GENOME REPLICATION AND REGULATION OF VIRAL ......DLBCLs. In contrast, we found that TET2 is expressed in normal naive tonsil B cells, in EBV-positive BLs with type III latency (which

Restricted TET2 Expression in GerminalCenter Type B Cells Promotes StringentEpstein-Barr Virus Latency

Coral K. Wille,a Yangguang Li,b Lixin Rui,b Eric C. Johannsen,b,c

Shannon C. Kenneyb,c

Departments of Medical Microbiology and Immunology,a Medicine,b and Oncology,c University of WisconsinSchool of Medicine and Public Health, Madison, Wisconsin, USA

ABSTRACT Epstein-Barr virus (EBV) latently infects normal B cells and contributes tothe development of certain human lymphomas. Newly infected B cells support ahighly transforming form (type III) of viral latency; however, long-term EBV infectionin immunocompetent hosts is limited to B cells with a more restricted form of la-tency (type I) in which most viral gene expression is silenced by promoter DNAmethylation. How EBV converts latency type is unclear, although it is known thattype I latency is associated with a germinal center (GC) B cell phenotype, and typeIII latency with an activated B cell (ABC) phenotype. In this study, we have examinedwhether expression of TET2, a cellular enzyme that initiates DNA demethylation byconverting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulatesEBV latency type in B cells. We found that TET2 expression is inhibited in normal GCcells and GC type lymphomas. In contrast, TET2 is expressed in normal naive B cellsand ABC type lymphomas. We also demonstrate that GC type cell lines have in-creased 5mC levels and reduced 5hmC levels in comparison to those of ABC typelines. Finally, we show that TET2 promotes the ability of the EBV transcription factorEBNA2 to convert EBV-infected cells from type I to type III latency. These findingsdemonstrate that TET2 expression is repressed in GC cells independent of EBV infec-tion and suggest that TET2 promotes type III EBV latency in B cells with an ABC ornaive phenotype by enhancing EBNA2 activation of methylated EBV promoters.

IMPORTANCE EBV establishes several different types of viral latency in B cells. How-ever, cellular factors that determine whether EBV enters the highly transformingtype III latency, versus the more restricted type I latency, have not been well charac-terized. Here we show that TET2, a cellular enzyme that initiates DNA demethylationby converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regu-lates EBV latency type in B cells by enhancing the ability of the viral transcriptionfactor EBNA2 to activate methylated viral promoters that are expressed in type III(but not type I) latency. Furthermore, we demonstrate that (independent of EBV)TET2 is turned off in normal and malignant germinal center (GC) B cells but ex-pressed in other B cell types. Thus, restricted TET2 expression in GC cells may pro-mote type I EBV latency.

KEYWORDS 5hmC, EBNA2, EBV, Epstein-Barr virus, TET2, germinal center B cell,latency

Epstein-Barr virus (EBV) is human gammaherpesvirus that infects over 90% of theworld population. EBV is the causative agent of infectious mononucleosis and isalso associated with several malignancies of B cell and epithelial cell origin, includingBurkitt lymphoma (BL), Hodgkin’s disease, posttransplant lymphoproliferative disorder,diffuse large B cell lymphoma (DLBCL), nasopharyngeal carcinoma (NPC), and gastriccarcinoma (1, 2). EBV establishes latent infection in normal B cells, in which the virus

Received 3 October 2016 Accepted 14December 2016

Accepted manuscript posted online 21December 2016

Citation Wille CK, Li Y, Rui L, Johannsen EC,Kenney SC. 2017. Restricted TET2 expressionin germinal center type B cells promotesstringent Epstein-Barr virus latency. J Virol91:e01987-16. https://doi.org/10.1128/JVI.01987-16.

Editor Richard M. Longnecker, NorthwesternUniversity

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Shannon C.Kenney, [email protected].

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persists as an episome and is replicated once per cell cycle by the cellular DNApolymerase in conjunction with the viral EBNA1 protein (3). There are at least threedifferent forms of EBV latency, which differ with regard to the number of viral genesexpressed. Type III latency (in which all 9 EBV latency proteins are expressed) is the onlylatency form sufficient to transform B cells in vitro (reviewed in reference 4). However,since type III latency is highly immunogenic, it occurs in immunocompetent humansonly during the initial stage of viral infection. Subsequently, EBV infection in B cellsconverts to a more stringent form of viral latency (type I latency), in which only theEBNA1 protein is expressed (in addition to noncoding viral encoded RNAs (reviewed inreferences 5 and 6). The cellular factors that regulate EBV latency type in infected B cellsremain poorly understood.

Following EBV infection of B cells, the virus initially establishes type III latency. Thefirst latent viral transcript expressed is derived from the EBV Wp promoter and isbiscistronic, encoding both EBNA2 and EBNA-LP (2). EBNA2, a transcription factor, thenactivates expression of the EBV Cp promoter, which drives expression of all EBNA genesduring type III latency (including EBNA2), and promoters for the EBV latent membraneproteins (LMPs) (1, 2). The divergent LMP1/LMP2B promoter drives expression of theLMP1 and LMP2B genes, and the LMP2A promoter drives the expression of LMP2A.

EBNA2 does not bind to DNA directly but instead activates EBNA gene transcriptionby interacting with the cellular transcription factor RBP-J�, which binds to sites in theC promoter (7). EBNA2 also interacts with RBP-J� to activate LMP2Ap (8). In the case ofthe LMP1/LMP2B promoter, EBNA2 interacts with RBP-J� as well as the cellular tran-scription factor PU.1 to activate transcription (9). In addition, many EBNA2 binding sitesin the cellular genome have been shown to colocalize with binding sites for theessential B cell differentiation factor EBF1 as well as other cellular transcription factors(10).

