H3K27me3 ChIP-Seq in LCLs cells with conditionally activated EBNA3C
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE109221 | GEO | 2018/01/17
SECONDARY ACCESSION(S): PRJNA430249
REPOSITORIES: GEO
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Project description:Epstein-Barr Virus (EBV) encoded Nuclear Antigens (EBNAs) and virus activated NF-kB subunits mostly bind to enhancers in EBV transformed lymphoblastoid cells lines (LCLs). Using LCL 3D genome organization map that links EBV enhancers to promoters, we built the most comprehensive virus regulome. EBV regulome contained 1992 genes and enhancers directly linked to them. ~30% of genes essential for LCL growth were linked to EBV enhancers. CRISPR knock out of EBNA2 sites significantly reduced their target gene expression. Additional EBV super-enhancer (ESE) targets including MCL1, IRF4, and EBF were identified. MYC ESEs looping to MYC TSS was dependent on EBNAs. CRISPR deletions of MYC ESEs greatly reduced MYC expression and LCL growth. EBNA3A/3C altered CDKN2A/B spatial organization to suppress senescence. EZH2 inhibition decreased the looping at the CDKN2A/B loci and reduced LCL growth. This study defines the most comprehensive host-pathogen interactions on the spatial organization of chromatin during infection and cancer.
2017-09-01 | GSE101426 | GEO
Project description:Epstein-Barr Virus (EBV) encoded Nuclear Antigens (EBNAs) and virus activated NF-B subunits mostly bind to enhancers in EBV transformed lymphoblastoid cells lines (LCLs). Using LCL 3D genome organization map that links EBV enhancers to promoters, we built the most comprehensive virus regulome. EBV regulome contained 1992 genes and enhancers directly linked to them. ~30% of genes essential for LCL growth were linked to EBV enhancers. CRISPR knock out of EBNA2 sites significantly reduced their target gene expression. Additional EBV super-enhancer (ESE) targets including MCL1, IRF4, and EBF were identified. MYC ESEs looping to MYC TSS was dependent on EBNAs. CRISPR deletions of MYC ESEs greatly reduced MYC expression and LCL growth. EBNA3A/3C altered CDKN2A/B spatial organization to suppress senescence. EZH2 inhibition decreased the looping at the CDKN2A/B loci and reduced LCL growth. This study defines the most comprehensive host-pathogen interactions on the spatial organiz ation of chromatin during infection and cancer.
2019-07-01 | GSE101666 | GEO
Project description:Epstein Barr virus (EBV) nuclear antigen 3C (EBNA3C) is an essential transcription factor for initiating and maintaining human B lymphocyte transformation to lymphoblastoid cell lines (LCLs). To comprehensively identify EBNA3C regulated cell genes in LCLs, oligonucleotide arrays were used to compare RNA abundances in 3 different LCLs transformed by an EBV that conditionally expresses EBNA3C. Cell RNA levels were assessed in actively growing LCLs, under non-permissive or permissive conditions or under non-permissive conditions after transcomplementation with wild type EBNA3C. A two-way ANOVA model with covariates including the 3 different clone effects and the 3 EBNA3C expression levels, identified 550 EBNA3C regulated genes, with False Discovery Rate <0.01 and >1.5 fold change. A seeded Bayesian network analysis of the 80 most significantly EBNA3C regulated genes that changed >1.5 fold, positioned RAC1, LYN and TNF upstream of other EBNA3C regulated genes. Further, Gene Set Enrichment Assay (GSEA) identified EBNA3C regulated genes to be enriched for MAP kinase signaling, cytokine-cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecule effects, implicating these pathways in LCL growth or survival. Moreover, 106 EBNA3C regulated genes could be placed in protein interaction networks. Since CXCL12 and CXCR4 signaling are implicated in LCL growth and were EBNA3C up-regulated, up-regulation of CXCL12 was validated by qRT-PCR and effects on induced LCL migration were confirmed. EBNA3C regulated genes significantly overlapped with EBNA2 and EBNA3A regulated genes, consistent with a central role for RBP/CSL in these effects.
