Abstract
ChIP-seq performed on lymphoblastoid cell lines (LCLs), expressing epitope-tagged EBNA3A, EBNA3B or EBNA3C from EBV-recombinants, revealed important principles of EBNA3 binding to chromatin. When combined with global chromatin looping data, EBNA3-bound loci were found to have a singular character, each directly associating with either EBNA3-repressed or EBNA3-activated genes, but not with both. EBNA3A and EBNA3C showed significant association with repressed and activated genes. Significant direct association for EBNA3B loci could only be shown with EBNA3B-repressed genes. A comparison of EBNA3 binding sites with known transcription factor binding sites in LCL GM12878 revealed substantial co-localization of EBNA3s with RUNX3—a protein induced by EBV during B cell transformation. The beta-subunit of core binding factor (CBFβ), that heterodimerizes with RUNX3, could co-immunoprecipitate robustly EBNA3B and EBNA3C, but only weakly EBNA3A. Depletion of either RUNX3 or CBFβ with lentivirus-delivered shRNA impaired epitope-tagged EBNA3B and EBNA3C binding at multiple regulated gene loci, indicating a requirement for CBF heterodimers in EBNA3 recruitment during target-gene regulation. ShRNA-mediated depletion of CBFβ in an EBNA3C-conditional LCL confirmed the role of CBF in the regulation of EBNA3C-induced and -repressed genes. These results reveal an important role for RUNX3/CBF during B cell transformation and EBV latency that was hitherto unexplored.
Highlights
Despite being associated with various cancers––including several B cell lymphomas––Epstein-Barr Virus (EBV) infects, persistently and asymptomatically, >90% of the human population [1,2]
chromatin immunoprecipitation (ChIP)-seq was performed to study the localization of EBNA3A, EBNA3B and EBNA3C across the host genome
The lymphoblastoid cell lines (LCLs) used were created by infecting primary B cells from a single donor with recombinant EBV expressing epitope-tagged EBNA3A, EBNA3B or EBNA3C
Summary
Despite being associated with various cancers––including several B cell lymphomas––Epstein-Barr Virus (EBV) infects, persistently and asymptomatically, >90% of the human population [1,2]. The EBV life cycle is closely linked to the normal B cell differentiation pathway (reviewed in 3,4). Infection of mature B cells by EBV initially leads to their activation and differentiation into proliferating B blasts. Latency-associated genes expressed at this stage, termed latency III, encode six EBV nuclear antigen proteins, three latent membrane proteins, two small non-coding RNAs and several microRNAs. The viral nuclear antigens expressed include EBNA3A, EBNA3B and EBNA3C––a family of related, but non-redundant EBV proteins, expressed from three genes arranged in tandem within a complex transcription unit (reviewed in 5). In vitro, infected mature B cells give rise to lymphoblastoid cell lines (LCLs) that carry the EBV genome as an extra-chromosomal episome and remain in the latency III state expressing all latency associated EBV genes and resembling cycling, antigen-activated B blasts. The ease of obtaining continuously proliferating LCLs from virtually any genetic background has led to LCLs being used in diverse studies with extensive data being generated on genome-wide transcription factor localization, global chromatin dynamics and analyses of the global epigenetic landscape (see below)
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