Role of the histone H3 lysine 9 methyltransferase Suv39 h1 in maintaining Epsteinn–Barr virus latency in B95–8 cells Kenichi Imai1,2, Noriaki Kamio1,2, Marni E. Cueno1, Yuko Saito1, Hiroko Inoue3, Ichiro Saito3 and Kuniyasu Ochiai1,2 1 Department of Microbiology, Nihon University School of Dentistry, Tokyo, Japan 2 Division of Immunology and Pathobiology, Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan 3 Department of Pathology, Tsurumi University School of Dental Medicine, Yokohama, Japan

Keywords BZLF1; EBV; histone methylation; latency; Suv39 h1 Correspondence K. Imai, Department of Microbiology, Nihon University School of Dentistry, 1–8–13 Kanda-Surugadai, Chiyoda–ku, Tokyo 101–8310, Japan Fax: +81 3 3219 8317 Tel: +81 3 3219 8125 E–mail: [email protected] (Received 18 October 2013, revised 12 January 2014, accepted 26 February 2014) doi:10.1111/febs.12768

The ability of Epstein–Barr Virus (EBV) to establish latent infection is associated with infectious mononucleosis and a number of malignancies. In EBV, the product of the BZLF1 gene (ZEBRA) acts as a master regulator of the transition from latency to the lytic replication cycle in latently infected cells. EBV latency is primarily maintained by hypoacetylation of histone proteins in the BZLF1 promoter by histone deacetylases. Although histone methylation is involved in the organization of chromatin domains and has a central epigenetic role in gene expression, its role in maintaining EBV latency is not well understood. Here we present evidence that the histone H3 lysine 9 (H3K9) methyltransferase suppressor of variegation 3–9 homolog 1 (Suv39 h1) transcriptionally represses BZLF1 in B95–8 cells by promoting repressive trimethylation at H3K9 (H3K9me3). Suv39 h1 significantly inhibited basal expression and ZEBRA-induced BZLF1 gene expression in B95–8 B cells. However, mutant Suv39 h1 lacks the SET domain responsible for catalytic activity of histone methyl transferase and thus had no such effect. BZLF1 transcription was augmented when Suv39 h1 expression was knocked down by siRNA in B95–8 cells, but not in Akata or Raji cells. In addition, treatment with a specific Suv39 h1 inhibitor, chaetocin, significantly enhanced BZLF1 transcription. Furthermore, chromatin immunoprecipitation assays revealed the presence of Suv39 h1 and H3K9me3 on nucleosome histones near the BZLF1 promoter. Taken together, these results suggest that Suv39 h1–H3K9me3 epigenetic repression is involved in BZLF1 transcriptional silencing, providing a molecular basis for understanding the mechanism by which EBV latency is maintained.

Introduction Chromatin is a highly dynamic structure of nucleosomes, which are composed of DNA wrapped around core histones. Post-translational modifications of the N– terminal region of each core histone, such as acetylation, methylation, phosphorylation and ubiquitination, have

important roles in controlling the structural organization of chromatin and its transcriptional status [1,2]. Several studies have demonstrated that, among histone modifications, acetylation and methylation of histones 3 and 4 (H3 and H4) have central epigenetic roles in

Abbreviations BL, Burkitt’s lymphoma; DZNep, 3–deazaneplanocin A; EBV, Epsteinn–Barr Virus; Ezh2, enhancer-of-zeste homolog 2; HDAC, histone deacetylase; HMT, histone methyltransferase; HP1, heterochromatin protein 1; SAHA, suberoylanilide hydroxamic acid; SET, su(var)3–9 enhancer-of-zeste, trithorax; Suv39 h1, suppressor of variegation 3–9 homolog 1.

