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.

Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

“…In total, we identified 6,791 distinct genomic sites bound by EBNA3 proteins in LCLs. Although this estimate is comparable to that reported for EBNA3 binding in Mutu III cells (33), only 1,466 (21%) of the sites are in common (see Fig. S1 in the supplemental material).…”

“…For EBNA3B, 21% are shared with EBNA3A, 22% with EBNA3C, and 37% with EBNA2. McClellan et al found that 80% of the genes closest to an EBNA3-bound site were also the closest genes to an EBNA2-bound site (33). Using their approach, we found only 36% of EBNA3-bound genes to be EBNA2 cobound, though this proportion rises to 50% for EBNA3B-bound genes (see Table S4 in the supplemental material).…”

“…Chromatin immunoprecipitation (ChIP) experiments have led to a rapid advance in our understanding of this problem. McClellan et al demonstrated that EBNA3 proteins bound to sites distinct from those of EBNA2 in the Mutu III Burkitt lymphoma cell line (33). However, due to cross-reactivity of the chromatin immunoprecipitation with deep sequencing (ChIP-seq) antibody for multiple EBNA3 proteins, they could only distinguish between EBNA3A, EBNA3B, and EBNA3C at specific EBNA3-bound sites by ChIP-quantitative PCR (qPCR).…”

A Single Amino Acid in EBNA-2 Determines Superior B Lymphoblastoid Cell Line Growth Maintenance by Epstein-Barr Virus Type 1 EBNA-2

“…The best characterized of these cell proteins is RBP-J (also known as CSL), but PU.1 (7,8), ATF/CRE (9), and EBF1 binding sites (10) have also been shown to mediate EBNA-2 function at certain promoters. Early studies of EBNA-2 function fo-cused on viral promoters or artificial promoters where the binding sites are close to the transcription start site (TSS), but recent chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) studies in human B cell lines (10,11) have shown that EBNA-2 binding sites at cellular gene targets are predominantly located far away from gene TSSs, at distances up to 100 kb up-or downstream.…”

Alzheimer’s Disease of the Immune System: A New Variant of Immune Deficiency

“…In contrast to allergic disease, it has generally been agreed that autoimmune conditions are mediated by an overactive Th1 arm of the immune system. More recently, there has been considerable debate about the role of Th2 in autoimmune conditions and the relative Th1/Th2 balance, especially in those mediated by autoantibodies such as systemic lupus erythematosis (SLE) and rheumatoid arthritis [13].…”