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• W3016787401 abstract "•CRISPR/Cas9 screen highlights host epigenetic suppressors of the EBV lytic cycle•EBV senses MYC abundance to maintain B cell latency•MYC depletion alters three-dimensional EBV genomic architecture•FACT is a druggable target for Burkitt B cell EBV latency reversal Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets. Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets. Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide. To accomplish this remarkable task, EBV uses a biphasic life cycle, in which the viral genome switches between latent and lytic programs. Establishment of latency in a long-lived cellular niche and subsequent lytic reactivation in response to environmental cues is a hallmark of herpesvirus infection, yet much remains to be learned about how EBV accomplishes these important roles. EBV is transmitted between hosts through saliva, from which it translocates across tonsillar epithelium to reach the B cell compartment. Upon B cell infection, the ∼170 kb linear double-stranded EBV genome is delivered to the nucleus, where it is circularized and chromatinized. Rapid histone deposition serves to initially restrict expression of the nearly 70 EBV lytic antigens (Lieberman, 2013Lieberman P.M. Keeping it quiet: chromatin control of gammaherpesvirus latency.Nat. Rev. Microbiol. 2013; 11: 863-875Crossref PubMed Scopus (96) Google Scholar). Rather than produce infectious particles, EBV instead uses a series of latency programs to navigate the B cell compartment. Epstein-Barr nuclear antigens (EBNA) initially escape silencing. EBNA1 tethers viral genomes to host chromosomes, while EBNA 2, LP, 3A, 3B, and 3C serve as transcription factors to regulate host and viral expression. This viral genome program strongly induces MYC and converts resting B cells into proliferating blasts (Pich et al., 2019Pich D. Mrozek-Gorska P. Bouvet M. Sugimoto A. Akidil E. Grundhoff A. Hamperl S. Ling P.D. Hammerschmidt W. First Days in the Life of Naive Human B Lymphocytes Infected with Epstein-Barr Virus.MBio. 2019; 10: e01723-19Crossref PubMed Scopus (7) Google Scholar, Price and Luftig, 2015Price A.M. Luftig M.A. To be or not IIb: a multi-step process for Epstein-Barr virus latency establishment and consequences for B cell tumorigenesis.PLoS Pathog. 2015; 11: e1004656Crossref PubMed Scopus (78) Google Scholar, Wang et al., 2019Wang L.W. Shen H. Nobre L. Ersing I. Paulo J.A. Trudeau S. Wang Z. Smith N.A. Ma Y. Reinstadler B. et al.Epstein-Barr-Virus-Induced One-Carbon Metabolism Drives B Cell Transformation.Cell Metab. 2019; 30: 539-555Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Shortly thereafter, the viral genome switches to the latency III program, where these EBNA together with EBNA3 antigens and two latent membrane proteins (LMP) further promote B cell growth, survival, and germinal center entry. Immune pressure and incompletely characterized epigenetic re-programing further restrict latency gene expression, until Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein expressed. This latency I program is observed in memory B cells, the reservoir for lifelong EBV infection, and also in most EBV-infected Burkitt lymphoma (BL) tumors. Because EBNA1 has evolved low immunogenicity, B cells with EBV latency I evade immune detection. Peripheral resting memory B cells maintain EBV latency even in the absence of EBNA1 expression (Babcock et al., 2000Babcock G.J. Hochberg D. Thorley-Lawson A.D. The expression pattern of Epstein-Barr virus latent genes in vivo is dependent upon the differentiation stage of the infected B cell.Immunity. 2000; 13: 497-506Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar), suggesting that epigenetic mechanisms involving host factors are important for the silencing of EBV lytic antigens. By incompletely characterized mechanisms, plasma cell differentiation triggers EBV lytic reactivation (Laichalk and Thorley-Lawson, 2005Laichalk L.L. Thorley-Lawson D.A. Terminal differentiation into plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo.J. Virol. 