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chromatin remodeling and up- and down-regulation of gene expression [1,2], and that histone modification and chromatin remodeling have important roles in regulating gene expression and viral latency and reactivation cycles [3–5]. The herpesvirus Epstein–Barr virus (EBV) is present in more than 90% of the world population, and is associated with several diseases in humans, including infectious mononucleosis and malignancies such as Burkitt’s lymphoma (BL), Hodgkin’s disease and nasopharyngeal carcinoma [6,7]. Similar to other herpesviruses, EBV establishes persistent infection in the human host, and its life cycle has lytic and latent phases [6,8]. The switch from latency to the lytic replication cycle in latently infected B cells is coordinated by the viral transcription and replication factor ZEBRA (also known as Z, Zta or EB1), which is the product of the immediateearly EBV gene BZLF1 [5,8]. ZEBRA transactivates early and late EBV genes, thereby inducing the lytic cycle cascade. Expression of BZLF1 is tightly controlled at the transcriptional level, and neither BZLF1 mRNA nor ZEBRA protein are detectable during latency [8,9]. EBV reactivation is associated with progeny virus production, transmission to a new host, and several human diseases, as noted above. Thus, elucidation of the molecular mechanism by which EBV is transcriptionally silenced in latently infected cells and reactivated is crucial in understanding the pathophysiological process of EBV infection and developing preventive measures and new therapies. Recent studies have demonstrated that post-translational modification of DNA-associated histone proteins by histone deacetylases (HDACs) and histone acetyltransferase (HAT) in the BZLF1 promoter is important in maintaining and disrupting EBV latency [5,10,11]. For example, negative transcription factors such as Jun dimerization protein 2, myocyte enhancer binding factor 2 and the Sp1/Sp3 protein complex are associated with HDACs and recruit these molecules to the BZLF1 promoter [10–12]. Formation of these complexes leads to hypoacetylation of local histones and establishment of transcriptional latency. In contrast, HDAC inhibitors such as butyric acid and trichostatin A induced reactivation of EBV by histone acetylation of the BZLF1 promoter in a variety of cultured cell lines derived from BL cells [13–15]. We previously demonstrated that culture supernatant from periodontopathic bacteria, which contained high concentrations of butyric acid, inhibited HDACs and thus increased the histone acetylation and transcriptional activity of BZLF1 [16]. In addition to histone acetylation and deacetylation, histone lysine methylation has an epigenetic role in 2

organizing chromatin domains and regulating gene expression [17–19]. Methylation of histone H3 at lysine 4 (H3K4me) and methylation of histone H3 at lysine 26 (H3K36me) are principally associated with transcriptional stimulation, whereas methylation of histone H3 at lysines 9 (H3K9me) and methylation of histone H3 at lysine 27 (H3K27me) is a marker of heterochromatin formation and transcriptional repression [17–19]. In addition with this, H3K9me was found to be strongly associated with formation of transcriptionally silent chromatin [20,21]. H3K9 methylation may be mediated by histone methyltransferases (HMTs), which contain a conserved SET domain (Suv39, enhancer-ofzeste, trithorax) that has mono-, di- or trimethylation activity [20,21]. The most studied H3K9 HMT is the trimethylase suppressor of variegation 3–9 homolog 1 (Suv39 h1), also known as lysine methyltransferase 1A, which mainly contributes to facultative heterochromatin formation and gene silencing [22,23]. Recent research has revealed the importance of histone methylation by HMTs in establishing and maintaining viral latency [24]. du Chene et al. [25] demonstrated that H3K9 methylation induced by Suv39h1 is responsible for HIV–1 gene repression and establishment of transcriptional latency. We previously described another H3K9 methyl transferase (G9) that is involved in maintaining HIV–1 latency [26]. With regard to the role of histone methylation by HMTs in EBV, a report found that the HMT enhancer-of-zeste homolog 2 (Ezh2; also known as lysine methyltransferase 6), the catalytic subunit of Polycomb repressive complex 2 and Suv420 h1, which are primarily involved in H3K27me3 and H4K20me3, respectively, is responsible for BZLF1 transcriptional silencing [27]. Although H3K9 methylation is known to play a crucial role in chromatin-mediated transcriptional silencing [20,21], the role of H3K9 methylation catalyzed by Suv39 h1 in maintaining EBV latency has not been elucidated. It was shown that H3K9me exhibited more definitive transcriptional repression compared with H3K27me and H4K20me [17–19]. In this study, we investigated the role of Suv39 h1 in BZLF1 gene expression, and found that it was responsible for maintaining chromatin-mediated BZLF1 silencing via histone modification of H3K9me3 in B95–8 cells.

