EBNA1 binding and epigenetic regulation of gastrokine tumor suppressor genes in gastric carcinoma cells
© Lu et al.; licensee BioMed Central Ltd. 2014
Received: 28 October 2013
Accepted: 17 January 2014
Published: 24 January 2014
Epstein-Barr Virus (EBV) latently infects ~10% of gastric carcinomas (GC). Epstein-Barr Nuclear Antigen 1 (EBNA1) is expressed in EBV-associated GC, and can bind host DNA, where it may impact cellular gene regulation. Here, we show that EBNA1 binds directly to DNA upstream of the divergently transcribed GC-specific tumor suppressor genes gastrokine 1 (GKN1) and gastrokine 2 (GKN2).
We use ChIP-Seq, ChIP-qPCR, and EMSA to demonstrate that EBNA1 binds directly to the GKN1 and GKN2 promoter locus. We generate AGS-EBV, and AGS-EBNA1 cell lines to study the effects of EBNA1 on GKN1 and GKN2 mRNA expression with or without 5′ azacytidine treatment.
We show that gastrokine genes are transcriptionally silenced by DNA methylation. We also show that latent EBV infection further reduces GKN1 and GKN2 expression in AGS gastric carcinoma cells, and that siRNA depletion of EBNA1 partially alleviates this repression. However, ectopic expression of EBNA1 slightly increased GKN1 and GKN2 basal mRNA levels, but reduced their responsiveness to demethylating agent.
These findings demonstrate that EBNA1 binds to the divergent promoter of the GKN1 and GKN2 genes in GC cells, and suggest that EBNA1 contributes to the complex transcriptional and epigenetic deregulation of the GKN1 and GKN2 tumor suppressor genes in EBV positive GC.
Epstein-Barr virus (EBV) is a human gammaherpesvirus found in a wide range of lymphoid and epithelial cell malignancies, including Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma (NPC), and post-transplant lymphoproliferative disease (reviewed in [1, 2]). More recently, EBV has been found in ~10% of all gastric carcinoma (GC) cases worldwide [3, 4]. EBV-associated GC has been shown to be a monoclonal outgrowth of EBV-infected gastric epithelial cells and is considered to be a distinct subtype of GC [5, 6]. Because the incidence of GC is close to 900,000 people per year , EBV-associated GC may be among the most prevalent EBV-associated cancers.
In EBV positive gastric carcinoma cells, EBV establishes a variant type I latency, where EBV transcription is limited to the canonical type I genes EBNA1, EBERs, BART family non-coding RNA and miRNAs, but with some additional expression of LMP2A [6, 8–11]. Among these latency genes, EBNA1 is the only viral nuclear protein that is detected in EBV-associated GC. EBNA1 is required for the establishment of the latent episomal infection and for the long-term survival of latently infected cells [12–15]. EBNA1 is a DNA binding protein that binds to both viral and host chromosomal sites. The binding sites in the viral genome have been characterized for essential functions in replication and transcriptional control of viral gene expression. However, the function of EBNA1 sequence-specific binding to the host chromosome is less well understood. While EBNA1 can bind to the promoter regions of several host genes, it remains unclear whether these genes are subject to EBNA1 regulation [12, 16, 17]. Overexpression of the EBNA1 DNA binding domain, which functions as a dominant negative in EBV infected cells, can inhibit cell viability in uninfected cells, suggesting that EBNA1 binds to and regulates cellular genes important for cell survival . Ectopic expression of EBNA1 has been shown to effect host cell mRNA expression , but it is not clear whether these effects are direct or indirectly related to specific EBNA1 binding sites in the cellular genome.
