survival, NCOR H3K27ac relevant ChIP-seq tracks are presented for the MYC-regulated H3K4me3 LP HA95 cell cycle entry gene ccnd2, the MYC proliferation-associated H3K9ac cell survival genes, bcl2 and igf2r, the MYC-induced cell senes- NCOR H2Az cence genes, cdkn2a and cdkn2b, and 1 of the 23 LP affected Fig. 5. Model. LP occupies mostly promoters, whereas E2 is found at mediator components, med26 (Fig. S7 A–E) (50–58). enhancers (17). Enhancers regulate transcription via long-distance DNA Thus, the data presented here position LP as a key component interactions mediated by CTCF, RAD21, and SMC3. Several transcription of EBV’s control of the cell transcription, proliferation, and factors are associated with LP sites and E2 sites and likely help mediate the survival-related gene transcription (Fig. S7 A–E). The genome- interactions between LP and E2 (blue box). The genes associated with LP wide approaches used to generate these data enabled the dis- 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). covery of unique aspects of LP roles, including functional asso- ciations with B-cell TFs to affect key pathways in B-cell growth, survival, and gene expression. whereas cluster 10 included ∼25% of LP sites that had sub- Fig. S7A shows the bcl2, promoter and enhancer, which has LP stantially less prevalent cell TF occupancies. at the promoter and at two distal enhancers, with E2, RBPJ, Not surprisingly, the genome-wide distribution of most LP TBLR1, ZNF143, CTCF, YY1, IRF4, BATF, ETS1, PU.1, EBF, clusters was similar to LP overall (Fig. S4). However, cluster 3 PAX5, SP1, NFκB, H3K27ac, RNAPII, H3K4me1, H3K9ac, (PAX5) was >57% promoter associated, substantially higher and H3k4me3. than LP. At the other extreme, cluster 9 was only 16% promoter Fig. S7B shows the igf2r locus, with strong LP signals at the associated, had higher intron and intergenic localization, and was promoter along with YY1, ETS1, PAX5, SP1, H3K27ac, RNAPII, highly occupied with YY1, CTCF, RAD21, SMC3, and ZNF143. H3K4me1, H3K9ac, and H3K4me3. YY1, PAX5, and BATF/ETS/IRF4 were abundant compo- Fig S7C shows the ccnd2 promoter with strong LP signals, nents of most LP clusters and are essential B-cell developmental weak E2, ZNF143, CTCF, RAD21, SMC3, YY1, BATF, ETS1, TFs that affect cell growth and gene expression (Table 1 and Fig. SP1, NFκB, H3K27ac, RNAPII, H3K9ac, and H3K4me3. 4A) (43, 45). DNA looping factors CTCF, RAD21, and SMC3 Fig. S7D shows the cdkn2a and 2b promoters with strong LP, were characteristic of LP clusters 2, 7, 8, and 9, which were also ZNF143, CTCF, RAD21, SMC3, YY1, SP1, signals, ZNF143, rich in ZNF143, E2, and RBPJ. Clusters 2, 4, 5, and 6 were rich weak and strong YY1, SP1, NFκB, H3K27ac, RNAPll, H3K9ac, in TBLR1, a ubiquitin ligase that is likely activated by LP- and H3K4me3. mediated NCOR removal leading to transcription derepression Fig. S7E shows the med26 locus with promoter-associated (46). LP cluster 5 includes the hes1 locus, which is less NCOR LP, ZNF143, RAD21, SMC3, YY1, ETS1, strong PAX5, SP1, occupied and derepressed when LP is expressed in BJAB H3K27ac, RNAPll, H3K4me1, and H3K4me3 signals at the pro- δ B-lymphoma cells (9) (Fig. S5A). Interestingly, PKC (prckd), moter. Notably, stronger signals are also apparent at the med26 the protein kinase that activates TBLR1 to degrade NCOR is up- < enhancer for LP, E2, RBPJ, TBLR1, RAD21, SMC3, YY1, IRF4, regulated 1.9-fold in LCLs [P 0.05 (47)]. Furthermore, LP/E2 BATF, ETS1, PU.1, EBF, PAX5, SP1, H3K27ac, RNAPII, prckd B occupied three sites in the locus (Fig. S5 ), indicative of H3K4me1, H3K9ac, and H3K4me3. a role for LP/E2 in regulating prckd expression. These results support the model shown in Fig. 5, that LP, To investigate the relationship between gene transcription and predominantly at or near promoters, and E2 at enhancer sites, LP site clusters, LCL RNA-seq data annotated to