Abstract
Epstein-Barr virus (EBV) causes a persistent infection in human B cells by establishing specific transcription programs to control B cell activation and differentiation. Transcriptional reprogramming of EBV infected B cells is predominantly driven by the action of EBV nuclear antigens, among them the transcriptional repressor EBNA3A. By comparing gene expression profiles of wt and EBNA3A negative EBV infected B cells, we have previously identified a broad array of cellular genes controlled by EBNA3A. We now find that genes repressed by EBNA3A in these cells are significantly enriched for the repressive histone mark H3K27me3, which is installed by Polycomb group (PcG) proteins. This PcG-controlled subset of genes also carries H3K27me3 marks in a variety of other tissues, suggesting that the commitment to PcG silencing is an intrinsic feature of these gene loci that can be used by EBNA3A. In addition, EBNA3A targets frequently reside in co-regulated gene clusters. To study the mechanism of gene repression by EBNA3A and to evaluate the relative contribution of PcG proteins during this process, we have selected the genomic neighbors CXCL10 and CXCL9 as a model for co-repressed and PcG-controlled genes. We show that EBNA3A binds to CBF1 occupied intergenic enhancers located between CXCL10 and CXCL9 and displaces the transactivator EBNA2. This impairs enhancer activity, resulting in a rapid transcriptional shut-down of both genes in a CBF1-dependent manner and initiation of a delayed gain of H3K27me3 marks covering an extended chromatin domain. H3K27me3 marks increase gradually and are maintained by EBNA3A. Our study provides direct evidence that repression by EBNA3A requires CBF1 and that EBNA3A and EBNA2 compete for access to CBF1 at identical genomic sites. Most importantly, our results demonstrate that transcriptional silencing by EBNA3A precedes the appearance of repressive PcG marks and indicate that both events are triggered by loss of enhancer activity.
Highlights
Epstein Barr virus (EBV) is a ubiquitous human herpesvirus that establishes a persistent latent infection in more than 90% of the adult human population
We report that cellular genes repressed by the Epstein-Barr virus (EBV) nuclear antigen 3A (EBNA3A) in EBV infected B cells frequently form contiguous clusters in the human genome and are committed to epigenetic silencing by Polycomb group (PcG) proteins
We show that EBNA3A binds to intergenic enhancers located between CXCL10 and CXCL9 and displaces the transactivator EBNA2
Summary
Epstein Barr virus (EBV) is a ubiquitous human herpesvirus that establishes a persistent latent infection in more than 90% of the adult human population. EBV infection of primary human B cells causes cell cycle entry of the infected cells This process is controlled by the concerted action of 6 latent EBV nuclear antigens (EBNAs) and 3 latent membrane proteins (LMPs), which mimic cellular functions required for B cell proliferation and differentiation [3,4]. The latent viral gene expression program is dynamic It switches to at least two additional distinct viral gene expression patterns (latency I and II), which reprogram the differentiation state of the infected host B cells to become resting memory B cells that serve as a life-long reservoir for the virus [5]. The infected B cells convert into permanently proliferating lymphoblastoid B cell lines (LCLs), which phenocopy activated B cell blasts and are frozen at that state of differentiation as long as all 9 latent proteins are expressed (latency III)
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