Epstein-Barr virus oncoprotein super-enhancers control B cell growth

Abstract

Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ∼1,800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals, characteristic of super-enhancers, and were designated "EBV super-enhancers." EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, which enable LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had high co-occupancy of STAT5 and NFAT transcription factors (TFs). EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare they have no competing financial interests.

Copyright © 2015 Elsevier Inc. All rights reserved.

Figures

Figure 1. EBNA2 Super-enhancers

(A) Rank-order of EBNA2 ChIP-seq signals for all EBNA2 sites. Overall, 888 EBNA2 super-enhancer sites have >23 fold higher ChIP-seq signals than the average signals of ~17,000 other EBNA2 sites. These EBNA2 super-enhancer sites are annotated to their nearest cell gene. EBNA2 super-enhancer-associated cell genes particularly important for LCL growth or survival are indicated. (B) EBNA2, other TFs, and histone modification ChIP-seq signals at super-enhancers near the MYC locus are shown. Numbers near gene names indicate tag density. A red arrow indicates the MYC TSS. Magnified views of EBNA2 super-enhancers (−525 and −428kb) of MYC are shown (blue arrows and red boxes). (C) Average ChIP-seq signals for H3K27ac and H3K4me1 in 80kb windows around EBNA2 super and typical enhancers are shown. Red indicates EBNA2 super- enhancers and blue indicates typical enhancers.

Figure 2. EBV super-enhancers

EBV super-enhancers are defined by high H3K27ac signals and the presence of all EBNAs and NF-κB subunits. (A) EBNA2, EBNALP, EBNA3A, EBNA3C, NF-κB subunits RELA, RELB, cRel, p50, and p52 are all significantly present at the EBNA2 super-enhancer −525kb of MYC. (B)1771 sites with significant EBV oncoproteins and NF-κB subunit binding are ranked by H3K27ac signals. 187 EBV super-enhancers with 4 fold higher H3K27ac signals than EBV typical enhancers are annotated to their nearest genes. Genes important for LCL growth and survival are indicated. (C) Anchor plots for H3K27ac, BRD4, and PolII show substantially higher and broader signals (normalized coverage) at EBV super-enhancers than at EBV typical enhancers. (D) ChIP-seq signals for virus and cell TFs and histone modifications at the BCL2 locus.

Figure 3. Transcription Factors that differentiate EBV super-enhancers from EBV typical enhancers

(A) TF Motifs enriched at EBV super-enhancers over other EBV enhancers. Other EBV enhancers have more than 1 EBV TF or NF-κB subunit, but less than all 9. (B) Boxplots of TF ChIP-seq signals density at EBV super-enhancers and typical enhancers. Signals for NFAT, STAT5, YY1, and ETS1 at EBV super-enhancers are significantly higher than typical enhancers (Wilcoxon Rank-Sum Test p values NFATc1: p−16, STAT5: p<2×10−16, YY1: p<1×10−14, and ETS1: p<3.8×10−6, * indicates p<10−6). (C) Anchor plots of normalized TF ChIP-seq signals (coverage) around EBV super-enhancers and EBV typical enhancers. (D) Boxplots of EBNA2, RBPJ, and EBNA3A ChIP-seq signals (density) at EBV super-enhancers and typical enhancers. EBNA2 and RBPJ ChIP-seq signals at EBV super-enhancers are significantly higher than EBV typical enhancers (Wilcoxon Rank-Sum Test p values: EBNA2 p<7.3×10−12, RBPJ p<1.4×10−10, EBNA3A p<0.019). For boxplots, Middle line indicates the median. The edges indicate the first and the third quartile. The whiskers indicate minimum and maximum.

Figure 4. EBV super-enhancer-associated genes have higher expression levels than typical enhancer associated genes

Boxplots of LCL EBV super-enhancer associated, EBV typical enhancer associated, and other EBV enhancer associated RNA-seq gene expression levels (Fragments Per Kilobase Of Exon Per Million Fragments Mapped (FPKM)) are shown. EBV super- enhancer associated genes are expressed significantly higher than EBV typical enhancer associated genes (Wilcoxon Rank- Sum Test p−10). EBV Super-Enhancer associated genes or EBV typical enhancer associated genes are expressed significantly higher than EBV other enhancer associated genes (Wilcoxon Rank- Sum Test p<2×10−16). Middle line indicates the median. The edges indicate the first and the third quartile. The whiskers indicate minimum and maximum.

Figure 5. H3K27ac signals at EBV super-enhancer and typical enhancers in LCLs and RBLs

(A) GM12878 LCL and tonsil RBL H3K27ac normalized coverage +/− 4kb of EBV super- enhancers, typical enhancers, and other enhancers are shown. (B) Overlap between RBL, LCL, and EBV super-enhancer associated genes. Genes important for B cell or LCL functions are indicated.

Figure 6. Perturbation of super-enhancer constituents reduces cell growth, gene expression, and H3K27ac signals

All Error bars represent SD. (A) Left: 24 hours post DMSO or 500nM JQ1 treatment of GM12878 LCLs, MYC mRNA levels were measured using qRT-PCR and normalized to GAPDH. MYC mRNA levels in DMSO treated cells were set to 1. Right: CFSE staining of GM12878 LCLs treated with DMSO or 500nM JQ1 for one or three days. (B) Left: Normalized MYC and MIR21 RNA levels, in conditional EBNA2 LCLs, grown under permissive (+) or non-permissive (−) conditions for EBNA2 expression. Right: H3K27ac levels at the −525kb MYC and MIR21 EBV super-enhancers, as determined by ChIP qPCR. EBNA2 (+) condition was set to 1. (C) EBV super-enhancers associate with RUNX3, MIR21, and MIR155. Super-enhancers are high-lighted by red lines with TFs signals indicated on the left. (D) Left: Normalized BCL2, RUNX3, and MIR155 mRNA levels in LCLs with high NF-κB activity (hi) versus low NF-κB activity (lo). Right: H3K27ac ChIP qPCR at BCL2, RUNX3, and MIR155 EBV super-enhancers. The NF-κB hi condition was set to 1.

Figure 7. EBV super-enhancer Model

RBLs have broad enhancer regions with moderate H3K27ac signals. These enhancers are occupied by a limited repertoire of cell TFs to maintain chromatin accessibility. Upon EBV infection, EBNA and LMP1 oncoproteins are expressed. LMP1 activates NF-κB. All EBNAs and NF-κB subunits then co-occupy primed B cell enhancer sites, recruit additional cell TFs, and nucleate EBV super-enhancers to up-regulate transcription.