The chromatin loop release factor WAPL regulates EBV latency status by restricting LMP production [HiChIP]. The chromatin loop release factor WAPL regulates EBV latency status by restricting LMP production [HiChIP]

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that is etiologically linked to several cancers and has been connected to multiple sclerosis. EBV persists in its human hosts through a biphasic replication cycle that depends on three latency stages. Understanding the host mechanisms that contribute to the tight regulation of viral gene expression in the discrete states of latency III, latency II, and latency I is important for developing therapeutic approaches for treating EBV positive cancers. Regulation of the chromatin structure of the EBV genome by cohesin and CTCF plays a role in this latency type switching. However, the function of the cohesin release factor WAPL had not previously been understood. In this study, we employ RNA-seq combined with immunofluorescence analysis to determine that the cohesin release factor WAPL plays a role specifically in inhibiting the expression of the oncogenic latent membrane proteins LMP-1 and LMP-2A. Through a combination of Hi-ChIP and ChIP-qPCR, we uncover that WAPL loss alters the looping interactions within the EBV genome and alters the histone modifications present at the LMP promoter. We propose that EBV coopts WAPL to maintain a latency I state and, without WAPL, leaky expression of the LMP proteins occurs. Overall design: Hi-ChIP was performed as previously descibed. WAPL was knocked out in MUTU I cells via CRISPR-Cas9 or an sgControl guide RNA was used. ChIP for H3K27Ac was performed.

爱泼斯坦-巴尔病毒（Epstein-Barr virus, EBV）是一种广泛分布的人类病原体，与多种癌症存在明确的病因学关联，同时也与多发性硬化症相关。EBV通过依赖三种潜伏阶段的双相复制周期在人体宿主中持续存在。阐明在潜伏III型、潜伏II型与潜伏I型这几种离散潜伏状态中，严格调控病毒基因表达的宿主机制，对于开发EBV阳性癌症的治疗策略具有重要意义。黏连蛋白（cohesin）与CCCTC结合因子（CTCF）对EBV基因组染色质结构的调控，在潜伏类型转换过程中发挥关键作用，但此前人们对黏连蛋白释放因子WAPL的功能尚未明确。本研究结合RNA测序（RNA-seq）与免疫荧光分析，证实黏连蛋白释放因子WAPL可特异性抑制致瘤性潜伏膜蛋白LMP-1和LMP-2A的表达。通过联合运用Hi-ChIP与染色质免疫沉淀定量PCR（ChIP-qPCR）技术，本研究发现WAPL缺失会改变EBV基因组内的染色质环相互作用，并改变LMP启动子区域的组蛋白修饰。本研究提出，EBV会劫持WAPL以维持潜伏I型状态；当缺乏WAPL时，LMP蛋白会出现渗漏性表达。整体实验设计：Hi-ChIP实验按照此前已发表的方法开展。本研究通过CRISPR-Cas9技术在MUTU I细胞中敲除WAPL，同时设置sgRNA对照（sgControl）组。此外还开展了针对H3K27乙酰化（H3K27Ac）的染色质免疫沉淀实验。