Results Suv39 h1 represses BZLF1 gene expression To examine whether Suv39 h1 affects BZLF1 gene expression, we examined the effect of Suv39 h1 overFEBS Journal (2014) ª 2014 FEBS

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expression on BZLF1 transcription. A plasmid expressing the luciferase reporter under the control of the BZLF1 promoter was co-transfected with the Suv39 h1 expression vector into HeLa cells. We used the
221 to +12 region of the BZLF1 promoter relative to the transcriptional initiation site of the BZLF1 gene because these elements are sufficient for basal 30 1

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Fig. 1. Repression of BZLF1 gene expression by Suv39 h1. (A) Effects of Suv39 h1 on gene expression from a transiently transfected construct containing the BZLF1 promoter. The Suv39 h1 expression plasmid (0.1 or 0.3 lg) was co-transfected into HeLa cells with 0.1 lg of a BZLF1–luciferase reporter construct that expresses the luciferase gene under the control of the BZLF1 promoter. Effects of Suv39 h1 on the basal level of BZLF1 gene expression (left) and the level of BZLF1 gene expression upon co-transfection with 0.1 lg of the ZEBRAexpressing plasmid pCI–Zta (right). Twenty-four hours after transfection, the cells were harvested, and luciferase activity was measured. Values are fold increases in luciferase activity (means  SD) relative to the control transfection for three independent experiments. (B) Repression of BZLF1 transactivation by Suv39 h1 in B95–8 cells stably transfected with a construct containing the BZLF1 promoter. The effects of Suv39 h1 in B95–8– 221 Luc cells were evaluated at the basal level (left) and upon co-transfection with 0.3 lg of the the ZEBRA-expressing plasmid pCI–Zta (right). The amounts of Suv39 h1 plasmid co-transfected were 0.3 and 0.9 lg per transfection. Twenty-four hours after transfection, luciferase assays were performed as in (A).

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activity and induced promoter activity [28]. As shown in Fig. 1A (left), basal transcription from the BZLF1 promoter was inhibited by Suv39 h1 in a dose-dependent manner. Expression of ZEBRA further activates the BZLF1 promoter by binding to its own promoter [5,28]. The results presented in the right panel of Fig. 1A show that Suv39 h1 inhibited ZEBRA-induced BZLF1 gene expression in these cells. Butyric acidinduced BZLF1 transcription was also repressed by Suv39 h1 (data not shown). To confirm the negative effect of Suv39 h1 on BZLF1 gene expression under more physiological conditions, we performed a luciferase assay using B95–8–221 Luc cells, which were stably transfected with a construct containing the BZLF1 promoter into B95–8 B cells [16]. As shown in Fig. 1B, Suv39 h1 expression resulted in a dose-dependent decrease in basal and ZEBRA-stimulated BZLF1 transcription in B95–8–221 Luc cells. These results demonstrate that Suv39 h1 represses BZLF1 gene expression in cultured cells in vivo.

Suv39 h1 contains a SET domain in the C–terminus, exhibiting HMT activity against H3K9 [22,23]. Thus, to determine the role of the SET domain of Suv39 h1 in down-regulating BZLF1 gene expression, we examined the effect of Suv39 h1 mutants from which the SET domain has been deleted (Suv39 h1ΔSET). The results showed that over-expression of Suv39 h1ΔSET abolished the repressive action of Suv39 h1 on basal and ZEBRA-induced BZLF1 expression (Fig. 2). These results indicate that HMT activity is involved in Suv39 h1-mediated repression of BZLF1 gene expression. Effect of Suv39 h1 knockdown on BZLF1 gene expression To examine the effect of endogenous Suv39 h1, we used an siRNA technique to knockdown Suv39 h1 expression, and examined the BZLF1 transcriptional level when endogenous Suv39 h1 was depleted. We found that depletion of Suv39 h1 protein by siRNA (Fig. 3A) resulted in increased expression of BZLF1 mRNA in B95–8 cells (3.7 times that of the control siRNA vector) (Fig. 3B). We also examined the effect of Suv39 h1 knockdown on BZLF1 gene expression in other EBV-positive BL cell lines, namely Akata and Raji cells. As shown in Fig. 3B, siRNA treatment did not alter BZLF1 mRNA levels in these cell lines. These results indicate that endogenous Suv39 h1 acts 3

