Epstein–Barr virus nuclear antigen leader proteinlocalizes to promoters and enhancers with celltranscription factors and EBNA2Daniel 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, HarvardUniversity, Boston, MA 02115; eDepartment of Biostatistics and Computational Biology, Dana Farber Cancer Institute, Boston, MA 02115; and fLaboratoryof 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(E2) are coexpressed in EBV-infected B lymphocytes and are criticalfor lymphoblastoid cell line outgrowth. LP removes NCOR andRBPJ repressive complexes from promoters, enhancers, and ma-trix-associated deacetylase bodies, whereas E2 activates transcrip-tion from distal enhancers. LP ChIP-seq analyses identified 19,224LP 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 enrichedfor LP-associated cell TFs and were more highly occupied by RBPJand EBF. LP sites were highly marked by H3K4me3, H3K27ac,H2Az, H3K9ac, RNAPII, and P300, indicative of activated transcrip-tion. LP sites were 29% colocalized with E2 (LP/E2). LP/E2 siteswere more similar to LP than to E2 sites in associated cell TFs,RNAPII, P300, and histone H3K4me3, H3K9ac, H3K27ac, and H2Azoccupancy, and were more highly transcribed than LP or E2 sites.Gene affected by CTCF and LP cooccupancy were more highlyexpressed than genes affected by CTCF alone. LP was at mycenhancers and promoters and of MYC regulated ccnd2, 23 medcomplex components, and MYC regulated cell survival genes, igf2rand bcl2. These data implicate LP and associated TFs and DNAlooping factors CTCF, RAD21, SMC3, and YY1/INO80 chromatin-remodeling complexes in repressor depletion and gene activationnecessary for lymphoblastoid cell line growth and survival.

genome-wide ChIP-seq analysis | gene expression

Epstein–Barr virus (EBV) nuclear antigens EBNALP (LP) andEBNA2 (E2) are EBV-encoded transcription factors (TFs)

that are coordinately expressed within hours after EBV infectionof resting B lymphocytes (RBLs) and are important for B-lym-phocyte conversion to lymphoblastoid cell lines (LCLs) (1–7).However, the biochemical mechanisms by which LP and E2 co-ordinately affect RBL transformation to LCLs are largely un-known. LP coactivates transcription by heterodimerizing withHA95 and Hsp70/72 to relocate HDAC4 from the nucleus to thecytoplasm, displaces Sp100 and Hp1α from ND10 bodies, anddisrupts matrix-associated deacetylase (MAD) bodies, broadlyaffecting repressor localization in cell nuclei (8–16). LP alsodecreases repressive NCOR and RBPJ occupancy at E2 sites,without altering E2 occupancy (9).E2 enhances gene expression by localizing to cell TF sites

through RBPJ or ZNF143 (17–19). E2/RBPJ sites localize in sixclusters of EBF, ETS1, ZNF143, PU.1, NFκB, and RUNX1 sites.Encyclopedia of DNA elements (ENCODE) ChIP-sequencingexperiments (ChIP-seqs) indicated high-level cell TF cooccu-pancy at E2 sites, consistent with these sites being open to cell orvirus TF occupancy. Indeed, E2 chromatin sites in LCLs are openchromatin sites in RBLs, before EBV infection, consistent withEBF and RBPJ as pioneering factors that displace nucleosomes

(17). E2 increases H3K4me1 signals allowing E2 and cell TFoccupancy and transcription activation. The E2 activation do-main recruits basal and activation-related cell TFs, includingTAF40, TFIIH, TFIIE, and histone acetylases P300, CBP, andPCAF (17–21). Focusing on 88 dynamically E2-regulated genes,using ENCODE chromosome conformation capture (3C) data,the transcriptional start site (TSS) of 50 E2 dynamically regu-lated genes are in proximity to approximately three E2 enhancersper gene. These enhancers are 61% on the same chromosome andat a median distance of ∼330 kb from their affected genes. Thecombined effect of three E2 enhancers accounts for E2’s strong up-regulatory effects (17). E2 induces MYC expression within 24 h ofRBL infection. MYC then drives RBL cell cycle entry and pro-liferation (14, 22). However, the nearest RBPJ and E2 sites are>100 kb from myc (17). FISH and 3C assays connect the mycTSS to an E2 site at −428 kb from myc (17).LP and E2 cooperatively activate virus and cell gene tran-

scription following transient or stable B-lymphocyte transfection(9, 23, 24). The experiments described here were undertaken toidentify the mechanisms through which LP and E2 affect cellgene transcription in LCLs.