2005; 79: 1296-1307Crossref PubMed Scopus (380) Google Scholar), where infectious virions are produced for spread to nearby target cells. The EBV immediate early factor BZLF1 is a master regulator of the B cell lytic cycle, and its induction is therefore highly regulated. While host factors that regulate BZLF1 expression have been identified, including the plasma cell master regulator Blimp1 (Reusch et al., 2015Reusch J.A. Nawandar D.M. Wright K.L. Kenney S.C. Mertz J.E. Cellular differentiation regulator BLIMP1 induces Epstein-Barr virus lytic reactivation in epithelial and B cells by activating transcription from both the R and Z promoters.J. Virol. 2015; 89: 1731-1743Crossref PubMed Scopus (58) Google Scholar), much remains to be learned about how the EBV genome senses and responds to B cell differentiation cues to induce BZLF1. Once expressed, BZLF1 triggers expression of ∼30 early lytic genes, including the viral DNA polymerase, the processivity factor BMRF1, kinases and other factors important for lytic DNA replication. Newly replicated genomes serve as the template for most of the nearly 30 EBV late genes, whose expression allows DNA encapsidation, virion assembly, and secretion. Potent immune responses are directed at EBV lytic antigens (Taylor et al., 2015Taylor G.S. Long H.M. Brooks J.M. Rickinson A.B. Hislop A.D. The immunology of Epstein-Barr virus-induced disease.Annu. Rev. Immunol. 2015; 33: 787-821Crossref PubMed Scopus (315) Google Scholar). Available anti-EBV small molecule therapies target EBV lytic cycle kinase and DNA polymerase (Meng et al., 2010Meng Q. Hagemeier S.R. Fingeroth J.D. Gershburg E. Pagano J.S. Kenney S.C. The Epstein-Barr virus (EBV)-encoded protein kinase, EBV-PK, but not the thymidine kinase (EBV-TK), is required for ganciclovir and acyclovir inhibition of lytic viral production.J. Virol. 2010; 84: 4534-4542Crossref PubMed Scopus (103) Google Scholar). While lytic infection is increasingly implicated in the pathogenesis of EBV-associated cancers, latent infection is instead observed in most human B cell and epithelial cancers malignancies. EBV is associated with 200,000 human cancers per year, including Burkitt and Hodgkin’s lymphoma, post-transplant lymphoproliferative disease, and nasopharyngeal and gastric carcinomas. Therefore, there is considerable interest in strategies to reverse EBV latency in order to sensitize tumor cells to agents such as ganciclovir, whose phosphorylation by an EBV lytic cycle kinase induces toxicity to infected and even neighboring cells (Meng et al., 2010Meng Q. Hagemeier S.R. Fingeroth J.D. Gershburg E. Pagano J.S. Kenney S.C. The Epstein-Barr virus (EBV)-encoded protein kinase, EBV-PK, but not the thymidine kinase (EBV-TK), is required for ganciclovir and acyclovir inhibition of lytic viral production.J. Virol. 2010; 84: 4534-4542Crossref PubMed Scopus (103) Google Scholar). To identify epigenetic factors critical for the maintenance of EBV latency, we performed a genome scale CRISPR/Cas9 loss-of-function screen in EBV+ Burkitt lymphoma cells, the available B cell latency I infection model. Our results identify MYC as a major regulator of the B cell EBV lytic switch. The EBV late lytic gene product gp350 decorates the plasma membrane of B cells undergoing viral replication, but is absent from cells with latent EBV infection (Thorley-Lawson, 2015Thorley-Lawson D.A. EBV Persistence--Introducing the Virus.Curr. Top. Microbiol. Immunol. 2015; 390: 151-209PubMed Google Scholar). We reasoned that gp350 abundance could be used as a physiologically relevant reporter of CRISPR/Cas9 targets, whose knockout (KO) induced EBV lytic reactivation. Biological replicates of an EBV-infected Burkitt lymphoma (BL) KO library were generated by transducing Cas9+ P3HR-1 cells with the Avana lentiviral single-guide RNA (sgRNA) library. Avana contains four independent sgRNAs that target distinct regions of each human protein coding gene (Figure 1A) (Doench et al., 2016Doench J.G. Fusi N. Sullender M. Hegde M. Vaimberg E.W. Donovan K.F. Smith I. Tothova Z. Wilen C. Orchard R. et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.Nat. Biotechnol. 