During type I latency, Cp promoter and EBNA2 expression is turned off, and EBNA1transcription is instead regulated by the viral Q promoter (Qp). The EBV genomebecomes highly methylated during the establishment of type I latency, and stringenttype I gene expression is enforced in part by CpG methylation of the viral Cp, LMP1/2B,and LMP2A promoters (11, 12). In contrast, the Cp, LMP1/LMP2B, and LMP2A promotersremain unmethylated in cells with type III latency. Treatment of cells exhibiting type Ilatency with demethylating agents is sufficient to convert cells to type III latency (13).Furthermore, the ability of the Cp and LMP1/LMP2B promoters to be activated byEBNA2 in transient reporter gene assays has been previously shown to be inhibited bypromoter methylation (14, 15). Of note, methylation of Cp does not prevent EBNA2/RBPJ�-mediated activation but instead prevents binding of the cellular AUF1 protein tothe EBNA2 enhancer site within Cp (14, 16).

Nevertheless, how methylation of the type III latency promoters is established, orreversed, during normal B cell infection is not clear. Interestingly, type I EBV latency inEBV-infected lymphomas (including Burkitt lymphomas and diffuse large B cell lym-phomas) is almost always found in tumors that have a germinal center (GC) phenotype(17–19). In contrast, type III latency is almost exclusively confined to tumors with anactivated B cell (ABC) phenotype. Furthermore, there is some evidence that EBVsuperinfection of EBV-negative DLBCLs in vitro converts these lines to a ABC phenotype,but only when the EBV infection assumes the type III latency form (18).

We hypothesized that a cellular environment unique to GC B cells may promotestringent type I EBV latency by enhancing de novo methylation, and/or decreasingdemethylation, of the Cp, LMP1/LMP2B, and LMP2A promoters following EBV infection.Reversal of promoter methylation is mediated by the cellular TET family of enzymes thathydroxylate 5-methylcytosine (5mC), producing 5-hydroxymethylcytosine (5hmC) (re-viewed in reference 20). 5hmC is an intermediate for DNA demethylation and is alsostably maintained on the genome in some cell types, where it is commonly associatedwith gene activation (21).

Here we demonstrate that TET2 expression is turned off in normal GC tonsil cells, aswell as in EBV-infected GC type BLs with type I latency and EBV-negative GC type

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DLBCLs. In contrast, we found that TET2 is expressed in normal naive tonsil B cells, inEBV-positive BLs with type III latency (which have an ABC phenotype), and in ABC typeDLBCLs. Furthermore, we show that BLs and DLBCLs with an ABC phenotype haveglobally increased 5hmC-modified cellular DNA, and decreased 5mC-modified cellularDNA, compared to those of BLs and DLBCLs with a GC phenotype. Importantly, wedemonstrate that TET2 enhances EBNA2’s ability to activate the methylated form of theLMP1 promoter in reporter gene assays and increases its ability to activate LMP1expression from the endogenous methylated viral genome in BL cells with type Ilatency. Finally, we show that the EBV C promoter is 5hmC modified in TET2-expressingcells with type III latency and, conversely, is highly methylated and silenced in TET2-deficient cells that maintain type I EBV latency. These results are the first to show thatTET2 is downregulated in B cells with the GC phenotype, and they suggest that TET2expression plays a critical role in determining the EBV latency type in B cells.

RESULTSTET2 is not expressed in Burkitt lymphoma cell lines with type I latency and a

GC type but is expressed in BL cells with type III latency and an ABC phenotype.Given that EBV infection in GC type Burkitt lymphomas is associated with a restrictedlatency gene expression program called type I latency, in which expression of the EBVtype III latency genes is silenced by promoter methylation, we explored whethercellular TET proteins might regulate EBV genome CpG methylation in BLs with type Iversus type III latency. TET proteins are 5-methylcytosine (5mC) hydroxylases thatinitiate DNA demethylation by converting 5mC into 5-hydroxymethylcytosine (5hmC)(20). Importantly, TET2/TET3 in vivo knockout in the mouse B cell lineage was recentlyshown to decrease B cell maturation and inhibit the germinal center reaction (22).Therefore, levels of TET2 and TET3 expression were compared in isogenic EBV� BL celllines with type I versus type III latency. Interestingly, reverse transcription-quantitativePCR (RT-qPCR) analysis of the TET2 transcript indicated that EBV� BL cell lines that haddrifted to the type III EBV latency program all expressed the TET2 transcript, while thelines that maintained a type I EBV latent infection did not express TET2 (Fig. 1A). Incontrast, levels of TET3 expression were similar in all cells lines (Fig. 1A). EBV latencytype was determined by EBNA3C expression (Fig. 1A), which is expressed in type III butnot type I latency. Immunoblot analysis of cell lines derived from the same BL tumor(Kem) that had either type I latency (Kem I) versus type III latency (Kem III) confirmedthat TET2 protein is expressed in Kem III cells but not Kem I cells (Fig. 1B). Similar resultswere obtained with Mutu I versus Mutu III cells (data not shown). As previouslyreported, type I lines had a GC phenotype (BCL6�/IRF4�), whereas type III lines had anABC phenotype (BCL6�/IRF4�) (Fig. 1B). These results suggest that EBV proteinsspecifically expressed in type III (but not type I) latency, and/or differences associatedwith the GC versus ABC phenotype, regulate TET2 expression in Burkitt lymphoma cells.