LP, LP/E2, or cooperatively affect LCL gene expression. LP’s presence at DNA E2 promoter sites were used. All LP-affected clusters were sig- niﬁcantly more highly expressed than random control genes (P < sites dismisses repressive NCOR complexes. Affected genes are − 2 × 10 16) (Fig. 4B). Cluster 8, which included YY1, PAX5, up-regulated by LP-mediated derepression and long-distance CTCF, RAD21, SMC3, and ZNF143 and cluster 9, which in- DNA interactions through CTCF, RAD21, and SMC3. cluded YY1, CTCF, RAD21, SMC3, and ZNF143, had relatively Materials and Methods lower expression levels, compared with other clusters (Fig. 4B). Overall, LP positively affected genes with CTCF sites (Fig. S6), ChIP-seq was performed as described (17). IB4 cells were grown in RPMI likely by removing repressors from these sites (Fig. S6). Compari- medium, 10% (vol/vol) FBS. Detailed methods for analysis of ChIP-seq data, dataset access, and ChIP-seq protocol are available in SI Materials and Methods. son of RNA-seq expression data from genes having a promoter- associated CTCF sites without LP with those having a promoter- ACKNOWLEDGMENTS. This research was supported by Grants R01CA131354, associated CTCF sites with LP, revealed genes with overlapping R01CA170023, and R01CA047006 from the National Cancer Institute of the CTCF/LP sites to be signiﬁcantly more highly expressed than genes National Institutes of Health of the US Public Health Service.

1. Cohen JI, Kieff E (1991) An Epstein-Barr virus nuclear protein 2 domain essential for 3. Cohen JI, Wang F, Mannick J, Kieff E (1989) Epstein-Barr virus nuclear protein 2 is a key transformation is a direct transcriptional activator. J Virol 65(11):5880–5885. determinant of lymphocyte transformation. Proc Natl Acad Sci USA 86(23):9558–9562. 2. Cohen JI, Wang F, Kieff E (1991) Epstein-Barr virus nuclear protein 2 mutations de- 4. Mannick JB, Cohen JI, Birkenbach M, Marchini A, Kieff E (1991) The Epstein-Barr virus ﬁne essential domains for transformation and transactivation. JVirol65(5): nuclear protein encoded by the leader of the EBNA RNAs is important in B-lympho-

2545–2554. cyte transformation. J Virol 65(12):6826–6837. CELL BIOLOGY

Portal et al. PNAS | November 12, 2013 | vol. 110 | no. 46 | 18541 Downloaded by guest on September 23, 2021 5. Dambaugh T, Hennessy K, Chamnankit L, Kieff E (1984) U2 region of Epstein-Barr 32. Chaumeil J, Skok JA (2012) The role of CTCF in regulating V(D)J recombination. virus DNA may encode Epstein-Barr nuclear antigen 2. Proc Natl Acad Sci USA 81(23): Curr Opin Immunol 24(2):153–159. 7632–7636. 33. Gillen AE, Harris A (2011) The role of CTCF in coordinating the expression of single 6. Dambaugh T, et al. (1986) Expression of the Epstein-Barr virus nuclear protein 2 in gene loci. Biochem Cell Biol 89(5):489–494. rodent cells. J Virol 59(2):453–462. 34. Rhodes JM, et al. (2010) Positive regulation of c-Myc by cohesin is direct, and evolu- 7. Wang F, Petti L, Braun D, Seung S, Kieff E (1987) A bicistronic Epstein-Barr virus mRNA tionarily conserved. Dev Biol 344(2):637–649. encodes two nuclear proteins in latently infected, growth-transformed lymphocytes. 35. Dorsett D, Merkenschlager M (2013) Cohesin at active genes: A unifying theme for J Virol 61(4):945–954. cohesin and gene expression from model organisms to humans. Curr Opin Cell Biol 8. Ling PD, et al. (2005) Mediation of Epstein-Barr virus EBNA-LP transcriptional co- 25(3):327–333. – activation by Sp100. EMBO J 24(20):3565 3575. 36. Dorsett D (2011) Cohesin: Genomic insights into controlling gene transcription and 9. Portal D, et al. (2011) EBV nuclear antigen EBNALP dismisses transcription repressors development. Curr Opin Genet Dev 21(2):199–206. fi NCoR and RBPJ from enhancers and EBNA2 increases NCoR-de cient RBPJ DNA 37. Hou C, Zhao H, Tanimoto K, Dean A (2008) CTCF-dependent enhancer-blocking by – binding. Proc Natl Acad Sci USA 108(19):7808 7813. alternative chromatin loop formation. Proc Natl Acad Sci USA 105(51):20398–20403. 