Most adults are infected by Epstein-Barr virus (EBV), which establishes persistent infection in B cells. EBV nuclear antigen 3C (EBNA3C) is a latent viral protein that co-regulates many cellular genes, but little is known about the mechanisms by which EBNA3C acts as repressor or activator of gene expression. This study aimed to further explore molecular mechanisms that underlie EBNA3C host gene regulation. For this, four EBNA3C target genes – three repressed (COBLL1, ADAM28 and ADAMDEC1) and one induced (AICDA) – were selected based on a previous exon microarray on EBV-recombinant lymphoblastoid cell lines (LCL) carrying conditional EBNA3C (3CHT).
Using EBV recombinants to infect primary B cells, EBNA3C was shown to be the main regulator of all four genes. Surprisingly, regulation by EBNA3C over orders of magnitude followed highly exponential activation or repression profiles and required ~30 days after infection. This could be reproduced efficiently in the 3CHT LCLs by activating EBNA3C, proving the utility of these cells to replicate EBNA3C gene regulation. Analysis of chromatin immunoprecipitation (ChIP) coupled to deep-sequencing identified EBNA3C binding to distal regulatory elements at all four genes and detailed ChIP analysis of histone modifications and cellular factors was performed. Unexpectedly, recruitment and/or stabilisation of the DNA-sequence binding factor RBPJ was observed at the EBNA3C binding sites only when EBNA3C was functional. This challenges existing models of how RBPJ functions in EBV-regulated gene expression. EBNA3C failed to regulate all four genes when it is unable to bind to RBPJ. Recruitment of a polycomb protein, generally linked to gene repression, was observed at all four genes irrespective of whether repression or activation by EBNA3C occurred. Changes to histone modifications at these loci correlated well with gene expression and indicate a two-step mechanism for EBNA3C-mediated repression and suggest the activation is linked to a very complex pattern of chromatin looping.