Histone methylation in Epstein–Barr virus-associated diseases

Abstract

Epstein-Barr virus (EBV) infection is linked to various human diseases, including both noncancerous conditions like infectious mononucleosis and cancerous diseases such as lymphoma and nasopharyngeal carcinoma. After the initial infection, EBV establishes a lifelong presence and remains latent in specific cells. This latent infection causes changes in the epigenetic marks known as histone methylation. Many studies have examined the role of histone methylation in different EBV-associated diseases, and understanding how EBV affects histone methylation can help us identify potential targets for epigenetic therapies. This review focuses on the research progress made in understanding histone methylation in well-studied EBV-associated diseases, intending to provide insights into potential strategies based on histone methylation to combat EBV-related ailments.

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Plain language summary

Article Highlights.

• Epstein–Barr virus (EBV), a DNA virus, is classified as a member of the gammaherpesvirus family, over 90% of adults are chronically infected.

• Epigenetics is involved in regulating the EBV life cycle.

Overview of histone modifications

• Introduces concepts related to histone modification and histone methylation.

EBV infection

• Histone methylation plays an important role in maintaining latent EBV infection.

Histone methylation & EBV-associated tumors

• Aberrant histone methylation promotes EBV-associated tumors development by suppressing the expression of the viral genome, silencing antioncogenes, driving the expression of oncogenes, promoting the transcriptional activation of autophagy genes and inhibiting DNA damage repair.

Histone methylation & EBV-associated non-neoplastic diseases

• Histone methylation associated with infectious mononucleosis, chronic active EBV infection, EBV-associated hemophagocytic syndrome and autoimmune diseases.

Prospect of histone methylation modification in the diagnosis & treatment of EBV-associated diseases

• Describing recent advances in histone methylation sheds light on its pivotal role in both diagnosing and intervening in EBV-related diseases.

Future perspective

• Histone methylation is intricately linked to EBV-related diseases and holds promise as a potential drug target.

1. Introduction

Epstein-Barr virus (EBV), a linear, double-stranded DNA virus, is classified as a member of the gammaherpesvirus family, and over 90% of adults are chronically infected [1]. The viral genome circularizes shortly after infection and throughout latency. EBV is known to cause various diseases, which impose a heavy burden on patients and their families [2,3]. Lots of diseases fall within the scope of EBV infection, such as infectious mononucleosis (IM), chronic active EBV infection (CAEBV) and several malignant tumors, including nasopharyngeal carcinoma (NPC), gastric cancer (GC), post-transplant lymphoproliferative disorder (PTLD), Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) [4,5]. Additionally, the association between EBV and the development and progression of autoimmune diseases, such as multiple sclerosis (MS) and rheumatoid arthritis (RA), has been established [6,7].

In the pathogenesis of EBV-related diseases, histone methylation plays a crucial role by impacting the transcription and replication of EBV's genome, following the pattern observed in other gamma herpesviruses [8,9]. The significance of histone methylation lies in its ability to influence viral gene expression, thereby contributing to the diverse spectrum of EBV-associated diseases. This review provides a comprehensive summary of how histone methylation participates in the development of EBV-related diseases.

2. Overview of histone modifications

Chromosomes mainly consist of core histones H2A, H2B, H3 and H4, which combine to form nucleosome subunits [10]. These subunits have N-terminal domains that serve as targets for enzymes that modify histones. Common histone post-translational modifications include acetylation, phosphorylation and methylation.

Acetylation of histones is a process in which acetyl groups are added to histone lysine residues by histone acetyltransferase (HATs). This alteration increases the electrostatic attraction and spatial resistance between DNA and histones, weakening their bond. Consequently, DNA can be easily unwound and chromatin becomes more accessible for transcription, allowing transcription factors to bind to the DNA and activate gene expression. On the contrary, histone deacetylases (HDACs) remove acetyl groups from histones, causing histones to bind tightly to DNA due to their positive charge. This leads to a condensed and repressive chromatin structure that suppresses gene transcription. The balance between histone acetylation and deacetylation in the nucleus is crucial for controlling gene expression, DNA replication, repair and even cellular memory [11,12].