Results and DiscussionLP, LP/E2, and E2 Genome Distributions. Duplicate LCL LP and anE2 ChIP-seq dataset (17) were analyzed using HOMER, witha false discovery rate of P < 0.001. LP localized to 19,224 sites

Significance

Epstein–Barr virus nuclear antigen (EBNA) leader protein (LP)and EBNA2 (E2) up-regulation of virus and cell gene expressionis important for human B-lymphocyte conversion to continu-ous, potentially malignant, lymphoblast cell lines. Although themolecular mechanism(s) underlying LP and E2 regulation of cellgene expression have been partially elucidated, LP ChIP-sequencing studies have now revealed that LP and LP/E2 interact,genome-wide, with human B-cell transcription factors, mostly ator near prepatterned promoter sites, to increase cell transcriptionfactor occupancies, increase activation-associated histonemarks, and positively affect cell gene transcription.

Author contributions: D.P. and B.Z. designed research; D.P. and E.L. performed research;P.V.K., L.W., J.Q., D.H., S.J., and Y.L. contributed new reagents/analytic tools; D.P., H.Z.,S.J., and E.K. analyzed data; and D.P. and E.K. wrote the paper.

The authors declare no conflict of interest.

Data deposition: All information regarding access to data is included in SI Materials andMethods.1D.P. and H.Z. contributed equally to this work.2To whom correspondence should be addressed. E-mail: ekieff@rics.bwh.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1317608110/-/DCSupplemental.

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and E2 localized to 19,845 sites (Fig. 1A) (17). In contrast to E2sites without LP, which are mostly at enhancers (17) and only10% at promoters (defined as −1 to +0.1 kb from a TSS), LPsites without E2 were 34% at promoters and more than 50% ofLP sites without E2 were within 2 kb of a TSS (Fig. S1), in-dicating that LP is much more promoter localized than E2 (Fig.S1). The 5,605 LP/E2 sites were also 31% promoter associated,similar to the 13,602 LP-only sites, which were 34% promoterassociated (Fig. 1A). These data indicate that LP is substantiallypromoter localized and dominantly maintains a similar level ofpromoter localization with E2 cooccupancy.

Cell TF Sites Associated with LP or E2. LP sites (±100 bp) weresignificantly enriched (from P < 0.01 to <1 × 10−958) for cell TFbinding sites important in lymphocyte development, includingCTCF, ETS1, PU.1, IRF4, SP1, YY1, ZNF143, NFκB, andRUNX1 sites (Fig. 1B). LP-associated cell TF sites were unaffectedby increasing the LP site search window to ±250 bp.The LP site-associated TFs are remarkable for their impor-

tance in B-cell development and mature B-cell responses toantigen. CTCF is a transcription insulator, which associates withYY1, RAD21, and SMC3 to mediate long-range chromatininteractions (25–38). YY1-associated INO80 chromatin-remod-eling complexes and PU.1 have prominent roles in development,immune responses (39), and chromatin domain transcription(40). ZNF143 and RBPJ mediate Notch or E2 interaction withcognate DNA (41, 42) in tissue development and in EBV RBLconversion to LCLs (42). BATF/JUN/FOS/ETS family proteinsheterodimerize with IRF4 or IRF8 and are essential for matureB-lymphocyte immune responses (43).Most cell TF binding sites at LP sites were also at E2 and LP/

E2 sites (Fig. 1B, compare LP, E2, and LP/E2 columns), consis-tent with the hypothesis that LP and E2 evolved to cooperatively

up-regulate transcription through RBL genome-wide sites thatare prepatterned for up-regulation of B-cell growth and survival.HOMER did not recognize a de novo LP DNA sequence,which may indicate that LP is not a DNA sequence-specificbinding protein.

Cell TF Cooccupancy Levels at LP, E2, and LP/E2 Sites. ENCODEChIP-seq data were used to determine cell TF occupancies atLP, LP/E2, and E2 sites. Cell TFs with statistically significantenrichment at LP, E2, or LP/E2 sites were YY1, SP1, PAX5,BATF, IRF4, ETS1, RAD21, PU.1, CTCF, RBPJ, ZNF143,SMC3, NFκB, and TBLR1. LP sites were more occupied withmost (9 of 15) of these factors than E2 sites, except for RBPJ andEBF, which were more highly occupied at E2 sites. E2 stabilizesRBPJ interaction with DNA (Table 1) (17). However, 11 of 15LP/E2 sites were more highly occupied by cell TFs than LP or E2only sites (Table 1). These data are consistent with LP’s der-epressive and cooperative effects with E2 being important intranscription activation.Although LP’s frequent localization to promoters and E2’s

