3D genome study to elucidate chromosome reorganisation and epigenetic dysregulation in Epstein-Barr virus-associated nasopharyngeal carcinoma (NPC)

Abstract

Nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) infection. More than 95% of the NPC cases have detectable EBV at the primary tumour site in the endemic regions. During the latency stage, EBV expresses a limited number of viral proteins, with EBNA1 in maintaining EBV episomes on human chromatin. Despite the rarity of EBV genome integration into the host genome, questions persist on the role of the double-stranded DNA EBV virus in inducing high-ordered epigenomic alterations in human chromatin. Recent studies have demonstrated that 3D genome folding is established by topologically associating domains (TADs) and chromatin loops. The chromatin loops are insulated by CTCF and cohesin proteins, affecting transcription. The importance of 3D chromatin architecture in cancer evolution involves enhancer-promoter contacts for activating transcription by disrupting TADs. However, it remains unclear whether EBV could epigenetically remodel the human 3D genome to activate cancer-relevant pathways, thereby facilitating tumorigenesis and tumour progression. Previously, we reported that CTCF, a master regulator for chromosome organisation, was often dysregulated with hemizygous loss and reduced protein level in NPC, and EBV-associated differentially methylated regions (EBV-DMRs) were often enriched at CTCF binding sites. Therefore, we hypothesise that EBV could reorganise the spatial chromatin to inactivate tumour suppressors and activate oncogenes in NPC. In this study, we integrated 1) modified Hi-C, 2) the assay for transposase-accessible chromatin with sequencing (ATAC-Seq), 3) cleavage under targets & release using nuclease with sequencing (CUT&RUN-Seq) for normal immortalized nasopharyngeal cell lines, EBV+/EBV- NPC cell lines, EBV+ patient-derived xenografts (PDXs) cells and a clinical sample, 4) bulk RNA-Seq data for cell lines, 5) three publicly available NPC single-cell RNA sequencing (scRNA-Seq) data, 6) spatial transcriptomes for NPC patients, to systematically profile 3D genome, and investigate EBV-human chromatin interactions associated with histone modifications and chromatin accessibility as well as their potential functional impacts. This study discovered EBV-associated genome reorganisation, where the high-ordered tertiary structure maintained by CTCF was dysregulated. The chromatin loops are exclusively reduced in EBV-associated NPC, leading to the unification and reduced diversity of the genomic compartments, suggesting the crucial role of EBV in remodelling the 3D genome. EBV-human chromatin interactions were frequently detected in EBV+ cell lines and PDXs. Apart from the oriP region, two novel regions covering EBNA1 and RPMS1 were found to frequently engage with human chromatin. These interactions could form cis-regulatory region bindings, especially at NFKB1 binding sites. Moreover, H3K27ac enhancer and H3K27me3 suppressor modifications were reorchestrated at EBV-interacting regions for enhancer activation and inactivation to facilitate EBV-associated open and closed chromatin. In particular, CD74, a chaperone for MHC class II, was upregulated by EBV latent infection through NF-κB binding and super-enhancer activation, which promoted immune suppression by elevating LAG3+ T cells. Treatment of SMARCA2/4 inhibitor targeting the chromatin remodeller SWI/SNF complexes partially rescued NF-κB activation by reducing chromatin accessibility at selective EBV-interacting regions and promoted cancer cell death in EBV+ NPC cells. This study provides evidence that the EBV genome mediates human chromosome reorganisation to reprogram epigenetic landscapes, regulate NF-κB signalling and facilitate immune suppression.