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Fig. 2. Effects of Suv39 h1 SET domain deletion on BZLF1 gene expression. B95–8–221 Luc cells were transfected with various amounts of an Suv39 h1 deletion mutant lacking the SET domain (Suv39 h1ΔSET) (0.3 and 0.9 lg per transfection) with or without co-transfection of pCI–Zta. Twenty-four hours after transfection, luciferase assays were performed as described in the legend to Fig. 1A. Values are fold increases in luciferase activity (means  SD) relative to the control transfection for three independent experiments.

as a negative regulator of BZLF1 gene expression in B95–8 cells. The specific Suv39 h1 inhibitor chaetocin activates BZLF1 transcription To confirm the negative effect of Suv39 h1 on BZLF1 gene expression, we examined the effect of chaetocin, a specific inhibitor of Suv39 h1, on ZEBRA expression in B95–8 cells. Chaetocin is a fungal mycotoxin that specifically inhibits Suv39 h1 HMT activity and H3K9me3 [29]. As shown in Fig. 4A (left), treatment with chaetocin clearly transactivated BZLF1 gene expression in a time-dependent manner in B95–8–221 Luc cells. Treatment with increasing concentrations of chaetocin increased BZLF1 gene promoter activity in a dose-dependent manner (Fig. 4A, right). In addition, Fig. 4B shows that ZEBRA expression was increased by chaetocin in B95–8 cells. However, chaetocin did not induce ZEBRA expression in Akata or Raji cells (data not shown). Under the same conditions, when purified histone fractions were prepared to examine the level of histone H3 methylation, H3K9me3, but not H3K27me3, was down-regulated by chaetocin in B95–8 cells. Because chaetocin had potent induction effects on BZLF1 gene transcription, we also tested BIX01294, a specific inhibitor of G9a, and 3–deazaneplanocin A (DZNep), and an inhibitor of H3K27me3 and H4K20me3 [27,30,31]. BIX01294 selectively inhib4

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Fig. 3. Effects of Suv39 h1 siRNA on ZEBRA expression. (A) Confirmation of siRNA-mediated Suv39 h1 depletion. B95–8, Akata and Raji cells were transfected with siRNA vectors directed against Suv39 h1 or LucGL3 (control) mRNAs. Thirty-six hours after transfection, cells were lysed and the Suv39 h1 level was assessed by immunoblotting using specific antibodies. Then the immunoblot membrane was stripped and re-probed with anti-b– actin antibody as a control. (B) Augmentation of BZLF1 gene expression by Suv39 h1 knockdown. psiRNA-hSuv39 h1- or psiRNA-LucGL3-transfected cells were harvested, and the level of BZLF1 mRNA was determined by real-time PCR. BZLF1 mRNA levels were normalized to GAPDH mRNA levels.

ited G9a HMT activity and H3K9me2, without affecting other HMTs such as Suv39 h1 [30]. DZNep, which inhibits S–adenosylhomocysteine hydrolase, a carbocyclic analog of adenosine, inhibited H3K27me3 and H4K20me3 but not H3K9me [31]. As shown in Fig. 4C, BIX01294 and DZNep did not alter BZLF1 gene transcription in B95–8–221 Luc cells. Neither of these inhibitors had any effect on ZEBRA expression in B95–8 cells (data not shown). These results clearly show that trimethylation at H3K9 by Suv39 h1 is involved in the repression of ZEBRA expression in B95–8 cells. Chaetocin facilitates BZLF1 transcription via chromatin remodeling As shown above, chaetocin down-regulated H3K9me3 by inhibiting Suv39 h1 and inducing expression of ZEBRA at the transcriptional level. To further clarify FEBS Journal (2014) ª 2014 FEBS

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Fig. 4. Effects of the Suv39 h1 inhibitor chaetocin on BZLF1 replication. (A) Activation of BZLF1 replication by chaetocin. B95– 8–221 Luc cells were incubated with chaetocin (150 nM) for the indicated times (left panel) and for 48 h at the indicated doses (right panel), after which luciferase activity was measured. (B) Chaetocin-induced expression of ZEBRA. B95–8 cells were incubated with chaetocin (0, 75 or 150 nM) for 48 h. The cells were lysed, and levels of ZEBRA, H3K9me3 and H3K27me3 were assessed by immunoblotting using specific antibodies. The unmodified H3 protein was used as a control. (C) Effects of BIX01294 and DZNep on BZLF1 gene expression. B95–8–221 Luc cells were treated with BIX01294 (0, 2.5, 5 or 10 lM) or DZNep (0, 10, 50 or 100 lM) for 48 h, and luciferase activity was measured.

