Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

Abstract

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets ( WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors.

Author Summary

Epstein-Barr virus (EBV) is associated with numerous cancers. The ability of the virus to infect B-cells and convert them from short-lived into immortal cells is the key to its cancer-promoting properties. A small number of EBV transcription factors are required for immortalization and act in concert to drive cell growth by deregulating the expression of cellular genes through largely unknown mechanisms. We have demonstrated that four of these key transcription factors function cooperatively by targeting common genes via long-range enhancer elements and modulating their looping interactions with gene promoters. Specifically we show that gene repression by the EBV EBNA 3 family of proteins can be mediated through the modulation of enhancer-promoter looping. Our results also reveal that different subsets of EBNA 3 proteins are bound at different genes and that this differential binding can vary in lymphoma cells compared to cells immortalized in culture, indicating that cell-background-specific gene regulation may be important in lymphoma development. Our results demonstrate how cellular genes can be deregulated by an oncogenic virus through modulation of enhancer-promoter looping with the specificity of binding by viral transcription factors controlling cellular reprogramming in a gene and cell-type specific manner.