2016; 34: 184-191Crossref PubMed Scopus (1831) Google Scholar). Fluorescence-associated cell sorting (FACS) was used to purify live gp350+ cells at 6 and 9 days post-transduction (Figure 1A). Two time points were used, because hits might induce lytic induction with distinct kinetics. Input versus sorted cell sgRNA abundances were quantitated by PCR amplification and next-generation sequencing. The STARS algorithm, which integrates data from independent guides targeting the same gene, was used to identify statistically significant hits (Doench et al., 2016Doench J.G. Fusi N. Sullender M. Hegde M. Vaimberg E.W. Donovan K.F. Smith I. Tothova Z. Wilen C. Orchard R. et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.Nat. Biotechnol. 2016; 34: 184-191Crossref PubMed Scopus (1831) Google Scholar). Using a p value <0.05 and fold change >1.5 cutoff, 85 and 72 hits were identified at Day 6 versus 9 post-transduction, respectively (Figures 1B and 1C; Table S1). Screen hits were highly enriched for nuclear epigenetic regulators (29.6% of total Days 6 and 9 common hits), including genes encoding the transcription factor MYC, the histone acetyltransferase EP300, multiple components of the mediator and the STAGA/GCN5 acetyltransferase complex, both FACT complex subunits, two cohesin subunits, and a cohesin loader. Notably, Mediator scored more strongly on Day 6, whereas STAGA subunits all scored more strongly on Day 9, perhaps reflecting different rates of decay of these multiprotein complexes following gene KO and effects on cell fitness (Figure S1A). Selected hits from each complex were validated by immunoblot and FACS assays (Figure 1). sgRNAs targeting genes encoding STAGA, FACT, Mediator, or cohesin subunits each induced EBV early lytic gene BMRF1 expression (Figure S1B). Similarly, gp350 plasma membrane expression was upregulated by screen hit sgRNAs (Figure S1C). Collectively, these results raised the possibility that multiple screen hits are nuclear factors that have interrelated roles in the maintenance of EBV latency in BL. Bioinformatic analysis highlighted that many top screen hits could be organized into an interaction network, centered on MYC (Figure 2A). This result raised the question of whether MYC, which is highly EBV-induced upon primary B cell infection, could be a central regulator of EBV latency. By contrast, using a temporal proteomic map of EBV lytic replication (Ersing et al., 2017Ersing I. Nobre L. Wang L.W. Soday L. Ma Y. Paulo J.A. Narita Y. Ashbaugh C.W. Jiang C. Grayson N.E. et al.A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells.Cell Rep. 2017; 19: 1479-1493Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), we observed that MYC protein abundance was significantly diminished in BL cells induced for EBV lytic replication (Figures 2B and S2A), which we confirmed by immunoblot (Figure 2C). To gain insights into how MYC may repress B cell EBV lytic reactivation, we first tested the effects of independent screen hit sgRNAs on MYC and EBV lytic gene expression in P3HR-1 and Akata BL cell lines. P3HR-1 harbors type II EBV, whereas Akata has the more prevalent type I EBV strain. Expression of either sgRNA depleted MYC and robustly induced immediate early BZLF1 and early BMRF1 expression by 72 h post-puromycin selection in both cell lines, a time point prior to when MYC depletion triggers BL cell death (Figures 2D and S2B). MYC KO induced plasma membrane gp350 late antigen expression in live BL cells (Figures 2E and 2F). MYC binds DNA as a heterodimer with one of several transcription factor partners, most commonly as a transcription activator when in complex with MAX, or instead as a