the role of Suv39 h1 in BZLF1 gene expression and the effects of chaetocin in B95–8 cells, we performed chromatin immunoprecipitation (ChIP) assays using antibodies against Suv39 h1, H3K9me3 and H3. We observed that Suv39 h1 and H3K9me3 bound to the core promoter region (from
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11) within BZLF1 when these cells were maintained in the latent state (without stimulation). However, when B95–8 cells were treated with chaetocin, Suv39 h1 and H3K9me3 readily dissociated from the BZLF1 promoter (Fig. 5). A recent report indicated that Ezh2 and Suv420 h1 are responsible for BZLF1 transcriptional silencing in another BL cell line, Raji cells [27]. Although the presence of Ezh2 on the BZLF1 promoter was faintly detected, Ezh2 and H3K27me3 levels were not altered by chaetocin in B95–8 cells (Fig. 5). In addition, Suv420 h1 was not observed in the BZLF1 promoter. These results are consistent with a previous finding FEBS Journal (2014) ª 2014 FEBS

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that DZNep did not alter BZLF1 gene expression in B95–8 cells (Fig. 4C). Synergistic activation of BZLF1 expression by chaetocin and the HDAC inhibitors SAHA and butyric acid As the Suv39 h1 HMT inhibitor chaetocin and the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and butyric acid independently induced BZLF1 expression in several cell lines [13–15], we examined whether the effect of chaetocin synergized with that of either of these HDAC inhibitors in inducing BZLF1 expression. As shown in Fig. 6A, when B95–8–221 Luc cells were treated with chaetocin alone or in combination with SAHA, we observed clear synergism between chaetocin and SAHA. Whereas treatment with SAHA alone and chaetocin alone (10 nM) induced BZLF1 transcription by 4.6- and 7.2-fold, 5

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respectively, treatment with both chaetocin and SAHA increased BZLF1 transcription 18.9-fold. Similar synergism was observed when B95–8–221 Luc cells were treated with both chaetocin and butyric acid (Fig. 6B). Synergism was also observed when chaetocin and trichostatin A were used (data not shown). Taken together, these findings suggest that methylation and acetylation of histones are independently involved in BZLF1 gene expression.

Discussion The ability of EBV to establish latent infection is essential in progression of diseases associated with EBV. In a healthy host, infected cells form a reservoir of chronic viral infection that is tightly controlled by the immune system. However, immunosuppression may trigger reactivation of latently infected cells, leading to development of diseases such as infectious mononucleosis and malignancy. Thus, identifying the molecular mechanisms that regulate gene expression during EBV latency is a central concern in EBV infection. Although appreciation of the importance of epigenetic regulation in EBV latency is increasing, the role of histone methylation by HMT in maintaining EBV latency has not been adequately clarified. In this study, we found that Suv39 h1 acts as a negative regulator of BZLF1 gene expression via H3K9me3 in B95–8 cells. Several research groups have shown that EBV quiescence is closely associated with recruitment of negative transcriptional factors and HDAC complexes to the BZLF1 promoter [10–12]. HDAC is the counterpart of HAT, which catalyzes the hydrolytic removal of acetyl groups [2]. Histone deacetylation leads heterochroma6

