CTCF-mediated Genomic Effects of BART Region on Epstein-Barr Virus Chromatin 3D Structure in Gastric Carcinoma Cells

Abstract

AbstractEBV latent infection in gastric carcinoma (GC) cells is characterized by distinct viral gene expression programs. CCCTC-binding factor (CTCF) is a chromatin structural factor that has been involved in coordinated chromatin interactions between multiple loci of Epstein-Barr virus (EBV) genes. Here, we investigate the role of CTCF in regulating EBV gene expression and chromosome conformation in model of EBV-associated gastric carcinoma (EBVaGC). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) against CTCF revealed 16 CTCF binding sites (BS) in EBV genome of EBVaGC, SNU719 cells. Among the CTCF BSs, one site named as BARTp (BamHI A right transcript promoter) CTCF BS is located at upstream of 11.8-kb BART region (EBV genome: 139724-151554) and was not yet defined its biological functions in EBV life cycle. EBV BART encodes a complex miRNA cluster of highly spliced transcripts that is implicated in EBV cancer pathogenesis. This present study investigated the functional role of the CTCF binding site at BARTp (BARTp CTCF BS) in regulating EBV gene transcription and EBV three-dimensional (3D) genome structure as DNA loop maker. Circular chromatin confirmation capture (4C)-seq and chromatin confirmation capture (3C)-semi-quantitative(sq)PCR assays using SNU719 cells revealed that BARTp CTCF BS interacts with CTCF BSs of LMP1/2, Cp/OriP, and Qp in EBV genome. We generated mutations in BARTp CTCF BS (S13) in bacmids with (BART+) or without (BART−) the 11.8-kb BART transcript unit (B(+/−)). ChIP-qPCR assay demonstrated that CTCF binding was ablated from BARTp in EBV B(+/−) S13− genomes (mutant S13), elevated at several other sites such as LMP1, OriP, and Cp in EBV B(-) (BART−) S13− genome, and decreased at the same sites in EBV B(+) S13− genome. Infection assay showed that BARTp CTCF BS mutation reduced infectivity, while BART transcript deletion has no detectable effects. Gene expression tests showed that EBNA1 was highly downregulated in B(+/−) S13− EBVs related to B(+/−) S13+ EBVs (wild-type S13). LMP1 and BZLF1 were more downregulated in B(-) S13− EBV than B(+) S13− EBV. Taken together, these findings suggest that the CTCF binding and BART region contribute to EBV 3D genome structure via a cluster of DNA loops formed by BARTp CTCF BS (S13) and are important for coordinated viral gene expression and EBV infectivity.