EBV Infection Is Associated with Histone Bivalent Switch Modifications in Squamous Epithelial Cells

EBV infection is associated with histone bivalent switch modifications in squamous epithelial cells

Merrin Man Long Leonga,1, Arthur Kwok Leung Cheunga,1, Wei Daia, Sai Wah Tsaob,c, Chi Man Tsangb, Christopher W. Dawsond, Josephine Mun Yee Koa, and Maria Li Lunga,c,2

aDepartment of Clinical Oncology, University of Hong Kong, Hong Kong SAR, People’s Republic of China; bSchool of Biomedical Sciences, University of Hong Kong, Hong Kong SAR, People’s Republic of China; cCentre for Nasopharyngeal Carcinoma Research, University of Hong Kong, Hong Kong SAR, People’s Republic of China; and dDepartment of Microbiology & Infection, Warwick Medical School, The University of Warwick, CV4 7HL Coventry, United Kingdom

Edited by Tak W. Mak, University Health Network, Toronto, ON, Canada, and approved May 23, 2019 (received for review December 22, 2018)

Epstein−Barr virus (EBV) induces histone modifications to regulate addition to EBNA3C contribute to EBV-associated histone signaling pathways involved in EBV-driven tumorigenesis. To date, modifications in EBV-infected epithelial cells. the regulatory mechanisms involved are poorly understood. In this Recent studies support the premise that changes in histone study, we show that EBV infection of epithelial cells is associated modifications are crucial in modulating gene expression (5, 6). A with aberrant histone modification; specifically, aberrant histone well-studied histone modification mark, trimethylation of histone bivalent switches by reducing the transcriptional activation histone H3 lysine 4 (H3K4me3), which is usually found in actively tran- mark (H3K4me3) and enhancing the suppressive mark (H3K27me3) scribed promoters, is proposed to actively regulate gene expres- – at the promoter regions of a panel of DNA damage repair members sion by recruiting the chromatin remodeling factors (7 10). On the in immortalized nasopharyngeal epithelial (NPE) cells. Sixteen DNA other hand, another histone mark, H3 lysine 27 trimethylation damage repair family members in base excision repair (BER), homol- (H3K27me3), works to repress the expression of targeted genes ogous recombination, nonhomologous end-joining, and mismatch (11). The balance between these two histone modification marks is repair (MMR) pathways showed aberrant histone bivalent switches. important for mammalian gene expression. These two histone Among this panel of DNA repair members, MLH1, involved in MMR, modification marks work together to create a bivalent switch to form a tightly regulated mechanism to control gene expression, was significantly down-regulated in EBV-infected NPE cells through

despite the presence of the H3K4me3 mark. This control mech- MEDICAL SCIENCES aberrant histone bivalent switches in a promoter hypermethylation- anism provides restricted control of a large proportion of pro- independent manner. Functionally, expression of MLH1 correlated moter regions of developmental genes, which are simultaneously closely with cisplatin sensitivity both in vitro and in vivo. Moreover, marked by these bivalent H3K4me3 and H3K27me3 marks (12) seven BER members with aberrant histone bivalent switches in the to form bivalent domains (13) in embryonic stem cells (ESCs) EBV-positive NPE cell lines were significantly enriched in pathway (Fig. 1A)(14). analysis in a promoter hypermethylation-independent manner. This In this current study, the role of EBV in regulating host gene observation is further validated by their down-regulation in EBV- expression profiles was examined in the EBV-associated epi- infected NPE cells. The in vitro comet and apurinic/apyrimidinic site thelial malignancy, NPC, which has a comparatively low muta- assays further confirmed that EBV-infected NPE cells showed re- tion rate, while harboring extensive hypermethylation (15). Our duced DNA damage repair responsiveness. These findings suggest the importance of EBV-associated aberrant histone bivalent switch Significance in host cells in subsequent suppression of DNA damage repair genes in a methylation-independent manner. Epstein−Barr virus (EBV) infection is implicated in the development of certain cancers; however, the mechanisms by which EBV Epstein−Barr virus | histone bivalent switch | DNA methylation | DNA contributes to the pathogenesis of epithelial cell malignancies damage repair pathway | MLH1 remains unclear. This study highlights the association of EBV − infection with aberrant modifications in histone bivalent marks, pstein Barr virus (EBV), a ubiquitous human oncogenic H3K4me3 and H3K27me3, in nasopharyngeal epithelial cells. Eherpesvirus, infects more than 90% of the adult population Down-regulation of the DNA damage repair pathway genes, worldwide. EBV infection is strongly associated with lymphoid including MLH1, associated with aberrant histone bivalent marks ’ ’ tumors such as Burkitt s lymphoma (BL), Hodgkin sdisease, in a promoter hypermethylation-independent manner, was ob- and T cell lymphoma, as well as epithelial cancers, including served after EBV infection. This finding provides strong evidence nasopharyngeal carcinoma (NPC) and ∼10% of cases of gastric linking EBV infection to epigenetic modifications in epithelial cancer (1). cells. This study also suggests that the level of MLH1 may be Several studies have highlighted the contribution of EBV in- useful as a potential biomarker to evaluate the responsiveness of fection to epigenetic modification in latently infected B lym- nasopharyngeal carcinoma patients to cisplatin-based therapies. phocytes (2, 3). EBV encodes a number of latent oncogenic proteins that are involved in epigenetic regulation of host cell Author contributions: M.M.L.L., A.K.L.C., and M.L.L. designed research; M.M.L.L. and gene expression. The EBV-encoded latent proteins, EBNA1 and A.K.L.C. performed research; S.W.T., C.M.T., and C.W.D. contributed new reagents/analytic EBNA3C, among others, regulate the host chromatin-binding tools; M.M.L.L., A.K.L.C., W.D., and J.M.Y.K. analyzed data; M.M.L.L., A.K.L.C., and M.L.L. wrote the paper; S.W.T. and C.M.T. provided the immortalized and EBV-infected nasopha- protein and histone modifiers to control host gene expression. ryngeal epithelial cell lines; and C.W.D. provided the EBNA1 plasmid. These EBV latent proteins control the expression of specific histone modifiers, whose modification of histone molecules The authors declare no conflict of interest. results in aberrant histone methylation and acetylation (2–6), This article is a PNAS Direct Submission. supporting the role of EBV in regulating host gene expression Published under the PNAS license. profile via histone modifications. However, in an epithelial cell 1M.M.L.L. and A.K.L.C. contributed equally to this work. background, EBV typically establishes a Type II latency pro- 2To whom correspondence may be addressed. Email: [email protected]. gram, expressing a limited subset of latent proteins, which in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. clude EBNA1, LMP1, and LMP2 (4), but not EBNA3C. This 1073/pnas.1821752116/-/DCSupplemental. raises the possibility that other latency-associated proteins in