Histone kinases are enzymes that facilitate the phosphorylation of histones through the transfer of phosphate groups from ATP to specific serine, threonine or tyrosine residues, and phosphatases reverse this modification. This chemical modification is vital for activities like gene transcription, DNA repair, apoptosis and chromosome condensation. When combined with histone acetylation, histone phosphorylation controls various cellular processes such as cell growth and division. The primary subtypes of histone phosphorylation encompass histone H1, H2A/H2B, H3 and H4. Similar to histone acetylation, phosphorylation can modify histone charge, impacting chromatin structure and regulating cellular functions [11–14].

Histone methylation is a crucial process that takes place on residues such as lysine, arginine and histidine. This modification is catalyzed by enzymes called histone methyltransferases (HMTs) (Table 1), which are responsible for transferring methyl groups from S-adenosyl methionine to specific residues. There are two main types of HMTs: histone lysine methyltransferases (HKMT) and protein arginine methyltransferases (PRMT), each with distinct catalytic residues. HKMTs add methyl groups to the ϵ-amino group of lysine, while PRMTs add methyl groups to the ω-guanidino group of arginine. Some well-known HMTs include mixed lineage leukemia (MLL), suppressor of variegation 3–9 homolog 1 (SUV39H1), suppressor of variegation 3–9 homolog 2 (SUV39H2), Zeste homologous enhancer 2 (EZH2, a catalytic subunit of the polycomb repression complex 2 [PRC2]), disruptor of telomeric silencing 1-like (DOT1L), SET domain-containing 2 (SETD2), co-factor associated arginine methyltransferase (CARM1), PRMT5 and others. Histone methylation was initially considered irreversible, the discovery of the H3K4 demethylase, lysine-specific demethylase 1A (KDM1A or LSD1), in 2004 proved that histone methylation is indeed reversible, leading to the expansion of the histone demethylase family [15]. As the most widely studied histone methylation, Lysine residues can be mono-, di-methylated, or trimethylated (me1, me2, me3), serving as marks for either active or repressive gene expression. Currently, several residues in the N-terminal tails of histones H3 and H4, including H3K4, H3K9, H3K27, H3K36, H3K79 and H4K20, have been functionally characterized regarding methylation [16]. H3K4me1/2/3, H3K9me1, H3K27me1, H3K36me2/me3 and H3K79me2/me3 are active marks associated with actively transcribed gene regions in chromatin. In contrast, H3K9me2/3, H3K27me3 and H4K20me2/3 are referred to as inhibition marks, usually linked to silent gene expression and concentrated chromatin [17]. Imbalances in histone modification have been implicated in metabolic diseases and tumors [10,18], with recent studies suggesting that H3K27me3 is associated with resistance to antineoplastic drugs [19,20].

Table 1.

Overview of histone lysine methyltransferase (KMTs) and demethylases (KDMs).

Residues methylated	Lysine methyltransferase	Lysine demethylases
H3K4	MLL1/KMT2A, MLL2/KMT2B, MLL3/KMT2C, MLL4/KMT2D, MLL5/KMT2E SET1A, SET1B ASH1L	LSD1/BHC110/KDM1A, JARID1B/KDM5B, LSD2/KDM1B/AOF1
H3K9	SUV39H1/KMT1A, SUV39H2/KMT1B, G9a/EHMT2/KMT1C, ESET/SETDB1, RIZ1	JHDM2A/JMJD1A/KDM3A, JHDM3A/JMJD2A/KDM4A, JMJD2B/KDM4B, JMJD2C/GASC1/KDM4C, JMJD2D/KDM4D
H3K27	EZH1/KMT6B, EZH2/KMT6A	UTX/KDM6A, JMJD3/KDM6B
H3K36	NSD, SYMD2	JHDM1A/KDM2A, JHDM1B/KDM2B, JHDM3A/JMJD2A/KDM4A, JMJD2C/GASC1/KDM4C
H3K79	DOT1L/KMT4	JHDM1B/KDM2B
H4K20	SUV4-20H/KMT5	LSD1n, hHR23A/B

Numerous histone modifications are involved in EBV infection, and the epigenetic regulation of the EBV life cycle was comprehensively reviewed by Guo et al. [21]. Among the various enzymes responsible for histone modifications, methyltransferases demonstrate the highest degree of specificity. This specificity may explain why histone methylation is one of the most well-studied modifications. This study will therefore emphasize the significance of histone methylation in EBV infection, specifically examining its impact on EBV-associated diseases.