frequent localization to enhancers might limit their cooperationin transcription activation, their high occupancies with many ofthe same cell TFs, including YY1, SP1, PAX5, BATF, IRF4,ETS1, PU1, CTCF, RBPJ, RAD21, SMC3, NFκB, and TBLR1,enable multiple dynamic interactions among E2, LP, and theirassociated cell TFs (9, 23, 24). E2 interactions at enhancer sitesand LP at promoter sites, with the similar interacting cell TFs,positions LP and E2 in proximity to each other to mediate DNAlooping and transcription activation (17).LP, E2, and LP/E2 sites are differentially occupied by cell TFs. To assesscell TF occupancies at LP, LP/E2, and E2 sites, ENCODE LCLChIP-seq data were used to quantify average occupancy orcoverage of cell TFs at LP, E2, and LP/E2 sites (Fig. 2). Themost frequent pattern applied to YY1, SP1, PAX5, BATF, IRF4,ETS1, PU.1, ZNF143, NFκB, and TBLR1 sites, where LP/E2coverages (red lines) were significantly higher than LP coverages(green lines) or E2 coverages (Fig. 2, blue lines) indicating thatLP/E2 sites are more highly occupied by these factors than LP orE2 sites (Fig. 2). However, EBF occupancy was higher at E2 thanat LP or LP/E2 sites, consistent with E2 and RBPJ or ZNF143stabilization of EBF coverage at promoter sites, as previouslyobserved for E2. A third pattern was represented by CTCF,SMC3, and RAD21, whose coverages were highest at LP sites,consistent with their important role in promoter derepression,enhancer-mediated chromatin looping, and INO80/YY1 tran-scription boundary effects at CTCF sites (31–33, 36–38, 44). At

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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 ofall 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 DNAbinding sequences at LP, E2, and LP/E2 sites (±100 b) (P < 0.01).

Table 1. Cell TF cooccupancy at LP, LP/E2, or E2 sites

TF

LP (13,602) LP/E2 (5,605) E2 (14,195)

No. sites % Total No. sites % Total No. sites % Total

YY1 8,059 59 3,965 71 4,812 34SP1 7,506 55 4,423 79 5,305 37PAX5 7,055 52 4,199 75 5,220 37BATF 5,618 41 3,987 71 5,607 39IRF4 5,526 41 3,870 69 4,753 33ETS1 4,822 35 2,662 47 2,172 15RAD21 4,529 33 1,822 33 2,495 18PU1 4,064 30 2,999 54 4,276 30CTCF 3,800 28 686 12 822 6RBPJ 3,737 27 4,434 79 11,529 81ZNF143 3,417 25 1,113 20 916 6SMC3 3,127 23 955 17 923 7NFκB 3,074 23 2,828 50 3,614 25TBLR1 2,594 19 2,472 44 2,130 15EBF 1,285 9 1,920 34 5,030 35

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the myc promoter, high CTCF and LP cooccupancies likelyprime these sites for distal E2/RBPJ enhancer looping to myc(17) (Fig. S2).LP, LP/E2, and E2 sites are associated with RNAPII, P300, and activation-related promoter or enhancer chromatin marks. Because LP, LP/E2,and E2 sites were highly occupied by cell TFs, the relationship ofcell TF occupancies to canonical epigenetic chromatin activationmarks at LP, LP/E2, and E2 sites were evaluated using high-quality ENCODE histone mark datasets to impute relative ac-tivities (Table 2 and Fig. 3 A and B).The 13,602 LP-only sites were highly associated with chromatin

marks characteristic of promoter-associated, activated transcrip-tion effects, including high-level H3K4me3, H3K27ac, H2Az,H3K9ac, as well as RNAPII and P300 signals. The 5,605 LP/E2sites had even higher promoter-associated H3K4me3, H3K27ac,H2Az, H3K9ac, RNAPII, and P300 coverage, albeit lowerH3K4me1, consistent with their promoter localization, whereas the14,195 E2, mostly enhancer sites, had substantially lower H3K4me3promoter marks, higher H3K4me1 enhancer activation marks, and

lower H3K27ac, H2Az, H3K9ac, RNAPII, and P300 levels. Over-all, LP, LP/E2, and E2 sites had active promoter or enhancer epi-genetic marks and occupancies by RNAPII and P300 (Table 2),indicative of active transcription.

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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 TFcoverage for DNA looping factors SMC3, RAD21, and CTCF at LP sites than at E2 or LP/E2 sites.