tin formation and thereby accessibility of transcriptional factors to nucleosomal DNA, resulting in repressing gene expression [2]. Although accumulating evidence suggests that HDACs are critical regulators of EBV latency, a number of studies have reported that histone deacetylation is not sufficient for repression of gene transactivation, including for the BZLF1 gene [9,32–34]. Treatment with the HDAC inhibitor trichostatin A and butyric acid alone did not reactivate gene expression if genes were still methylated [32]. Similarly, an HDAC inhibitor alone did not efficiently induce BZLF1 transcription in some EBV-infected BL cell lines [9,33,34]. In addition, it was shown that histone acetylation is not sufficient to active the EBV lytic cycle [9,13]. These observations suggest that other, unknown suppressive mechanisms are present in addition to histone deacetylation. For example, histone methylation may be involved establishing EBV latency. Several studies have reported that H3K9 methylation of viral immediate-early genes is involved in the latency of a–herpesviruses (herpes simplex virus and varicella zoster virus), and that this repressive state is modulated by recruitment of histone H3K9 demethylase to immediate-early gene promoters [35–37]. In addition, using a viral genome-wide analysis, Tempera et al. [38] observed high levels of H3K9me3 at the EBV Q promoter in two BL cell lines. Furthermore, in addition to HDAC, methylation of H3K9 is catalyzed by Suv39 h1, which is responsible for HIV–1 transcriptional silencing in latently infected cells [25]. These results prompted us to investigate whether Suv39 h1 and H3K9me are involved in establishing EBV latency. We observed that when Suv39 h1 is over-expressed BZLF1 gene expression is greatly suppressed. In FEBS Journal (2014) ª 2014 FEBS

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contrast, Suv39 h1 knockdown mediated by siRNA increased BZLF1 transcription in B95–8 cells, but not in Akata or Raji cells. Moreover, chaetocin, a specific inhibitor of Suv39 h1, down-regulated H3K9me3 and reactivated EBV in B95–8 cells. These results are the first evidence of involvement of Suv39 h1 in maintaining transcriptional latency in EBV. Among the HMTs that methylate H3K9, Suv39 h1 is the main HMT for H3K9me3 in heterochromatin, whereas G9a is the main HMT for H3K9me2 in the silenced region within euchromatin [39,40]. Recent studies have shown that Suv39 h1 also plays a role in repression of several euchromatic genes [41–43]. It is possible that Suv39 h1 and G9a HMT have different localization patterns in nuclei and distinct roles in chromatin modification, and may have different molecular partners [23,39,40]. Our results showed that the Suv39 h1-specific inhibitor chaetocin, but not the G9a-specific inhibitor BIX01294, induces BZLF1 transcription and ZEBRA expression in B95–8 cells. In addition, no effect of BIX01294 on BZLF1 activation was observed in another BL cell line (Raji cells) [27]. Thus, Suv39 h1-mediated chromatin silencing may play a critical role in establishment of latent EBV infection, at least in B95–8 cells. Similarly, the HMTs, Ezh2 and Suv420h1 are responsible for BZLF1 transcriptional silencing [27]. Murata et al. [27] demonstrated that knockdown of Ezh2 and Suv420h1 and treatment with DZNep, an inhibitor of H3K27me3 and H4K20me3, markedly increased BZLF1 induction in Raji cells treated with trichostatin A. However, in contrast to findings in Raji cells, B95–8 and Akata cells did not respond to DZNep [27]. We also observed that ZEBRA expression was increased by chaetocin in B95–8 cells, but not in Akata or Raji cells. Taken together, these observations suggest that the mechanism of epigenetic silencing involved in histone modification of the BZLF1 promoter is cell type-dependent. Methylation of histone proteins affects binding of histone modification enzymes to chromatin, which then induces other post-translational modifications [17–19]. H3K9 methylation by Suv39 h1 recruits members of the heterochromatin protein 1 (HP1) family of heterochromatic adaptor molecules involved in both transcriptional repression and formation of transcriptionally silent chromatin [25,44,45]. HP1 proteins associate with many other proteins, including HDACs [46–48]. Although the role of HP1 in EBV latency is unclear, it is possible that when the BZLF1 gene is suppressed by recruitment of Suv39 h1, and subsequently by H3K9me3 (which is recognized by a heterochromatin complex containing HP1), recruitment of HDAC results in formation of silent chromatin. FEBS Journal (2014) ª 2014 FEBS