3. EBV infection

The EBV life cycle can be characterized by three distinct phases: initial binding of the EBV glycoprotein GP350/220 to the complement receptor CD21 on B cells, followed by internalization and delivery of its linear DNA genome to the nucleus. Shortly after infection, a variety of viral lytic genes are activated alongside latent genes. Approximately 10 days post-infection, EBV enters a latent phase characterized by the predominant expression of latent genes. Upon specific stimulation, such as the activation of memory B-cell surface immunoglobulins, a signaling cascade is triggered, resulting in the expression of the viral transcription factor BZLF1. This event serves as a precursor to the reactivation of lytic genes and subsequent viral synthesis [5]. The promoters encoding the EBV nuclear antigens EBNAs include W promoter (Wp), C promoter (Cp), and Q promoter (Qp), where Wp and Cp are common transcriptional promoters for EBNAs that transcriptionally synthesize all six EBNAs mRNAs and Qp is a proprietary promoter for EBV nuclear antigen 1 (EBNA1) that selectively transcribes and synthesizes EBNA1 mRNA. Epigenetics plays a significant role in the successful establishment and maintenance of EBV latency, as highlighted by many scholars [21–24]. In this discussion, we will specifically explore the role of histone methylation in both EBV latency and reactivation. Upon EBV infection of target cells, EBV establishes latent infection by binding to host chromatin through EBNA1. It has been observed that the enrichment of H3K4me2 occurs at the active Qp of the EBV genome, leading to the promotion of EBNA1 expression in EBV latent type I and II infections (Figure 1). In contrast, the active histone acetylation mark was significantly clustered on the active Cp [25]. Moreover, the chromatin insulator, CCCTC binding factor (CTCF), can bind to the upstream region of the Qp transcription start site. This binding inhibits H3K9me3-mediated epigenetic silencing of EBNA1 expression [26]. Furthermore, in lymphocytes with EBV latent type III infections, H3K4me2 modification of the latent membrane protein 2A (LMP2A) promoter enhances its expression [27]. Notably, the expression of the lytic gene BamHIZleftfragment1 (BZLF1) is also influenced by histone methylation [28,29], in addition to the virus protein associated with latent infection. Evidence suggests that the transcriptional repression of BZLF1 in B95-8 cells is mediated by the histone H3 lysine 9 methyltransferases, Suv39h1, which promotes repressive trimethylation of H3K9 (H3K9me3), knockdown of Suv39 h1 protein by siRNA and use of chaetocin, a specific inhibitor of Suv39 h1, resulted in increased expression of BZLF1 mRNA in B95-8 cells [30]. The epigenetic enzymes ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) and DNA methyltransferase 1 (DNMT1) work together to maintain the inheritance of DNA methylation. Guo et al. conducted a study showing that UHRF1 interacts with methylated H3K9, hemimethylated DNA, and potentially histone ubiquitination, all of which are crucial for the maintenance of latency I. Meanwhile, UHRF1 plays a role in suppressing the promoters C, W, and LMP in latency I [31]. In Raji cells, the inhibition of H3K27me3 and H4K20me3, as well as the knockdown of histone methyltransferases of H3K27me3 or H4K20me3, leads to increased BZLF1 levels. This further confirms that histone methylation inhibits the BZLF1 promoter, Zp [32]. The findings demonstrate that the maintenance of EBV latency is heavily influenced by histone methylation, specifically in relation to the BZLF1 gene's silencing. In the context of EBV-infected cells undergoing lytic reactivation, the removal of repressive chromatin marks on BZLF1 occurs, with H3K27me3 on histones being erased while H3K9me3 remains unaffected. In Akata Burkitt lymphoma cells, the interaction between BZLF1 and BZLF1 response elements within the H3K9me3-associated chromatin can counteract the latency-associated repressive chromatin's influence on EBV early lytic promoters [29]. This counteraction results in the activation of the RNA polymerase II (RNAP II) multiprotein complex, which then initiates the transcriptional machinery, creating an open chromatin environment conducive to early lytic promoters in lymphoblastoid cell lines (LCLs, latency III). Another histone mark, H3K4me3, known for its activity, has also been found to be suitable for early lytic promoters in LCLs [33]. In essence, the combined effect of various histone methylations plays a critical role in triggering the transition of EBV from latency to activity. Furthermore, EBV infection has been observed to influence the histone methylation of the host genome [34–36], subsequently contributing to the occurrence and progression of the disease.