www.pnas.org/cgi/doi/10.1073/pnas.1821752116 PNAS Latest Articles | 1of10 Downloaded by guest on October 6, 2021 Fig. 1. Comparison of the levels of H3K4me3 histone marks at the global level for targeted genes in parental and EBV-infected cell lines and their association with DNA damage repair candidate genes. (A) Simplified schematic diagram of bivalent switch control of H3K4me3 and H3K27me3 marks in the regulation on the human gene expression. (B) The ChIP-Seq analysis shows the reduction of H3K4me3 in the targeted gene region. (C) At the global level, reduction of H3K4me3 level near the TSS was observed in the 361-EBV. (D) The WB shows the reduction of H3K4me3 in the EBV-infected cell lines. *P < 0.05; **P < 0.01.

findings suggest a unique and important characteristic of EBV in Western blot (WB), revealing a lower level of H3K4me3 in the terms of aberrant epigenetic regulation and provide us a good EBV-infected NPE cells (Fig. 1D). model to further study the role of EBV in regulation of epithelial From the ChIP-Seq results, a total of 628 promoter regions cell gene expression. showed reduction of H3K4me3 levels in both sets of EBV- infected NPE cells (SI Appendix, Fig. S2A and Datasets S1–S3). Results Since this transcriptional activation mark is commonly enriched Identification of DNA Damage Repair Pathway Members, Whose in the activated promoter regions, genes showing reduction of Expression Is Modulated by EBV Through Suppression of the H3K4me3 in the promoter regions were further analyzed, and the results suggested that 90% of the enriched promoter regions Transcriptional Activation Histone Mark, H3K4me3. Two pairs of im- are protein-coding genes (SI Appendix, Fig. S2B). Interestingly, a mortalized, nontumorigenic nasopharyngeal epithelial (NPE) cell total of 16 DNA damage repair signaling members (Table 1), lines, stably infected with EBV, or their uninfected counterparts which belong to base excision repair (BER), homologous re- (361/361-EBV and 550/550-EBV) (16), were used to examine the combination (HR), nonhomologous end-joining (NHEJ), and functional role of EBV in the regulation of H3K4me3 status in mismatch repair (MMR) pathways, showed significant reduction host cells. The presence of EBV was firstly determined in both of H3K4me3 in either one or both EBV-infected NPE cell lines. 361-EBV and 550-EBV by confirming the expression of green fluorescent protein (GFP), given that the recombinant EBV used EBV Infection Is Associated with Down-Regulation of a MMR Pathway SI Appendix in these studies was engineered to express GFP ( , Fig. Member, MLH1, via Aberrant H3K4me3 and H3K27me3 Bivalent Histone S1). The histone modification status of a transcriptional activation Switches in a Hypermethylation-Independent Manner. Among these histone mark, H3K4me3, in these two pairs of EBV-infected NPE 16 DNA damage repair members, MLH1, from the MMR path- cells was then confirmed by chromatin immunoprecipitation se- way, showed a significant reduction of H3K4me3 in both 361-EBV quencing (ChIP-Seq). From the H3K4me3 ChIP-Seq results, a and 550-EBV (Fig. 2A). In fact, epigenetic silencing of MLH1 significant reduction of H3K4me3 in both 361-EBV and 550-EBV has been reported in various cancers (17–19). Therefore, the cells was identified in the transcription start sites (TSS) of a panel ChIP-Seq results regarding this gene were selected for further of target genes (Fig. 1B); globally, only 361-EBV and not the 550- investigation. The recent studies suggested that H3K4me3 alone EBV showed a significant reduction of H3K4me3 at the TSS (Fig. might not fully explain the regulation of gene expression (20). 1C). The expression level of H3K4me3 was further investigated by H3K4me3 and H3K27me3 work together to form a bivalent

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821752116 Leong et al. Downloaded by guest on October 6, 2021 Table 1. Sixteen DNA damage repair genes identified from ChIP-Seq results with reduction of H3K4me3 in promoter regions of the EBV-positive NPE cell lines ID no. Gene symbol Gene name DNA damage repair pathways