TET2 is turned off in EBV-negative DLBCL lines with a GC phenotype but isexpressed in DLBCL cell lines with the ABC phenotype. To determine whetherphenotypic differences between GC type and ABC type B cells are associated withaltered TET2 expression in the absence of EBV infection, we examined the level of TET2protein in a series of EBV-negative DLBCL cell lines that had either GC or ABCphenotypes (23). As shown in Fig. 2, TET2 protein was not expressed in four differentGC type EBV-negative DLBCL lines (HT, SUDHL4, RL, and DB) but was expressed in fourdifferent EBV-negative ABC DLBCL cell lines (HBL1, OCI-Ly10, OCI-Ly3, and SUDHL2).The ABC DLBCL cell lines were confirmed to have an ABC phenotype by the presenceof IRF4 expression (Fig. 2). These results suggest that TET2 is specifically turned off inB cells with a GC phenotype rather than activated by EBV infection per se.

Normal human GC cells do not express TET2. Given that DLBCL and BL cell lineswith a GC phenotype express little, if any, TET2, we hypothesized that TET2 is turned off innormal human GC B cells. To examine this, naive and GC type B cells were isolated fromhuman tonsils, and RNA was extracted from each cell type, as previously described (24).Isolated naive B cells were confirmed by fluorescence-activated cell sorter (FACS) analysis to

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be IgD� CD38low CD27�, and isolated GC B cells (centroblasts) were confirmed to beCD77� CD38high (data not shown). TET2 transcript levels (relative to glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were quantitated by RT-qPCR and were compared tothose in Kem I (TET2-negative) versus Kem III (TET2-positive) cell lines.

As shown in Fig. 3, TET2 was highly expressed in primary naive tonsillar B cells(similar to the level expressed in Kem III BL cells). In contrast, little, if any, TET2expression was observed in either primary GC tonsillar B cells or Kem I BL cells. SinceTET2 has been previously demonstrated to be highly expressed in CD19� B cells (apan-B cell marker) (25), TET2 expression appears to be specifically inhibited in normalGC B cells, rather than activated in naive B cells and ABC type DLBCLs.

Loss of TET2 expression correlates with globally decreased 5hmC-modifiedDNA, and globally increased 5mC-modified DNA, in BL and DLBCL cell lines. Todetermine if TET2 expression correlates with the amount of globally 5mC- and/or

FIG 1 TET2 is not expressed in EBV� BL cell lines with a GC phenotype and type I latency. (A) RNA wasisolated from EBV� BL cell lines (Kem I and Mutu I) with a GC phenotype that maintain type I EBV latency,and isogenic EBV� BL cell lines (Kem III and Mutu III) with an ABC phenotype that maintain type III EBVlatency. Expression of TET2 and TET3, and the type III latency viral gene for EBNA3C, was quantified withRT-qPCR. Transcript levels were quantified relative to �-actin expression, and Kem III transcript levelsrelative to �-actin were set to 1. Error bars indicate �1 standard deviation calculated from triplicatesamples. N.D., not detected. (B) Immunoblot analysis was performed to examine TET2 protein expressionin Kem I and Kem III cells, and the B cell phenotype was analyzed by probing for IRF4 (ABC marker) andBCL-6 (GC marker). Tubulin served as a loading control.

FIG 2 TET2 is not expressed in EBV-negative DLBCLs with a GC phenotype. Immunoblots were performedto compare TET2 protein expression in EBV-negative diffuse large B cell lymphoma (DLBCL) cell lines witha GC phenotype (HT, SUDHL4, RL, and DB) versus an ABC phenotype (HBL1, OCI-Ly10, OCI-Ly3, andSUDHL2); IRF4 served as a marker for ABC phenotype and �-actin as the loading control.






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5hmC-modified DNA, total DNA was isolated from BL and DLBCL cell lines that hadbeen treated or not for several days with vitamin C (enhances TET enzyme activity)(26–28), and dot blots were performed to quantify global 5mC- and 5hmC-modifiedDNA in TET2-positive versus TET2-negative cell lines. As shown in Fig. 4A, EBV-infectedBL cells with type I latency (Kem I) have globally higher levels of cytosine methylationthan isogenic cells that had type III latency (Kem III). Furthermore, Kem I cells have lessglobal 5hmC-modified DNA than Kem III cells. Similar results were obtained with MutuI versus Mutu III cells (data not shown). Additionally, EBV-negative GC-type DLBCL celllines have less global 5hmC-modified DNA than EBV-negative ABC-type DLBCL cell lines(Fig. 4B). These results suggest that loss of TET2 expression in normal GC B cells, as wellas in GC-type BL and DLBCL lymphomas, inhibits global 5hmC modification whilepromoting global 5mC modification of DNA.

The EBV genome is 5hmC modified at a type III latency promoter in BL cellswith an ABC phenotype. To determine if EBV promoters that are active in cells withtype III, but not type I, latency can be 5hmC modified, we quantitated the amount of5hmC- and 5mC-modified DNA on the type III Cp using the DNA immunoprecipitation(DIP) technique. Mutu I and Mutu III cells were treated for 4 days with acyclovir (toprevent lytic EBV DNA replication, which results in loss of 5hmC and 5mC on the EBVgenome), and isolated total DNA was immunoprecipitated using antibodies that spe-cifically detect 5hmC- or 5mC-modified DNA. As expected, the EBV Cp promoter ishighly methylated in Mutu I cells (where the promoter is silent) (Fig. 5) and much lessmethylated in Mutu III cells (where the promoter is active). In contrast, the Cp had littleor no 5hmC modification in Mutu I cells (in which TET2 is not expressed) but haddetectable 5hmC modification in Mutu III cells (which express TET2) (Fig. 5). These datasuggest that TET2 expressed in EBV-infected cell lines can target methylated type III

FIG 3 TET2 is turned off in normal human GC B cells. RNA was harvested from separated normal humantonsil naive and GC B cells, and Kem I and Kem III EBV-positive BL cells, and converted to cDNA by RT-PCR.TET2 transcript levels relative to GAPDH were determined by qPCR. The level of TET2 relative to GAPDHin naive B cells was set to 1, and error bars indicate �1 standard deviation calculated from duplicatesamples.

