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Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

ABSTRACT: Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

SUBMITTER: Karlsson QH

PROVIDER: S-EPMC2267006 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

Karlsson Questa H QH Schelcher Celine C Verrall Elizabeth E Petosa Carlo C Sinclair Alison J AJ

PLoS pathogens 20080307 3

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-e ...[more]

PMID: 18369464

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Project description:Ikaros is a zinc finger DNA-binding protein that regulates chromatin remodeling and the expression of genes involved in the cell cycle, apoptosis, and Notch signaling. It is a master regulator of lymphocyte differentiation and functions as a tumor suppressor in acute lymphoblastic leukemia. Nevertheless, no previous reports described effects of Ikaros on the life cycle of any human lymphotropic virus. Here, we demonstrate that full-length Ikaros (IK-1) functions as a major factor in the maintenance of viral latency in Epstein-Barr virus (EBV)-positive Burkitt's lymphoma Sal and MutuI cell lines. Either silencing of Ikaros expression by small hairpin RNA (shRNA) knockdown or ectopic expression of a non-DNA-binding isoform induced lytic gene expression. These effects synergized with other lytic inducers of EBV, including transforming growth factor ? (TGF-?) and the hypoxia mimic desferrioxamine. Data from chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) and ChIP-sequencing (ChIP-seq) analyses indicated that Ikaros did not bind to either of the EBV immediate early genes BZLF1 and BRLF1. Rather, Ikaros affected the expression of Oct-2 and Bcl-6, other transcription factors that directly inhibit EBV reactivation and plasma cell differentiation, respectively. IK-1 also complexed with the EBV immediate early R protein in coimmunoprecipitation assays and partially colocalized with R within cells. The presence of R alleviated IK-1-mediated transcriptional repression, with IK-1 then cooperating with Z and R to enhance lytic gene expression. Thus, we conclude that Ikaros plays distinct roles at different stages of EBV's life cycle: it contributes to maintaining latency via indirect mechanisms, and it may also synergize with Z and R to enhance lytic replication through direct association with R and/or R-induced alterations in Ikaros' functional activities via cellular signaling pathways.This is the first report showing that the cellular protein Ikaros, a known master regulator of hematopoiesis and critical tumor suppressor in acute lymphoblastic leukemia, also plays important roles in the life cycle of Epstein-Barr virus in B cells.

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Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

Publications

Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

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