repressor when in complex with MIZ1. Independent sgRNAs depleted MAX expression and induced early gene BMRF1 and late gene gp350 expression (Figure 2G). Similarly, the small molecule inhibitor KJ-PYR-9, which blocks MYC and MAX heterodimerization (Hart et al., 2014Hart J.R. Garner A.L. Yu J. Ito Y. Sun M. Ueno L. Rhee J.K. Baksh M.M. Stefan E. Hartl M. et al.Inhibitor of MYC identified in a Kröhnke pyridine library.Proc. Natl. Acad. Sci. USA. 2014; 111: 12556-12561Crossref PubMed Scopus (91) Google Scholar), induced gp350 expression in P3HR-1 cells (Figure S2C). These data suggest that a MYC/MAX heterodimer is important for the maintenance of EBV latency in BL cells. To further characterize the effects of MYC depletion on BL EBV lytic gene expression, we performed RNA sequencing (RNA-seq) analysis of Akata transcripts following expression of control or MYC targeting sgRNAs. 77 EBV lytic cycle genes were significantly induced and were among the most strongly upregulated by MYC depletion (Figure 2H; Table S2). While EBNA1 can inhibit EBV lytic induction, MYC depletion increased EBNA1 mRNA and protein abundance (Figure S2D). Induction of EBV lytic gene expression by independent MYC-targeting sgRNAs was validated by qPCR (Figure S2E). To determine whether MYC depletion stimulates viral genome replication, viral load analysis was done using BL with control or anti-MYC sgRNAs. MYC targeting increased EBV genome copy number, with intracellular and DNase-treated extracellular viral loads approaching levels achieved by anti-IgG cross-linking (Figure 2I). Resistance of extracellular EBV genomes to DNase treatment suggests that these genomes were encapsidated and likely secreted as virus particles. We next asked whether MYC suppresses lytic gene expression in lymphoblastoid B cells (LCLs) with latency III expression, where EBV super-enhancers induce MYC expression (Jiang et al., 2017Jiang S. Zhou H. Liang J. Gerdt C. Wang C. Ke L. Schmidt S.C.S. Narita Y. Ma Y. Wang S. et al.The Epstein-Barr Virus Regulome in Lymphoblastoid Cells.Cell Host Microbe. 2017; 22: 561-573Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) to lower levels than in BL. MYC targeting in GM12878 LCLs, which have an untranslocated MYC locus, induced BZLF1, and to a lesser extent BMRF1 expression (Figure S2F). Notably, LCL TET2 demethylase limits the ability of BZLF1 to induce EBV early genes (Lu et al., 2017Lu F. Wiedmer A. Martin K.A. Wickramasinghe P.J.M.S. Kossenkov A.V. Lieberman P.M. Coordinate Regulation of TET2 and EBNA2 Controls the DNA Methylation State of Latent Epstein-Barr Virus.J. Virol. 2017; 91: e00804-17Crossref PubMed Scopus (19) Google Scholar, Wille et al., 2017Wille C.K. Li Y. Rui L. Johannsen E.C. Kenney S.C. Restricted TET2 Expression in Germinal Center Type B Cells Promotes Stringent Epstein-Barr Virus Latency.J. Virol. 2017; 91: e01987-16Crossref PubMed Scopus (15) Google Scholar). We then tested effects of conditional MYC inactivation in p493 LCLs, which have conditional MYC and EBNA2 alleles (Schuhmacher et al., 2001Schuhmacher M. Kohlhuber F. Hölzel M. Kaiser C. Burtscher H. Jarsch M. Bornkamm G.W. Laux G. Polack A. Weidle U.H. Eick D. The transcriptional program of a human B cell line in response to Myc.Nucleic Acids Res. 2001; 29: 397-406Crossref PubMed Scopus (265) Google Scholar). Cells were grown in the absence of doxycycline or 4-hydroxytamoxifen (4HT), where high levels of the exogenous MYC allele simulate BL physiology (Schuhmacher et al., 2001Schuhmacher M. Kohlhuber F. Hölzel M. Kaiser C. Burtscher H. Jarsch M. Bornkamm G.W. Laux G. Polack A. Weidle U.H. Eick D. The transcriptional program of a human B cell line in response to Myc.Nucleic Acids Res. 2001; 29: 397-406Crossref PubMed Scopus (265) Google Scholar) (Figures 2J and S2G). Doxycycline addition suppressed MYC and induced EBV lytic transcripts (Figure S2H). To confirm that on-target MYC editing caused lytic re-activation, we stably expressed sgRNA-resistant rescue