10. Portal D, Rosendorff A, Kieff E (2006) Epstein-Barr nuclear antigen leader protein 38. Splinter E, et al. (2006) CTCF mediates long-range chromatin looping and local histone coactivates transcription through interaction with histone deacetylase 4. Proc Natl modification in the beta-globin locus. Genes Dev 20(17):2349–2354. Acad Sci USA 103(51):19278–19283. 39. Cai Y, et al. (2007) YY1 functions with INO80 to activate transcription. Nat Struct Mol 11. Han I, et al. (2002) Protein kinase A associates with HA95 and affects transcriptional Biol 14(9):872–874. coactivation by Epstein-Barr virus nuclear proteins. Mol Cell Biol 22(7):2136–2146. 40. Lin YC, et al. (2012) Global changes in the nuclear positioning of genes and intra- and 12. Han I, et al. (2001) EBNA-LP associates with cellular proteins including DNA-PK and interdomain genomic interactions that orchestrate B cell fate. Nat Immunol 13(12): HA95. J Virol 75(5):2475–2481. 1196–1204. 13. Harada S, Kieff E (1997) Epstein-Barr virus nuclear protein LP stimulates EBNA-2 acidic 41. Wang H, et al. (2011) Genome-wide analysis reveals conserved and divergent features domain-mediated transcriptional activation. J Virol 71(9):6611–6618. of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells. Proc 14. Alfieri C, Birkenbach M, Kieff E (1991) Early events in Epstein-Barr virus infection of – human B lymphocytes. Virology 181(2):595–608. Natl Acad Sci USA 108(36):14908 14913. 15. Mannick JB, Tong X, Hemnes A, Kieff E (1995) The Epstein-Barr virus nuclear antigen 42. Halbig KM, Lekven AC, Kunkel GR (2012) The transcriptional activator ZNF143 is es- fi leader protein associates with hsp72/hsc73. J Virol 69(12):8169–8172. sential for normal development in zebra sh. BMC Mol Biol 13:3. 16. Peng CW, et al. (2007) Hsp72 up-regulates Epstein-Barr virus EBNALP coactivation 43. Li P, et al. (2012) BATF-JUN is critical for IRF4-mediated transcription in T cells. Nature – with EBNA2. Blood 109(12):5447–5454. 490(7421):543 546. 17. Zhao B, et al. (2011) Epstein-Barr virus exploits intrinsic B-lymphocyte transcription 44. Amouyal M (2010) Gene insulation. Part II: Natural strategies in vertebrates. Biochem programs to achieve immortal cell growth. Proc Natl Acad Sci USA 108(36): Cell Biol 88(6):885–898. 14902–14907. 45. Donohoe ME, Silva SS, Pinter SF, Xu N, Lee JT (2009) The pluripotency factor Oct4 18. Grossman SR, Johannsen E, Tong X, Yalamanchili R, Kieff E (1994) The Epstein-Barr interacts with Ctcf and also controls X-chromosome pairing and counting. Nature virus nuclear antigen 2 transactivator is directed to response elements by the J kappa 460(7251):128–132. recombination signal binding protein. Proc Natl Acad Sci USA 91(16):7568–7572. 46. Perissi V, et al. (2008) TBL1 and TBLR1 phosphorylation on regulated gene promoters 19. Henkel T, Ling PD, Hayward SD, Peterson MG (1994) Mediation of Epstein-Barr virus overcomes dual CtBP and NCoR/SMRT transcriptional repression checkpoints. Mol Cell EBNA2 transactivation by recombination signal-binding protein J kappa. Science 29(6):755–766. 265(5168):92–95. 47. Price AM, et al. (2012) Analysis of Epstein-Barr virus-regulated host gene expression 20. Tong X, Wang F, Thut CJ, Kieff E (1995) The Epstein-Barr virus nuclear protein 2 acidic changes through primary B-cell outgrowth reveals delayed kinetics of latent mem- domain can interact with TFIIB, TAF40, and RPA70 but not with TATA-binding pro- brane protein 1-mediated NF-κB activation. J Virol 86(20):11096–11106. tein. J Virol 69(1):585–588. 