KSHV chromatin looping facilitates effective gene expression: high-resolution mapping of long-range chromatin interactions in the KSHV genome [Hi-C]

The three-dimensional structure of chromatin via formation of genomic loops allows cellular RNA polymerase II to access distal promoters, thus it has a significant impact on the outcome of gene expression. The Kaposi’s sarcoma-associated herpesvirus (KSHV) entire lytic transcriptional program of 80 plus genes is initiated by a single viral transactivator, K-Rta. Here we examined the relationship between the KSHV genomic structure and K-Rta recruitment sites with unbiased Capture Hi-C analyses. The studies showed that KSHV forms a unique genomic structure, which coordinates a domain-specific viral gene expression program via K-Rta recruitment, and identified a number of direct physical, long-range, and dynamic genomic interactions. Chromatin immunoprecipitation-sequencing analyses identified a limited number of K-Rta recruitment sites in the KSHV genome, including the K12 and PAN RNA promoters. KSHV engineered to harbor point mutations in the K-Rta-responsive elements (RE) at these promoters significantly attenuated not only the direct downstream gene, but also distal viral gene expression in a domain-specific manner. Despite the long distance between two RE sites in the linear orientation, efficient recruitment of K-Rta to either promoter required intact K-Rta REs at both promoters. Finally, inducible genomic loops generated during KSHV reactivation occurred preferentially at K-Rta recruitment sites. Mutation of a K-Rta RE inhibited formation of inducible genomic loops, gene expression of its destination of looping genes, and KSHV replication in de novo infected human gingival epithelial cells. These results suggest that the KSHV genome is programed to form both stable and inducible chromatin hubs for effective KSHV gene expression, and partly explains why K-Rta has such a dominant effect on KSHV lytic gene transcription. The overall goal of these studies was to define the long-range chromatin interactions occurring within the KSHV genome as well as between the KSHV and human host cell genomes both during latency and viral reactivation. These were defined in an unbiased manner using Hi-C analysis, which is chromosome conformation capture (3C) combined with next-generation sequencing (NGS). A modified protocol, referred to as Capture Hi-C, was utilized to selectively enrich interactions involving KSHV by performing target enrichment with a custom-designed KSHV genomic capture probe library. Experiments were performed in two cell lines, TREx-(F3H3)-K-Rta BCBL-1 and iSLK.r219 cells, in which KSHV can be reactivated via doxyclyine-induced expression of K-Rta. Accordingly, Capture Hi-C sequencing libraries were prepared from replicate cultures of each cell line cultured in the absence (i.e., latency) or presence (i.e., reactivation) of doxycycline for 24 hours.

染色质通过基因组环折叠形成的三维构象，可使细胞RNA聚合酶II（RNA polymerase II）结合远端启动子，进而对基因表达的最终结果产生显著影响。卡波西肉瘤相关疱疹病毒（Kaposi’s sarcoma-associated herpesvirus, KSHV）拥有一套包含80余个基因的完整裂解期转录程序，该程序仅由单一病毒反式激活因子K-Rta启动。本研究通过无偏的捕获Hi-C（Capture Hi-C）分析，探究了KSHV基因组构象与K-Rta招募位点之间的关联。研究结果显示，KSHV可形成独特的基因组构象，该构象通过K-Rta的招募协调域特异性的病毒基因表达程序，并鉴定出了一批直接物理性、长距离且动态的基因组相互作用。染色质免疫沉淀测序（Chromatin immunoprecipitation-sequencing, ChIP-seq）分析在KSHV基因组中鉴定出了少量K-Rta招募位点，其中包括K12与PAN RNA启动子。针对上述启动子处的K-Rta应答元件（K-Rta-responsive elements, RE）引入点突变的工程化KSHV，不仅会显著下调其直接下游基因的表达，还会以域特异性的方式抑制远端病毒基因的表达。尽管两个RE位点在线性基因组上相距较远，但要让K-Rta有效结合任一启动子，必须保证两个启动子处的K-Rta RE均保持完整。此外，KSHV复活过程中产生的诱导性基因组环，优先富集于K-Rta招募位点区域。K-Rta RE的突变会抑制诱导性基因组环的形成、其环化靶基因的表达，以及初次感染的人牙龈上皮细胞内的KSHV复制。上述结果表明，KSHV基因组被编程形成稳定与诱导性兼具的染色质枢纽（chromatin hub），以保障高效的KSHV基因表达，这也部分解释了为何K-Rta对KSHV裂解期基因转录具有如此显著的调控作用。本研究的整体目标是，明确潜伏感染与病毒复活阶段中，KSHV基因组内部以及KSHV与人类宿主细胞基因组之间的长距离染色质相互作用。本研究采用无偏的Hi-C分析方法完成该目标——Hi-C是将染色体构象捕获（chromosome conformation capture, 3C）与下一代测序（NGS）相结合的技术。本研究采用了一种被称为捕获Hi-C（Capture Hi-C）的改良实验方案，通过定制化设计的KSHV基因组捕获探针文库进行靶标富集，从而选择性富集涉及KSHV的染色质相互作用。实验在两种细胞系中开展：TREx-(F3H3)-K-Rta BCBL-1与iSLK.r219细胞，这两种细胞系均可通过强力霉素（doxycycline）诱导K-Rta的表达，进而实现KSHV的复活。据此，我们分别收集了每种细胞系在不含强力霉素（即潜伏感染状态）或含强力霉素培养24小时（即病毒复活状态）下的重复培养物，以此制备捕获Hi-C测序文库。