Role oF epigeNeticS iN eBv Regula-tioN & pathogeNeSiS
Hans Helmut Niller1, Zsófia Tarnai2, Gábor Decsi3, Ádám Zsedényi2, Ferenc Bánáti4 & Janos Minarovits*,2
1Department of Microbiology & Hygiene, University of Regensburg, Franz-Josef-Strauss Allee 11, D-93053 Regensburg, Germany 2Department of Oral Biology & Experimental Dental Research, University of Szeged, Faculty of Dentistry, H-6720 Szeged,
Tisza Lajos krt. 64, Hungary 3Department of Oral Surgery, University of Szeged, Faculty of Dentistry, H-6720 Szeged, Tisza Lajos krt. 64., Hungary 4RT-Europe Nonprofit Research Ltd, Pozsonyi u. 88, H-9200 Mosonmagyaróvár, Hungary
AbstrAct: Epigenetic modifications of the viral and host cell genomes regularly occur in EBV-associated lymphomas and carcinomas. The cell type-dependent usage of latent EBV promoters is determined by the cellular epigenetic machinery. Viral oncoproteins interact with the very same epigenetic regulators and alter the cellular epigenotype and gene-expression pattern: there are common gene sets hypermethylated in both EBV-positive and EBV-negative neoplasms of different histological types. A group of hypermethylated promoters may represent, however, a unique EBV-associated epigenetic signature in EBV-positive gastric carcinomas. By contrast, EBV-immortalized B-lymphoblastoid cell lines are characterized by genome-wide demethylation and loss and rearrangement of heterochromatic histone marks. Early steps of EBV infection may also contribute to reprogramming of the cellular epigenome.
Keywords • CpG island • DNA methylation • epigenetic regulation • histone modification • hit and run oncogenesis • methylome • pioneer transcription factor • Polycomb repressor complex • tumor suppressor
epigenetic regulationEpigenetic regulatory mechanisms ensure the inheritance of cell type-specific gene-expression pat-terns from cell generation to cell generation. The major epigenetic regulatory mechanisms and their complex interactions were discussed in recent reviews [1–7]. Thus, here we wish to give only a brief outline of epigenetic control mechanisms. In mammals, the best characterized epigenetic regulators include DNA methyltransferases, which mark cytosines at certain positions within the genome, and histone acetylases and histone methyltransferases, which covalently modify the tails of both histone H3 and histone H4 molecules. Other epigenetic regulators like the complexes formed by Polycomb and Trithorax group proteins also modify histone tails and form stable complexes with DNA, even in mitotic chromosomes. Pioneer transcription factors do not have an enzymatic activity: they bind to compact, repressive chromatin areas and ‘bookmark’ the genes to be activated. Pioneer factors remain associated with their recognition sequences in mitotic chromatin, contributing thereby to epigenetic memory.
The epigenetic marks deposited on the chromatin affect promoter activity. DNA methylation at the C-5 position of cytosines, deacetylation of histone tails, methylation of certain lysine or arginine residues of core histones and binding of Polycomb repressor complexes (PRCs) favors the establishment of heterochromatin and facilitates promoter silencing. By contrast, active promoters are frequently located in ‘open’ chromatin domains characterized by hypomethylated or unmethyl-ated CpG dinucleotides in promoter regulatory regions. In addition, active promoters are marked by euchromatic histone modifications including acetylation of histone H3 and histone H4 proteins, as
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well as methylation of histone tails at ‘activating’ positions. They are frequently associated with the histone variant H2A.Z or Trithorax group complexes, too. Transition of condensed chro-matin to a more relaxed conformation and vice versa is affected by ‘reader’ factors like 5-methyl-cytosine-binding proteins, transcription factors and chromatin remodeling complexes that rec-ognize and interpret the euchromatic or hetero-chromatic marks ‘written’ to the chromatin. The reversibility of epigenetic changes is ensured by ‘eraser’ mechanisms: members of the Fe(II)- and α-ketoglutarate-dependent dioxigenase family oxidize the methyl group of 5-methylcytosine whereas other family members target histones and demethylate mono-, di- and trimethylated lysine or arginine residues. Passive DNA dem-ethylation can occur by inhibition of DNMT1, a maintenance methyltransferase that establishes the methylation pattern of daughter strands dur-ing DNA replication. Histone acetylation, an activating modification, is reverted by histone deacetylases (HDACs).