9156 EXO1 Exonuclease 1 MMR/HR 4292 MLH1 MutL homolog 1 MMR 5111 PCNA Proliferating cell nuclear antigen MMR/BER 6742 SSBP1 Single-stranded DNA binding protein 1 MMR/HR 4361 MRE11 Meiotic recombination 11 homolog A HR/NHEJ 5889 RAD51C RAD51 homolog C HR 25788 RAD54B RAD54 homolog B HR 7979 SEM1 26S proteasome complex subunit HR 4968 OGG1 8-oxoG DNA glycosylase BER 27301 APEX2 AP endonuclease 2 BER 328 APEX AP endonuclease 1 BER 55247 NEIL3 Nei endonuclease VIII-like 3 BER 143 PARP4 Poly (ADP ribose) polymerase family, member 4 BER 5423 POLB DNA polymerase beta BER 5424 POLD1 DNA polymerase delta 1 BER/HR/MMR 6996 TDG Thymine DNA glycosylase BER

switch to regulate gene expression in ESCs and cancer devel- role of MLH1 expression in the sensitivity of cells to cisplatin opment (Fig. 1A) (12, 20). Hence, the H3K27me3 levels in the treatment. To elucidate the association of MLH1 expression to the promoter of MLH1 gene in two pairs of EBV-infected NPE cells cisplatin sensitivity, MLH1 was knocked out in MLH1-positive were also examined. HONE1, whereas it was overexpressed in the MLH1-negative Expression of MLH1 in the EBV-infected NPE cells was val- C666 cells to compare the changes of the sensitivity to cisplatin idated to be down-regulated in the EBV-positive NPE cells (Fig. treatment. The MLH1 single-guide RNA 2 (sgRNA2) showed the 2 B and C) and in a panel of paired NPC clinical specimens (Fig. highest knockout efficiency in the MLH1 knockout among the MEDICAL SCIENCES 2D). As the association of microsatellite instability (MSI) with three sets of knockout oligos used and was chosen to establish the expression of MLH1 has been reported (21, 22), microsatellite the MLH1-knockout HONE1 cell line (HONE1-MLH1-KO) (Fig. typing was performed for the EBV-infected and the uninfected NPE 3D). On the other hand, MLH1 was reexpressed using the lenti- cells. However, MSI was not observed in the EBV-positive NPE virus system in C666 to establish C666-MLH1−expressing cells to cells, even though expression of MLH1 is down-regulated (SI Ap- examine their sensitivity to cisplatin treatment (Fig. 3D). The IC50 pendix,Fig.S3). The H3K4me3 and H3K27me3 modification levels of these NPC cell lines was determined for the cisplatin treatment were then further confirmed in the EBV-infected NPE cells. In- (SI Appendix,Fig.S7). Knocking out MLH1 in the HONE1 sig- terestingly, the EBV-associated bivalent switch was detected in both nificantly reduced HONE1 sensitivity after 5 μM cisplatin treat- NPE cell lines; the results indicated the reduction of H3K4me3 and ment, compared with the parental HONE1 LacZ control (Fig. 3E). enhanced H3K27me3 in the MLH1 promoter of EBV-infected NPE On the other hand, with 15 μM cisplatin treatment, reexpression of cells (Fig. 2E). On the other hand, promoter hypermethylation is MLH1 significantly increased the sensitivity of C666-MLH1 (Fig. usually associated with down-regulation of the relevant genes, and 3E) compared with the parental C666 vector alone (VA) control. the genes with bivalent marks are often strong targets of DNA In addition, the MLH1-KO− and MLH1-reexpressing NPC cells methylation in cancers. Surprisingly, our bisulfite sequencing results were also used for in vivo study. Consistent with the in vitro study, suggested that the promoter CpG islands remained unmethylated HONE1-MLH1-KO cells were more resistant to cisplatin treat- regardless of the status of EBV infection of the NPE cells (Fig. 2F), ment compared with HONE1-LacZ, while C666-MLH1 exhibited which is consistent with the previous study results (23). This finding a significant sensitivity to the cisplatin treatment compared with further supports the hypothesis that MLH1 down-regulation is as- VA–cisplatin (Fig. 3F). The tumor sizes in day 12 among VA–PBS, sociated with bivalent switch of these two histone marks and is in- VA–cisplatin,andMLH1-PBSinC666arenotstatisticallydiffer- dependent of the promoter hypermethylation. ent (P > 0.05). The MLH1 knock-out and overexpressed status in The importance of MLH1 down-regulation in epithelial ma- HONE1 and C666, respectively, were confirmed in the mouse lignancy was further confirmed by functional studies. MLH1 tumors by immunohistochemical (IHC) staining (SI Appendix, Fig. expression is reported to correlate with clinical response to S4). These findings further support the role of EBV in the regu- platinum drugs including cisplatin, and MLH1-proficient cells lation of host cell DNA damage repair members and, thus, its are more sensitive to cisplatin in colon cancer and head and neck important association in the tumor development. cancer (24, 25). As expression of MLH1 in EBV-positive NPE cells is down-regulated, it is expected that EBV-positive NPE The BER Pathway Is Significantly Enriched with Aberrant Bivalent cells are more resistant to cisplatin, which is borne out by our H3K4me3 and H3K27me3 Switches After EBV Infection. After the current studies (Fig. 3A); both pairs of EBV-infected NPE cells functional study for MLH1, it is also interesting to elucidate the showed greater resistance to cisplatin throughout the treatment DNA damage pathway enriched with EBV-associated aberrant period. The concentrations of cisplatin applied to the two pairs bivalent switches. Pathway analysis was performed to identify DNA of NPE cells were selected according to the concentrations damage repair pathways that show loss of the H3K4me3 mark; closest to their corresponding half-maximal inhibitory concen- seven members (APEX, POLB, POLD1, PCNA, TDG, OGG1,and tration (IC50) values (SI Appendix, Fig. S7). NEIL3) from the BER pathway were significantly enriched [false As the two sets of NPE cells are nontumorigenic, to further discovery rate (FDR) = 0.0709, cutoff < 0.1] in both EBV-infected examine the importance of MLH1 in mediating cisplatin cyto- NPE cell lines (SI Appendix,Fig.S5), and the down-regulation was toxicity in this epithelial malignancy, two tumorigenic cell lines, further validated at the RNA and protein levels (Fig. 4A and SI HONE1 (EBV-negative) with observable high MLH1 expression Appendix,Fig.S6). These results suggested that EBV infection and C666 (EBV-positive) with relatively lower MLH1 expres- could alter host cell H3K4me3 modification and is associated with sion, were utilized for functional studies (Fig. 3 B and C). These down-regulation of these seven BER members in the EBV-infected two cell lines provided a good model to investigate the critical 361 and 550 cell lines. The clinical significance of down-regulation