cDNA encoding HA-tagged MYC or GFP. HA-MYC blocked induction of BZLF1, BMRF1, or gp350 upon Cas9 targeting of endogenous MYC in Akata BL or GM12878 LCL (Figures 2K, 2L, and S2I–S2K). Consistent with prior reports that MYC cDNA overexpression suppresses leaky lytic antigen expression in LCLs (Cutrona et al., 1995Cutrona G. Ulivi M. Fais F. Roncella S. Ferrarini M. Transfection of the c-myc oncogene into normal Epstein-Barr virus-harboring B cells results in new phenotypic and functional features resembling those of Burkitt lymphoma cells and normal centroblasts.J. Exp. Med. 1995; 181: 699-711Crossref PubMed Scopus (52) Google Scholar, Fais et al., 1996Fais F. Cutrona G. Ulivi M. Roncella S. Gagliardi M.C. Cornaglia-Ferraris P. Rowe M. Barnaba V. Ferrarini M. Lymphoblastoid cells transfected with c-myc: downregulation of EBV-lytic antigens and impaired response of autologous CD4+ T cells in vitro.Int. J. Cancer. 1996; 68: 810-816Crossref PubMed Scopus (8) Google Scholar), stable MYC expression significantly impaired BZLF1 and gp350 induction by anti-IgG cross-linking of Akata cells (Figure S2L). We next asked whether BZLF1 is required for induction of EBV lytic gene expression by MYC depletion. Functional BZLF1 KO Akata cells were generated by CRISPR, validated by T7 assay and by demonstration of impaired lytic response to anti-IgG challenge (Figures 2M and S2M), and then challenged by MYC KO. MYC depletion triggered gp350 expression in control, but not BZLF1-edited Akata (Figure 2N), suggesting that MYC acts at the level of BZLF1 to control the EBV lytic cycle. BZLF1 can activate its own promoter, the immediate early EBV BRLF1 promoter, or together with BRLF1 EBV, early gene promoters (Kenney and Mertz, 2014Kenney S.C. Mertz J.E. Regulation of the latent-lytic switch in Epstein-Barr virus.Semin. Cancer Biol. 2014; 26: 60-68Crossref PubMed Scopus (173) Google Scholar). Because MYC can block BZLF1-mediated transcriptional activation in epithelial cells (Lin et al., 2004Lin Z. Yin Q. Flemington E. Identification of a negative regulatory element in the Epstein-Barr virus Zta transactivation domain that is regulated by the cell cycle control factors c-Myc and E2F1.J. Virol. 2004; 78: 11962-11971Crossref PubMed Scopus (17) Google Scholar), we used two conditional BZLF1 expression systems to gain insights into the level at which MYC inhibits BZLF1 effects on BL EBV lytic genes. First, we tested the effect of lentivirus-driven MYC on BL with doxycycline-inducible HA-epitope-tagged BZLF1 expression. Conditional HA-BZLF1 upregulated BMRF1 and gp350 (Figure S3A) to similar levels in BL cells with GFP versus MYC expression, consistent with the bypass of EBV genomic BZLF1 promoter inhibition by MYC. We then asked whether lentivirus-driven MYC can block BZLF1 autoactivation of the EBV genome BZLF1 promoter, using P3HR-1 cells that stably express a BZLF1 4HT-binding domain fusion protein (BZLF1-HT). In the absence of 4HT, BZLF1-HT is retained in the cytosol, whereas it translocates to the nucleus and activates target genes upon 4HT addition. Conditional BZLF1-HT activation by 4HT induced untagged EBV genomic BZLF1 and BMRF1 at similar levels in cells with control GFP and MYC overexpression (Figure S3B). 4HT also significantly induced gp350 expression in cells with GFP versus MYC overexpression, albeit to a lesser extent in the presence of HA-MYC. These results suggest MYC acts at the level of the BZLF1 promoter. The cohesins SMC1A and RAD21 are implicated in control of Kaposi sarcoma-associated herpesvirus lytic reactivation (Chen et al., 2012Chen H.S. Wikramasinghe P. Showe L. Lieberman P.M. Cohesins repress Kaposi’s sarcoma-associated herpesvirus immediate early gene transcription during latency.J. Virol. 2012; 86: 9454-9464Crossref PubMed Scopus (61) Google Scholar, Li et al., 2014Li D.J. Verma D. Mosbruger T. Swaminathan S. CTCF and Rad21 act as host cell restriction factors for Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.PLoS Pathog. 