FIG 4 GC B cells have less 5hmC, and more 5mC, than B cells with an ABC phenotype. Global levels of5hmC-modified and 5mC-modified DNA in EBV� BL cell lines with a type III EBV latency (TET2�, ABC)versus type I EBV latency (TET2�, GC) (A) or EBV-negative DLBCL cell lines with a GC-like phenotypeversus ABC-like phenotype (B) were assessed using dot blotting. Cells were treated or not, as indicated,for 4 days with vitamin C (Vc), which enhances TET2 activity prior to harvesting cellular DNA. DNA wasspotted on a nitrocellulose membrane (1,000 ng of DNA for 5hmC and 62.5 ng for 5mC), and equalloading of DNA was confirmed by methylene blue staining.






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latency promoters for 5hmC modification, which subsequently may promote demethy-lation and activation of these promoters.

TET2 enhances EBNA2-mediated activation of the LMP1 promoter. The EBVoncoprotein latent membrane protein 1 (LMP1) is expressed in cells with type III EBVlatency, but not type I latency, and the activity of the LMP1 promoter has been shownto be regulated in part by CpG methylation (11, 12, 15). Since EBNA2 activates LMP1expression, and LMP1 expression is associated with LMP1p demethylation, we exploredwhether TET2 enhances EBNA2-mediated activation of LMP1 when the promoter ismethylated. The LMP1 ED-L1 promoter (the primary LMP1 promoter used in B cells [29])was cloned upstream of luciferase in a reporter gene vector that lacks any CpGs motifs(pCpGL-Basic), thus preventing nonspecific silencing due to methylation of the lucif-erase gene itself. The LMP1p luciferase vector was methylated or mock treated in vitroand transfected into the EBV-negative B cell line BJAB, with or without an EBNA2expression vector, in the presence or absence of a cotransfected TET2 expressionvector.

As shown in Fig. 6, in the absence of cotransfected TET2, EBNA2 efficiently activatedthe unmethylated, but not the methylated, form of the LMP1 promoter. CotransfectedTET2 had little effect on the ability of EBNA2 to activate the unmethylated form of the

FIG 5 The EBV type III latency C promoter is methylated in Mutu I cells, and 5hmC modified, but notmethylated, in Mutu III cells. A DIP assay was performed to quantitate 5mC and 5hmC modificationof the Cp in Mutu I (TET2�, GC) versus Mutu III (TET2�, ABC) BL cells. Cells were treated for 4 dayswith acyclovir, and then DNA was harvested, sonicated, and immunoprecipitated with anti-5mC,anti-5hmC, or appropriate Ig isotype controls. Promoter immunoprecipitation was quantified usingqPCR with primers spanning the Cp. Promoter modification is shown as fold pulldown relative to theisotype control (set to 1).

FIG 6 TET2 restores EBNA2 activation of the methylated LMP1 promoter. The ability of EBNA2 to activatethe unmethylated, methylated, and 5hmC-modified forms of the EBV LMP1 promoter was analyzed usingreporter gene assays. LMP1p was cloned upstream of the luciferase gene in the CpG-free vectorpCpGL-basic and was methylated (dark gray) or mock treated (light gray) in vitro. The construct wastransfected into EBV� BJAB cells with and without TET2, which converts 5mC into 5hmC (black), in thepresence and absence of EBNA2. Luciferase assays were performed 2 days posttransfection. The foldluciferase activation is shown relative to the activity of the unmethylated vector transfected with controlvectors, which is set to 1. Error bars indicate standard errors calculated from 2 replicate experimentsconsisting of triplicate conditions.






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LMP1 promoter. Importantly, however, coexpression of TET2 greatly increased theability of EBNA2 to activate the methylated form of LMP1p (Fig. 6). These resultssuggest that TET2 is required for efficient activation of methylated LMP1 promoter byEBNA2.

TET2 expression enhances EBNA2-mediated type III latency gene expressionfrom the endogenous viral genome in cells with type I latency. To determine if TET2enhances the ability of EBNA2 to induce gene expression from the endogenous highlymethylated viral genome in cells with type I latency, Mutu I cells were transfected withan EBNA2 expression vector in the presence and absence of a cotransfected (consti-tutively active) TET2 vector. EBNA2-mediated activation of the type III latency programwas determined by immunoblot analysis for LMP1. As shown in Fig. 7, transfectedEBNA2 alone induced a very low level of LMP1, while cotransfection of EBNA2 and TET2greatly enhanced EBNA2-mediated LMP1 expression. These results suggest that thelack of TET2 expression in EBV-infected B cells with a GC phenotype helps to maintainEBV type I latency by suppressing the ability of low-level EBNA2 to induce type III genetranscription.

DISCUSSION

EBV-infected cells can enter the most stringent form of viral latency (type I) whencritical viral promoters active in type III latency are silenced via cytosine methyl-ation (11, 12). However, exactly why these promoters become methylated in certaincells but not others is not clear. The cellular TET family of enzymes hydroxylates5-methylcytosine (5mC), producing an intermediate for DNA demethylation,5-hydroxymethylcytosine (5hmC) (20), and enhances gene transcription. Here weshow that TET2 expression is extremely low both in normal GC cells isolated fromhuman tonsils and in B cell lymphomas with a GC phenotype, including Burkittlymphomas and GC type DLBCLs. Furthermore, we found that TET2 expression inEBV-infected B cells is strongly correlated with the type of viral latency anddemonstrated that TET2 enhances the ability of EBNA2 to transcriptionally activatethe methylated form of the LMP1 promoter. These results suggest that a lack ofTET2 expression in GC cells contributes to the tendency of EBV to convert to typeI latency in this cell type, and conversely, TET2 expression in ABC type B cellspromotes type III latency both by reversing 5mC modification of the EBV genomeand by increasing the ability of EBNA2 to activate methylated viral promoters.