Leong et al. PNAS Latest Articles | 3of10 Downloaded by guest on October 6, 2021 Fig. 2. Down-regulation of MLH1 through EBV-associated bivalent histone marks regardless of the promoter methylation. (A) The ChIP-Seq result shows a reduction of H3K4me3 in the promoter region of MLH1 in the EBV-positive NPE cells. (B and C) The real-time qPCR and WB analysis further validates the down-regulation of MLH1 in the EBV-infected cells and (D) the down-regulation of MLH1 in 18 paired NPC biopsies (P < 0.05). (E) ChIP-qPCR validated the reduction of H3K4me3 levels at the promoter region of MLH1 in the EBV-positive NPE cells. The same procedure was used to show the gain of H3K27me3 in the promoter region of MLH1.(F) The bisulfite sequencing results show the unmethylated CpG sites in the promoter region of the MLH1 in both EBV-positive and EBV-negative NPE cell lines. *P < 0.05; **P < 0.01; ***P < 0.001.

of BER genes in NPC specimens was also examined. Significant Furthermore, as there is a panel of DNA damage repair down-regulation of APEX, POLB TDG, PCNA,andOGG1 can be members down-regulated after EBV infection, it is reasonable to observed in a panel of clinical NPC paired nontumor and NPC infer that EBV infection induces greater host cell susceptibility tissues (Fig. 4B). to DNA damage. Therefore, hydrogen peroxide was used to The aberrant bivalent H3K4me3 and H3K27me3 switch of induce DNA damage to the cells by 2-h treatment, and the comet these seven BER pathway members was also examined. assay was used to compare the DNA damage for the EBV- H3K4me3 signals in ChIP-Seq data were found near the CpG positive and EBV-negative NPE cells. The comet tails indicate islands of promoter regions across these seven BER members in the DNA damage in the cells. After a 24-h recovery, the DNA EBV-negative NPE cell lines (Fig. 4C). In contrast, there was a damage from both EBV-negative NPE cells returned to a similar reduction of H3K4me3 signals in corresponding regions of the level to that without treatment. Nevertheless, the DNA damage in the EBV-positive cells is significantly higher than that in the EBV- EBV-positive NPE cell lines. The ChIP quantitative PCR (ChIP- A qPCR) results from both sets of the EBV-infected NPE cells are negative cells (Fig. 5 ). In addition, an apurinic/apyrimidinic (AP) consistent with the ChIP-Seq results, showing reduction of site is one of the intermediates produced during BER. An AP site assay was performed to compare the efficiency of the BER in the H3K4me3 in the candidate genes in the EBV-positive NPE cell EBV-infected and noninfected cell lines (Fig. 5B). The number of lines, while, in contrast, there is gain of the H3K27me3 mark in 5 D AP sites per 10 nucleotides was estimated by utilizing a standard EBV-positive NPE cells across the candidate genes (Fig. 4 ). curve (SI Appendix,Fig.S8). EBV-infected cells showed a lower This suggested that the association of the expression of these level of AP site accumulation after H2O2 treatment than the pa- seven BER members and the bivalent histone switches. The rental cells. After 24-h recovery, the noninfected cells could sig- hypermethylation status in the promoter regions of these seven nificantly reduce the number of AP sites from ∼48 to ∼38 AP per BER pathway members was examined (Fig. 4E), and there is no 105 nucleotides and ∼43 to ∼35 AP per 105 nucleotides for 361 and significant difference in the promoter methylation status be- 550, respectively. However, with regard to the EBV-infected cells, tween the EBV-positive and EBV-negative cells; this provides no significant reduction of the number of AP sites could be ob- solid evidence that the down-regulation of the BER members is served after 24-h recovery. Taken together with the comet assay, highly associated with the histone bivalent switch and indepen- the results indicate the impaired DNA damage response in the dent of the promoter hypermethylation. EBV-positive NPE cells and further confirm the role of EBV in