2014; 10: e1003880Crossref PubMed Scopus (51) Google Scholar). To investigate a possible common gamma-herpesvirus role, we validated that Avana SMC1A sgRNAs induced EBV lytic genes (Figures S3C–S3F). SMC1A depletion induced expression of 77 EBV lytic genes (Figure 3A) and increased intracellular and DNase-resistant extracellular EBV genome copy number, suggestive of lytic replication (Figure 3B; Table S3). SMC1A depletion effects on BL fitness likely account for the lower CRISPR screen signal at Day 9, although surviving cells continued to expressed gp350 at Day 9 (Figure S3C). SMC1A depletion down-modulated MYC mRNA by nearly 2-fold (Figure 3A), consistent with cohesin roles in MYC promoter regulation (Rhodes et al., 2011Rhodes J.M. McEwan M. Horsfield J.A. Gene regulation by cohesin in cancer: is the ring an unexpected party to proliferation?.Mol. Cancer Res. 2011; 9: 1587-1607Crossref PubMed Scopus (30) Google Scholar). Notably, MYC mRNA and protein are highly labile, with half-lives of 20–30 min (Farrell and Sears, 2014Farrell A.S. Sears R.C. MYC degradation.Cold Spring Harb. Perspect. Med. 2014; 4: a014365Crossref PubMed Scopus (263) Google Scholar). Even in BL with stabilizing MYC mutations (Gregory and Hann, 2000Gregory M.A. Hann S.R. c-Myc proteolysis by the ubiquitin-proteasome pathway: stabilization of c-Myc in Burkitt’s lymphoma cells.Mol. Cell. Biol. 2000; 20: 2423-2435Crossref PubMed Scopus (365) Google Scholar), MYC half-life remains short, and MYC must be continually replenished. We therefore tested whether lentivirus-driven MYC cDNA expression could prevent lytic induction triggered by SMC1A depletion. MYC overexpression diminished BZLF1, BMRF1, and gp350 levels in SMC1a-edited cells (Figures 3C–3E). While additional roles in EBV latency maintenance are plausible, these data suggest that SMC1A controls EBV latency at least in part by supporting MYC expression. The facilitated chromatin transcription (FACT) complex, comprised of SUPT16H and SSRP1 subunits, enables RNA polymerase processivity by remodeling histones at sites of active transcription (Belotserkovskaya et al., 2003Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. FACT facilitates transcription-dependent nucleosome alteration.Science. 2003; 301: 1090-1093Crossref PubMed Scopus (645) Google Scholar, Saunders et al., 2003Saunders A. Werner J. Andrulis E.D. Nakayama T. Hirose S. Reinberg D. Lis J.T. Tracking FACT and the RNA polymerase II elongation complex through chromatin in vivo.Science. 2003; 301: 1094-1096Crossref PubMed Scopus (232) Google Scholar). Interestingly, cross-talk between FACT and MYC has been identified in multiple cell types as drivers of each other’s expression (Carter et al., 2015Carter D.R. Murray J. Cheung B.B. Gamble L. Koach J. Tsang J. Sutton S. Kalla H. Syed S. Gifford A.J. et al.Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma.Sci. Transl. Med. 2015; 7: 312ra176Crossref PubMed Scopus (95) Google Scholar). It was therefore of interest that the SSRP1 and SUPT16H FACT subunits each scored as CRISPR screen hits (Figures 1, 2A, S4A, and S4B). To gain insights into FACT roles in BL host and viral gene expression, we performed RNA-seq on control versus SUPT16H-depleted P3HR-1 cells. SUPT16H sgRNA suppressed MYC expression by 65% and induced 67 EBV lytic mRNAs (Figure 4A; Table S4) and lytic proteins (Figures S4D and S4E). SUPT16H sgRNA increased EBV genome copy number (Figure 4B), suggesting that FACT activity on host and/or viral genomes was important for the maintenance of EBV latency. SUPT16H depletion in GM12878 LCLs induced EBV lytic gene expression (Figure 4C), suggesting a more general FACT role in cells with wild-type MYC loci. Notably, our recent proteomic analysis demonstrated that EBV upregulates SUPT16H and SSRP1 protein abundance upon primary B cell infection (Figure S4C), consistent with an important FACT role in establishment of EBV latency, perhaps even from the earliest stages of B