Infecting over 95% of the population, the Epstein-Barr Virus (EBV) is a herpesvirus associated with many EBV-related diseases. However, EBV-associated gastric carcinoma (EBVaGC) is the most common malignancy resulting from EBV infection. All EBV-associated tumors express Epstein Barr Virus nuclear antigen 1 (EBNA1), a viral protein necessary to replicate and maintain the viral genome, but what distinguishes EBVaGCs from other malignancies is their extreme hypermethylation phenotype. Methylation is an epigenetic process, regulating gene expression through the enzymatic processes of DNA methyltransferases (DNMTs). Abnormal methylation levels can be a marker for tumor progression due to the silencing of certain genes like tumor suppressor and cell cycle genes. Currently, it is unknown if EBNA1 associates with DNMTs or if EBNA1 is necessary for hypermethylation seen in EBVaGCs. We hypothesize that EBNA1 and the DNMTs contribute to the hypermethylation of EBVaGCs. 

We first sought to determine the significance of DNMTs on the methylation landscape of EBV-positive gastric carcinoma cells. Reduced representation bisulfite sequencing of EBV-positive cancer cells undergoing siRNA knockdowns of DNMT1, DNMT3A, and DMNT3B revealed that DNMT1 significantly reduces methylation in 48% of the genome. Amongst that, 22% of promoter regions and 49% of intergenic regions also showed reduced methylation when DNMT1 expression is reduced. To identify EBNA1’s involvement in EBV-associated hypermethylation, dot blot analysis of EBV-negative cells transfected with EBNA1 suggests that EBNA1 expression may facilitate elevated global methylation levels. Knowing that DNMT1 expression and EBNA1 expression may both contribute to hypermethylation, we tested for potential interactions between the two proteins. Immunoprecipitation of EBNA1 followed by liquid chromatography with tandem mass spectrometry revealed a 13.01-fold change in EBNA1-DNMT1 association when comparing wild-type EBNA1 vs its empty vector. The interaction was further validated by a co-immunoprecipitation of DNMT1 in EBV-negative cells. To ascertain whether the EBNA1-DNMT1 interaction is necessary for mediating hypermethylation, their binding loci would have to be located. Consequently, we sought to map their binding domains via co-immunoprecipitation and discovered that DNMT1 may exhibit partial binding on the 400-440 and DNA binding domain of EBNA1. The results suggest that DNMT1 binds toward the C-terminal region of EBNA1.

In conclusion, EBNA1 and DNMT1’s impact on methylation is a promising discovery that could help elucidate how EBV-positive gastric carcinomas methylate the human genome. Prospective plans involve ascertainment of the complete binding domains between EBNA1 and DNMT1 via mutational analysis. Construction of the non-binding mutants and a DNA methylation analysis would then be performed to review the importance of the EBNA1-DNMT1 interaction in the hypermethylation of EBV-positive gastric carcinomas. RNA-seq analysis of viral and host genes methylated by DNMT1 would also be conducted to evaluate which genes DNMT1 is responsible for expressing.