Although the role of EBV latency promoter methylation in enforcing type I EBVlatency has been known for some time, how such methylation is regulated remainslargely unclear. Methylation of the EBV genome first becomes apparent around 2 weeksafter infection of B cells (30). Regulation of viral genome methylation appears to becomplex, since inactivation of single DNA methyltransferases (DNMTs) or a combinationof DNMT1 and DNMT3B is not sufficient to induce Cp demethylation or activation oftype III latency gene expression (31). Two different EBV latency proteins, LMP1 andLMP2A, have been reported to increase expression of cellular DNA methyltransferases

FIG 7 EBNA2 activation of LMP1 in an EBV� BL cell line with a type I latency is augmented by TET2expression. Mutu I cells were transfected with a control vector, or an EBNA2 expression vector with andwithout a TET2 expression vector, as indicated. Two days later, immunoblotting was performed toexamine transfected EBNA2 protein expression and induced LMP1 protein expression. �-Actin served asa loading control.






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(32, 33). Furthermore, the viral latency proteins EBNA3A and EBNA3C induce promotermethylation of the cellular proapoptotic Bim gene through an unknown mechanism(34). Thus, EBV-encoded proteins expressed during type III latency seem to promoteEBV (and cellular) genome cytosine methylation, allowing the virus to switch to themost stringent form of viral latency and better evade the host immune response.

Nevertheless, at least in tissue culture, it is clear that certain EBV-infected Burkittlymphoma lines with type I latency can drift into type III latency. Burkitt cells thatconvert to type III latency express more anti-apoptotic proteins and have a survivaladvantage (reviewed in reference 4) and thus tend to overgrow cells with type I latencyin vitro. Such lines provide definitive evidence that type I latency, and methylation oftype III latency promoters, is reversible. In addition, cells with type III latency arecommonly found in immunosuppressed patients (1, 2), although in this case it cannotbe ruled out that such cells are newly infected and have not yet undergone viralgenome methylation. Given the ability of type I Burkitt lines to switch to type III latency,and to lose cytosine methylation of the latency III promoters, we hypothesized that thedemethylating functions of cellular TET protein(s) might be involved in this phenom-enon.

In this study, we have unexpectedly discovered that B cell lymphomas with a GCphenotype (regardless of whether they are EBV infected or not) express little, if any,TET2 (Fig. 1 and 2) and that, likewise, normal tonsillar GC cells express much less TET2than normal naive B cells (Fig. 3). Furthermore, we found that lymphoma cells with aGC phenotype have globally decreased 5hmC-modified DNA, and globally increased5mC modified DNA, in comparison to those of their ABC counterparts (Fig. 4) and thatthis effect is EBV independent. In support of our findings in this study, examination ofthe TET1, -2, and -3 transcript levels reported in a recently published transcriptomesequencing (RNA-seq) data set from four human GC DLBCL cell lines and four humanABC DLBCL cell lines (half of which were not used in this study) (35) revealed that TET2expression was increased more than 4-fold in ABC lines compared to GC DLBCL lines.In contrast, ABC and GC DLBCLs expressed similar levels of TET3 and expressed little, ifany, TET1 (35).

Since TET2 is expressed in many tissue types (36) and has been shown to be highlyexpressed in pooled CD19� B cells (a pan-B cell marker) (25), our finding that TET2 isexpressed very poorly (if at all) in normal tonsillar GC cells and GC type lymphomaslikely results from loss of TET2 expression in GC type B cells rather than a gain ofexpression in ABC type B cells. TET2 plays a critical role in early stages of B celldevelopment, since in vivo knockout of TET2/TET3 in the mouse B-cell lineage preventsdemethylation of B cell-specific enhancers and key regulatory sites, such as the Ig�locus, that occurs normally beginning in early in B cell development, at the pro-B cellstage (22). Phenotypically, TET2/TET3 knockout inhibits B cell maturation and renders Bcells unable to mount an immune response and form germinal centers (22). Since themaster regulator of the GC phenotype, BCL-6, is a transcriptional repressor (reviewed inreference 37), it is possible that BCL-6 itself, or other transcription factors activateddownstream of BCL-6, inhibits transcription of TET2 once B cells have become GC cells.As BCL-6 expression is lost when B cells exit the GC phenotype, this could explain whyTET2 is expressed when BL cells convert to an ABC type. Alternatively, cellular tran-scription factors activated in ABCs (such as IRF4 [23]) may be required for TET2transcription in B cells. Conversely, TET2 expression may influence B cell phenotype byactivating genes involved in GC exit.

Like other groups (19), we found that type III latency in BL cells is associated with anABC phenotype, while type I latency is associated with a GC phenotype (Fig. 1). Sincethe EBV EBNA2 latency protein is the key transcription factor required for activation oftype III latency viral promoters, we examined how methylation of the LMP1 promoter(the major viral oncoprotein) affects its ability to be activated by EBNA2. We found thatcytosine methylation of the LMP1 promoter greatly decreases the ability of EBNA2 toactivate the promoter in reporter gene assays and that cotransfection with a constitu-tively active TET2 vector reverses this effect (Fig. 6). Furthermore, TET2 greatly enhances






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the ability of EBNA2 to activate expression of the methylated LMP1 promoter from theendogenous viral genome in BL cells with type I latency (Fig. 7). Thus, a lack of TET2expression in GC cells may promote type I latency both by increasing methylation ofviral promoters and by attenuating EBNA2’s ability to activate methylated viral pro-moters.