epigenetic regulation of latent eBv genomes: epigenetic regulation of the viral oncogenesEBV, a human gammaherpesvirus, was discov-ered 50 years ago in cultures of Burkitt’s lym-phoma (BL) cells [8]. EBV is associated with a series of malignant tumors, including lym-phomas, carcinomas and leiomyosarcoma, and in vitro infection of B lymphocytes yields immor-talized lymphoblastoid cell lines (LCLs) [9,10]. EBV was also implicated in the initiation and progression of autoimmune diseases [11]. After initial productive replication in epithelial cells and B lymphocytes the virus establishes latency in resting, memory B cells. Latent, episomal EBV genomes co-replicate with the cellular DNA once per cell cycle using oriP, the latent origin of DNA replication [12]. The expression of the latent viral genome is highly restricted in resting B cells, neoplastic cells and LCLs. Different cell types express various combinations of latent EBV pro-teins and nontranslated RNAs, forming distinct EBV latency types [13]. The products of ‘classi-cal’ viral latency genes include six Epstein–Barr nuclear antigens (EBNAs) and three latent membrane proteins (LMPs) [10]. In addition, the EBV-encoded small RNAs (EBERs) and viral miRNAs also contribute to oncogenesis [14,15].
The host cell phenotype-dependent activity of latent EBV promoters is determined by epigenetic
regulatory mechanisms [16–19]. In LCLs (type III latency), transcripts for six nuclear antigens (EBNAs) are initiated at the C promoter (Cp), located in the vicinity of unmethylated regula-tory sequences on an ‘acetylation island’, which is enriched in activating, euchromatic histone mod-ifications such as acetylated histone H3 and H4. By contrast, Cp is silenced by CpG methylation and heterochromatic histone marks in carcino-mas and the majority of EBV-positive lympho-mas (latency type I and II). In these neoplasms Cp is replaced by an alternative promoter, the Q promoter (Qp). At Qp, which is unmethylated independently of its activity, only transcripts for a single nuclear protein, EBNA1, are initiated. Qp silencing in LCLs is accompanied with het-erochromatic histone marks, but also involves the binding of repressor proteins [20,21]. Qp meth-ylation and silencing is prevented by a close by upstream CTCF binding site [22]. A third pro-moter, Wp, is used for the initiation of EBNA transcripts in BLs carrying EBNA2-deleted virus genomes [23,24] and at the initial stage of B-cell infection by EBV [25]. In late-passage LCLs and the majority of BLs, Wp is switched off and hypermethylated [26]. Three transcriptional ele-ments have been defined for Wp. Besides binding of CREB/ATF, additional B-cell-specific factors are important for Wp regulation [27,28]. Because Wp is multiply present in each W repeat, pro-gredient LCL transformation largely silences Wp transcripts, but with unmethylated Wp repeats remaining, a very low Wp activity frequently remains detectable. Because Wp methylation is incomplete, but silencing very efficient, other fac-tors than methylation play a crucial role in silenc-ing Wp [29]. Upon infection of tonsillar germinal center B cells, LMP1-induced DNMT3A has been shown to bind to the methylated Wp [30].
Similar epigenetic modifications regulate the activity of the promoters where transcripts encoding LMPs (LMP1 and LMP2B; LMP2A) are initiated [17]. Chromatin loops formed between oriP, a long-range enhancer, and the latency promoters Cp and Qp, and the LMP loci were also implicated in the regulation of the viral oncogenes [22,31–32]. Latent EBV episomes attach to the nuclear scaffold at a region including the latent origin of EBV replication, oriP [33]. OriP-bound EBV nuclear antigen EBNA1 [12] directs the viral episomes to transcriptionally active regions of the host cell nucleus [34]. Because both oriP and the adjacent host chromatin were enriched in euchromatic histone marks, it was
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suggested that the transcriptionally active host chromatin may influence the epigenetic pattern at oriP [34]. Alternatively, chromatin-attached EBV episomes may affect the local methylation pattern of the host genome [35].