48. Peng CW, Zhao B, Kieff E (2004) Four EBNA2 domains are important for EBNALP 21. Tong X, Drapkin R, Yalamanchili R, Mosialos G, Kieff E (1995) The Epstein-Barr virus coactivation. J Virol 78(20):11439–11442. nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that 49. Peng CW, et al. (2004) Direct interactions between Epstein-Barr virus leader protein – can interact with TFIIE. Mol Cell Biol 15(9):4735 4744. LP and the EBNA2 acidic domain underlie coordinate transcriptional regulation. Proc 22. Nikitin PA, et al. (2010) An ATM/Chk2-mediated DNA damage-responsive signaling Natl Acad Sci USA 101(4):1033–1038. pathway suppresses Epstein-Barr virus transformation of primary human B cells. Cell 50. Mushinski JF, et al. (1999) Myc-induced cyclin D2 genomic instability in murine B cell – Host Microbe 8(6):510 522. neoplasms. Curr Top Microbiol Immunol 246:183–189; discussion 190–192. 23. Peng R, Tan J, Ling PD (2000) Conserved regions in the Epstein-Barr virus leader 51. Bouchard C, et al. (2001) Regulation of cyclin D2 gene expression by the Myc/Max/ protein define distinct domains required for nuclear localization and transcriptional Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cooperation with EBNA2. J Virol 74(21):9953–9963. cyclin D2 promoter. Genes Dev 15(16):2042–2047. 24. Peng R, Moses SC, Tan J, Kremmer E, Ling PD (2005) The Epstein-Barr virus EBNA-LP 52. Sinclair AJ, Palmero I, Holder A, Peters G, Farrell PJ (1995) Expression of cyclin D2 in protein preferentially coactivates EBNA2-mediated stimulation of latent membrane Epstein-Barr virus-positive Burkitt’s lymphoma cell lines is related to methylation proteins expressed from the viral divergent promoter. J Virol 79(7):4492–4505. status of the gene. J Virol 69(2):1292–1295. 25. Phillips JE, Corces VG (2009) CTCF: Master weaver of the genome. Cell 137(7): 53. Zinkel S, Gross A, Yang E (2006) BCL2 family in DNA damage and cell cycle control. 1194–1211. Cell Death Differ 13(8):1351–1359. 26. Merkenschlager M, Odom DT (2013) CTCF and cohesin: Linking gene regulatory el- 54. Harris LK, Westwood M (2012) Biology and significance of signalling pathways acti- ements with their targets. Cell 152(6):1285–1297. – 27. Remeseiro S, Cuadrado A, Gómez-López G, Pisano DG, Losada A (2012) A unique role vated by IGF-II. Growth Factors 30(1):1 12. of cohesin-SA1 in gene regulation and development. EMBO J 31(9):2090–2102. 55. Lewis BA, Reinberg D (2003) The mediator coactivator complex: Functional and – 28. Cuadrado A, Remeseiro S, Gómez-López G, Pisano DG, Losada A (2012) The specific physical roles in transcriptional regulation. J Cell Sci 116(Pt 18):3667 3675. contributions of cohesin-SA1 to cohesion and gene expression: Implications for 56. Maruo S, et al. (2011) Epstein-Barr virus nuclear antigens 3C and 3A maintain lym- cancer and development. Cell Cycle 11(12):2233–2238. phoblastoid cell growth by repressing p16INK4A and p14ARF expression. Proc Natl 29. Degner SC, et al. (2011) CCCTC-binding factor (CTCF) and cohesin influence the ge- Acad Sci USA 108(5):1919–1924. nomic architecture of the Igh locus and antisense transcription in pro-B cells. Proc Natl 57. Rayess H, Wang MB, Srivatsan ES (2012) Cellular senescence and tumor suppressor Acad Sci USA 108(23):9566–9571. gene p16. Int J Cancer 130(8):1715–1725. 30. Degner SC, Wong TP, Jankevicius G, Feeney AJ (2009) Cutting edge: Developmental 58. Sinclair AJ, Palmero I, Peters G, Farrell PJ (1994) EBNA-2 and EBNA-LP cooperate to stage-specific recruitment of cohesin to CTCF sites throughout immunoglobulin loci cause G0 to G1 transition during immortalization of resting human B lymphocytes during B lymphocyte development. J Immunol 182(1):44–48. by Epstein-Barr virus. EMBO J 13(14):3321–3328. 31. Herold M, Bartkuhn M, Renkawitz R (2012) CTCF: Insights into insulator function 59. Li Y, et al. (2006) A novel histone deacetylase pathway regulates mitosis by modu- during development. Development 139(6):1045–1057. lating Aurora B kinase activity. Genes Dev 20(18):2566–2579.

18542 | www.pnas.org/cgi/doi/10.1073/pnas.1317608110 Portal et al. Downloaded by guest on September 23, 2021