Figure 1.

Histone methylation in EBV latency I. In EBV latency I, DNA methylation and repressive histone methylation act synergistically, and UHRF1 is involved in this process, silencing LMPp, Cp, Wp and BZF1 Zp, and activating Qp by active histone methylation to stimulate EBNA1 expression.

4. Histone methylation & EBV-associated tumors

There have been associations found between EBV infection and the occurrence of various tumors, such as NPC, EBVaGC, Burkitt lymphoma and Hodgkin's lymphoma. The development of EBV-related tumors involves multiple viral gene products, including LMP1, LMP2A, EBNA1 and associated signaling pathways. This process also includes the interaction between the viral genome and the host genome. During EBV latent infection, histone methylation is believed to play a significant role in tumorigenesis. It suppresses the expression of the viral genome, silences antioncogenes, drives the expression of oncogenes, promotes the transcriptional activation of autophagy genes and inhibits DNA damage repair [37–41].

4.1. Histone methylation & nasopharyngeal carcinoma

In Southeast Asia and North Africa, NPC is a malignant tumor of the nasopharyngeal epithelium that is commonly associated with EBV infection [42–45]. The regulation of TP53 mutations, DNA repair and chromatin modifications by histone methylation plays a crucial role in the development of nasopharyngeal carcinoma [46–48].

Significantly higher expression of H3K27me3 was observed in NPC tissues compared with paraneoplastic tissues, as shown by Duan et al. This expression was also found to be correlated with cisplatin resistance, indicating the important involvement of H3K27me3 in the initiation of NPC and the development of drug resistance [49]. EZH2, a catalytic subunit of the polycomb repression complex 2(PRC2) [50], is responsible for triggering lysine trimethylation at position 27 of histone H3. This process leads to the suppression of TP53 tumor suppressor gene expression (Figure 2). High levels of EZH2 are strongly associated with a more aggressive phenotype and an unfavorable prognosis in NPC [51]. EBV viral gene LMP1 indirectly affects EZH2 expression possibly by impacting the NF-κB signaling pathway through various mechanisms (Table 2) [36]. Similarly, EZH1, a homolog of EZH2, is involved in H3K27 trimethylation as a component of PRC2 [52,53]. However, the exact mechanism by which EBV modulates EZH1 expression remains unclear.

Figure 2.

Histone methylation in Epstein-Barr virus (EBV)-associated tumors. (A) Histone methylation in nasopharyngeal carcinoma. (B) Histone methylation in EBVaGC. (C) Histone methylation in EBV-associated lymphoma.

Table 2.

Histone methylation features in Epstein-Barr virus-associated diseases.

Diseases	Involved EBV gene or protein	Histone methylation alterations	Functions	Ref.
IM	Not reported	Not reported	Not reported	Not reported
CAEBV	Not reported	Not reported	Not reported	[54]
HLH	Not reported	H3K4me3 ↑	Upregulating the expression of UNC13D in NK cells and effector CD8+ T cells	[55]
MS	EBNA1	H3K27me3 ↓ H3K4me3 ↑	Associated with MS immune activity status, mitochondrial alterations	[56–58]
RA	EBNA2	H3K4me3 ↑	Identifying the importance of T cells in the pathogenesis of RA	[59]
NPC	LMP1	H3K27me3 ↑ H3K4me3 ↓	Silencing of TP53 antioncogene; inhibition of DNA damage repair gene expression	[49,50,60]
EBVaGC	LMP2A	H3K9me3 ↓ H3K27me3 ↓ H3K4me1 ↑ H3K4me3 ↑	Acting on FZD5 gene; driving cancer development	[61–63]
B-cell lymphoma	LMP1/EBNA3C	H3K27me3 ↑ H3K9me3 ↑ H3K4me1 ↑ H3K4me3 ↑ S2Me-H3R8 ↑ S2Me-H4R3 ↑	Silencing antioncogenes such as DOK; driving oncogene expression; promoting autophagy gene transcriptional activation; maintaining latent infection	[34,35,64–67]
NK/T-cell lymphoma	Not reported	H3K27me3 ↑	Silencing antioncogenes, driving cancer development	[68,69]
PBL	Not reported	Not reported	Not reported	[70]

The summary of histone methylation and Epstein-Barr virus (EBV)-associated diseases.