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821752116 Leong et al. Downloaded by guest on October 6, 2021 regulating DNA damage repair by down-regulating the BER family hand, a study performed on EBV-infected B cells identified that member genes. the levels of the H3K4me3 mark was low in latently infected B To further confirm the role of EBV in regulating the expres- cells, but was very high after reactivation, especially in the lytic sion of the candidate genes, NPE cells stably expressing the EBV promoter region including BZLF1 (31). In this study, it is also genome maintenance protein, EBNA1, were established (Fig. evident that EBV infection contributes to the aberrant histone 5C). Results from analysis revealed that overexpression of H3K4me3 and H3K27me3 bivalent switches in epithelial cells and, EBNA1 induced down-regulation of the DNA damage repair consequently, impacts the regulation of the host cell transcription members at the transcriptional level (Fig. 5D), and, with the profile independently from promoter hypermethylation. EBV in- exception of the PCNA and OGG1, down-regulation can also fection was confirmed to induce reduction of the H3K4me3 marks be detected at the translational level (SI Appendix, Fig. S6). The in the host cell. The degree of reduction of the H3K4me3 mark results support the important role of EBNA1 in regulating the globally in the host cell genome shows variation between the two expression of these BER pathway members and MLH1 in tested EBV-infected cell lines and is likely attributed to the dif- the NPE cells. ferences in genetic backgrounds of these two cell lines. Clearly, consistent H3K4me3 down-regulation can be detected in specific Discussion candidate gene regions. Therefore, this further supports the cru- EBV is etiologically linked to tumorigenesis in a number of cial role of EBV in the regulation of this histone mark to particular cancer types. In EBV-associated carcinoma, including NPC, EBV regions in host cells. infection in precancerous nasopharyngeal epithelium is postulated A slower proliferation rate in EBV-infected NPE cells was to be an early event during tumor development (26). Previously, previously reported compared with the uninfected NPE cells (16, NPC was characterized by relatively low somatic mutation rates, 32), suggesting a role of EBV in contributing to the slower but displaying higher percentages of gene hypermethylation growth rate in the host cell. As POLD1 and PCNA are not only compared with other cancer types (15). It is reasonable to assume participating in BER pathway but also in DNA replication and that EBV plays an important role in the aberrant epigenetic cell cycle progression (33, 34), down-regulation of these two modification of infected cells to promote tumorigenesis. candidate genes implicates a certain influence on the cell cycle Previous studies have established a role for EBV in promoting progression and, hence, slower proliferation rate. This provides host cell epigenetic control of transcription in epithelial cells hints to explain the slower proliferation rate in the EBV-infected through a mechanism involving global hypermethylation, achieved, cells by the down-regulation of POLD1 and PCNA. For the other in part, through effects on the activity of the DNA methyl- BER candidate genes, they are majorly involved in BER path- transferases DNMT1, DNMT3A, and DNMT3B (15, 27–29). The way, indicating the down-regulation of those candidate genes clinical significance of the H3K4me3 mark was proposed given a associated with dysregulation of BER pathway and cell pro- MEDICAL SCIENCES poor prognosis in colorectal cancer (CRC), where expression of liferation. In fact, to maintain DNA integrity, the DNA repair the H3K4me3 mark was significantly decreased (30). On the other mechanisms must be conserved. BER is one of the fundamental

Fig. 3. The levels of MLH1 are associated with cellular responses to cisplatin. (A) Comparison of the cytotoxicity upon 10 μM and 20 μM cisplatin treatment for 361/361-EBV and 550/550-EBV, respectively. A significant cisplatin resistance can be observed in both 361-EBV and 550-EBV NPE cell lines. (B) The qPCR shows the expression of MLH1 in C666 is down- regulated compared with HONE1. (C) WB shows down-regulation of MLH1 protein expression in C666, compared with HONE1, where p84 was used as an equal loading control. (D) WB confirmed the MLH1 knockout in HONE1 by CRISPR/Cas9 and MLH1 overexpression in C666. For MLH1 knockout in HONE1, three oligos of sgRNA were utilized. The sgRNA2 provides the most effective knockout for MLH1.(E) The MTT assay reveals that the MLH1-KO in HONE1 shows more significant resistance to cisplatin compared with the LacZ control in response to treatment with 5 μM cisplatin. Overexpression of MLH1 in C666 induced greater sensitivity to cisplatin compared with the VA after treatment with 15 μM cisplatin. (F) The in vivo nude mouse s.c. injection validates the in vitro MTT results. Cisplatin at 2.5 mg/kg and 5 mg/kg were injected intraperitoneally into mice carrying the HONE1 and C666 tumors, respectively. PBS was injected as a vehicle control. The in vivo study suggests tumors with MLH1 expression are more sensitive to cisplatin treatment. LacZ ctrl, nontargeting control oligo, downward triangle, the time point for cisplatin treatment. *P < 0.05; **P < 0.01; ***P < 0.001.

Leong et al. PNAS Latest Articles | 5of10 Downloaded by guest on October 6, 2021 Fig. 4. EBV infection is associated with bivalent histone modification of members of the BER pathway independent of promoter hypermethylation. (A and B) The real-time qPCR validated the down-regulation of the seven BER candidate genes in the EBV-infected NPE cells, and the down-regulation was observedin the 18 paired NPC biopsies. Reduced expression of APEX, POLB, PCNA, TDG, and OGG1 is statistically significant (P < 0.05). (C) The ChIP-Seq results show a reduction of H3K4me3 in the promoter regions of the EBV-positive NPE cells for the seven BER pathway members. (D) ChIP-qPCR validated the reduction of H3K4me3 and gain of H3K27me3 of the six BER pathway members. A suitable primer set for OGG1 could not be designed, so the status of histone H3K4me3 and H3K27me3 was not examined by ChIP-qPCR. However, the qPCR and ChIP-Seq results support that the expression of this gene is associated with EBV infection. (E) The bisulfite sequencing results exhibit unmethylated CpG sites in the promoter region of the seven BER candidate genes in both EBV- positive and EBV-negative NPE cell lines. *P < 0.05; **P < 0.01; ***P < 0.001.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821752116 Leong et al. Downloaded by guest on October 6, 2021 MEDICAL SCIENCES

