KSHV chromatin looping facilitates effective gene expression: high-resolution mapping of K-Rta binding sites in the KSHV genome [ChIP-seq]. Homo sapiens

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. Overall design: The goal of these studies was to perform genome-wide mapping of K-Rta binding sites across the KSHV genome using ChIP-Sequencing (ChIP-Seq). This was performed in the context of the BCBL-1 cell line model, which is derived from a KSHV-infected human primary effusion lymphoma (PEL) and contains latent KSHV genomes. For these studies, we utilized an engineered subline, referred to as TREx-K-Rta BCBL-1, which contains a Tetracycline/Doxycycline (Dox)-inducible Flagx3- and HAx3-tagged K-Rta expression cassette. K-Rta expression was induced in individual TREx-K-Rta BCBL-1 cultures by treatment with Dox for 24 hours. Following treatment, the cells were then processed for ChIP-Seq analyses by formaldehyde cross-linking, chromatin solubilization, and chromatin immunoprecipitation (ChIP) with anti-FLAG M2 monoclonal antibody. Subsequently, DNA was isolated from the ChIP and total chromatin (Input) samples, and followed by library preparation and next-generation sequencing (NGS).