epigenetic regulation of immediate early genesMultiple cis-acting positive and negative tran-scriptional control elements influence the expres-sion of the BZLF1 gene that codes for the imme-diate early protein BZLF1 (also called Zta, EB1 and ZEBRA) that initiates productive, lytic EBV replication in latently infected host cells [36,37]. In addition to the silencers situated close to the transcriptional start site of the BZLF1 promoter, protein–DNA interaction studies defined addi-tional repressive elements, a distal one with Sp1 and NF1 binding sites [38] and several E-box elements which are located both proximal and distal [39]. BZLF1, a member of the bZIP fam-ily of transactivators, binds to oriLyt, the lytic origin of viral replication, a sequence present in two copies in most EBV genomes. In addition, BZLF1 binds to ZIII, a positive regulatory ele-ment of the BZLF1 promoter, and associates with the promoter of BRLF1, a gene coding for the immediate early protein Rta (BRLF1). BZLF1 and BRLF1 transactivate a series of early EBV promoters in concert.
Although according to the traditional view BZLF1 expression is confined to the lytic cycle, recently it has been demonstrated that BZLF1 and several early viral proteins involved in pro-ductive EBV replication are transiently expressed in newly infected B cells during the establish-ment of latent infection, too [40]. BHRF1 and BALF1 block apoptosis whereas BCRF1 (vIL-10) may contribute to immune evasion. Early, tran-sient BZLF1 expression may support the tran-sition of resting B cells to cycling B cells. It is vital, however, to switch off BZLF1 transcrip-tion within several days of infection, otherwise the cells die [36]. The mechanism of killing as discussed by Yu et al. remains unclear. It is not due to lytic virus production, and also not due to the lack of latency transcripts. The latency mRNAs are expressed at the same level, whether BZLF1 is expressed or not [36]. Thus, silencing of BZLF1 and maintenance of its silent state is crucial for the establishment of EBV latency, at least in cultured cells. Contrary to cell culture, in humanized mice, BZLF1 overexpression does not prevent the establishment of latency [41]. In
EBV-associated tumors, the immediate early pro-moters Rp and Zp are mostly hypermethylated [42]. Cellular repressor proteins (ZEB1, ZEB2 and MEF-2D), HDACs and histone methyl-transferases depositing heterochromatic histone marks kept the BZLF1 promoter silent during latency [43,44]. By contrast, lytic cycle activa-tion by the HDAC inhibitor TSA enriched the activating histone modifications H3K4me2 and H3K8ac at the promoter [45]. BZLF1 is an unusual transcription factor preferentially bind-ing to methylated BZLF1-responsive elements within its own promoter and other lytic promot-ers [46,47]. Thus, it was suggested that methyla-tion of EBV episomes during latency might be a prerequisite for successful reactivation [40,48]. However, the immediate early protein BRLF1 was capable to initiate the transcription of lytic EBV genes and productive EBV replication of a largely unmethylated EBV genome. In addition, BRLF1 induced H3K9 histone acetylation at unmethylated lytic promoters [49] with the con-sequence that the interplay of BRLF1 and BZLF1 may offer a way for EBV to reactivate latent viral genomes regardless of their methylation state.
pathoepigenetics of eBv-associated lymphomasEBV is associated with a series of lymphomas including BL, Hodgkin’s lymphoma (HL), T/NK-cell lymphoma, AIDS-associated lym-phoma, diffuse large B-cell lymphoma (DLBCL), and it is the causative agent of post-transplant lymphoproliferative disease (PTLD) [9,50]. The role of viral oncoproteins in the reprogramming of the host cell epigenome in EBV-associated lymphomas was summarized recently [51]. Here we wish to outline only the recent developments with special regard to genome-wide epigenetic studies.