In addition to inhibiting the activity of tumor suppressor genes, histone methylation also triggers the development of nasopharyngeal carcinoma by suppressing the expression of DNA damage repair genes through a process known as ‘bivalent histone conversion’. Lin DC et al. conducted a study on immortalized nasopharyngeal epithelial cells (NPE) and found that EBV infection promotes nasopharyngeal carcinogenesis by reducing the levels of H3K4me3 and increasing the levels of H3K27me3 in the promoter regions of DNA damage repair genes. This alteration interferes with important DNA repair mechanisms such as base excision repair (BER), mismatch repair (MMR), homologous recombination and non-homologous end joining, leading to an imbalance in the expression of DNA damage repair genes. Notably, this dysregulation is not dependent on DNA methylation [60].

4.2. Histone methylation & EBV-associated gastric cancer

Approximately 9% of gastric cancers have been linked to EBV [71,72]. In EBV-associated gastric cancer (EBVaGC) and NPC, the pattern of EBV gene expression exhibits latency II infection, and the lymphoepithelial subtype of EBVaGC shares similarities with NPC in terms of histology. However, unlike NPC, when infecting EBVaGC cells, EBV alters histone marks in promoter and enhancer regions, such as H3K4me1, H3K4me3, H3K9me3 and H3K27me3 [61]. In EBVaGC, there is a significant decrease in H3K9me3 and H3K27me3 activation at incorrectly activated enhancers, while H3K4me1, H3K4me3 and other active marks show significant increases [61]. Li et al. demonstrated that LMP2A stimulates E26 transformation-specific homologous factor (EHF) by phosphorylating STAT3, thereby activating the Wnt/β-catenin pathway through binding with active enhancers enriched in H3K4me1, H3K4me3 on the frizzled protein-5 (Frizzled-5, FZD5) gene, thereby promoting EBVaGC [62].

Histone methylation patterns undergo dynamic and synergistic changes with DNA methylation in EBVaGC. Genes sensitive to DNA methylation exhibit significant association with loss of the H3K27me3 pre-mark or reduced levels of histone activity marks like H3K4me3 and H3K27ac, while genes related to apoptosis are prominently enriched in these epigenetically suppressed alterations. On the other hand, the DNA methylation-resistant genes and the unmethylated genes were marked with both or one of the active histone marks, with genes associated with the karyokinesis cell cycle and DNA repair being significantly enriched in these epigenetically activated changes. In summary, EBV is implicated in the development of EBVaGC through enhancer activation via histone activity modifications and dynamic coordinated action with DNA methylation [63].

4.3. Histone methylation & lymphoma

Several studies have established a connection between EBV and the emergence of different types of lymphomas [73–76]. It is worth noting that specific EBV-positive lymphomas exhibit significant changes in histone methylation in comparison to EBV-negative lymphomas [77].

4.3.1. Histone methylation & B-cell lymphoma

EZH2 aberrations were found in various types of B and T-cell lymphoid malignancies, including diffuse large B-cell lymphoma, classical HL and Burkitt lymphoma [78–80]. The recruitment of EZH2 to the tumor suppressor gene DOK1 promoter, induced solely by the viral oncoprotein LMP1, leads to trimethylation of histone H3 at lysine 27 (H3K27) and the subsequent silencing of gene expression [64]. LMP1 also activates host oncogenes by inhibiting the suppressive histone modification, H3K27me3, through the activation of poly ADP-ribose polymerase 1 (PARP1) [65]. Moreover, EBNA3C, another viral protein, interacts with histone lysine demethylase KDM2B and enriches the histone mark H3K27me3, facilitating lymphoma development [34,35]. Additionally, EBNA3C plays a crucial role in the activation of autophagy genes, particularly autophagy-related gene 7 (ATG7), autophagy-related gene 5 (ATG5), and autophagy-related gene 3 (ATG3), which are vital for autophagosome formation in EBV-associated B-cell lymphoma. To achieve this, EBNA3C recruits several histone-activating epigenetic marks such as H3K4me3, H3K4me1, H3K9ac and H3K27ac, facilitating transcriptional activation and inhibiting apoptosis, thereby supporting cellular growth [81]. In addition to the histone methylation marks described above, Hanneke Vlaming et al. discovered that conserved crosstalk between histone deacetylation and H3K79 methylation is relevant in a lymphoma context, since heterozygous deletion of DOT1L gene, which encodes DOT1L, catalyzing the methylation of histone H3 lysine 79, prolongs the survival of mice that develop Hdac1-deficient thymic lymphomas, most commonly Hodgkin's lymphoma, large B-cell lymphoma [82].

