KSHV Topologically Associating Domains in Latent Chromatin and Regulation of Reactivation

Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 base pair resolution and constructed a 3D KSHV genomic structural model. The latent KSHV genome formed very similar TAD structures among three different naturally infected PEL cell lines. When KSHV reactivation was triggered, genomic loops within TADs were dramatically decreased, while contacts extending outside of TAD borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D structural model proposes that the immediate-early promoter region is localized on the periphery of the 3D viral genome, while highly inducible non-coding RNA regions moved toward the inner space of the structure, resembling the coordination of a "bird cage" during reactivation. Finally, inhibition of the initial burst of lytic gene expression by stop codon insertion in the viral transactivator reduced genomic loops, while supplementing K-Rta expression in trans during establishment of latency attenuated the defect. Our studies suggest that the latent 3D genomic structural information is embedded in the lytic gene transcription program.

真核基因组通过形成多个环结构实现组织化，这些环构成了基因表达调控单元——拓扑关联结构域（topologically associating domains, TADs）。本研究揭示了分辨率达500碱基对的卡波西肉瘤相关疱疹病毒（KSHV，Kaposi's sarcoma-associated herpesvirus）的TAD结构，并构建了KSHV基因组的三维结构模型。在3株不同的自然感染原发性渗出性淋巴瘤（PEL，Primary Effusion Lymphoma）细胞系中，潜伏态KSHV基因组形成了高度相似的TAD结构。当KSHV再激活被触发时，TAD内部的基因组环显著减少，而跨越TAD边界的染色质互作水平上升，进而形成更大的调控单元，同时染色质区室从抑制型（B区室）向激活型（A区室）发生转变。三维结构模型显示，即刻早期启动子区域定位于病毒三维基因组的外周区域，而高诱导性非编码RNA区域则向结构内部迁移，这一过程类似于再激活过程中“鸟笼”结构的协同重排。最终，通过在病毒反式激活因子中插入终止密码子以阻断裂解性基因表达的初始爆发，可减少基因组环的形成；而在潜伏感染建立阶段通过反式补充K-Rta的表达，则可减轻该缺陷。本研究表明，潜伏态的三维基因组结构信息嵌入于裂解性基因的转录程序之中。