Table 2. Basal TF cooccupancy and epigenetic marks at LP,LP/E2, or E2 sites

Histonemark/TF

LP (13,602) LP/E2 (5,605) E2 (14,195)

No. sites % Total No. sites % Total No. sites % Total

H3K4me3 7,539 55 3,404 61 4,176 29H3K27ac 7,513 55 4,162 74 5,687 40H2AZ 7,412 54 3,172 57 4,083 29H3K9ac 6,905 51 3,441 61 4,049 29RNA Pol II 4,798 35 2,361 42 2,148 15H3K4me1 2,300 17 1,960 35 4,154 29P300 1,986 15 2,233 40 2,121 15

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Consistent with LP’s association with active promoters, LPsites were highly occupied by RNAPII (Fig. 3A, green line) P300,H3K9ac, and H3K27ac, whereas, consistent with E2’s enhancerlocalization, E2 and LP/E2 sites had higher H3K4me1 levelsthan LP. LP/E2 sites were overall most coincident with activa-tion-associated histone marks and highest RNAPII and P300levels (Fig. 3A, red lines). The strong differences in cell TF andepigenetic mark anchor plots at LP, LP/E2, and E2 sites wereless evident when LP, E2, and LP/E2 sites in promoter regionswere assessed (Fig. S3).To correlate LP-, E2-, and LP/E2-associated cell TF occu-

pancies and chromatin activation marks with activated tran-scription, high-quality ENCODE LCL RNA-seq data for geneswith LP, LP/E2, or E2 sites (−1/+0.1 kb from a TSS) indicatedthat LP, LP/E2, and E2 annotated genes are more highlyexpressed than random genes (P < 1 × 10−16) (Fig. 3B).

However, LP-annotated genes were less expressed than E2 (P <5.6 × 10−4) or LP/E2 (P < 2.7 × 10−12) annotated genes (Fig. 3B),and LP/E2-annotated genes were more highly expressed than E2annotated genes (P < 0.05), despite substantial differences in cellTF occupancies, as well as RNAPII, P300, and activating histoneepigenetic marks (Fig. 3B). These data highlight a complexity ofLP and E2 activation of proliferation and survival pathways thatremains to be deconstructed using shRNAs for individual LP-,E2-, and LP/E2-associated cell TFs.

LP Sites Clusters Differed in Cell TF Composition and TranscriptionEffects. To better understand the range of LP site cell TF occu-pancies, a K-means clustering segregation analysis of LP sites wasundertaken (Fig. 4). Clusters 1–7 were highly occupied by B-celldevelopmental TFs, YY1, PAX5, BATF, IRF4, and PU.1. Cluster2 had more YY1, PAX5, BATF, and IRF4, less PU.1, and highRAD21, CTCF, SMC3, E2, RBPJ, and ZNF143 levels, TFs char-acteristic of B-cell enhancers and promoter looping factors. Cluster3 was uniformly PAX5, an EBF-induced early B-cell transcriptionactivator, and ∼50% YY1. Cluster 4 was uniformly RBPJ and al-most 50% E2. Cluster 5 was solidly PU.1, an activating B-cell de-velopmental TF, and TBLR1, an NCOR/SMRT repressivecomplex component that may be an LP target. Cluster 5 wasenriched for E2 and RBPJ, which enhance MYC and MYC-drivencell 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, andIRF4 occupied, consistent with B-cell developmental transcriptionactivation. Cluster 7 was also high in RAD21, CTCF, SMC3, andZNF143; ZNF143 is a potential alternative mediators of E2 orNotch interaction with DNA. Cluster 8 was uniformly PAX5, anactivating B-cell TF, and RAD21, CTCF, and SMC3, loopingfactors that mediate enhancer interactions with promoters. Cluster9 was YY1, CTCF, RAD21, SMC3, and ZNF143 occupied,

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Fig. 3. Anchor plots for cell epigenetic marks and affected gene expressiondata. (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 plotsof RNA-seq at LP, E2, or LP/E2 annotated peaks for the nearest promoter versusa control (Ctrl) random gene set. LP, E2, and LP/E2 genes were significantlymore 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 thanLP/E2 (P < 0.05). The box plots indicate the data distribution in percentiles, withthe horizontal line being the median. The top of the box represents upper25% 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 linesat the ends of the dotted lines are the maximum observed values.

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Fig. 4. LP site-associated cell TFs separated into 10 K-means clusters and geneexpression. (A) Cell TFs YY1, PAX5, BATF, IRF4, and PU.1 were prominent com-ponents ofmost clusters, consistent with their 30–59% LP site association. Notably,E2, RBPJ, and TBLR1 clustered separately from looping factors CTCF, SMC3, andRAD21. (B) Box plots of RNA-seq gene expression from LP-affected promoters.All LP-affected genes weremore highly expressed than random control genes (P<2 × 10−16). The box plots indicate the data distribution in percentiles, with thehorizontal 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 lowerquartile (25% of the data below that value). The horizontal lines at the ends ofthe dotted lines are the maximum observed values, excluding the outliers.

