Epigenetic Repression of p16INK4A by Latent Epstein-Barr Virus Requires the Interaction of EBNA3A and EBNA3C with CtBP

Abstract

As an inhibitor of cyclin-dependent kinases, p16INK4A is an important tumour suppressor and inducer of cellular senescence that is often inactivated during the development of cancer by promoter DNA methylation. Using newly established lymphoblastoid cell lines (LCLs) expressing a conditional EBNA3C from recombinant EBV, we demonstrate that EBNA3C inactivation initiates chromatin remodelling that resets the epigenetic status of p16INK4A to permit transcriptional activation: the polycomb-associated repressive H3K27me3 histone modification is substantially reduced, while the activation-related mark H3K4me3 is modestly increased. Activation of EBNA3C reverses the distribution of these epigenetic marks, represses p16INK4A transcription and allows proliferation. LCLs lacking EBNA3A express relatively high levels of p16INK4A and have a similar pattern of histone modifications on p16INK4A as produced by the inactivation of EBNA3C. Since binding to the co-repressor of transcription CtBP has been linked to the oncogenic activity of EBNA3A and EBNA3C, we established LCLs with recombinant viruses encoding EBNA3A- and/or EBNA3C-mutants that no longer bind CtBP. These novel LCLs have revealed that the chromatin remodelling and epigenetic repression of p16INK4A requires the interaction of both EBNA3A and EBNA3C with CtBP. The repression of p16INK4A by latent EBV will not only overcome senescence in infected B cells, but may also pave the way for p16INK4A DNA methylation during B cell lymphomagenesis.

Epstein-Barr virus nuclear antigens 3C and 3A maintain lymphoblastoid cell growth by repressing p16 INK4A and p14 ARF expression

“…S2 indicate that the CDKN2A p16 INK4A and p14 ARF locus in LCLs is already deficient in H3K27me3 and has over-background Pol II, as well as promoterassociated H3K4me3, consistent with significant potential for further derepression and activation. Indeed, EBNA3C inactivation further reduced H3K27me3 at the p16 INK4A promoter and increased histone H3 acetylation and H3K4me3 consistent with recent findings of EBNA3C-and EBNA3A-related histone modification at the p16 INK4A locus (8).…”

“…The experiments described here investigate the underlying biochemical mechanisms and significance of initial findings that conditional EBNA3A or EBNA3C inactivation in LCLs induces G 1 growth arrest, which for EBNA3C is associated with induction of CDKN2A p16 INK4A expression, consistent with the possibility that EBNA3A and EBNA3C's essential role is to repress CDKN2A p16 INK4A (5)(6)(7)(8). EBNA3A, EBNA3B, and EBNA3C likely arose as a consequence of gene triplication, after the evolution of EBNA2 and EBNALP (9).…”

“…Because conditional E3AHT inactivation in LCLs has similar effects on LCL growth and p16 INK4A is also elevated in EBNA3A knockout LCLs relative to wild-type LCLs (5,7,8), EBNA3A may also have a role in p16 INK4A and p14 ARF repression. Indeed, incubation of E3AHT LCLs in media without 4HT, resulted at day 6 in substantially reduced E3AHT, slowed cell growth, increased p16 INK4A and p14 ARF protein expression, and ∼1.4-fold significantly increased p16 INK4A and p14 ARF RNA levels ( Fig.…”

“…Also, EBNA3B, which interacts with RBPJ is not required for LCL proliferation (17). EBNA3C and EBNA3A association with CtBP has been implicated in epigenetic repression of p16 INK4A (8), although deletion of the EBNA3C or EBNA3A binding sites for CtBP is only associated with slower LCL growth, whereas even point mutations in the EBNA3C or EBNA3A binding sites for RBPJ are null for LCL growth (22)(23)(24). EBNA3C and/or EBNA3A may cooperatively modulate transcription of regulators of the CDKN2A locus or more directly regulate suppression of p16 INK4A and p14 ARF through another interaction with RBPJ.…”

How virus persistence can initiate the tumorigenesis process

“…These cells develop virus induced chromosome damage and can undergo abnormal mitosis (Table 2), both in vitro and [64] E6, E7 [66,67] Naturally occurring pre-S mutants [68] --EBNA-3C v-CYC [65] LMP-1 [63] Lytic proteins BZLF-1 [69] -E1, E2 [71,72] HBx [73,74] Core Tax [76,77] BGLF-5 [70] NS3 [23,62] NS5 [75] EBV: Epstein-Barr virus; HHV-8: Human herpesvirus 8, also named Kaposi sarcoma virus; HPV: Human papillomavirus; HBV: Hepatatis B virus; HCV: Hepatitis C virus; HTLV-1: Human T-cell leukemia virus 1. EBNA-3A, EBNA-3C [78] LANA-1 [81] E6 [83] HBx [85,86] Core [87] Tax [88] LMP-1 [79] microRNA [82] E7 [84] LMP-2 [80] EBV: Epstein-Barr virus; HHV-8: Human herpesvirus 8, also named Kaposi sarcoma virus; HPV: Human papillomavirus; HBV: Hepatatis B virus; HCV: Hepatitis C virus; HTLV-1: Human T-cell leukemia virus 1.…”