PTLD is a serious complication in organ transplant and bone marrow recipients undergo-ing immunosuppressive treatment [9]. The phe-notype and EBV latency type of most early-onset PTLD cells is similar to that of LCLs derived from peripheral B cells immortalized by EBV in vitro. Thus, LCLs provide a suitable in vitro model for PTLD development. EBV infection of B cells induced hypomethylation of 256 cellular genes [52]. Most of the demethylated genes carried B-cell-specific trancription factor binding sites in their promoters, half of them for NF-κB, and showed an increased transcription. Some of the upregulated genes were known inducers of B-cell
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proliferation. Furthermore, hypomethylated genes were involved in the immune response, cell adhesion, inflammation, B-cell signaling and chemotaxis. Another study determined that hypomethylated genes in EBV-transformed LCLs were involved in the regulation of tran-scription, cell cycle and the immune response [53]. EBV-mediated transformation also caused a genome-wide decrease and redistribution of heterochromatic histone marks and increased the endonuclease accessibility in thousands of genomic sites, including the HOX gene clus-ter [54]. Recombinant viruses with deletions in EBNA2 or LMP1 coding sequences elicited simi-lar changes but failed to immortalize the infected B cells. Because stimulation of B-cell prolifera-tion using CD40/IL4 did not induce heterochro-matin loss, Hernando et al. suggested that the decrease of heterochromatic marks is associated with B-cell transformation, a process distinct from cell cycle entry of resting B cells [54].
EBV-induced immortalization also caused extensive changes in the B-cell methylome: whole-genome bisulfite sequencing revealed widespread demethylation affecting 2.18 GB of the genome and one-third of genes [55]. Overlapping hypomethylated blocks of strik-ing 1.72 GB were observed in the epigenomes of various EBV-negative carcinoma types including colon, lung, breast and thyroid car-cinomas and Wilms’ tumors [56]. There was a consistency between the block boundaries charted specifically in colon cancer and EBV-immortalized LCLs [55], and there was a signifi-cant overlap between the blocks, too. 462 MB hypomethylated as well as 96 MB hypermethyl-ated sequences were unique, however, for EBV-transformed B cells amounting to 25% differ-ences between colon cancer and LCLs. Besides cancer cells, hypomethylated blocks in LCLs also corresponded with lamin-associated domains (LADs), heterochromatin-associated lysine-methylated regions (large organized chromatin K [lysine] modifications; LOCKs), reprogramming domains in embryonic and induced pluripotent stem cells, indicating that especially these large genomic blocks are reactive and essentially mal-leable. Small differentially methylated regions (DMRs) were also observed in LCLs, extending from 250 to 2500 bp in length and encompassing altogether 1 MB. There were 1502 hypermeth-ylated and 1468 hypomethylated small DMRs, some of them located in the vicinity of promoter regions [55].
Deposition of a heterochromatic histone mark by PRC2 to the promoters of tumor suppressor genes at the INK4b-ARF-INK4a and BIM loci was also observed during EBV-mediated B-cell immortalization [57]. The viral oncoproteins EBNA3A and EBNA3C recruited PRC2 to these loci, preventing the activation of tumor suppres-sor genes involved in senescence and apoptosis induction during B-cell transformation [57–60]. In latency type III cells like LCLs and early-onset PTLDs the EBV oncoproteins LMP1 and EBNA2 may upregulate a plethora of cellular genes [61]. For example, LMP1 induced HIF-1α, a regulator of cellular responses to hypoxia [62]. HIF-1α, by inducing VEGF production, may contribute to tumor progression. Other angio-genic and tumor invasion factors were also induced by LMP1 [62]. It has not been exam-ined, but is likely that initially increased gene expression may locate to permanently hypometh-ylated blocks of LCLs. In EBV-infected germi-nal center B cells LMP1 may cause cellular gene silencing by upregulating DNMT3A, a de novo DNA methyltransferase [30], and may contrib-ute to the genesis of EBV-positive HL. Contrary, DNMT1 and DNMT3B were downregulated upon EBV-infection of germinal center B cells. Hypomethylation was significantly enriched at genes for cell surface signaling, cell adhesion signaling and mainly G-protein-mediated sign-aling pathways [30]. Interestingly, high CpG den-sity seems to correspond with hypermethylation, while low CpG density concurs with hypometh-ylation (see Table 2 in reference [30] for direct and indirect epigenetic effects of viral gene products).