The effective reprogramming of the viral genome by EBV has been shown to involve the utilization of various histone pathways, thereby facilitating its establishment and persistence in a latent state. One noteworthy pathway in this process is associated with the chromatin assembly factor-1 (CAF1), which serves as a molecular chaperone for histones. Perturbing CAF1 reduces the occupancy of histones 3.1 and 3.3, as well as the suppressive histone 3 lysine 27 and 9 trimethyl marks (H3K27me3 and H3K9me3), at different regulatory elements of the viral genome during the lytic phase. It is worth noting that CAF1 expression is significantly upregulated in newly EBV-infected primary human B-cells prior to the first cell division, indicating its early involvement in the process. Notably, at this particular time point, the EBV genome has already been loaded with histones 3.1 and 3.3 [66].

EBV infection can have severe consequences in immunodeficient patients, with plasmablastic lymphoma (PBL), a rare and aggressive tumor, being particularly common in immunocompromised individuals [83]. A study conducted by Leeman-Neill et al. aimed to investigate the correlation between EBV infection and PBL following transplantation. Results showed that tumor cells from 6 out of 11 patients (67%) with post-transplant PBL were EBER-positive, underscoring the involvement of EBV in tumor development. Additionally, targeted genomic sequencing revealed gene mutations in histone methylation-related genes, including KMT2A, KMT2C, KMT2D and KDM5C, in patients with post-transplant PBL. These findings suggest a potential role of histone methylation in the pathogenesis of PBL [70].

Furthermore, apart from lysine residue methylation, arginine residue methylation also contributes to lymphoma pathogenesis. The tumor suppressor gene PTPROt, a protein tyrosine phosphatase gene, undergoes silencing during EBV-driven B-cell transformation. Notably, PRMT5, a type II arginine methyltransferase, was found to catalyze symmetric dimethylation of histones H3 (S2Me-H3R8) and H4 (S2Me-H4R3). This modification leads to chromatin structure alterations that promote PTPROt silencing, B-cell transformation, and maintenance of a malignant phenotype. Consequently, this process contributes to lymphoma development [67].

4.3.2. Histone methylation & NK/T-cell lymphoma

Our laboratory has previously conducted studies on EBV-positive NK/T-cell lymphoma, in which we compiled the clinical features [2]. Similar to other tumor types, the presence of PRC2-mediated H3K27me3 enrichment is a crucial characteristic of NK/T-cell lymphoma [68,69,77,84,85]. Additionally, defective H3K36me3 has been found to be associated with intestinal T-cell lymphoma [86]. Several studies have indicated that the mutations of KMT2D, KMT2C, KMT2B, SETD2 and KDM6A genes, which are associated with histone methylation and demethylation, can be observed in patients with this type of lymphoma. This implies that histone methylation, alongside H3K27me3, may have a significant role in the progression of NK/T-cell lymphoma [74].

5. Histone methylation & EBV-associated non-neoplastic diseases

5.1. Histone methylation & IM

IM is a self-limiting acute disease characterized by fever, pharyngitis, enlarged lymph nodes, hepatosplenomegaly and increased proportion of peripheral lymphocytes and atypical lymphocytes [87]. However, the current understanding of how histone methylation regulates IM pathogenesis is still incomplete, warranting further in-depth investigations into the connection between histone methylation and IM pathogenesis.