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whereas cluster 10 included ∼25% of LP sites that had sub-stantially less prevalent cell TF occupancies.Not surprisingly, the genome-wide distribution of most LP

clusters was similar to LP overall (Fig. S4). However, cluster 3(PAX5) was >57% promoter associated, substantially higherthan LP. At the other extreme, cluster 9 was only 16% promoterassociated, had higher intron and intergenic localization, and washighly occupied with YY1, CTCF, RAD21, SMC3, and ZNF143.YY1, PAX5, and BATF/ETS/IRF4 were abundant compo-

nents of most LP clusters and are essential B-cell developmentalTFs that affect cell growth and gene expression (Table 1 and Fig.4A) (43, 45). DNA looping factors CTCF, RAD21, and SMC3were characteristic of LP clusters 2, 7, 8, and 9, which were alsorich in ZNF143, E2, and RBPJ. Clusters 2, 4, 5, and 6 were richin TBLR1, a ubiquitin ligase that is likely activated by LP-mediated NCOR removal leading to transcription derepression(46). LP cluster 5 includes the hes1 locus, which is less NCORoccupied and derepressed when LP is expressed in BJABB-lymphoma cells (9) (Fig. S5A). Interestingly, PKCδ (prckd),the protein kinase that activates TBLR1 to degrade NCOR is up-regulated 1.9-fold in LCLs [P < 0.05 (47)]. Furthermore, LP/E2occupied three sites in the prckd locus (Fig. S5B), indicative ofa role for LP/E2 in regulating prckd expression.To investigate the relationship between gene transcription and

LP site clusters, LCL RNA-seq data annotated to LP, LP/E2, orE2 promoter sites were used. All LP-affected clusters were sig-nificantly more highly expressed than random control genes (P <2 × 10−16) (Fig. 4B). Cluster 8, which included YY1, PAX5,CTCF, RAD21, SMC3, and ZNF143 and cluster 9, which in-cluded YY1, CTCF, RAD21, SMC3, and ZNF143, had relativelylower expression levels, compared with other clusters (Fig. 4B).Overall, LP positively affected genes with CTCF sites (Fig. S6),

likely by removing repressors from these sites (Fig. S6). Compari-son of RNA-seq expression data from genes having a promoter-associated CTCF sites without LP with those having a promoter-associated CTCF sites with LP, revealed genes with overlappingCTCF/LP sites to be significantly more highly expressed than genes

having CTCF sites without LP (P < 2 × 10−16). These data indicatethat LP localized with CTCF at promoter sites increases tran-scription. LP-associated transcription increases are most likelymediated by LP dismissal of CTCF-, SMC3-, or RAD21-associ-ated NCOR or HDACs and may also be affected by long-distanceenhancer interaction with CTCF, RAD21, and SMC3 at CTCF/LPsites. Overall, LP, localized with CTCF at or near promoters hadderepressive effects comparable to E2’s activating effects (9, 10,24, 48, 49).To illustrate LP and associated TFs roles in up-regulating bi-

ologically important genes for LCL proliferation and survival,relevant ChIP-seq tracks are presented for the MYC-regulatedcell cycle entry gene ccnd2, the MYC proliferation-associatedcell survival genes, bcl2 and igf2r, the MYC-induced cell senes-cence genes, cdkn2a and cdkn2b, and 1 of the 23 LP affectedmediator components, med26 (Fig. S7 A–E) (50–58).Thus, the data presented here position LP as a key component

of EBV’s control of the cell transcription, proliferation, andsurvival-related gene transcription (Fig. S7 A–E). The genome-wide approaches used to generate these data enabled the dis-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.Fig. S7A shows the bcl2, promoter and enhancer, which has LP

at the promoter and at two distal enhancers, with E2, RBPJ,TBLR1, ZNF143, CTCF, YY1, IRF4, BATF, ETS1, PU.1, EBF,PAX5, SP1, NFκB, H3K27ac, RNAPII, H3K4me1, H3K9ac,and H3k4me3.Fig. S7B shows the igf2r locus, with strong LP signals at the

promoter along with YY1, ETS1, PAX5, SP1, H3K27ac, RNAPII,H3K4me1, H3K9ac, and H3K4me3.Fig S7C shows the ccnd2 promoter with strong LP signals,

weak E2, ZNF143, CTCF, RAD21, SMC3, YY1, BATF, ETS1,SP1, NFκB, H3K27ac, RNAPII, H3K9ac, and H3K4me3.Fig. S7D shows the cdkn2a and 2b promoters with strong LP,

ZNF143, CTCF, RAD21, SMC3, YY1, SP1, signals, ZNF143,weak and strong YY1, SP1, NFκB, H3K27ac, RNAPll, H3K9ac,and H3K4me3.Fig. S7E shows the med26 locus with promoter-associated