In a previous study, we used ChIP-seq methods to analyze the genome-wide enrichment sites of EBNA1 in latently infected Raji Burkitt’s lymphoma cells and identified numerous cellular sites bound by EBNA1 . Among those EBNA1 cellular enrichment sites we identified a significant EBNA1 binding peak located at the gastrokine 1 (GKN1) and gastrokine 2 (GKN2, also known as trefoil factor interacting protein (TFIZ1)) gene cluster. GKN1 and GKN2 have been identified based on their frequent loss of expression in neoplastic gastric carcinoma epithelial cells, compared to normal gastric tissue [20–22] (reviewed in ). Several recent studies have described anti-proliferative and anti-invasive activity for GKN1 in gastric epithelial cells, which, together with its frequent expression loss in cancer, suggests it functions as tumor suppressor specific to gastric epithelium [21, 24–28]. GKN1 can inhibit cell migration and invasion in wound healing, transwell and Matrigel assay, as well as alter cell markers associated with the epithelial-mesenchymal transition . GKN1 and GKN2 genes are located in close proximity and transcribed in opposite directions, suggesting that they likely share a bi-directional promoter, and are subject to coordinate regulation by shared transcription regulatory factors (reviewed in ).
In this study, we demonstrated the direct binding between EBNA1 and GKN1-GKN2 loci and investigated GKN1 and GKN2 gene expression modulation by EBV infection and EBNA1 protein. Our findings suggest that EBV infection can further inhibit GKN1 and GKN2 expression, and that loss of EBNA1 can facilitate epigenetic de-repression of GKN2 transcription. We also observed elevated DNA methylation levels at GKN1 and GKN2 promoter regions, and a potential role for EBNA1 in the deregulation and epigenetic repression of this tumor suppressor locus.
Identification of a high-occupancy EBNA1 binding site at GKN1-GKN2 locus by ChIP-Seq
GKN1 and GKN2 mRNA are highly expressed in primary stomach tissue
EBV latency reduces GKN1 and GKN2 mRNA expression in GC cell lines
EBV increases DNA methylation dependent repression of GKN1 and GKN2 mRNA in GC cell lines
To better understand the mechanism of GKN1 and GKN2 epigenetic repression, we examined the DNA methylation levels using methyl cytosine-specific antibody directed DNA immunoprecipitation (MeDIP) assay. We assayed the enrichment of methylated DNA in the GKN1-GKN2 control region in AGS and AGS-EBV cells with or without Aza treatment (Figure 5C). We observed a relative enrichment of methylated CpG at a region between the EBNA1 binding site and GKN2 transcription start site (GKN2_A). Aza treatment led to a decrease in MeDIP signal in most regions where a signal was detected, suggesting that Aza treatment led to a general reduction in DNA methylation. Similar loss of CpG methylation was observed at the alpha-globin gene (which does not bind EBNA1), and at several EBNA1 binding sites, including HDAC3 and MAP3K7IP (Figure 5D). We also noted that MeDIP signals were generally higher in EBV-AGS than in AGS cells, suggesting that EBV latent infection may promote or stabilize DNA methylation throughout the host genome.
EBNA1 depletion activates GNK1 and GKN2 mRNA expression in EBV positive epithelial cells
EBNA1 inhibits GKN1 and GKN2 transcription after DNA demethylation
In this study, we identified a high-occupancy EBNA1 binding site in the 5′ promoter control region of the divergently transcribed GKN1 and GKN2 genes. EBNA1 binding sites were observed in two independent ChIP-Seq data sets from EBV positive lymphoid BL cells Raji and EBV positive epithelial nasopharyngeal carcinoma cells C666-1 (Figure 1A). We confirmed these binding sites by conventional ChIP–qPCR in both cell lines (Figure 1B and C). EBNA1 was also shown to bind directly to these sites by EMSA with purified recombinant EBNA1 DBD protein (Figure 1D). We show that GKN1 and GKN2 mRNA levels are highly repressed in most cell lines relative to primary gastric tissue (Figure 2). To study the potential role of EBV and EBNA1 in the transcriptional control of GKN1 and GKN2, we generated an EBV positive AGS gastric carcinoma cell line. We show that EBV adopts a variant type I latency pattern in AGS cells (Figure 3), and that EBNA1 can bind to the GKN1/GKN2 promoter region in the cellular chromosome (Figure 4C). We also found that GKN1 and GKN2 mRNA were further suppressed in EBV positive AGS cells relative to control EBV negative AGS cells (Figure 4D). We then showed that Aza-treatment led to the increase expression of GKN1 and GKN2 (Figure 5A), and that EBV latent infection inhibits Aza activation of GKN2 (Figure 5B). We found that siRNA depletion of EBNA1 in EBV positive AGS cells leads to transcription activation of GKN2 (Figure 6). We also show that EBNA1 ectopic expression moderately increases basal, but inhibits the Aza-induced levels of GKN1 and GKN2 transcription (Figure 7). Taken together, these findings indicate that EBNA1 binds to the GKN1-GKN2 promoter control region in multiple cell types, and raise the possibility that EBNA1 contributes to the transcriptional and epigenetic repression of the GKN1 and GKN2 tumor suppressor genes in EBV positive GC.
