Epstein–Barr Virus Nuclear Antigen 3C binds to BATF/IRF4 or SPI1/IRF4 composite sites and recruits Sin3A to repress CDKN2A Sizun Jianga,b,c,d, Bradford Willoxa,c, Hufeng Zhoua,c, Amy M. Holthausa,c, Anqi Wange, Tommy T. Shia,c, Seiji Maruof, Peter V. Kharchenkod,g, Eric C. Johannsene, Elliott Kieffa,b,c,1, and Bo Zhaoa,c aDepartment of Medicine, Brigham and Women’s Hospital, bProgram in Virology, cDepartment of Microbiology and Immunobiology, and dCenter for Biomedical Informatics, Harvard Medical School, Boston, MA 02115; eDepartment of Medicine and McArdle Laboratory for Cancer Research, University of Wisconsin–Madison, Madison, WI 53706; fDepartment of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan; and gDivision of Hematology/Oncology, Children’s Hospital, Boston, MA 02115 Contributed by Elliott Kieff, November 20, 2013 (sent for review October 3, 2013) Epstein–Barr virus nuclear antigen 3C (EBNA3C) repression of LCL growth, indicating that EBNA3C is specifically required for CDKN2A p14ARF and p16INK4A is essential for immortal human LCL growth (6). Similar experiments reveal EBNA3C N-termi- B-lymphoblastoid cell line (LCL) growth. EBNA3C ChIP-sequencing nal amino acids 50–400 to be essential for LCL growth (3, 7, 8). identified >13,000 EBNA3C sites in LCL DNA. Most EBNA3C sites EBNA3C also up-regulates EBV LMP1 and cell CXCR4 and were associated with active transcription; 64% were strong CXCL12 gene expression (9–12), which are required for LCL H3K4me1- and H3K27ac-marked enhancers and 16% were active growth in nude mice (13). EBNA3C and EBNA3A joint re- p14ARF p16INK4A promoters marked by H3K4me3 and H3K9ac. Using ENCODE LCL pression of and expression is essential for LCL ARF INK4A p16INK4A transcription factor ChIP-sequencing data, EBNA3C sites coincided growth and knock down of p14 and p16 or null ± mutations allow LCL growth in the absence of EBNA3C, in- ( 250 bp) with RUNX3 (64%), BATF (55%), ATF2 (51%), IRF4 (41%), p14ARF p16INK4A MEF2A (35%), PAX5 (34%), SPI1 (29%), BCL11a (28%), SP1 (26%), dicating that EBNA3C repression of and is an essential EBNA3C function (14, 15). Both EBNA3A and EBNA3C TCF12 (23%), NF-κB (23%), POU2F2 (23%), and RBPJ (16%). EBNA3C have repressive activities that correlate with cell histone mod- sites separated into five distinct clusters: (i)Sin3A,(ii) EBNA2/RBPJ, MICROBIOLOGY iii iv v ifications: EBNA3A induces histone modifications at the CXCL10 ( ) SPI1, and ( )strongor() weak BATF/IRF4. EBNA3C signals and CXCL9 chemokine genes (16), whereas EBNA3C induces ARF were positively affected by RUNX3, BATF/IRF4 (AICE) and SPI1/IRF4 histone modifications which are important for p14 and INK4A (EICE) cooccupancy. Gene set enrichment analyses correlated p16 repression (14, 17). However, the detailed mecha- ARF INK4A EBNA3C/Sin3A promoter sites with transcription down-regulation nism through which EBNA3C represses p14 and p16 (P < 1.6 × 10−4). EBNA3C signals were strongest at BATF/IRF4 and ARF expression is unknown. SPI1/IRF4 composite sites. EBNA3C bound strongly to the p14 In contrast to EBNA2, which is tethered to DNA by RBPJ, promoter through SPI1/IRF4/BATF/RUNX3, establishing RBPJ-, Sin3A-, EBNA3C binding to RBPJ prevents RBPJ binding to DNA in and REST-mediated repression. EBNA3C immune precipitated with electrophoretic mobility-shift assays (EMSAs) and blocks EBNA2 Sin3A and conditional EBNA3C inactivation significantly decreased activation effects (18, 19). EBNA3C affects cell and EBV gene ARF Sin3A binding at the p14 promoter (P < 0.05). These data support expression through cell TFs. Chromatin immunoprecipitation a model in which EBNA3C binds strongly to BATF/IRF4/SPI1/RUNX3 (ChIP) followed by quantitative PCR (qPCR) found EBNA3C sites to enhance transcription and recruits RBPJ/Sin3A- and REST/ boundtovirusandcellpromotersites,includingtheEBV NRSF-repressive complexes to repress p14ARF and p16INK4A expression. LMP1, cell BIM, and ITGA4 promoters (20, 21, 22), whereas EBV | tumor suppressor | resting B lymphocyte | lymphoma Significance pstein–Barr virus (EBV) is a highly prevalent γ-herpesvirus Epstein–Barr virus (EBV) is an important causative agent of Ethat causes B and T lymphomas, Hodgkin disease, naso- B-cell lymphomas and Hodgkin disease in immune-deficient pharyngeal carcinoma, and gastric carcinoma. EBV-associated people, including HIV-infected people. The experiments de- B lymphomas and Hodgkin disease are more prevalent in T-cell scribed here were undertaken to determine the mechanisms immune-deficient people and are major causes of mortality in through which the EBV-encoded nuclear protein EBNA3C ARF INK4A HIV-infected people. EBV infection of B lymphocytes, in vitro, blocks the cell p14 and p16 tumor suppressor-mediated results in continuously proliferating lymphoblastoid cell lines inhibition of EBV-infected B-cell growth, thereby unfettering (LCLs). LCLs and EBV-infected cells in T-cell immune-deficient EBV-driven B-cell proliferation. The experiments also identify people express six EBV-encoded nuclear proteins (EBNA1, the molecular basis for diverse EBNA3C enhancer interactions EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNALP), three with cell DNA-binding proteins and cell DNA to regulate MYC, latent infection-associated membrane proteins (LMP1, LMP2A, pRB, BCL2, and BIM expression. Surprisingly, EBNA3C’s role in and LMP2B), microRNAs, and EBER 1 and 2 RNAs. Genetic enhancer-mediated cell gene transcription up-regulation is analyses indicate that EBNA1, EBNALP, EBNA2, EBNA3A, primarily mediated by combinatorial effects with cell tran- EBNA3C, and LMP1 are essential for LCL outgrowth (1, 2). scription factors, most notably AICEs, EICEs, and RUNX3. EBNA3A, EBNA3B, and EBNA3C are similar proteins, com- posed of ∼1,000 aa. Each has a near N-terminal site that binds Author contributions: S.J., E.C.J., E.K., and B.Z. designed research; B.W., A.M.H., T.T.S., and RBPJ, the cell sequence specific transcription factor (TF) that B.Z. performed research; A.M.H., A.W., S.M., and E.C.J. contributed new reagents/analytic mediates EBNA2 or Notch binding to DNA. EBNA3A and tools; S.J., H.Z., P.V.K., and B.Z. analyzed data; and S.J., E.K., and B.Z. wrote the paper. EBNA3C are both required for LCL growth, whereas EBNA3B is The authors declare no conflict of interest. not (3–5). When conditional, hydoxytamoxifen (HT)-dependent, Data deposition: The ChIP sequence data reported in this paper has been deposited in the EBNA3C (EBNA3CHT)-expressing LCLs are grown in medium GenBank database (accession no. GSE52632). without HT, the cells stop growing (6). HT addition or comple- 1To whom correspondence should be addressed. E-mail: [email protected]. mentation with wild-type (WT) EBNA3C-expression plasmids, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. but not with EBNA3A- or EBNA3B-expression plasmids, restores 1073/pnas.1321704111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1321704111 PNAS | January 7, 2014 | vol. 111 | no. 1 | 421–426 Downloaded by guest on September 25, 2021 A binding sites, using input DNA controls. Sequencing reads were mapped to the human genome using Bowtie with two mismatches (28). Phantom peak calling was used to evaluate ChIP-seq quality. Both EBNA3C ChIP-seq replicates had a Quality tags >1, in ac- cordance with the Encyclopedia of DNA Elements (ENCODE) high quality data standard (29). The ChIP-seq processing pipeline (SPP) identified over 13,000 EBNA3C peaks with irreproducible discovery rates (IDRs) <0.01 (29, 30). EBNA3C peaks were an- notated using the ENCODE GM12878 LCL epigenetic landscape, which divides the genome into seven functional domains defined by unique histone modifications (31). EBNA3C sites were 38% (4,969 sites) at strong enhancers, marked by high H3K4me1 and H3K27ac; 25% (3,287 sites) at weak enhancers, marked by in- termediate H3K4me1 and little H3K27ac; 8% (1,021 sites) at B active promoters, marked by high H3K4me3 and H3K9ac; 8% (1,108 sites) at weak or poised promoters, marked by high H3K4me3 and low H3K27ac; or high H3K27me3, and 16% (2,093 sites) at heterochromatin, marked by the absence of detectable histone modifications (Fig. 1A). EBNA3C peaks were significantly enriched over background controls at weak promoters, poised promoters, and strong and weak enhancers (Fisher’sexacttest: − P < 1 × 10 5)(Fig. S1A), identifying EBNA3C as a TF that significantly affects enhancer and promoter activity. EBNA3C and EBNA2/RBPJ Peaks Overlap. Reanalysis of previous EBNA2 and RBPJ data (32) using IDR identified the top 5,000 EBNA2 and 10,000 RBPJ sites. Overall, 9% of EBNA3C sites overlapped with EBNA2/RBPJ sites and 7% overlapped with RBPJ sites lacking EBNA2 (Fig. 1B). EBNA3C was 84% at DNA sites without significant RBPJ, whereas most EBNA2 sites coincided with RBPJ. EBNA2-associated RBPJ sites had two- fold higher signals than RBPJ sites that lacked EBNA2, in- dicating a strong EBNA2 effect on RBPJ association with DNA Fig. 1. Genome-wide distribution of EBNA3C sites and EBNA3C site overlap with EBNA2/RBPJ, BATF/IRF4, SPI1/IRF4, and RUNX3. (A) Genome-wide distribu- tion of EBNA3C DNA binding by chromatin states (%). EBNA3C distribution calculated based on the chromatin state of GM12878 cells; 12,992 EBNA3C sites within annotated genomic regions were used to identify the genome-wide distribution of EBNA3C sites. (B) Venn diagram showing EBNA3C site overlap with RUNX3, SPI1/IRF4, EBNA2/RBPJ, or BATF/IRF4 (±250bpofEBNA3Csites). ChIP-sequencing (ChIP-seq) using an antibody that immune pre- cipitates EBNA3A, EBNA3B, and EBNA3C, found an EBNA3 at around 7,000 sites in a Burkitt lymphoma cell line (23), consistent with the hypothesis that EBNA3C may affect transcription through interactions with other cell TFs (11, 23–27). Recently, EBNA3C residues 130–159wereshowntobindtoIRF4orIRF8,TFsim- portant for lymphopoiesis (24). EBNA3C can also coactivate the EBV LMP1 promoter with EBNA2 through a SPI1 site (10, 11). Overall, these findings are consistent with the hypothesis that EBNA3C affects transcription through interactions with cell TFs.