Fig. 5. Association of EBV infection with defective DNA damage repair response in the NPE cell lines and the involvement of EBV latent protein in the down- regulation of the DNA damage repair candidate genes. (A) The comet assay assessed the DNA damage induced by hydrogen peroxide. The comet tails indicate the DNA damage. While both EBV-positive and EBV-negative NPE cell lines show long comet tails after 2 h of hydrogen peroxide treatment, only EBV-positive NPE cells retain observable comet tails after 24 h of recovery; the comet DNA tails were quantified. The results show the defective DNA damage repair response induced by hydrogen peroxide in 361-EBV and 550-EBV after 24-h recovery; the observable fraction tail DNA is more significant in the EBV-infected cells compared with those without treatment (P < 0.05). *P < 0.05; **P < 0.01; ***P < 0.001. (B) AP sites assay was utilized to determine the BER efficiency by quantifying the number of AP sites after 2-h treatment of hydrogen peroxide and 24-h recovery. The results indicate the high number of AP sites in the EBV-infected cells even after 24-h recovery, but that in the noninfected cells reduced observably after 24-h recovery. (C) Overexpression of the EBNA1 in the NPE cells was validated by qPCR. (D) The qPCR results show down-regulation of the candidate genes in both the EBNA1 overexpressing NPE cells compared with the VA control.

housekeeping systems that preserves the integrity of DNA. In NEIL3, to excise the lesion of nucleobase such as 8-oxoG and addition to the environmental factors, oxidative respiration gen- results in leaving an AP site for the subsequent BER process (37, erates reactive oxygen species (ROS), such as hydrogen peroxide, 38). OGG1 from the BER system is responsible for the excision superoxide, and hydroxyl radicals, as by-products of cellular of 8-oxoG and, hence, helps to maintain the integrity of the metabolism. The excessive production of ROS has been linked to DNA. As the expression of OGG1 is down-regulated in the the initiation and progression of cancer (35). These by-products EBV-infected cells, this may contribute to the lower ability in render DNA susceptible to oxidation. One of the major oxidized forming the AP sites compared with the parental cells with high bases in DNA is 8-oxoguanine (8-oxoG). This oxidized base is expression of the DNA glycosylase members after 2-h H2O2 highly mutagenic due to its tendency to pair with adenine and treatment. Furthermore, down-regulation of the BER members cytosine (36). The efficacy in inducing AP sites in BER lies in the may contribute to the accumulation of 8-oxoG in EBV-infected expression of the DNA glycosylases, including OGG1, TDG, and NPE cells, which makes it more susceptible to DNA damage,