LP, ZNF143, RAD21, SMC3, YY1, ETS1, strong PAX5, SP1,H3K27ac, RNAPll, H3K4me1, and H3K4me3 signals at the pro-moter. Notably, stronger signals are also apparent at the med26enhancer for LP, E2, RBPJ, TBLR1, RAD21, SMC3, YY1, IRF4,BATF, ETS1, PU.1, EBF, PAX5, SP1, H3K27ac, RNAPII,H3K4me1, H3K9ac, and H3K4me3.These results support the model shown in Fig. 5, that LP,

predominantly at or near promoters, and E2 at enhancer sites,cooperatively affect LCL gene expression. LP’s presence at DNAsites dismisses repressive NCOR complexes. Affected genes areup-regulated by LP-mediated derepression and long-distanceDNA interactions through CTCF, RAD21, and SMC3.

Materials and MethodsChIP-seq was performed as described (17). IB4 cells were grown in RPMImedium, 10% (vol/vol) FBS. Detailed methods for analysis of ChIP-seq data,dataset access, and ChIP-seq protocol are available in SI Materials andMethods.

ACKNOWLEDGMENTS. This research was supported by Grants R01CA131354,R01CA170023, and R01CA047006 from the National Cancer Institute of theNational 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

transformation is a direct transcriptional activator. J Virol 65(11):5880–5885.2. Cohen JI, Wang F, Kieff E (1991) Epstein-Barr virus nuclear protein 2 mutations de-

fine essential domains for transformation and transactivation. J Virol 65(5):

2545–2554.

3. Cohen JI, Wang F, Mannick J, Kieff E (1989) Epstein-Barr virus nuclear protein 2 is a key

determinant of lymphocyte transformation. Proc Natl Acad Sci USA 86(23):9558–9562.4. Mannick JB, Cohen JI, Birkenbach M, Marchini A, Kieff E (1991) The Epstein-Barr virus

nuclear protein encoded by the leader of the EBNA RNAs is important in B-lympho-

cyte transformation. J Virol 65(12):6826–6837.

HA95

CTCF

RAD21 SMC3

RBPJ EBF

E2

RNAPII

INO80-YY1/PAX5/SP1/ETS IRF4/BATF/ZNF143

LP

NCOR

NCOR

NCORLP

NCOR

LP

++++

HA95

HA95

H3K27acH3K4me1H3K9acH2Az

H3K27acH3K4me3H3K9acH2Az

CTCF

RAD21 SMC3

Fig. 5. Model. LP occupies mostly promoters, whereas E2 is found atenhancers (17). Enhancers regulate transcription via long-distance DNAinteractions mediated by CTCF, RAD21, and SMC3. Several transcriptionfactors are associated with LP sites and E2 sites and likely help mediate theinteractions between LP and E2 (blue box). The genes associated with LPsites are highly expressed and rich in activation histone marks (Fig. 3). LP andHA95 regulate the dismissal of the NCOR repressive complex (9, 59).

Portal et al. PNAS | November 12, 2013 | vol. 110 | no. 46 | 18541

CELL

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ded

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Oct

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202

1

5. Dambaugh T, Hennessy K, Chamnankit L, Kieff E (1984) U2 region of Epstein-Barrvirus DNA may encode Epstein-Barr nuclear antigen 2. Proc Natl Acad Sci USA 81(23):7632–7636.

6. Dambaugh T, et al. (1986) Expression of the Epstein-Barr virus nuclear protein 2 inrodent cells. J Virol 59(2):453–462.

7. Wang F, Petti L, Braun D, Seung S, Kieff E (1987) A bicistronic Epstein-Barr virus mRNAencodes two nuclear proteins in latently infected, growth-transformed lymphocytes.J Virol 61(4):945–954.

8. Ling PD, et al. (2005) Mediation of Epstein-Barr virus EBNA-LP transcriptional co-activation by Sp100. EMBO J 24(20):3565–3575.

9. Portal D, et al. (2011) EBV nuclear antigen EBNALP dismisses transcription repressorsNCoR and RBPJ from enhancers and EBNA2 increases NCoR-deficient RBPJ DNAbinding. Proc Natl Acad Sci USA 108(19):7808–7813.

10. Portal D, Rosendorff A, Kieff E (2006) Epstein-Barr nuclear antigen leader proteincoactivates transcription through interaction with histone deacetylase 4. Proc NatlAcad Sci USA 103(51):19278–19283.

11. Han I, et al. (2002) Protein kinase A associates with HA95 and affects transcriptionalcoactivation by Epstein-Barr virus nuclear proteins. Mol Cell Biol 22(7):2136–2146.