2011-01-06 | GSE24362 | GEO
Project description:Epstein Barr virus (EBV) nuclear antigen 3C (EBNA3C) is an essential transcription factor for initiating and maintaining human B lymphocyte transformation to lymphoblastoid cell lines (LCLs). To comprehensively identify EBNA3C regulated cell genes in LCLs, oligonucleotide arrays were used to compare RNA abundances in 3 different LCLs transformed by an EBV that conditionally expresses EBNA3C. Cell RNA levels were assessed in actively growing LCLs, under non-permissive or permissive conditions or under non-permissive conditions after transcomplementation with wild type EBNA3C. A two-way ANOVA model with covariates including the 3 different clone effects and the 3 EBNA3C expression levels, identified 550 EBNA3C regulated genes, with False Discovery Rate <0.01 and >1.5 fold change. A seeded Bayesian network analysis of the 80 most significantly EBNA3C regulated genes that changed >1.5 fold, positioned RAC1, LYN and TNF upstream of other EBNA3C regulated genes. Further, Gene Set Enrichment Assay (GSEA) identified EBNA3C regulated genes to be enriched for MAP kinase signaling, cytokine-cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecule effects, implicating these pathways in LCL growth or survival. Moreover, 106 EBNA3C regulated genes could be placed in protein interaction networks. Since CXCL12 and CXCR4 signaling are implicated in LCL growth and were EBNA3C up-regulated, up-regulation of CXCL12 was validated by qRT-PCR and effects on induced LCL migration were confirmed. EBNA3C regulated genes significantly overlapped with EBNA2 and EBNA3A regulated genes, consistent with a central role for RBP/CSL in these effects. RNAs from three different Lymphoblastoid Cell Lines(LCLs) expressing conditional EBNA3C grown under permissive or non-permissive conditions for 7 days; the same LCLs transcompletemented with EBNA3C expressed in trans at full wild-type level were used to identify EBNA3C regualted cellular genes. Total cell RNAs were hybrdized to Affymetrix U-133 Plus 2.0 microarrays. A two way ANOVA model was developed with covariates including the 3 different clone effects and the 3 EBNA3C expression levels and identified 550 EBNA3C regulated genes.
2011-01-06 | E-GEOD-24362 | biostudies-arrayexpress
Project description:Epstein-Barr virus (EBV) is able to drive the transformation of B-cells, resulting in the generation of lymphocyte cell lines (LCLs) in vitro. EBV nuclear proteins EBNA3A and EBNA3C are necessary for efficient transformation, while EBNA3B is dispensable. We describe a transcriptome analysis of LCLs generated with EBNA3B knockout (3BKO) EBV (made in the B95-8 BAC generated by Delecluse and Hammerschmidt) and comparable LCLs made with the parental B95-8 BAC (wtBAC).<br>Peripheral blood cells from three donors were infected with two 3BKO virus preparations, and two wtBAC virus preps. LCLs were grown out, and RNA extracted after 3-6 weeks (depending how long the outgrowth took). Total RNA was ribominus treated and then labelled and hybridised to Affymetrix human exon 1.0ST microarrays.
2010-10-30 | E-MEXP-2768 | biostudies-arrayexpress
Project description:Deciphering the molecular pathogenesis of virally induced cancers is challenging due, in part, to the heterogeneity of both viral and host gene expression. Epstein-Barr Virus (EBV) is a ubiquitous herpesvirus prevalent in B-cell lymphomas of the immune suppressed. EBV infection of primary human B cells leads to their immortalization into lymphoblastoid cell lines (LCLs) serving as a model of these lymphomas. In previous studies, our lab has described a temporal model for immortalization with an initial phase characterized by expression of the Epstein-Barr Nuclear Antigens (EBNAs), high c-Myc activity, and hyper-proliferation in the absence of the Latent Membrane Proteins (LMPs), called latency IIb. This is followed by the long-term outgrowth of LCLs expressing the EBNAs along with the LMPs, particularly the NFkB-activating LMP1, defining latency III. LCLs, however, express a broad distribution of LMP1 such that a subset of these cells expresses LMP1 at levels seen in latency IIb, making it difficult to distinguish these two latency states. In this study, we performed mRNA-Seq on early EBV-infected latency IIb cells and latency III LCLs sorted by NFkB activity. We found that latency IIb transcriptomes clustered independently from latency III independent of NFkB. We identified and validated mRNAs defining these latency states. Indeed, we were able to distinguish latency IIb cells from LCLs expressing low levels of LMP1 using multiplex RNA-FISH targeting EBV EBNA2, LMP1, and human CCR7 or MGST1. This study defines latency IIb as a bona fide latency state independent from latency III and identifies biomarkers for understanding EBV-associated tumor heterogeneity
2019-06-04 | GSE132138 | GEO