5.2. Histone methylation & chronic active EBV infection

CAEBV is a lymphoproliferative disease that affects multiple organs, exhibiting recurrent or persistent IM-like symptoms such as fever, lymph node enlargement, and hepatosplenomegaly. Recent research has discovered mutations in the KMT2D and KDM6A genes among CAEBV patients. Interestingly, KMT2D mutations are observed in T, NK, and B cell lineages of the disease [16]. Moreover, systemic CAEBV patients also exhibit additional mutations in the EZH2 and SET structural domain-containing protein 2 (SETD2) gene [54]. These findings suggest that histone methylation may have a significant role in the development and progression of CAEBV. However, more research is necessary to comprehend the underlying mechanisms fully.

5.3. Histone methylation & EBV-associated hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome of great severity and life-threatening consequences. It is caused by the abnormal activation of macrophages and cytotoxic T cells [88,89]. EBV infection often triggers HLH and can contribute to both familial HLH and inherited susceptibility [90]. Autosomal recessive mutations in the gene UNC13D, which encodes Munc13-4, have been associated with HLH [88]. Cichocki et al. conducted a study on UNC13D in primary lymphocyte subsets and found that H3K4me3, an active histone mark, was extensively present on the conventional and intron 1 promoter of UNC13D in CD8⁺ T cells and NK cells. This was accompanied by increased binding of signal transducer and activator of transcription 4 (STAT4) to the Brahma-related gene 1 (BRG1) (BRG1) [55]. In recent years, several studies have explored the relationship between histone modifications and inflammatory responses. For instance, Daskalaki et al. summarized the impact of aberrant histone methylation in macrophages on the inflammatory response [91]. In macrophages, EZH2 silencing led to the upregulation of interleukin-1 receptor-associated kinase-m (IRAK-M) and resulted in increased H3K27me3 on the IRAK-M promoter [92]. Additionally, H4K20me3 was found to regulate the inflammatory response by influencing the Toll-like receptor (TLR4) response [93]. These findings suggest a potential connection between histone methylation and the inflammatory response in the context of HLH.

5.4. Histone methylation & EBV-associated autoimmune diseases

Autoimmune diseases refer to a set of disorders caused by the immune system's response to self-antigens, which results in damage or dysfunction of the body's tissues. In recent years, the connection between EBV and various autoimmune diseases, such as MS and RA, has been widely studied [7,94–98]. The relevance between histone methylation and autoimmune diseases has been subject to several hypotheses [99,100]. Th17 cells, which are involved in pro-inflammatory responses in various autoimmune disorders, play a critical role in this association [101]. Research has indicated that Th17 cell differentiation is influenced by H3K27 demethylation, mainly mediated by the H3K27 demethylase jumonji domain-containing 3 (Jmjd3). In vitro experiments have shown that the H3K27 demethylase-specific inhibitor GSK-J4 can hamper Th17 cell differentiation [102]. Therefore, targeting Jmjd3 with small-molecule inhibitors may have therapeutic potential for autoimmune diseases. Researchers like Meijer et al. have found that certain immune genes in MS exhibit bivalent histone modification states (H3K4me3/H3K27me3), suggesting a shift in immune activity status [56]. Moreover, decreased betaine homocysteine methyltransferase (BHMT) in cortical neurons of MS patients has been connected to altered H3K4me3 methylation, which may lead to down-regulation of oxidative phosphorylation genes in MS cortex and aspiratory defects. ChIP-seq data suggested that BHMT affects chromatin to increase local histone methyltransferase activity, thereby increasing H3K4me3 and activating the expression of genes that support neuronal energetics. This suggests another possible mechanism between histone methylation and MS, namely damage to axons through altered mitochondrial damage [57,58].

In the context of rheumatoid arthritis (RA), the presence of anti-citrullinated protein antibodies (ACPA) is crucial for diagnosis [103]. Previous findings have indicated that antibodies targeting guanosine peptides derived from EBV nuclear antigen are detected years before the onset of RA and demonstrate cross-reactivity with human guanosine fibronectin, indicating a potential molecular mimicry mechanism similar to MS pathogenesis [104]. Also, serum EBV DNA has been found to correlate with RA activity, and EBV has been found in synovial membranes of patients with RA [95,97,105]. These studies suggest that EBV infection is an important etiology of RA and that molecular mimicry exists for specific mechanisms that may be similar to MS pathogenesis. Genetic variants differentially associated with RA reveal that RA-specific non-coding variants were expressively enriched within H3K4me3 histone modification marks in CD4⁺ memory T primary cells and regulatory T primary cells [59]. The function of histone methylation in RA, especially the relationship with EBV, needs to be further explored.

