Histone Hyperacetylation Occurs on Promoters of Lytic Cycle Regulatory Genes in Epstein-Barr Virus-Infected Cell Lines Which Are Refractory to Disruption of Latency by Histone Deacetylase Inhibitors

Abstract

Activation of the Epstein-Barr virus (EBV) lytic cycle is mediated through the combined actions of ZEBRA and Rta, the products of the viral BZLF1 and BRLF1 genes. During latency, these two genes are tightly repressed. Histone deacetylase inhibitors (HDACi) can activate viral lytic gene expression. Therefore, a widely held hypothesis is that Zp and Rp, the promoters for BZLF1 and BRLF1, are repressed by chromatin and that hyperacetylation of histone tails, by allowing the access of positively acting factors, leads to transcription of BZLF1 and BRLF1. To investigate this hypothesis, we used chromatin immunoprecipitation (ChIP) to examine the acetylation and phosphorylation states of histones H3 and H4 on Zp and Rp in three cell lines, Raji, B95-8, and HH514-16, which differ in their response to EBV lytic induction by HDACi. We studied the effects of three HDACi, sodium butyrate (NaB), trichostatin A (TSA), and valproic acid (VPA). We also examined the effects of tetradecanoyl phorbol acetate (TPA) and 5-aza-2-deoxycytidine, a DNA methyltransferase inhibitor, on histone modification. In Raji cells, TPA and NaB act synergistically to activate the EBV lytic cycle and promote an increase in histone H3 and H4 acetylation and phosphorylation at Zp and Rp. Surprisingly, however, when Raji cells were treated with NaB or TSA, neither of which is sufficient to activate the lytic cycle, an increase of comparable magnitude of hyperacetylated and phosphorylated histone H3 at Zp and Rp was observed. In B95-8 cells, NaB inhibited lytic induction by TPA, yet NaB promoted hyperacetylation of H3 and H4. In HH514-16 cells, NaB and TSA strongly activated the EBV lytic cycle and caused hyperacetylation of histone H3 on Zp and Rp. However, when HH514-16 cells were treated with VPA, lytic cycle mRNAs or proteins were not induced, although histone H3 was hyperacetylated as measured by immunoblotting or by ChIP on Zp and Rp. Taken together, our data suggest that open chromatin at EBV BZLF1 and BRLF1 promoters is not sufficient to activate EBV lytic cycle gene expression.

Viral epigenomes in human tumorigenesis

“…Recently, studies of these histone marks in specific promoter regions in the EBV genome have yielded some interesting results from an analysis of the epigenetic patterns of viral genes, such as Cp, LMP2A and BZLF1, which code for essential latent proteins. As these histone marks are associated with open and accessible chromatin, these results suggested that, in coordination with DNA methylation, they have a major role in the modulation (activation or repression) of specific viral genes (Gerle et al, 2007;Countryman et al, 2008;Fejer et al, 2008).…”

“…This reactivation is initiated by the expression of the immediate-early gene BZLF1, which encodes the transcriptional activator Zta, probably by stimuli such as plasma cell differentiation, and has been found to be unmethylated (Fernandez et al, 2009). This protein, apart from affecting the expression of host genes (Chang et al, 2006), has the ability to bind to methylated sites and activate the expression of BRLF1, another of this virus's immediate-early genes, and both together then activate the expression of the remaining lytic genes, thereby inducing a lytic infection (Bhende et al, 2005;Countryman et al, 2008;Dickerson et al, 2009;Heather et al, 2009). …”

“…During latency, episomal DNA is associated with nucleosomes in arrays that are indistinguishable from bulk cellular DNA (Shaw et al, 1979;Dyson and Farrell, 1985) and, not surprisingly, the histone marks associated with particular nucleosome positioning affect the expression patterns of viral genes. Several studies have been published in relation to this that identify specific associations between the expression of viral genes and patterns of histone modification (Gerle et al, 2007;Countryman et al, 2008;Fejer et al, 2008).…”

Broadening Specificity and Enhancing Cytotoxicity of Adoptive T Cells for Nasopharyngeal Carcinoma Immunotherapy

“…As a first step, we used the TPA þ NaB combination as the most commonly used treatment to trigger EBV lytic reactivation (2,21). Healthy donor-derived LCLs treated for 48 hours with TPA þ NaB showed significantly increased BARF1 mRNA expression (2.5 mean fold-increase, P 0.05; Fig.…”

Transcriptional Repression by Sumoylation of Epstein-Barr Virus BZLF1 Protein Correlates with Association of Histone Deacetylase

“…Changes in histone acetylation in the BZLF1 promoter region results in activation of BZLF1 gene expression leading to reactivation from latency, as treatment with histone deacetylase (HDAC) inhibitors can activate viral lytic gene expression (7)(8)(9). It is reported that, in latently infected cells, the silent state of the virus genome is maintained, at least in part, by MEF2-mediated recruitment of HDAC proteins to the BZLF1 promoter region (10).…”