Interestingly, EBV superinfection of GC-type DLBCLs in vitro converts the cells to anABC phenotype in lines where EBV establishes type III latency, but not in lines whereEBV establishes type I latency, and both LMP1 and EBNA2 contribute to loss of the GCphenotype (18). Thus, EBV proteins expressed during type III latency may stabilize thisform of latency by inducing loss of the GC phenotype, with concomitant activation ofTET2 expression. Although silencing type III protein expression in EBV-infected B cellsnormally involves methylation of type III latency promoters, a subset of Burkitt lym-phomas has been found to have EBNA2 deletions that convert the virus to morestringent form of latency termed Wp restricted, even in an ABC environment (38);furthermore, some of these EBNA2-deleted tumors have been shown to express IRF4and thus do not have a strict GC phenotype (38). Thus, deletion of the EBNA2 geneserves as another mechanism by which the type III latency program could be shut downin BLs.

EBNA2 does not bind directly to DNA but is instead tethered to promoters via itsinteractions with cellular transcription factors, including RBP-J�, PU.1, and EBF1 (7, 9,10). Both PU.1 and EBF1 binding motifs in the LMP1 promoter contribute to itsactivation by EBNA2 (9, 10). Intriguingly, both PU.1 and EBF-1 were also recently shownto directly interact with TET2, but not the other TET enzymes (21). PU.1 targets TET2 toPU.1 sites in cellular promoters of genes required for osteoclast differentiation, and thisinteraction results in the promoters becoming 5hmC modified, and then demethylated,during osteoclast differentiation (39). Although the PU.1 DNA binding consensus motifdoes not contain a CpG motif, the interaction between TET2 and PU.1 results indemethylation of surrounding CpGs that inhibit promoter transcription when methyl-ated (39). Thus, it is likely that the PU.1/EBNA2 complex bound to the LMP1p inducesactivation of the promoter by recruiting TET2 and inducing demethylation of thepromoter. Similarly, the TET2/EBF1 interaction is thought to inhibit methylation ofpromoters, since TET2 and EBF1 directly interact and colocalize at EBF1 binding sitesthat are commonly located within promoters that become hypermethylated in cancerswith inactive TET function due to IDH1/2 mutations (40). Importantly, the previouslyreported findings showing that PU.1 and EBF1 specifically interact with TET2 but notother TET proteins help to explain the unique requirement for TET2 (versus TET1/TET3)in EBV-infected B cells for efficient EBNA2 transcriptional function.

It will be important to confirm whether loss of TET2 expression and 5hmC modifi-cation is an important part of normal GC cell biology. Somatic hypermutation andaffinity maturation of antibodies (which specifically occur in GC cells) are associatedwith immunoglobulin gene mutation and demethylation via deamination of cytosineinto uracil, and perhaps via deamination of 5mC into thymine, by activation-induceddeaminase (AID) (41, 42). Demethylation may also be mediated by the activation ofTET2 expression, which converts 5mC into 5hmC and the further oxidized forms5-formylcytosine (5fC) and 5-carboxycytosine (5caC) (20). 5caC-G base pairs wererecently shown to be targeted more efficiently for repair back to C-G by thymine-DNAglycosylase (TDG) than T-G mismatches (43). Possibly, the TET2 function interferesand/or competes with the AID function, and hence TET2 must be turned off in GC cells.Finally, it is also possible that TET2 is specifically turned off in malignant GC-typelymphomas and that loss of TET2 plays a critical “driver” role in these lymphomas.Although PU.1 is required at the first stage of B lymphoid development, and itsexpression level determines whether lymphoid progenitors differentiate into the my-eloid or B cell lineage (44), PU.1 also functions as a tumor suppressor during later stagesof B cell development and is often turned off in certain B cell malignancies (45, 46).Given the previous findings that TET2 is required for efficient PU.1 activation of genes






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(39), loss of TET2 expression in B cell malignancies may serve as another mechanism toinhibit PU.1 activity.

Importantly, TET2 loss has recently been implicated in the development of EBV�

nasopharyngeal carcinoma (NPC) (47). EBV exclusively, lytically infects more differenti-ated normal epithelial cells (48–53), and thus, the viral genome remains highly un-methylated (11). However, in the case of NPC, EBV maintains a latent infection and theviral genome becomes highly methylated (54). We recently showed that TET2 5hmCmodifies the EBV genome to promote lytic gene expression in epithelial cells and thatNPC tumors lose TET2 expression, which may enhance oncogenesis (47). Furthermore,TET2 was recently found to be a resistance factor of EBV-induced DNA methylation ingastric carcinoma cells, demonstrating that TET2 may have an important role inprotection against EBV� tumor development (55). Thus, regulation of TET2 activity mayplay critical roles in the development of both epithelial and B cell malignanciesassociated with EBV.

MATERIALS AND METHODSCell lines and culture. All cell lines were maintained in RPMI 1640 growth medium (Invitrogen)

supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen) at 37°Cwith 5% CO2 and 100% humidity. Mutu I and Kem I (gifts from Alan Rickinson [Birmingham, England] andJeff Sample [Penn State]) are EBV� Burkitt lymphoma cell lines that maintain a type I EBV latency. MutuIII and Kem III (also gifts from Alan Rickinson and Jeff Sample) originated from Mutu I and Kem I cell lines,respectively, but the EBV infection has drifted to type III latency in culture. EBV-negative human diffuselarge B cell lymphoma (DLBCL) cell lines with a germinal center (GC)-like phenotype (HT, SUDHL4, RL, DB,and SUDHL7) and activated B cell (ABC)-like phenotype (HBL1, OCI-Ly10, OCI-Ly3, SUDHL2, and RIVA)were acquired from Louis Staudt (NCI) via Lixin Rui (University of Wisconsin-Madison). BJAB (a kind giftfrom Janet Mertz, University of Wisconsin-Madison) is an EBV-negative B cell lymphoma line thought tobe derived from an EBV-negative BL tumor (56).