In recent years, the disease spectrum of EBV has expanded, and studies have suggested that EBV infection may be associated with the development of cervical and breast cancer [106,107]. However, the involvement of histone methylation in these diseases still requires further exploration.

6. Prospect of histone methylation modification in the diagnosis & treatment of EBV-associated diseases

The expression of histone marks is vital in assessing tumor stage progression, radiotherapy resistance and poor prognosis in several cancer types, such as NPC, GC and lymphoma [49,108,109]. In a study led by Mu-Yan Cai et al., the evaluation of H3K27me3 expression involved western blotting and immunohistochemistry. The analysis indicated a positive correlation between high H3K27me3 expression and advanced tumor stage, increased metastasis, chemoradioresistance and later T classification. These significant findings propose that H3K27me3 expression could potentially serve as an immunomarker for predicting radiotherapy response and patient prognosis in NPC. Integrating various clinicopathological prognostic models can be a promising approach to identifying patients with diverse clinical outcomes [49]. However, unfortunately, no further research has been conducted on this topic in the last decade.

Substantial advancements have been made in the development of drugs targeting histone methylation, including adenosine homolog DZnep and EZH2 inhibitors like tametostat and valmestat [110–114]. Tazemetostat, an EZH2 inhibitor, recently obtained FDA approval as the first lysine methyltransferase (KMT) inhibitor. Currently, this drug is indicated for hematologic malignancies and solid tumors [115]. To date, the drug's indications include hematologic malignancies and solid tumors [110,116,117], showcasing the broad therapeutic potential of KMT inhibitors in various diseases. Izutsu et al. reported that Tazemetostat demonstrated high efficacy and an acceptable and manageable safety profile in relapsed or refractory B-cell non-Hodgkin lymphoma [116].

7. Conclusion

Histone methylation plays a crucial role in EBV-associated diseases, influencing viral latency, reactivation, and disease development. Dysregulation of histone methylation marks contributes to EBV-related tumors and non-neoplastic diseases. Understanding these mechanisms offers insights into disease progression and may lead to innovative diagnostic and therapeutic strategies.

8. Future perspective

The process of gene expression and genome organization is significantly influenced by the intricate and resilient mechanisms of histone methylation. The regulation of EBV latency promoter activity in host cells and prevention of lytic replication are crucial functions of histone methylation. On the other hand, the proportion of repressive and active histone methylation marks on lytic genes, such as BZLF1, contributes to the promotion of viral reactivation. Dysregulation of histone methylation also impacts gene sets associated with tumorigenesis, as observed in lymphoma, NPC and GC, wherein EZH2-catalyzed H3K27me3 marks typically silence antioncogenes.

Akin to DNA methylation, histone methylation is a reversible process, as mentioned in the previous study by Zhang et al. [24]. Expanding research on the role of histone modifications in EBV-associated diseases, particularly non-tumor diseases, can reinforce our understanding of the pathogenesis of these diseases from an epigenetic standpoint. Moreover, this research can provide valuable data to support the identification of molecular marks for early diagnosis and treatment. Additionally, investigating the synergistic effects of antiviral drugs and drugs that restore abnormal histone methylation can facilitate the development of new treatments, offering promising options for individuals with EBV-associated diseases.

Funding Statement

This work was supported by the Capital's Funds for Health Improvement and Research (2024-4-1142), Beijing Hospitals Authority Innovation Studio of Young Staff Funding (202328), BCH Young Investigator Program (BCHYIP) and CAMS Innovation Fund for Medical Sciences (CIFMS) (2019-I2M-5-026).

Author contributions

G Chen and L Zhang drafted the manuscript. R Wang and Z Xie revised and edited the manuscript. All authors read and made final approval of the manuscript. G Chen and L Zhang contributed equally to this work.

Financial disclosures

This work was supported by the Capital's Funds for Health Improvement and Research (2024-4-1142), Beijing Hospitals Authority Innovation Studio of Young Staff Funding (202328), BCH Young Investigator Program (BCHYIP) and CAMS Innovation Fund for Medical Sciences (CIFMS) (2019-I2M-5-026). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity 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.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.