Role of Suv39 h1 in maintaining EBV latency

Disruption of the balance of epigenetic networks results in a number of intractable diseases, including various cancers, immuno-inflammatory diseases, and viral infectious diseases [49]. Because post-translational modifications of histone are readily reversible using epigenetic drugs such as HDAC inhibitors, epigenetic therapy has emerged as an effective chemotherapy for several intractable diseases [49–51]. An example of such an agent is SAHA, which was used in this study. It has been approved by the US Food and Drug Administration for treatment of cutaneous T–cell lymphoma, and was also found to be effective in an animal model of arthritis [50,52,53]. More recently, histone methyltransferases and demethylases have emerged as new therapeutic targets [54,55]. It is well known that HDACs and HMTs are responsible for the transcriptional quiescence of latent EBV, providing a potential molecular basis for the efficacy of combination therapy using conventional antiviral drugs and an HDAC inhibitor and/or HMT inhibitor for patients with EBV-positive cancers. Additional studies are required needed to clarify the role of Suv39 h1 and other repressor proteins in maintenance of EBV latency in vivo. It is likely that improving our understanding of the pathogenesis of EBV infection from the perspective of epigenetic regulation will lead to new treatments and superior methods of prevention.

Experimental procedures Reagents and plasmids Chaetocin was purchased from Merck Millipore (Billerica, MA, USA), was purchased DZNep from Cayman Chemical (Ann Arbor, MI, USA), and BIX01294, was purchased from Alexis Biochemicals (San Diego, CA, USA). SAHA, a conventional hydroxamate HDAC inhibitor, was synthesized as described previously [56]. The Suv39 h1 expression vector and its mutant (Suv39h1ΔSET) were generous gifts from Thomas Jenuwein (Max Planck Institute of Immunology and Epigenetics, Freiburg, Germany) [22]. A plasmid expressing the luciferase reporter under the control of the BZLF1 promoter (which contains the
221 to +12 region of the BZLF1 gene), and the ZEBRA expression vector (pCI–Zta) were as described previously [16,57].

Cell culture The EBV-positive BL cell lines (B95–8, Akata and Raji cells) and the B95–8–221 Luc cells were stably transfected with a construct containing the BZLF1 promoter [16] and maintained at 37 °C in RPMI–1640 (Sigma, St Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum

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(Thermo Scientific, Rockford, IL, USA), penicillin (100 unitsmL
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1) and streptomycin (100 mgmL
1).

Western blot analysis Immunoblotting was performed as described previously [26,58]. Briefly, cells were centrifuged to wash once with ice-cold NaCl/Pi at 4 for 5 min and lysed in 0.5 mL lysis buffer (25 mM HEPES/NaOH, pH 7.9, 150 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.3% Nonidet P-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). Cellular debris was removed by centrifugation at 15 000 g for 10 min. For western blotting, the proteins were separated by SDS/PAGE and transferred to a polyvinylidene fluoride membrane (Merck Millipore). The Pierce microplate BCA protein assay kit/reducing agent compatible kit (Thermo Scientific) was used to standardize the protein concentration in all samples used. For analysis of histone 3, cell lysates were prepared by acid extraction as described previously [26]. To detect EBV ZEBRA and Suv39 h1 proteins, the cell lysates were subjected to immunoblotting using antibodies against ZEBRA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or Suv39 h1 antibody (GeneTex, Irvine, CA, USA), respectively, and immunoreactive proteins were visualized by enhanced chemiluminescence (SuperSignal West Pico; Thermo Scientific). Antibody against b–actin (Santa Cruz Biotechnology) was used as an internal control. The levels of histone 3 and methylated forms were analyzed by immunoblotting with antibodies against H3K9me3 (Abcam, Cambridge, MA, USA), H3K27me3 (Merck Millipore) or histone 3 (Merck Millipore).

RNA interference A silencing vector expressing an siRNA targeting human Suv39 h1 (psiRNA-hSuv39 h1) and a control plasmid containing a sequence targeting LucGL3 (psiRNA-LucGL3) were obtained from InvivoGen (San Diego, CA, USA). For knockdown studies, EBV-positive BL cell lines cultured in six-well plates (1 9 106 cells) were transfected with 10 llg above psiRNA vectors using NEPAGENE (Nepa Gene Co. Ltd, Chiba, Japan). After 36 h, the cells were harvested by lysis buffer RNeasy mini kit (Qiagen, Frederick, MD, USA) 50 for real-time PCR analysis. To confirm knockdown of Suv39 h1 protein production, western blotting was performed using Suv39 h1 antibody.