cell infection. We investigated whether FACT supports EBV latency through effects on MYC expression. Lentivirus-driven HA-MYC substantially blocked EBV lytic gene induction by SUPT16H KO (Figures 4D–4F). FACT associates with MYC (Heidelberger et al., 2018Heidelberger J.B. Voigt A. Borisova M.E. Petrosino G. Ruf S. Wagner S.A. Beli P. Proteomic profiling of VCP substrates links VCP to K6-linked ubiquitylation and c-Myc function.EMBO Rep. 2018; 19: e44754Crossref PubMed Scopus (53) Google Scholar), and additional FACT roles at the protein level, may underlie the significant but incomplete rescue of lytic gene silencing by MYC overexpression. As was observed for MYC KO, SUPT16H sgRNA failed to induce gp350 in BZLF1 edited cells (Figure S4F), consistent with FACT and/or MYC roles in control of BZLF1. The MYC family member N-MYC is a major driver of glioblastoma, where the curaxin small molecule FACT inhibitor CBL0137 blocks N-MYC expression, tumor initiation, and progression in a mouse model (Carter et al., 2015Carter D.R. Murray J. Cheung B.B. Gamble L. Koach J. Tsang J. Sutton S. Kalla H. Syed S. Gifford A.J. et al.Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma.Sci. Transl. Med. 2015; 7: 312ra176Crossref PubMed Scopus (95) Google Scholar). To investigate whether FACT might be a druggable target in BL EBV latency reversal, we tested CBL0137 effects on MYC and EBV lytic gene expression. CBL0137 depleted MYC and induced lytic antigens in BL and in LCLs established from three individuals (Figures 4G, 4H, and S4G). Although CBL0137 has pleotropic effects on host cells, its BL and LCL effects on EBV lytic reactivation were suppressed by HA-MYC overexpression (Figure S4H), suggesting that MYC is the functionally relevant target. These data support a key FACT role in EBV latency. We tested CBL0137 using a BL xenograft model, because latency I expression cannot typically be achieved in current humanized mouse EBV infection models. Following establishment of MUTU I BL tumors, mice were treated either with dextrose vehicle or CBL0137 at 10 or 60 mg/kg by tail vein injection (Figure 4I). 48 h later, one group of mice were sacrificed and tumors were characterized by RNA-seq. Remarkably, a single CBL0137 dose significantly induced expression of 77 EBV lytic genes in tumor xenografts (Figure 4J; Table S5). Dose-dependent xenograft induction of EBV lytic mRNAs was validated by qPCR (Figure 4K), and BZLF1 protein expression in numerous BL cells was evident (Figure 4L). Sequence-specific transcription factors including MYC recruit the STAGA/GCN5 histone acetyltransferase (HAT) to promote transcription initiation (Helmlinger and Tora, 2017Helmlinger D. Tora L. Sharing the SAGA.Trends Biochem. Sci. 2017; 42: 850-861Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). MYC and STAGA co-occupy many DNA sites (Hirsch et al., 2015Hirsch C.L. Coban Akdemir Z. Wang L. Jayakumaran G. Trcka D. Weiss A. Hernandez J.J. Pan Q. Han H. Xu X. et al.Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming.Genes Dev. 2015; 29: 803-816Crossref PubMed Scopus (52) Google Scholar, Wang and Dent, 2014Wang L. Dent S.Y. Functions of SAGA in development and disease.Epigenomics. 2014; 6: 329-339Crossref PubMed Scopus (90) Google Scholar, Zhang et al., 2014Zhang N. Ichikawa W. Faiola F. Lo S.Y. Liu X. Martinez E. MYC interacts with the human STAGA coactivator complex via multivalent contacts with the GCN5 and TRRAP subunits.Biochim. Biophys. Acta. 2014; 1839: 395-405Crossref PubMed Scopus (29) Google Scholar). It was therefore of interest that genes encoding six STAGA subunits were screen hits (Figures 1, 5A , and S5A), mapping to three of four STAGA modules (Figures 5A an" @default.
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• W3016787401 date "2020-05-01" @default.
• W3016787401 modified "2023-10-16" @default.
• W3016787401 title "MYC Controls the Epstein-Barr Virus Lytic Switch" @default.
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