Epstein–Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation

Henar Hernando,Abul B M M K Islam,Ignasi Forné,Axel Imhof,Laura Ciudad,Esteban Ballestar,Claire Shannon-Lowe,Javier Rodríguez-Ubreva

doi:10.1093/nar/gkt886

Abstract

Epstein–Barr virus (EBV) infects and transforms human primary B cells inducing indefinite proliferation. To investigate the potential participation of chromatin mechanisms during the EBV-mediated transformation of resting B cells we performed an analysis of global changes in histone modifications. We observed a remarkable decrease and redistribution of heterochromatin marks including H4K20me3, H3K27me3 and H3K9me3. Loss of H4K20me3 and H3K9me3 occurred at constitutive heterochromatin repeats. For H3K27me3 and H3K9me3, comparison of ChIP-seq data revealed a decrease in these marks in thousands of genes, including clusters of HOX and ZNF genes, respectively. Moreover, DNase-seq data comparison between resting and EBV-transformed B cells revealed increased endonuclease accessibility in thousands of genomic sites. We observed that both loss of H3K27me3 and increased accessibility are associated with transcriptional activation. These changes only occurred in B cells transformed with EBV and not in those stimulated to proliferate with CD40L/IL-4, despite their similarities in the cell pathways involved and proliferation rates. In fact, B cells infected with EBNA-2 deficient EBV, which have much lower proliferation rates, displayed similar decreases for heterochromatic histone marks. Our study describes a novel phenomenon related to transformation of B cells, and highlights its independence of the pure acquisition of proliferation.

Highlights

Quiescent differentiated B lymphocytes, like many other types of differentiated cells, have a relatively restricted transcriptome generated through the differentiation process
To investigate whether the described global chromatin changes are directly associated with Epstein–Barr virus (EBV)-mediated transformation and are not just a mere effect of B cell activation or the acquisition of a proliferative phenotype, we focused on the acquisition of changes in H3K27me3, H3K9me3 and H4K20me3 in B cells activated/stimulated with IL-4 and CD40L
Our results provide evidence that EBV-mediated transformation of B cells associates with both global decrease in heterochromatin histone post-translational modifications (H3K9me3, H3K27me3 and H4K20me3) and increased chromatin accessibility to endonucleases

Summary

Introduction

Quiescent differentiated B lymphocytes, like many other types of differentiated cells, have a relatively restricted transcriptome generated through the differentiation process. Constitutive heterochromatin comprises genepoor DNA containing highly repetitive sequences, is characterized by H4K20me as well as H3 and H4 hypoacetylation [2], and is independent of cell differentiation and proliferation status, as well as the level of transcriptional activity. Both types of heterochromatin are highly condensed and are less accessible to endonucleases than euchromatin, characteristic of transcriptionally active regions. There is a general notion that certain histone modifications are associated with a particular chromatin structure One of such examples is H3K27me that is generally associated with condensed facultative heterochromatin, less accessible to nucleases, and both associated with low levels of expression

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