EBNA1 binding and epigenetic regulation of gastrokine tumor suppressor genes in gastric carcinoma cells

Background: Epstein-Barr Virus (EBV) latently infects similar to 10% of gastric carcinomas (GC). Epstein-Barr Nuclear Antigen 1 (EBNA1) is expressed in EBV-associated GC, and can bind host DNA, where it may impact cellular gene regulation. Here, we show that EBNA1 binds directly to DNA upstream of the divergently transcribed GC-specific tumor suppressor genes gastrokine 1 (GKN1) and gastrokine 2 (GKN2). Methods: We use ChIP-Seq, ChIP-qPCR, and EMSA to demonstrate that EBNA1 binds directly to the GKN1 and GKN2 promoter locus. We generate AGS-EBV, and AGS-EBNA1 cell lines to study the effects of EBNA1 on GKN1 and GKN2 mRNA expression with or without 5' azacytidine treatment. Results: We show that gastrokine genes are transcriptionally silenced by DNA methylation. We also show that latent EBV infection further reduces GKN1 and GKN2 expression in AGS gastric carcinoma cells, and that siRNA depletion of EBNA1 partially alleviates this repression. However, ectopic expression of EBNA1 slightly increased GKN1 and GKN2 basal mRNA levels, but reduced their responsiveness to demethylating agent. Conclusions: These findings demonstrate that EBNA1 binds to the divergent promoter of the GKN1 and GKN2 genes in GC cells, and suggest that EBNA1 contributes to the complex transcriptional and epigenetic deregulation of the GKN1 and GKN2 tumor suppressor genes in EBV positive GC.