The chromatin loop release factor WAPL regulates EBV latency status by restricting LMP production

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that is etiologically linked to several cancers and has been connected to multiple sclerosis. EBV employs a series of latency programs, including latency III, latency II, and latency I, to persistently colonize the B-cell compartment. Each of these programs is associated with human malignancies. However, our understanding remains incomplete of how these distinct latency programs are epigenetically restricted. While regulation of the chromatin structure of the EBV genome by cohesin and CTCF contributes to genome regulation, the function of the cohesin release factor Wings Apart-Like Protein Homolog (WAPL) had not previously been understood. In this study, we employ RNA-seq combined with immunofluorescence analysis to determine that loss of WAPL leads to aberrant expression of the oncogenic latent membrane proteins LMP1 and LMP2A in latency I Burkitt lymphoma cells. Through a combination of Hi-C, Hi-ChIP, and ChIP-qPCR, we uncover that WAPL loss causes alterations in looping across the EBV genome and specifically induces formation of loops between the LMP promoter and the oriLyt enhancers. We propose that EBV coopts WAPL to maintain a strict latency I state and, without WAPL, leaky expression of the LMP proteins occurs. Overall design: HiC sequencing of EBV genome in WAPL WT and KO cell lines

爱泼斯坦-巴尔病毒（Epstein-Barr virus, EBV）是一种广泛存在的人类病原体，其与多种癌症存在病因学关联，同时还与多发性硬化症密切相关。EB病毒借助包括潜伏III型、潜伏II型与潜伏I型在内的一系列潜伏程序，持续定植于B细胞区域，上述各类潜伏程序均与人类恶性肿瘤的发生发展相关。然而，目前学界对这些不同潜伏程序的表观遗传调控机制仍不完全明晰。尽管黏连蛋白（cohesin）与CCCTC结合因子（CTCF）对EB病毒基因组的染色质结构调控可参与基因组的表观遗传调控，但此前人们对黏连蛋白释放因子——翅膀分离样蛋白同源物（Wings Apart-Like Protein Homolog, WAPL）的功能尚未阐明。本研究结合RNA测序（RNA-seq）与免疫荧光分析，证实WAPL缺失会导致潜伏I型伯基特淋巴瘤细胞中致瘤性潜伏膜蛋白LMP1与LMP2A出现异常表达。通过联合运用染色体构象捕获技术（Hi-C）、高通量染色质免疫沉淀技术（Hi-ChIP）与染色质免疫沉淀定量PCR（ChIP-qPCR），本研究揭示WAPL缺失会引发EB病毒基因组范围内的染色质环结构改变，并特异性诱导LMP启动子与oriLyt增强子之间形成染色质环。我们推测EB病毒会劫持WAPL以维持严格的潜伏I型状态，而当WAPL缺失时，LMP蛋白会出现渗漏性表达。实验整体设计：对WAPL野生型（WT）与敲除型（KO）细胞系中的EB病毒基因组进行Hi-C测序。