Leong et al. PNAS Latest Articles | 7of10 Downloaded by guest on October 6, 2021 that is consistent with our findings showing that EBV-infected ulation of histone modifications, leading to the down-regulation cells display a reduced DNA repair ability, as assessed by the of the candidate genes. Moreover, a previous EBNA1 ChIP-Seq comet assay and AP site assay. Clinically, a study of a Chinese study showed that overlapping of EBNA1 binding sites with the population showed that patients with BER polymorphisms dis- H3K4me3 and H3K27me3 is detectable in a BL B cell line, and played an increased susceptibility to developing NPC (39). proposed the association of EBNA1 in the regulation of gene ex- Moreover, defective or variant BER members have been linked pression with these two bivalent histone modification marks (59). to the development of other types of human cancers (40, 41). However, whether EBNA1 directly contributes to the reduction of These findings further highlight the association of defects in H3K4me3 and gain of H3K27me3 in the DNA damage repair BER and cancer development. candidate genes still requires further investigation. Furthermore, in Another DNA damage repair member in the MMR pathway, addition to the bivalent marks, changing of other histone marks, MLH1 , was also found to be down-regulated in response to EBV including H3K27ac, H3K26me3, and H3K9me3, may concurrently infection. Mutation of the gene causes predisposition to hered- contribute to the aberrant expression of the candidate genes. The itary nonpolyposis CRC (42), characterized by MSI (43). Al- MLH1 current study shows a strong association between the alteration of though was found to be down-regulated in this current histone marks and changes of the candidate gene expression. Fu- study, no MSI was detected in the EBV-positive NPE cell lines. ture functional studies regarding the change of specific bivalent Since a microsatellite stable phenotype is observed in the EBV- marks and their association with the down-regulation of the DNA positive NPE cells, this suggests the low basal expression of MLH1 is sufficient to support the microsatellite stable phenotype damage repair candidate genes are necessary to prove a direct in the NPE cells. This is consistent with the previous NPC studies connection between these observations. reporting that MSI is relatively rare in this cancer (44). A cor- In conclusion, this current study elucidates the role of EBV in relation of EBV infection and the epigenetic regulation of the regulation of histone bivalent switches associated with the host MLH1 was reported earlier by Saha et al. (3); EBV-positive DNA damage repair and implicates EBNA1 having an important stomach cancer was shown to lack the MLH1 promoter hyper- role in NPC tumorigenesis. These current findings provide an methylation characteristic of MSI-associated CpG island meth- exciting and important direction for investigating the interaction ylator phenotype (23). Similarly, this current study shows that between EBV and the host cell transcription. down-regulation of MLH1 in EBV-infected NPE cells is associated with the bivalent histone switch and is independent of Materials and Methods promoter hypermethylation. Moreover, another study found that Cell Culture and Cell Lines. The immortalized NPE cell lines, NP361-htert- DNA methylation was antagonistic to H3K27me3 at promoters cyclinD, NP361-htert-cyclinD-EBV, NP550-htert-cyclinD, and NP550-htert- (45). This is consistent with findings of other studies that some cyclinD-EBV (361, 361-EBV, 550, 550-EBV), were established (16) and cul- genes can be silenced by their gaining H3K27me3 in the pro- tured with defined keratinocyte–serum-free medium and EpiLife (1:1) with moter region without promoter hypermethylation (46, 47). supplements and 10% FBS (GIBCO, Invitrogen). HONE1 was cultured in Head and neck squamous cell carcinoma (HNSCC), unlike Dulbecco’s modified eagle medium with 5% FBS and 5% newborn calf se- NPC, is usually not associated with EBV infection (48, 49), and rum (60). C666 was cultured in Roswell Park Memorial Institute with 10% FBS HNSCC cells with down-regulation of MLH1 are generally re- (61) (GIBCO, Invitrogen). The 293FT cell line was purchased from Invitrogen sistant to cisplatin (24), suggesting that other factors in addition and cultured as recommended for virus packaging. All cell lines were tested to EBV infection may contribute to the down-regulation of and proven to be mycoplasma negative. MLH1 in HNSCC, and hence resistance to cisplatin. Our current study further supports that down-regulation of MLH1 increases Clinical Samples. Specimens were collected by the NPC Area of Excellence resistance to cisplatin in vitro and in vivo. Clinically, some NPC (AoE) Research Tissue Bank with written consent from patients. The study patients are resistant to cisplatin-based chemotherapy (50, 51), a was approved by the Institutional Review Board of the University of Hong phenomenon that is probably linked to a deficiency in MLH1, Kong. Eighteen matched pairs of normal and NPC tumor samples were compromising the cisplatin-induced apoptotic signaling (52). randomly selected for the studies. Specimens were processed as previously A number of studies have shown that EBV infection is asso- described for the qPCR experiments (62). ciated with increased levels of DNA damage (53, 54). A previous ChIP and ChIP-Seq by Next-Generation Sequencing and Data Analysis. The study has demonstrated a role for LMP1 in DNA repair sup- chromatin samples were prepared by utilizing Covaris trueChIP Chromatin pression through the PI3K/AKT pathway and inactivation of Shearing Reagent for fragmentation by the M220 High Performance Ultra- FOXO3a and DDB1 (55). Coexpression of LMP1 and LMP2A in γ sonicator system. ChIP reaction was performed by utilizing ChIP-grade an- EBV-negative NPC cells is accompanied by a reduction in H2AX tibody against the H3K4me3, and H3K27me3 (Qiagen). The input and IgG phosphorylation in response to treatment with genotoxins, which isotype control were used for calculation of the fold of enrichment (63). A can promote DNA replication stress or single-strand breaks (56). total of 5 ng of ChIP-enriched DNA was used for next-generation sequencing Furthermore, in EBV-infected B cells, EBNA3C was reported to library construction, and sequencing reactions were performed on the Illu- suppress DNA damage repair responses (57, 58). These studies mina HighSeq 1500 in the Centre For Genomic Sciences (CGS), The University of provide strong evidence of the ability of EBV to contribute to Hong Kong (HKU). The DNA library was constructed using the KAPA Library genomic instability through deregulating the host DNA repair Preparation Kit (Roche) according to the manufacturer’s protocol. After adaptor mechanisms. However, neither the association of EBV infection and low-quality base trimming, the clean reads were aligned to human genome and DNA damage to precancerous epithelial cells nor a role for (hg19) by Burrows–Wheeler alignment (64). The broad peaks for H3K4me3 EBV in epigenetic regulation of DNA damage repair pathways were identified by model-based analysis of ChIP-Seq with FDR < 0.05 (65) and has been proposed. In this study, we demonstrated that expres- the peaks were subsequently annotated by hypergeometric optimization of sion of the EBV genome maintenance protein, EBNA1, is as- motif enrichment (66). sociated with down-regulation of seven BER members and a MMR member in the epithelial cells. At the protein level, only Real-Time qPCR and Western Blotting. The qPCR reactions were performed as PCNA and OGG1 did not show obvious down-regulation; this previously described (62). Each reaction was repeated in triplicate, and the may be caused by many different factors, including protein sta- data represent the mean, with error bars indicating SE (SEM). Western blotting bility, posttranslation modifications, and other causes. Further was performed as previously described (62). The antibodies, including APEX1 studies are required to understand this observation. After in- (1:1,000; Cell Signaling), POLB (1:1,000; GeneTex), POLD (1:1,000; GeneTex), fection of epithelial cells, EBV adopts a latency Type II program, PCNA (1:1,000, GeneTex), TDG (1:1,000, Abcam), OGG1 (1:1,000, Abcam), expressing EBNA1, LMP1, LMP2, and the EBER RNAs, but MLH1 (1:1,000; Cell Signaling), H3K4me3 (1:1,000; Cell Signaling), and not EBNA3A and EBNA3C, which are previously reported to total Histone H3 (1:10000; Cell Signaling), were used for detecting the contribute to histone modification. Given that EBNA1 is localized expression of the targeted proteins, while p84 (1:1,000; GeneTex) was used to the nucleus, it is possible that EBNA1 plays a role in the reg- as a loading control.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1821752116 Leong et al. Downloaded by guest on October 6, 2021 Construction of Plasmid Vectors and Lentiviral Infection. The full-length human and Penta D, were utilized for PCR reactions according to the MSI Analysis MLH1 gene in pCEP9 was obtained from Addgene (Addgene no. 16458), and System Version 1.2 (Promega). DNA extracted from normal and CRC speci- the coding region was subcloned into pLVX-EF1α lentiviral expression vector mens was used as negative and positive MSI controls, as we previously de- (modified from pLVX-CMV; Clontech). For the knockout experiment, three sets scribed (69). The fluorescent amplicons were sent to CGS for gene scanning, of MLH1 sgRNAs knockout oligos were designed according to Broad Institute and the results were analyzed by Gene Mapper 3.7. Genetic Perturbation Platform and were cloned into the lentiCRISPR vector (Addgene no. 52961). The EBNA1-pLenti6 lentivirus production and infection Comet Assay. The comet assay was performed according to Bio-protocol (70). were described previously (62, 67). For one group of the cells, a treatment with 50 μM hydrogen peroxide