It is interesting to note that temporary expression of the EBV genome may also induce epigenetic changes and stable alteration of gene-expression pattern and cell behavior, as demonstrated by transient EBV infection of a human lung carcinoma cell line [63]. These data support the idea that transient virus infec-tions may initiate ‘hit and run’ tumorigenesis, for example in case of EBV-negative BLs [64]. A ‘hit and hide’ scenario, when the initially active viral genes are switched off, is also plausible [37]. One may speculate that transient expression of EBNA1, a putative pioneer factor [65] that binds both to viral episomes and cellular genes, may induce permanent epigenetic alterations before the loss of EBV episomes during the generation of EBV-negative BLs.
BL is characterized by chromosomal translo-cations juxtaposing the MYC oncogene to one
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of the immunoglobulin loci. The association of BL with EBV is variable: approximately 20% of sporadic cases carry latent EBV genomes whereas the vast majority of endemic cases are EBV posi-tive. A microarray-based study showed that six out of the 767 genes studied were hypermethyl-ated in hematological neoplasms independently of the subtype: B-cell, T-cell and myeloid malig-nancies were characterized with highly methyl-ated DBC1, DIO3, FZD9, HS3ST2, MOS and MYOD1 sequences [66]. How silencing of these genes contributes to lymphomagenesis remains to be established. Hypermethylated gene sets were found in BLs, DLBCLs and a series of other B-cell lymphomas typically not associated with EBV. By contrast, in B-cell chronic lymphocytic leukemia there were more hypomethylated genes than hypermethylated ones. In BL, nearly 60% of the hypermethylated genes were targets of PRC2 in embryonic stem cells [66]. Since PRC2 enrichment of hypermethylated genes was found not only for BL, but for all hematological neoplasms except acute myeloid leukemia and multiple myeloma, a direct role for EBV in the hypermethylation of PRC2 targets is unlikely. Thus, PRC2 may pinpoint certain gene sets for de novo DNA methylation in BLs. Another study [67] identified a common set of de novo methyl-ated genes in mature agressive B-cell lymphomas including BLs and DLBCLs. Hypermethylation of EYA4, HOXA11, MT1A and HIC1 both in adult stem cells and in mature agressive B-cell lymphomas suggested that these neoplasms may originate from precursor cells with stem cell-like features [67]. Alternatively, constitutive Myc over-expression may confer a ‘stemness’ phenotype to BL cells [68]. Further experiments may illuminate whether EBV leaves a virus-specific epigenetic signature on the methylome of EBV-asociated lymphomas. Recently, Kreck et al. analyzed the DNA methylome of the BL line Daudi at base-pair resolution [69]. Daudi cells, originating from an endemic BL, are EBV positive and carry the
typical t(8;14) translocation. More than 90% of the 969 genes regularly hypermethylated in mature aggressive B-cell lymphomas, including BL [67], were found to be hypermethylated and silenced in Daudi cells, too.
The genome-wide, predominant epigenetic alterations observed in LCLs and BLs are sum-marized in table 1. Altogether, our 10-year-old, at first controversial [70,71] proposal of a sharp distinction of alternative pathogenetic pathways between BL-type tumors on one hand and LCL-type tumors on the other hand [72–74] is entirely supported by recently accumulating large-scale transcriptomic and epigenomic data.