12. Han I, et al. (2001) EBNA-LP associates with cellular proteins including DNA-PK andHA95. J Virol 75(5):2475–2481.

13. Harada S, Kieff E (1997) Epstein-Barr virus nuclear protein LP stimulates EBNA-2 acidicdomain-mediated transcriptional activation. J Virol 71(9):6611–6618.

14. Alfieri C, Birkenbach M, Kieff E (1991) Early events in Epstein-Barr virus infection ofhuman B lymphocytes. Virology 181(2):595–608.

15. Mannick JB, Tong X, Hemnes A, Kieff E (1995) The Epstein-Barr virus nuclear antigenleader protein associates with hsp72/hsc73. J Virol 69(12):8169–8172.

16. Peng CW, et al. (2007) Hsp72 up-regulates Epstein-Barr virus EBNALP coactivationwith EBNA2. Blood 109(12):5447–5454.

17. Zhao B, et al. (2011) Epstein-Barr virus exploits intrinsic B-lymphocyte transcriptionprograms to achieve immortal cell growth. Proc Natl Acad Sci USA 108(36):14902–14907.

18. Grossman SR, Johannsen E, Tong X, Yalamanchili R, Kieff E (1994) The Epstein-Barrvirus nuclear antigen 2 transactivator is directed to response elements by the J kapparecombination signal binding protein. Proc Natl Acad Sci USA 91(16):7568–7572.

19. Henkel T, Ling PD, Hayward SD, Peterson MG (1994) Mediation of Epstein-Barr virusEBNA2 transactivation by recombination signal-binding protein J kappa. Science265(5168):92–95.

20. Tong X, Wang F, Thut CJ, Kieff E (1995) The Epstein-Barr virus nuclear protein 2 acidicdomain can interact with TFIIB, TAF40, and RPA70 but not with TATA-binding pro-tein. J Virol 69(1):585–588.

21. Tong X, Drapkin R, Yalamanchili R, Mosialos G, Kieff E (1995) The Epstein-Barr virusnuclear protein 2 acidic domain forms a complex with a novel cellular coactivator thatcan interact with TFIIE. Mol Cell Biol 15(9):4735–4744.

22. Nikitin PA, et al. (2010) An ATM/Chk2-mediated DNA damage-responsive signalingpathway suppresses Epstein-Barr virus transformation of primary human B cells. CellHost Microbe 8(6):510–522.

23. Peng R, Tan J, Ling PD (2000) Conserved regions in the Epstein-Barr virus leaderprotein define distinct domains required for nuclear localization and transcriptionalcooperation with EBNA2. J Virol 74(21):9953–9963.

24. Peng R, Moses SC, Tan J, Kremmer E, Ling PD (2005) The Epstein-Barr virus EBNA-LPprotein preferentially coactivates EBNA2-mediated stimulation of latent membraneproteins expressed from the viral divergent promoter. J Virol 79(7):4492–4505.

25. Phillips JE, Corces VG (2009) CTCF: Master weaver of the genome. Cell 137(7):1194–1211.

26. Merkenschlager M, Odom DT (2013) CTCF and cohesin: Linking gene regulatory el-ements with their targets. Cell 152(6):1285–1297.

27. Remeseiro S, Cuadrado A, Gómez-López G, Pisano DG, Losada A (2012) A unique roleof cohesin-SA1 in gene regulation and development. EMBO J 31(9):2090–2102.

28. Cuadrado A, Remeseiro S, Gómez-López G, Pisano DG, Losada A (2012) The specificcontributions of cohesin-SA1 to cohesion and gene expression: Implications forcancer and development. Cell Cycle 11(12):2233–2238.

29. Degner SC, et al. (2011) CCCTC-binding factor (CTCF) and cohesin influence the ge-nomic architecture of the Igh locus and antisense transcription in pro-B cells. Proc NatlAcad Sci USA 108(23):9566–9571.

30. Degner SC, Wong TP, Jankevicius G, Feeney AJ (2009) Cutting edge: Developmentalstage-specific recruitment of cohesin to CTCF sites throughout immunoglobulin lociduring B lymphocyte development. J Immunol 182(1):44–48.

31. Herold M, Bartkuhn M, Renkawitz R (2012) CTCF: Insights into insulator functionduring development. Development 139(6):1045–1057.

32. Chaumeil J, Skok JA (2012) The role of CTCF in regulating V(D)J recombination.Curr Opin Immunol 24(2):153–159.

33. Gillen AE, Harris A (2011) The role of CTCF in coordinating the expression of singlegene loci. Biochem Cell Biol 89(5):489–494.

34. Rhodes JM, et al. (2010) Positive regulation of c-Myc by cohesin is direct, and evolu-tionarily conserved. Dev Biol 344(2):637–649.

35. Dorsett D, Merkenschlager M (2013) Cohesin at active genes: A unifying theme forcohesin and gene expression from model organisms to humans. Curr Opin Cell Biol25(3):327–333.

