Epigenetic silencing of tumor suppressor genes during in vitro Epstein–Barr virus infection

Abstract

DNA-methylation at CpG islands is one of the prevalent epigenetic alterations regulating gene-expression patterns in mammalian cells. Hypo- or hypermethylation-mediated oncogene activation, or tumor suppressor gene (TSG) silencing mechanisms, widely contribute to the development of multiple human cancers. Furthermore, oncogenic viruses, including Epstein–Barr virus (EBV)-associated human cancers, were also shown to be influenced by epigenetic modifications on the viral and cellular genomes in the infected cells. We investigated EBV infection of resting B lymphocytes, which leads to continuously proliferating lymphoblastoid cell lines through examination of the expression pattern of a comprehensive panel of TSGs and the epigenetic modifications, particularly methylation of their regulatory sequences. EBV infection of primary B lymphocytes resulted in global transcriptional repression of TSGs through engagement of hypermethylation. Therefore, CpG methylation profiles of TSGs may be used as a prognostic marker as well as development of potential therapeutic strategies for controlling acute infection and EBV-associated B-cell lymphomas.

Epigenetic Impact on EBV Associated B-Cell Lymphomagenesis

“…The list includes ABCB1, AKT1, ATM, BCL2, BRAF, CADM1, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CTNNB1, DAPK1, DIRAS3, EGFR, ERBB2, HIC1, HRAS, ING1, MDM2, MGMT, MLH1, MYCN, NF1, NF2, NME1, OPCML, PRDM2, PTCH1, RB1, RET, RUNX3, SCGB3A1, SFRP2, SH3PXD2A, SMARCB1, TGFB1, TGFBR2, THBS1, TP53 and TP73. While only six TSGs—APC, HOXA1, MYC, SFRP1, SOCS1, and WT1—demonstrated a decreasing trend in methylation, 25 TSGs, including ABL1, CAV1, CCND2, CDH13, CDX2, DKK3, DLC1, E2F1, GSTP1, IGF2, IGF2R, NFKB1, PTEN, PTGS2, RASSF1, SFN, SLC5A8, MEN1, TERT, TIMP3, TSC1, TSC2, VHL, WWOX, and ZMYND10, fall in rather an unusual category where methylation status was either significantly elevated by two days post-infection followed by down-regulation, or in reverse [82]. The importance of second-day post-infection was elaborated as EBV hyper-proliferation [83], which might explain this fluctuation during the course of EBV infection in RBLs.…”

“…Likewise, several TSGs’ hypermethylation are also demonstrated in EBV-associated cancers including multiple B-cell lymphomas [28]. Recently we have demonstrated that EBV infection of RBLs resulted in overall transcriptional repression of a panel of TSGs through recruiting hypermethylation activity [82]. Among 96 gene promoter sequences assessed, approximately 50% of the TSGs were shown to have an increasing trend of methylation in response to EBV infection.…”

Epstein–Barr Virus: Diseases Linked to Infection and Transformation

“…However, only EBNA3A and EBNA3C are essential for viral transformation of B-lymphocytes, and all appear to significantly contribute to maintaining the viability of transformed cells, suggesting an important role in oncogenesis (Tomkinson et al, 1993). EBNA3C has been reported to interact with many cellular factors (Robertson et al, 1995; Choudhuri et al, 2007; Saha et al, 2011, 2015; Banerjee et al, 2013, 2014; Jha et al, 2013a, 2014, 2015a,b,c). One of these cellular antigens is RBP-JK, which binds to all the EBNA3 proteins (Robertson et al, 1995, 1996).…”

“…The available database included cellular players that are significantly associated ( p -value is ∼10 -13 ) with transformation and proliferation of tumor cell lines ( p -value is ∼10 -12 ) ( Figure 2 ). Our lab and others have demonstrated that EBNA1, EBNA2, EBNA3C, and LMP1 are potent EBV antigens associated with a number of transcription factors including E2F1, Tp73, MDM2, Tp53, IRF4, Myc, HDAC1, and GMNN (Robertson et al, 1995, 1996; Kaiser et al, 1999; Ma et al, 2000; Plaxco et al, 2000; Zhou et al, 2000, 2005; Knight et al, 2005; Liu et al, 2005; Saridakis et al, 2005; Choudhuri et al, 2007; Ding et al, 2007; Chau et al, 2008; Allday, 2009; Forte and Luftig, 2009; Borestrom et al, 2012; Frappier, 2012a; Accardi et al, 2013; Banerjee et al, 2013; Tursiella et al, 2014; Jha et al, 2015b,c; Saha et al, 2015), kinases including Aurora Kinase B, Pim1, GSK3B (Saha et al, 2011; Jha et al, 2013a; Banerjee et al, 2014; Edwards et al, 2015), and forms complexes with cellular factors whcih includes the tumor suppressor and oncoproteins, Rb, Skp2, and CyclinD (Arvanitakis et al, 1995; Ruf and Sample, 1999; Prathapam et al, 2002; Knight et al, 2005; Choudhuri et al, 2007; Kang et al, 2011; Saha and Robertson, 2011; Saha et al, 2011, 2015; Sun et al, 2015; Figure 2 ). Cell transformation is regulated through a broad range of interacting partners including Rb, E2F1, Aurora Kinase B, Tp73, Mdm2, Pim1, Tp53, IRF4, Skp2, and Myc (Halazonetis and Kandil, 1991; Lahoz et al, 1994; Hoang et al, 1995; Pirollo et al, 1997; De Laurenzi et al, 1998; Givol et al, 1998; Mochizuki et al, 1999; Lin et al, 2000; Gstaiger et al, 2001; Kanda et al, 2005; Zhang et al, 2005; Xu et al, 2011).…”

“…In several studies utilizing primary or immortalized B-cells infected with wild type EBV isolated from BAC clones, we monitored the expression of genes at transcript levels during the early days of infection ( Figure 1 ; Halder et al, 2009; Saha et al, 2011, 2015; Banerjee et al, 2013; Jha et al, 2013a, 2015b,c). Further, we have used several epithelial primary and immortalized cells including primary neurons, Ntera-2, Sh-Sy5Y, and HEK-293 cells (Jha et al, 2015a), and clearly showed that during the initial days of EBV infection the latent gene expression was low, but the lytic gene expression resulted in active virus progeny produced in the culture medium.…”