epigenetic reprogramming in eBv-associated carcinomasEBV is associated with the anaplastic form of nasopharyngeal carcinoma (NPC), a lymphoe-pithelial tumor endemic in southeast Asia and North Africa, and gastric carcinoma (GC), which is EBV positive in approximately 10% of cases. The lymphoepithelial subtype of GC is mostly EBV positive and histologically resembles NPC, whereas the ordinary subtype is ususally EBV negative. Widespread hypermethylation in both NPC and EBV-positive GC suggests a CpG island (CGI) methylator phenotype for both epithelial cancers (reviewed in [9,35,51,64,75–76]). EBV gene-expression patterns in NPC and GC belong mostly to latency I (or II) with a variable expression of LMP1 in the case of NPC, and with expression of BARF1, BARF0 and a variable expression of LMP2A in addition to EBNA1 and the BART miRNAs in the case of GC (reviewed in [76]) [77]. The role of EBV, which is mono-clonal in the tumor tissue, and the sequential order of events in lymphoepithelial carcinogen-esis remain to be clarified. While chromosomal aberrations have been found in NPC precursor lesions, such as high-grade dysplasia and carci-noma in situ, virus-positive normal epithelia have so far not been found for NPC and GC [35,75].
table 1. predominant genome-wide epigenetic alterations in lymphoid and epithelial cells carrying latent eBv genomes.
cell type epigenetic mechanism
DNA methylation Heterochromatic histone marks Polycomb complexes
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Monoclonality and a peculiar case of a very rap-idly developing EBV-positive GC in an immune-suppressed patient suggest a direct role for EBV in GC carcinogenesis [78].
eBv-associated gcCGI hypermethylation in GC is global, but nonrandom. It may be induced as a defense mechanism against viral infection and start with the viral genome itself [79], be triggered by LMP2A activating DNMT1 expression via STAT3 phosphorylation [80], or just reflect selec-tion processes in the ‘evolution’ of the carcinoma tissue. A genome-wide screen demonstrated that approximately 400 genes expressed in normal gastric epithelia were silenced in GC cells [81]. CGIs in promoters of cancer-related genes, such as P16INK4A, P73 and CDH1 were frequently hypermethylated. A study of EBV-negative and EBV-positive subclones of a GC cell line indi-cated that EBV infection induced DNMT3B via LMP2A. EBV-positive subclones hyper-methylated a large number of genes, many of them involved in cancer-associated signaling pathways as tumor suppressors [82]. The tumor suppressor genes WNT5A [83] and SSTR1 [84] were also hypermethylated in EBV-positive GCs. Genome-wide methylation analysis of 51 GC samples yielded three different GC epi-genotypes, low- and high-methylation epigeno-types in EBV-negative cancer and EBV-positive-specific very-high-methylation epigenotype. While a surplus of 270 genes was methylated in the EBV-positive type, PRC target genes of embryonic stem cells were not enriched among the EBV-positive marker genes, and the DNA repair gene MLH1 was not methylated at all in the EBV-positive subtype. EBV infection of an EBV-negative low-methylated GC cell line led to a repeatedly consistent increase of DNA methylation, resembling methylation patterns of EBV-positive very-high-methylation epigeno-type. Thus, a different mechanism of aberrant methylation was postulated for the EBV-positive epigenotype in addition to the methylation mechanism active in the other subtypes, which was likely attributed to EBV infection. However, hypermethylation could not be simply attributed to one of the latent gene products [85].
Nasopharyngeal carcinomaThe ability of LMP1 to upregulate DNMTs may induce frequent hypermethylation of tumor sup-pressor genes in NPC tissue. NPC tissues were
examined in comparison with nontumor tissue for their methylation status and LMP1 expres-sion. LMP1 expression correlated with the degree of methylation of CGIs at ten preselected tumor suppressor genes [86]. CGI hypermethylation has practical consequences for diagnostics and therapy (reviewed in [51,75–76]). Tumor suppressor or marker genes [87–92] frequently methylated in NPCs may allow the use of methylation-sensitive PCR for the early detection of NPCs in high-risk groups besides high levels of anti-EBV-IgA in serum samples [93]. In addition, epigenetic alterations acquired by tumor cells during chemotherapy may play a role in the develop-ment of drug resistance. For example, a recent study identified a panel of 30 hypermethylated and 18 hypomethylated genes in taxol-resistant NPC cells. Affected gene loci were involved in tumor suppression, cell signaling, apoptosis and drug metabolism [94]. Epigenetic changes may contribute to the failure of 13-cis retinoic acid therapy [95] of NPCs, too. LMP1 was the viral protein responsible for the induction of RAR-β2 methylation in NPC cells. Furthermore, repressive histone marks and PCG proteins were indicators for late tumor stages, high chemora-dioresistance in NPC [96] and poor prognosis in both NPC, GC and even BL [97–99]. The pre-dominant, genome-wide epigenetic alterations observed in NPC and EBV-positive GC are summarized in table 1.