Project description:Epstein-Barr virus (EBV) is able to drive the transformation of B-cells, resulting in the generation of lymphocyte cell lines (LCLs) in vitro. EBV nuclear proteins EBNA3A and EBNA3C are necessary for efficient transformation, while EBNA3B is dispensable. We describe a transcriptome analysis of BL31 cells infected with a series of EBNA3-knockout EBVs, including one deleted for all three EBNA3 genes. BL31 cells were infected with a variety of EBV mutants and their revertants, generated in the EBV-BAC system developed by Henri-Jacques Delecluse and Wolfgang Hammerschmidt. These are mutants of the EBV EBNA3 genes - specifically individual knockouts of EBNA3A, EBNA3B and EBNA3C (in this case the first exon of EBNA3C was retained) and a deletion of the total EBNA3 locus (ie all three EBNA3 genes missing).<br>RNA from cultures of cell lines carrying these mutant EBVs (3-4 independent cell lines for each mutant; 2-3 for each revertant, plus three each for uninfected BL31 and parental B95-8 BAC-infected lines) was isolated using RNeasy midi kits (qiagen) and then ribominus-treated and then labelled and hybridised to Affymetrix Human Exon 1.0ST arrays. Array analysis was performed using the MMBGX algorithm developed by Ernest Turro and Alex Lewin. <br>
2010-10-30 | E-MEXP-2767 | biostudies-arrayexpress
Project description:The genome of Epstein-Barr virus (EBV) encodes 86 proteins but only a limited set is expressed in EBV-growth transformed B cells, termed lymphoblastoid cell lines (LCLs). These cells proliferate via the concerted action of EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), some of which are rate limiting to establish a stable homeostasis of growth promoting and anti-apoptotic activities. We show here that EBV mutants, which lack the EBNA-3A gene, are impaired but can still initiate cell-cycle entry and proliferation of primary human B cells in contrast to an EBNA-2-deficient mutant virus. Surprisingly and in contrast to previous reports, these viral mutants are attenuated in growth transformation assays but give rise to permanently growing EBNA-3A negative B cell lines which exhibit reduced proliferation rates and elevated levels of apoptosis. Expression profiles of EBNA-3A deficient LCLs are characterized by 129 upregulated and 167 downregulated genes, which are significantly enriched for genes involved in apoptotic processes or cell cycle progression like the tumor suppressor gene p16/INK4A or might contribute to essential steps in the viral life cycle. In addition EBNA-3A cellular target genes remarkably overlap with previously identified targets of EBNA-2. Experiment Overall Design: Comparison of gene expression profiles from lymphoblastoid cell lines (LCLs) infected with EBNA-3A positive or negative EBV. LCLs were generated from three donors. A total of five EBNA-3A positive and nine negative LCLs were analysed.
2009-09-13 | E-GEOD-17908 | biostudies-arrayexpress
Project description:ChIP-seq performed on lymphoblastoid cell lines (LCLs), expressing epitope-tagged EBNA3A, EBNA3B or EBNA3C from EBV-recombinants, revealed important principles of EBNA3 binding to chromatin. When combined with global chromatin looping data, EBNA3-bound loci were found to have a singular character, each directly associating with either EBNA3-repressed or EBNA3-activated genes, but not with both. EBNA3A and EBNA3C showed significant association with repressed and activated genes. Significant direct association for EBNA3B loci could only be shown with EBNA3B-repressed genes. A comparison of EBNA3 binding sites with known transcription factor binding sites in LCL GM12878 revealed substantial co-localization of EBNA3s with RUNX3 – a protein induced by EBV during B cell transformation. The beta-subunit of core binding factor (CBFβ), that heterodimerizes with RUNX3, could co-immunoprecipitate robustly EBNA3B and EBNA3C, but only weakly EBNA3A. Depletion of either RUNX3 or CBFβ with lentivirus-delivered shRNA impaired epitope-tagged EBNA3B and EBNA3C binding at multiple regulated gene loci, indicating a requirement for CBF heterodimers in EBNA3 recruitment during target-gene regulation. ShRNA-mediated depletion of CBFβ in an EBNA3C-conditional LCL confirmed the role of CBF in the regulation of EBNA3C-induced and -repressed genes. These results reveal an important role for RUNX3/CBF during B cell transformation and EBV latency that was hitherto unexplored.
2016-11-12 | GSE88729 | GEO
Project description:This study compares cellular and viral gene expression in a cord blood-humanized (CBH) mouse model infected with wild-type Epstein-Barr virus (WT), versus a mutant EBV deleted in the latent viral EBNA3C gene (∆3C). EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently infected cells, is required for EBV transformation of B cells in vitro. However, many EBV+ lymphomas in humans do not express this protein, suggesting that it may be less important in vivo or that cellular mutations and/or signaling pathways may obviate the need for EBNA3C. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C-deleted EBV transforms B cells containing a p16 germline mutation in vitro. We found that the ∆3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and ∆3C viruses induced monoclonal B-cell lymphomas (without somatic hyper-mutation) that were infiltrated by polyclonal T cells. In comparison to WT-infected tumors, ∆3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that ∆3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. Unexpectedly, ∆3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo.
2018-04-13 | GSE113070 | GEO