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.

爱泼斯坦-巴尔病毒（Epstein–Barr virus, EBV）在宿主细胞内的三维（3D）染色质结构，以及染色质互作与基因调控的潜在机制（尤其是涉及EB病毒非编码RNA（ncRNAs）的机制）至今尚未被完全阐明。本研究采用当前前沿的三维基因组图谱技术，包括结合配对末端标签测序的蛋白质关联染色质互作分析（ChIA-PET）、RNA关联染色质互作技术（RDD）以及超分辨率显微镜技术，以解析人类淋巴母细胞中EB病毒的空间构象。我们系统分析了与宿主蛋白及EB病毒RNA相关的EB病毒-EB病毒（E–E）、EB病毒-宿主（E–H）以及宿主-宿主（H–H）互作。研究结果显示，EB病毒借助宿主CCCTC结合因子（CTCF）与RNA聚合酶II（RNAPII）形成独特的染色质接触结构域（CCDs）及RNAPII关联互作结构域（RAIDs）。这些染色质结构的锚点可作为与宿主染色质广泛互作的平台，进而调控宿主基因表达。值得注意的是，EB病毒非编码RNA，尤其是EB病毒编码RNA（EBERs），可靶向结合宿主染色质中较难接近的区域，通过抑制RNAPII关联的染色质环来抑制部分基因的表达。该过程依赖于辅助因子核仁素（NCL）及其RNA识别基序，敲低NCL或EBERs均可改变宿主感染控制、免疫应答及细胞周期调控相关关键基因的表达水平。上述研究结果揭示了EB病毒与宿主染色质之间复杂的相互作用。实验整体设计如下：我们在GM12878细胞中针对非编码RNA（包括EBERs、v-snoRNA、miR-BART以及MALAT1）生成了RDD数据。其中EBERs的实验包含3个生物学重复：重复1包含3次独立实验（EBERs_RDD_rep1_exp1-3），重复2包含6次独立实验（EBERs_RDD_rep2_exp1-6），重复3包含10次独立实验（EBERs_RDD_rep3_exp1-10）。v-snoRNA与miR-BART各包含2个生物学重复，MALAT1则包含3个生物学重复。针对ChIA-PET数据，各实验组均设置2个生物学重复，包括EBERs敲低的GM12878细胞（EBERs-KD）以及作为阴性对照的乱序寡核苷酸处理组（EBERs-NC），同时包含核仁素敲低的GM12878细胞（NCL-KD）及其阴性对照（NCL-NC）。此外，我们还在HEK293T细胞中开展了ChIA-PET实验（NCL-KD、NCL-NC）。针对GM12878细胞中HSPA9与NCL的ChIP-seq数据，在EBERs-KD与EBERs-NC条件下各包含2个生物学重复。GM12878细胞的ATAC-seq实验中，EBERs-NC组设置3个生物学重复，EBERs-KD组设置2个生物学重复。GM12878细胞的RNA-seq实验中，EBERs-KD与EBERs-NC组各设置2个生物学重复，CLIP-seq实验同样在GM12878细胞中设置2个生物学重复。