conclusionLatent EBV genomes undergo epigenetic modi-fications that determine the host cell-dependent activity of EBV latency promoters and may pre-vent productive viral replication. The epigeno-type of cells in EBV-associated lymphomas and carcinomas differs from the epigenotype of their normal counterparts. Epigenetic dysregulation may affect gene sets implicated in tumorigenesis but also genes directly not involved in tumor ini-tiation or progression. In BL, NPC and GC there are significant changes in the host cell methyl-ome: typically a series of promoters located in CGIs become silenced by DNA methylation. Although certain gene sets were hypermethyl-ated in both EBV-positive and EBV-negative neoplasms, there were hypermethylated genes unique for EBV-associated GCs suggesting the existence of an EBV-specific ‘epigenetic signature’.
In contrast to most of the neoplasms carry-ing latent EBV genomes, LCLs established by
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in vitro EBV infection of peripheral B cells were characterized by genome-wide demethylation and a significant decrease and redistribution of heterochromatic histone marks, in parallel with gene activation. Gene silencing, mediated by latent viral oncoproteins, also contributed to the process of B-cell immortalization by switching off key genes related to senescence and apop-tosis induction. Although usually LCLs are polyclonal, and tumor biopsies contain nonma-lignant cells as well, hypomethylation in LCLs was so striking and hypermethylation markers in EBV-associated malignancies so coherent that the essential findings and differences of the cited studies appear to be highly meaningful.
Future perspectiveThe specificity of the mechanisms targeting hypermethylation or hypomethylation to cel-lular promoters in EBV-infected cells is most intriguing. It should be identified which meth-ylation mechanisms are involved in the hyper-methylation of PRC target genes of embryonic
stem cells in lymphomas, which mechanisms are involved in the hypermethylation of non-PRC target genes in EBV-positive high-methylation GC, and which mechanisms are involved in the establishment of widespread hypomethyl-ated blocks during the outgrowth of LCLs. Epigenetic marks and genes differentially meth-ylated in EBV-associated neoplasms may have a diagnostic and prognostic value. Because epige-netic changes are reversible, the drugs affecting the epigenotype of EBV-associated tumors may be exploited in therapy.
Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
executive summAryepigenetic regulation of latent eBv genomes: epigenetic regulation of the viral oncogenes
● Gene expression of latent viral genomes in resting memory B cells, lymphoid or epithelial tumor cells, or lymphoblastoid cells is highly restricted by epigenetic mechanisms.
● Different viral gene-expression patterns corresponding to different latency types are regulated by epigenetic marks.
epigenetic regulation of immediate early genes
● Immediate early protein BZLF1 preferentially recognizes methylated binding sites.
● Shutoff of initial lytic gene expression is essential for establishing viral latency.
pathoepigenetics of eBv-associated lymphomas
● EBV is associated with different lymphoma types.
● Cellular methylation patterns are widely different between EBV-associated lymphoma types and in vitro immortalized lymphoblastoid cell lines (LCLs).
● The genome of LCLs is characterized by large hypomethylated blocks and loss of heterochromatic histone marks.
● In Burkitt’s lymphoma (BL) hypermethylated genes are strongly enriched for Polycomb repressor complex (PRC) target genes of embryonic stem cells.
epigenetic reprogramming in eBv-associated carcinomas
● EBV-associated epithelial malignancies are characterized by a CpG island (CGI) methylator phenotype.
● Just like in BLs, epigenetic lesions are more common than genetic lesions.
● Many tumor suppressor genes are hypermethylated.
● PRC target genes of embryonic stem cells are not enriched among hypermethylated CGIs in EBV-positive highly methylated gastric carcinoma.
● Increased hypermethylation during chemotherapy may lead to drug resistance of nasopharyngeal carcinoma.
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