Plasmids and cloning. Plasmid DNA was purified with columns using the Qiagen midi/maxiprep kitaccording to the manufacturer’s instructions. pSG5 was acquired from Stratagene. SG5-EBNA2 (a giftfrom Paul Ling, Baylor College of Medicine) contains the EBNA2 gene under the control of the simianvirus 40 (SV40) promoter. pcDNA3.1(�) was obtained from Invitrogen. pcDNA3-FLAG-TET2 (a gift from YiZhang, Howard Hughes Medical Institute and Harvard Medical School) contains the FLAG-taggedcatalytic domain of the mouse Tet2 coding sequence under the control of cytomegalovirus (CMV)promoter (57).

pCpGL-basic (a gift from Micheal Rehli, Universitätsklinikum Regensburg) is a CpG-free, promoterlessluciferase reporter gene vector that was constructed as previously described (58). The proximal LMP1ED-L1 promoter was PCR amplified using the EBV B95.8 genome as the template with the primers5=-GCTACTAGTTCATGACACTCGCACAGCCCACACC-3= and 5=-CTGAGATCTCAGTGTGTCAGGAGCAAGGCAGTTG-3= and cloned upstream of the luciferase gene in pCpGL-basic using the SpeI and BglII restrictionsites.

Isolation of primary tonsillar naive and GC B cells. Isolation of GC B cells was done describedbefore (59). Briefly, magnetic cell separation with the MidiMACS system (Miltenyi Biotec) was used toisolate naive B cells and GC B cells from human tonsils according to the protocol published by Klein etal. (24). The purity of separated B cell subpopulations was confirmed with FACScan analysis (BectonDickinson) with the following surface markers: naive B cells were IgD� CD38low CD27� and GC B cells(centroblasts) were CD77� CD38high.

RNA isolation and RT-qPCR. RNA was isolated from the EBV-positive BL cell lines Mutu I, Mutu III,Kem I, and Kem III and primary tonsillar naive and GC B cells using the RNeasy Plus minikit (Qiagen)according to the manufacturer’s instructions. Equal amounts of RNA derived from EBV-positive BL celllines were DNase treated, and cDNA was made using the Improm-II reverse transcription (RT) system(Promega). Equal amounts of primary tonsillar B cell RNA were reversed transcribed with a first-strand cDNAsynthesis kit (Invitrogen). Relative transcript levels were quantified with iTaq universal SYBR green (Bio-Rad)using quantitative PCR (qPCR) analysis with primers for the following: TET2 (5=-AAAGATGAAGGTCCTTTTTATACCC-3= and 5=-TTTCAACTTCTGTCCAAACCTT-3=), TET3 (5=-CAGCAGCCGAGAAGAAGAAG-3= and 5=-GGACAATCCACCCTTCAGAG-3=), EBNA3C (5=-GGGCTGTCAAGCAATCGCAC-3= and 5=-GTGGTGCATTCCACGGGTAA-3=),�-actin (5=-GCCGGGACCTGACTGACTAC-3= and 5=-TTCTCCTTAATGTCACGCACGAT-3=), and GAPDH (5=-TGAAGGTCGGAGTCAACGGATTG-3= and 5=-GCCATGGAATTTGCCATGCCATGGGTGG-3=).

Immunoblotting. Immunoblotting was performed as previously described (60). Cells wereharvested in Sumo lysis buffer with 1� proteasome inhibitor cocktail (Roche). Lysate proteinconcentration was determined using the Sumo protein assay (Bio-Rad), and equal amounts ofprotein were loaded on 6%, 10%, or 4 to 20% gradient (Bio-Rad) sodium dodecyl sulfate (SDS)-polyacrylamide gels. Proteins were resolved and transferred to nitrocellulose membranes, whichwere blocked in a phosphate-buffered saline solution with 5% milk and 0.1% Tween 20. Themembranes were then incubated with primary antibody diluted in the blocking solution. Thefollowing antibodies were used: anti-�-actin AC-15 (Sigma; A5441; 1:5,000), anti-BCL-6 (Santa Cruz;0.N.26; 1:200), anti-EBNA2 PE2 (Abcam; ab90543; 1:250), anti-IRF4 (Santa Cruz; sc-6059; 1:200),






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anti-LMP1 S12 (Kerafast; ETU001; 1:5000), anti-TET2 (Proteintech; 21207-1-AP; 1:1,000), and anti-tubulin (Sigma; T5168; 1:2,000). The secondary antibodies used were horseradish peroxidase (HRP)-goat anti-mouse (Pierce; 1:5,000) and HRP-goat anti-rabbit (Pierce; 1:10,000).