EBV latent infection is known to increase the tumorigenic phenotype of gastric carcinoma cells [29–31]. GKN1 and GKN2 are reported to function as cell growth inhibitors and tumor suppressors in GC [20, 21, 23, 25–27]. Our mRNA expression data showing high-level mRNA expression only in primary normal gastric tissue are consistent with a role of GKN1 and GKN2 as a tumor suppressor. However, we were unable to show that over-expression of either or both GKN1 or GKN2 in AGS or AGS-EBV cause a cell cycle arrest or reduce viability (data not shown). This suggests that GKN1 and GKN2 function at earlier stages in tumor cell evolution, or in more complex tumor microenvironments. We speculate that EBNA1 may have a more pronounced effect on GKN1 and GKN2 expression in situations where EBV may infect primary gastric cells where basal expression of GKN1 and GKN2 are high and important for tumor suppression.
Previous published studies have shown that GKN1 and GKN2 transcription is subject to epigenetic suppression by DNA methylation in all forms of GC . Our studies are consistent with the role of DNA methylation in the epigenetic suppression of GKN1 and GKN2 in AGS cells. Treatment with Aza resulted in the 4-10 fold increase in GKN1 and GKN2 mRNA expression (Figure 5A), and MeDIP revealed enrichment of methylated DNA at the promoter regions (Figure 5C). AGS-EBV cells did show an increase in DNA methylation at several cellular sites, including regions surrounding the EBNA1 binding sites at the GKN1 promoter region (Figure 5C), and the HDAC3 and MAP3K7IP2 genes (Figure 5D). However, the presence of EBNA1 in AGS-EBV cells did not prevent Aza-induced demethylation at these sites. This suggests that EBNA1 may repress transcription from some promoters, like GKN2, through a mechanism distinct from DNA methylation. However, ectopic expression of EBNA1 alone produced a more complicated phenotype, causing a small increase in basal expression, but limiting the effects of Aza-induced demethylation (Figure 7). This may suggest that that EBNA1 may function differently when expressed ectopically, than when expressed in the context of the viral genome. Nevertheless, our findings suggest that EBNA1 perturbs the normal transcriptional regulation of the GKN1 and GKN2 genes.
The precise function of EBNA1 in transcription regulation remains unclear. EBNA1 has been implicated in the transcriptional activation and repression of both viral and cellular genes [32, 33]. EBNA1 can repress its own mRNA expression from the EBV Qp in type III latency, where repression has been linked to steric interference with RNA polymerase II binding to the transcription initiation site . On the other hand, EBNA1 can activate Cp and LMP1 promoters in type III latency where it may function as an enhancer-like factor [35–37]. EBNA1 has been implicated in transcription activation of some cellular genes, including the Nox2 gene involved in reactive oxygen species formation . EBNA1 may also affect host-cell transcription through a global remodeling of the host chromosome . Thus, EBNA1 may alter cellular transcription through multiple direct and indirect mechanisms.