36. Dorsett D (2011) Cohesin: Genomic insights into controlling gene transcription anddevelopment. Curr Opin Genet Dev 21(2):199–206.

37. Hou C, Zhao H, Tanimoto K, Dean A (2008) CTCF-dependent enhancer-blocking byalternative chromatin loop formation. Proc Natl Acad Sci USA 105(51):20398–20403.

38. Splinter E, et al. (2006) CTCF mediates long-range chromatin looping and local histonemodification in the beta-globin locus. Genes Dev 20(17):2349–2354.

39. Cai Y, et al. (2007) YY1 functions with INO80 to activate transcription. Nat Struct MolBiol 14(9):872–874.

40. Lin YC, et al. (2012) Global changes in the nuclear positioning of genes and intra- andinterdomain genomic interactions that orchestrate B cell fate. Nat Immunol 13(12):1196–1204.

41. Wang H, et al. (2011) Genome-wide analysis reveals conserved and divergent featuresof Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells. ProcNatl Acad Sci USA 108(36):14908–14913.

42. Halbig KM, Lekven AC, Kunkel GR (2012) The transcriptional activator ZNF143 is es-sential for normal development in zebrafish. BMC Mol Biol 13:3.

43. Li P, et al. (2012) BATF-JUN is critical for IRF4-mediated transcription in T cells. Nature490(7421):543–546.

44. Amouyal M (2010) Gene insulation. Part II: Natural strategies in vertebrates. BiochemCell Biol 88(6):885–898.

45. Donohoe ME, Silva SS, Pinter SF, Xu N, Lee JT (2009) The pluripotency factor Oct4interacts with Ctcf and also controls X-chromosome pairing and counting. Nature460(7251):128–132.

46. Perissi V, et al. (2008) TBL1 and TBLR1 phosphorylation on regulated gene promotersovercomes dual CtBP and NCoR/SMRT transcriptional repression checkpoints.Mol Cell29(6):755–766.

47. Price AM, et al. (2012) Analysis of Epstein-Barr virus-regulated host gene expressionchanges through primary B-cell outgrowth reveals delayed kinetics of latent mem-brane protein 1-mediated NF-κB activation. J Virol 86(20):11096–11106.

48. Peng CW, Zhao B, Kieff E (2004) Four EBNA2 domains are important for EBNALPcoactivation. J Virol 78(20):11439–11442.

49. Peng CW, et al. (2004) Direct interactions between Epstein-Barr virus leader proteinLP and the EBNA2 acidic domain underlie coordinate transcriptional regulation. ProcNatl Acad Sci USA 101(4):1033–1038.

50. Mushinski JF, et al. (1999) Myc-induced cyclin D2 genomic instability in murine B cellneoplasms. Curr Top Microbiol Immunol 246:183–189; discussion 190–192.

51. Bouchard C, et al. (2001) Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at thecyclin D2 promoter. Genes Dev 15(16):2042–2047.

52. Sinclair AJ, Palmero I, Holder A, Peters G, Farrell PJ (1995) Expression of cyclin D2 inEpstein-Barr virus-positive Burkitt’s lymphoma cell lines is related to methylationstatus of the gene. J Virol 69(2):1292–1295.

53. Zinkel S, Gross A, Yang E (2006) BCL2 family in DNA damage and cell cycle control.Cell Death Differ 13(8):1351–1359.

54. Harris LK, Westwood M (2012) Biology and significance of signalling pathways acti-vated by IGF-II. Growth Factors 30(1):1–12.

55. Lewis BA, Reinberg D (2003) The mediator coactivator complex: Functional andphysical roles in transcriptional regulation. J Cell Sci 116(Pt 18):3667–3675.

56. Maruo S, et al. (2011) Epstein-Barr virus nuclear antigens 3C and 3A maintain lym-phoblastoid cell growth by repressing p16INK4A and p14ARF expression. Proc NatlAcad Sci USA 108(5):1919–1924.

57. Rayess H, Wang MB, Srivatsan ES (2012) Cellular senescence and tumor suppressorgene p16. Int J Cancer 130(8):1715–1725.

58. Sinclair AJ, Palmero I, Peters G, Farrell PJ (1994) EBNA-2 and EBNA-LP cooperate tocause G0 to G1 transition during immortalization of resting human B lymphocytesby Epstein-Barr virus. EMBO J 13(14):3321–3328.

59. Li Y, et al. (2006) A novel histone deacetylase pathway regulates mitosis by modu-lating Aurora B kinase activity. Genes Dev 20(18):2566–2579.

18542 | www.pnas.org/cgi/doi/10.1073/pnas.1317608110 Portal et al.

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