Preparation of mRNA and real-time RT–PCR Total cellular RNA was prepared from each cell line using an RNeasy mini kit (Qiagen), and RT–PCR was performed as described previously [58]. For cDNA synthesis, 2 lg total RNA were reverse transcribed using an RNA PCR kit (PrimeScript; Takara Bio, Shiga, Japan). The resulting cDNA mixture was subjected to real-time PCR analysis using SYBR Premix Ex Taq solution (Takara Bio) containing 10 lM sense and antisense primers. The primer sequences for each amplified gene were as follows: BZLF1 forward, 50 -CCATACCAGGTGCCTTTTGT-30 ; reverse, 50 -GAGACTGGGAACAGCTGAGG-30 ; glyceraldehyde-3– phosphate dehydrogenase (GAPDH) forward, 50 -TGCAC CACCAACTGCTAGC-30 ; reverse, 50 -GGCATGGACTGT GGTCATGAG-30 . The PCR assays were performed using a Thermal Cycler Dice real-time system (TP–800; Takara Bio), and analyzed using the software provided by the manufacturer. The protocol for BZLF1 and GAPDH consisted of 40 cycles at 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 1 min. All real-time PCR experiments were performed in triplicate, and the specificity of each product was verified by melting curve analysis. Calculated gene expression levels were normalized to the levels of GAPDH mRNA.

Transfection and luciferase assay The process of transfection and the luciferase assay have been described previously [16,57]. HeLa cells cultured in 12-well plates (1 9 105 cellsmL
1) were transfected using FuGENE–6 transfection reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. B95–8–221 Luc cells (1 9 106) were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The transfected cells were harvested using passive lysis buffer (Promega, Fitchburg, WI, USA), and the extracts were subjected to a luciferase assay using the dual luciferase reporter assay system (Promega). All experiments were performed in triplicate, and the data are presented as fold increases in luciferase activity (means  SD) relative to control for three independent transfections.

8

Chromatin immunoprecipitation (ChIP) assay ChIP assays were performed as described previously using a ChIP assay kit (Magna ChIP; Merck Millipore) according to the manufacturer’s instructions with some modifications [26,58]. Briefly, B95–8 cells (2 9 106) were fixed with 1% formaldehyde at 37 °C for 10 min, washed twice with ice-cold NaCl/Pi containing protease inhibitors (Complete Protease Inhibitor Cocktail tablets; Roche, Basel, Switzerland) and phenylmethylsulfonyl fluoride, and then suspended in SDS lysis buffer (1% SDS, 50 mM Tris/HCl, pH 8.0, 16.7 mM NaCl, 1 mM phenylmethylsulfonyl fluoride and protease inhibitors). The cross-linked chromatin was sheared by sonication 10 times for 30 s each time at maxi-

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K. Imai et al.

mum power, with 30 s of cooling on ice between pulses using a Bioruptor (Cosmo Bio, Tokyo, Japan). Crosslinked and released chromatin fractions were cleared prior to further analysis using salmon sperm DNA and Protein G–agarose beads for 1 h, followed by immunoprecipitation with the specific antibodies overnight at 4 °C. AntiSuv39 h1, anti-H3K27me3 and anti-histone 3 antibodies were obtained from Merck Millipore. Anti-H3K9me3, anti-Ezh2 and anti-Suv420 h1 antibodies were purchased from Abcam, and anti-H4K20me3 antibody was purchased from Active Motif (Carlsbad, CA, USA). The precipitated DNA was analyzed by PCR (32–35 cycles) using HotStarTaq Master Mix (Qiagen) and primers for the BZLF1 promoter region (nucleotides
191 to
11: forward, 50 TTGACACCAGCTTATTTTAGACACTTCT-30 ; reverse, PCR 50 -TAACCTGTCTAACATCTCCCCTTTAAA-30 ). products were separated by electrophoresis on a 2% agarose gel. For each reaction, 10% of the original sheared chromatin DNA was reverse cross-linked and purified, and the recovered DNA was used as the input control.

Acknowledgments We thank Thomas Jenuwein (Max Planck Institute of Immunology and Epigenetics, Freiburg, Germany) for the Suv39 h1 and Suv39 h1ΔSET plasmids. This work was supported by Grants-in-Aid from the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan, and the Strategic Research Base Development Program for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan 2010–2014 (S1001024).

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