Epigenetic modifications are known to play an important role in EBV-associated gastric carcinoma . Interestingly, AGS cells carrying EBV bacmid genomes had higher levels of methylated DNA at many tested sites (Figure 5D). This is consistent with the proposed role of EBV in the methylation of host tumor suppressor genes . This is also consistent with the findings that EBV positive GC has elevated DNA methylation at promoter regions of several key GC tumor suppressors, including gastrokine genes [39, 41–45]. While EBNA1 bound near DNA methylated regions of the GKN2, we were unable to show that EBNA1 modulates DNA methylation at the GKN1 and GKN2 sites (data not shown). However, it is possible that EBNA1 in association with another viral encoded or induced factor may stabilize GKN1 and GKN2 transcriptional repression through a chromatin-dependent and structural mechanism that reinforces DNA methylation. It is also possible that EBNA1 may regulate GKN1 or GNK2 only in tissue or tumor microenvironments that are not readily recapitulated in cell culture. While the function of EBNA1 binding to host cell chromosome sites remains an important area of investigation, more sophisticated infection models may be required to elucidate its potential role in altering host cell gene expression and carcinogenesis.
Cells, plasmids, and lentivirus infection
EBV-positive Burkitt’s lymphoma Raji cells, EBV positive nasopharyngeal carcinoma C666-1 cells, and gastric carcinoma cell lines (a gift from Dr. Antonia R. Sepulveda, Columbia University) including GTL, MKN28, MKN74, SNU638 were maintained in RPMI containing 10% FBS and supplemented with antibiotics (penicillin and streptomycin). Gastric carcinoma AGS cells (ATCC No. CRL-1739) were maintained in F-12K containing 10% FBS. Primary mouth epithelial cells were provided by Dr. Manjunatha Benakanakere, University of Pennsylvania and cultured in Keratinocyte-SFM medium. EBV-LCL was established by primary infection of peripheral blood mononuclear cells (PBMC) with EBV BAC virions generated from stimulated 293-EBV cells [46, 47]. EBV-LCL contains a hygromycin B resistant EBV bacmid were maintained in RPMI containing 10% FBS, hygromycin B (100 μg/ml), glutamax (Invitrogen), and antibiotics. AGS-EBV cells were generated from AGS cells co-cultivated with EBV-LCL by adapting a previously published co-cultivation method described AGS cells infection with rEBV through cell-to-cell contact  with some modification. Briefly, EBV-LCL was induced by 20 ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1 mM sodium butyrate (NaB) 24 hours prior to co-cultivation. The induced EBV-LCL cells were washed with PBS twice to completely remove the inducing agents, resuspended with complete RPMI medium at 106 cells/ml before the co-incubation. AGS cells were plated in 6-well Plates 24 hours before co-cultivation, then 60 - 70% confluent AGS cells in 1 ml complete F-12K medium were incubated with 106 induced and washed EBV-LCL cells in 1 ml complete RPMI. 24 hours later, 2 ml of Serum free F-12K medium was added to each well to reduce the FBS concentration to 5% to prevent cell overgrowth. 3 days after co-incubation, EBV-LCL cells were removed from the co-cultures and the AGS cells were thoroughly washed with PBS at least 5 times to remove any of the donor EBV-LCL cells. The infected AGS cells was then incubated with fresh F-12K medium with 10% FBS and 100 μg/ml hygromycin B and the selection medium was changed every 2 to 3 days until the infected AGS cells formed Hyg B selection colonies with GFP expression (usually 3 to 4 weeks after co-cultivation). The selection colonies were then pooled and tested for EBNA1 expression and EBV genome copy number before subject to experiments. AGS-EBV cells were maintained in F-12K medium with 10% FBS and 100 μg/ml hygromycin B.
