Landscape of the Epstein-Barr virus-host chromatin interactome and gene regulation
The three-dimensional (3D) chromatin structure of Epstein–Barr virus (EBV) within host cells and the underlying mechanisms of chromatin interaction and gene regulation, particularly those involving EBV's noncoding RNAs (ncRNAs), have remained incompletely characterized. In this study, we employed state-of-the-art techniques of 3D genome mapping, including protein-associated chromatin interaction analysis with paired-end tag sequencing (ChIA-PET), RNA-associated chromatin interaction technique (RDD), and super-resolution microscopy, to delineate the spatial architecture of EBV in human lymphoblastoid cells. We systematically analyzed EBV-to-EBV (E–E), EBV-to-host (E–H), and host-to-host (H–H) interactions linked to host proteins and EBV RNAs. Our findings reveal that EBV utilizes host CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) to form distinct chromatin contact domains (CCDs) and RNAPII-associated interaction domains (RAIDs). The anchors of these chromatin domains serve as platforms for extensive interactions with host chromatin, thus modulating host gene expression. Notably, EBV ncRNAs, especially Epstein–Barr-encoded RNAs (EBERs), target and interact with less accessible regions of host chromatin to repress a subset of genes via the inhibition of RNAPII-associated chromatin loops. This process involves the cofactor nucleolin (NCL) and its RNA recognition motifs, and depletion of either NCL or EBERs alters expression of genes crucial for host infection control, immune response, and cell cycle regulation. These findings unveil a sophisticated interplay between EBV and host chromatin. Overall design: We generated RDD data for ncRNA, including EBERs, v-snoRNA, miR-BART, and MALAT1, in GM12878 cells. For EBERs, there are three biological replicates: replicate 1 consists of three experiments (EBERs_RDD_rep1_exp1-3), replicate 2 consists of six experiments (EBERs_RDD_rep2_exp1-6), and replicate 3 consists of ten experiments (EBERs_RDD_rep3_exp1-10). Both v-snoRNA and miR-BART have two biological replicates, and MALAT1 has three biological replicates. For ChIA-PET data, there are two biological replicates for each condition. These include GM12878 cells with EBERs knockdown (EBERs-KD) and scrambled oligos serving as a negative control (EBERs-NC), as well as siRNA knockdown for NCL (NCL-KD) and its scrambled control (NCL-NC). ChIA-PET experiments were also conducted on HEK293T cells (NCL-KD, NCL-NC). ChIP-seq data for HSPA9 and NCL has two biological replicates in GM12878 cells (EBERs-KD, EBERs-NC). The ATAC-seq experiments on GM12878 cells included three biological replicates for EBERs-NC and two for EBERs-KD. RNA-seq experiments were conducted with two biological replicates in GM12878 cells for both EBERs-KD and EBERs-NC, and CLIP-seq experiments also had two biological replicates in GM12878 cells.