(H2O2) was used to induce DNA damage. For the other group, the same The 3-(4,5-Cimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide Assay. The treatment was applied followed by 24-h recovery. The cells were fixed with 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was absolute ethanol for electrophoresis with 300 mM NaOH and 1 mM ethyl- 4 3 performed as described (62). A total of 2 × 10 cells for C666, 5 × 10 cells for enediaminetetraacetic acid electrophoresis solution. After DAPI staining, the × 3 HONE1, and 3 10 cells for the NPE cells was seeded in 96-well plates. The comet tail images were visualized with a fluorescence microscope and cisplatin (SelleckChem) was prepared with PBS and further diluted to the quantified by ImageJ (n = 16). desired concentration for cell treatment; 5 μM and 15 μM of cisplatin were used for the treatment of HONE1 and C666, respectively. The concentration AP Site Analysis. The DNA damage assay kit (AP sites, colorimetric) (Abcam) μ μ of 10 M was applied to 361 and 361-EBV, whereas 20 M was utilized for was utilized to determine the number of AP sites (71). Firstly, the cell lines 550 and 550-EBV. The different concentrations of cisplatin for NPC and NPE were treated with 50 μMH2O2 for 2 h, and the genomic DNA was extracted. cell lines were used according to the concentrations closest to their corre- The aldehyde groups, the open ring form of the AP sites, were labeled by ARP sponding IC (SI Appendix, Fig. S7), which was calculated by Prism8 50 reagent to tag with biotin residues. The biotin residues were quantified using (GraphPad Software). avidin-biotin assay, followed by a colorimetric detection by absorbance at OD450 nm. A standard curve with a range of 0 to 40 AP sites per 1 × 105 bp Nude Mouse Tumorigenicity Assay. Female nude mice (BALB/cAnN-nu) 6 wk to was constructed to estimate the number of AP sites (SI Appendix,Fig.S8). 8 wk of age were subcutaneously (s.c.) injected with 1 × 107 transduced HONE1 and C666 cells. When the tumors reached about 100 mm3, the mice IHC Staining. IHC staining for MLH1 was performed as previously described were divided into four groups (n = 4) for cisplatin treatment. For HONE1, the (72). The dilution for the MLH1 antibody is 1:200 (Genetex). HONE-LacZ and HONE1-MLH1-KO were treated with cisplatin (SelleckChem) by i.p. injection every 3 d (2.5 mg/kg). Similarly, intraperitoneal (i.p.) injection of cisplatin to C666-VA and C666-MLH1 was administered (5 mg/kg). Pathway Enrichment Analysis and Statistic Calculations. The pathway analysis Thirty percent PBS diluted by the corresponding culture medium was in- was performed by utilizing the database for annotation, visualization, and jected into the mice as separate control groups. The size of tumor (length × integrated discovery (73, 74). The Benjamini test was used to calculate the

< MEDICAL SCIENCES width × height) and the body weight were measured before each treatment. FDR for the pathway analysis; a FDR of 0.1 was used as the cutoff point to ’ The in vivo study was conducted under the animal license from the De- identify the significantly enriched pathways. Student s t test was utilized for partment of Health, Hong Kong, and approved by the Committee on the Use statistical analyses for experiments unless stated otherwise. A P-value of Live Animals in Teaching and Research of The University of Hong Kong. of <0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001). Bisulfite Sequencing. The bisulfite sequencing was performed using the SeqCap Epi Enrichment System (Roche) targeting the global CpG islands upon ACKNOWLEDGMENTS. This work was supported by the Research Grants ’ bisulfite conversion using EpiTect kit (Qiagen) in NP361 and NP550 with and Council of the Hong Kong Special Administrative Region, People s Republic without EBV infection. The data were analyzed using bisulfite sequence of China: Grant AoE/M-06/08 to M.L.L.; Collaborative Research Fund (C7027- 16G) and General Research Fund (106180141) to S.W.T.; and Seed Funding mapping program (68) for investigating the methylation status of the Programme for Basic Research of HKU: 201308159003 to A.K.L.C. We also candidate genes. acknowledge help from Dr. Agnes Chan and Mr. Cheng Wei Wu from the CGS, The University of Hong Kong, for sharing their experience on the ChIP- Microsatellite Analysis. Five microsatellite markers, including NR21, NR24, Seq experiment and consultations. The sequencing work in this current study BAT25, BAT26, MONO27, and two pentanucleotide repeat markers, Penta C was performed at CGS, The University of Hong Kong.

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