pLU-EBNA1 Lentivirus expression vector was constructed by PCR amplification of EBNA1 with primers (GCGGGATCCTCTGACGAGGGGCCAGGTACAGGACCT and ATCGTCGACTCACTCCTGCCCTTCCTCACCCTCATC) introducing a 5′ BamH I and 3′ Sal I site cloned in frame to pLU-TCMV-FMCS-pPURO. AGS or MKN74 cells were infected with Lentivirus expressing pLU-EBNA1 or pLU control vector generated freshly from 293T cells. Infected AGS or MKN74 cells were then selected with 2.5 μg/ml Puromycin for 10 to 14 days. The selected cells were pooled and treated with or without 5'-azacytidine for 48 hours then subjected to RT-PCR.
siRNA against EBNA1 and siRNA Control (Cat. No. D-001810-01-20) were all purchased from Dharmacon. siRNA directed against EBNA1 3′UTR were synthesized using the target sequence CGGAGAUGACGGAGAUGAAUU. Transfection of siRNA duplexes was conducted by using Oligofectamine (Dharmacon), following manufacturers specifications.
Chromatin immunoprecipitation (ChIP) assays
ChIP assays were performed as described previously . Quantification of precipitated DNA was determined using real-time PCR and the delta Ct method for relative quantification (ABI 7900HT Fast Real-Time PCR System; Applied Biosystems). Primers for ChIP assays are available upon request. The following antibodies were used for ChIP assays: rabbit anti-EBNA1 (305/10 wk), anti- rabbit IgG (Santa Cruz Biotechnology, sc-2027). Anti-Actin HRP (Sigma, A3854) and rabbit anti-EBNA1 (305/10 wk) were used for Western Blotting. EBNA1 specific antibody was raised against a bacterial EBNA1 protein lacking the GA-repeat region using New Zealand white rabbits and then affinity purified using the same bacterial protein coupled to sepharose beads.
DNA fragments were labeled using T4 polynucleotide kinase (NEB) in the presence of [γ-32p]ATP and purified using G-25 spin columns (GE Healthcare). Purified EBNA1 DNA binding domain  was incubated with probes at room temperature for 30 min in a total volume of 20 μl buffer containing 10mM TrisCl PH 8.0, 100 mM KCl, 1 mM EDTA, 10 mM MgCl2, 0.05 μg/μl poly(dI-dC), 0.5 μg/μl bovine serum albumin, 0.05% NP40, 35 mM β-mercaptoethanol, and 10% glycerol. The samples were then separated by electrophoresis on a native 5% polyacrymide gel. Gels were dried and analyzed using a Typhoon phosphorImager system. The forward probe sequences are gatccggatacagattaggatagcatatactaccca (FR), tttcttattttccaggcagcatatactatctcaagctcaccatgtc (GKN1/2) and cgccgccggggcctgcggcgcctcccgcccgggcatggggccgcgc (negative control, LANA binding sites at KSHV TR).
For cell lines, RNA was isolated from 2 × 106 cells using RNeasy Kit (Qiagen) and then further treated with DNase I. Reverse transcriptase PCR (RT-PCR) was performed as previously described . Real-time PCR was performed with SYBR green probe in an ABI Prism 7900 according to the manufacturer’s specified parameters. Primer sequences for RT-PCR are available upon request. Total human stomach RNA (Cat. No. 540037) was purchased from Agilent Technologies.
Genome copy number assay
Genome copy number assay was performed as described previously . The total cellular DNA was assayed by real-time PCR using primers for the EBV Ori-Lyt region and normalized by the cellular GAPDH DNA region. The primers for Ori-Lyt and GAPDH are available upon request.
Methyl-DNA immunoprecipitation (MeDIP) methods have been described previously . MeDIP DNA were analyzed by real-time PCR with delta Ct method for quantification. The primers for MeDIP are available upon request.
We thank Andreas Wiedmer for technical assistance. We thank H.J. Delecluse for kindly providing EBV bacmid. We acknowledge the support of the Wistar Institute Cancer Center Core Facilities in Flow Cytometry, Bioinformatic, and Genomics. This work was supported by Wistar Institute Cancer Center Core Grant (P30 CA10815), K99AI099153 award from the National Institute Of Allergy And Infectious Diseases to IT, and RO1 (CA085678, CA093606, and DE017336) to PML.
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