Dot blots. Cells were pretreated or not with vitamin C (50 �g/�l) daily for 4 days prior to DNA isolation.DNA was isolated using the GenElute mammalian genomic DNA miniprep kit (Sigma) according to themanufacturer’s instructions. NaOH was added at a final concentration of 0.2 M to 2 �g of DNA. Samples wereincubated at 95°C for 5 min, followed by immediate incubation on ice to denature the DNA. Ammoniumacetate was added to a final concentration of 0.8 M to neutralize the NaOH. A 0.22-�m nitrocellulosemembrane was presoaked in 2� SSC (0.3 M NaCl, 30 mM sodium citrate [pH 7.0]), and DNA was then spottedonto the membrane through a dot blot apparatus. One thousand nanograms of DNA was loaded to probefor 5hmC, and 62.5 ng was loaded to probe for 5mC. The DNA was cross-linked to the membrane with UVirradiation, and the membrane with incubated with blocking solution (Tris-buffered saline solution with 5%milk and 0.1% Tween 20) and then incubated with primary antibody diluted in the blocking solution. Thefollowing antibodies were used: anti-5-methylcytosine 33D3 (Active Motif; 39649; 1:1,000) and anti-5-hydroxymethylcytosine (Active Motif; 39769; 1:10,000). The secondary antibodies used were HRP-goat anti-mouse (Pierce; 1:2,000) and HRP-goat anti-rabbit (Pierce; 1:2,000). Equal loading of DNA was determined bystaining with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2).

DNA immunoprecipitation (DIP). Mutu I and Mutu III were treated with acyclovir (100 �g/ml) for 4days, and DNA was isolated using the GenElute mammalian genomic DNA miniprep kit (Sigma)according to the manufacturer’s instructions. DNA (10 �g in 500 �l of Tris-EDTA [TE]) was sonicated toan average size of 300 bp and denatured by heating at 95°C for 10 min followed by rapid chilling on ice.Fifty microliters (10%) was reserved to be used as input, and then 50 �l of 10� IP buffer (100 mM sodiumphosphate buffer [pH 7.0], 1.4 M NaCl, 0.5% Triton X-100) was added to the sample. The samples wereprecleared with protein A/G beads for 2 h and separated into four conditions of equal volume (one foreach antibody and isotype control). Samples were immunoprecipitated overnight with the followingantibodies or isotype controls: 1 �l of anti-5hmC (Active Motif; 39769, rabbit), 1 �g of rabbit isotype IgGcontrol (Cell Signaling; 39769), 1 �g of anti-5mC 33D3 (Active Motif; 39649; mouse), and 1 �g of mouseisotype IgG control (Santa Cruz). The antibody bound DNA was captured with protein A/G beads for 2h at 4°C, washed 5 times with 1� IP buffer, eluted overnight at 55°C in elution buffer (50 mM Tris-HCl[pH 8.0], 10 mM EDTA, 0.5% SDS, 0.25 mg/ml of proteinase K), and cleaned using a Qiagen gel extractionkit. qPCR was used to determine the presence and relative amount of specific DNA fragments modifiedby 5hmC and 5mC.

DIP qPCR. The relative amount of immunoprecipitated DNA fragments from DIP assays was measuredusing qPCR analysis with SYBR green (Bio-Rad) according to the manufacturer’s instructions. Samples wereanalyzed using an ABI Prism 7900 real-time PCR system (Applied Biosystems). The C promoter was amplifiedwith the following primer set: 5=-CCTAGGCCAGCCAGAGATAAT-3= and 5=-AGATAGCACTCGACGCACTG-3=. Adilution series of each input sample was utilized to create a standard curve of the threshold cycle (CT) valuefor each dilution. Percent input bound of each sample was calculated with the determined standard curve.Each condition and input dilution was loaded in triplicate.

In vitro methylation of reporter gene constructs. The EBV LMP1 ED-L1 promoter inserted into thepCpGL-basic reporter gene construct was methylated or mock treated in vitro using CpG methyltransferaseM.SssI (New England BioLabs [NEB]) according the manufacturer’s instructions. The DNA was cleaned byphenol-chloroform extraction and salt precipitation. Promoter CpG methylation was verified by enzymaticdigestion with two restriction enzymes (NEB) that share the same cut site: HpaII (methylation inhibitsdigestion) and MspI (cuts regardless of methylation status).

Reporter gene assays. BJAB cells were transfected using Lipofectamine 2000 transfection reagent(Invitrogen) according to the manufacturer’s instructions with 0.3 �g of LMP1p-ED-L1-pCpGL reporter vector,0.5 �g of SG5-EBNA2, 0.5 �g of TET2 or pcDNA3.1(�), and up to 1.6 �g of SG5. Cells were harvested in 1�reporter lysis buffer (Promega) 2 days posttransfection and subjected to one freeze-thaw cycle. Luciferaseactivity was quantified using a BD monolight 3010 (BD biosciences) with luciferase assay reagent (Promega).Each luciferase assay condition was performed in triplicate on two separate days.

Nucleofection. Mutu I cells were transfected with 1 �g of EBNA2 or SG5 and 1 �g of TET2 orpcDNA3.1(�) using an Amaxa nucleofector device (Lonza) according to the Raji cell protocol (Lonza).Briefly, 3 � 106 actively growing cells per condition were resuspended in buffer V (Lonza), aliquoted incuvettes with 2 �g of DNA, and nucleofected using program G-016. Cells were harvested for immunoblotanalysis 48 h postnucleofection.

ACKNOWLEDGMENTS

We thank Alan Rickinson, Jeff Sample, and Janet Mertz for cell lines, Yi Zhang for theTET2 expression vector, Paul Ling for the EBNA2 expression vector, Michael Rehli for thepCpGL-basic luciferase vector, and members of the Kenney, Johannsen, Rui, and Mertzlaboratories for suggestions and discussions.

This work was supported by HHS | NIH | National Cancer Institute (NCI) grants4P01CA022443-39 and R01-CA174462-04 and by HHS | NIH | National Institute of Dentaland Craniofacial Research (NIDCR) grant 4R01DE023939-04.






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GENOME REPLICATION AND REGULATION OF VIRAL ......DLBCLs. In contrast, we found that TET2 is expressed in normal naive tonsil B cells, in EBV-positive BLs with type III latency (which