Studies on Gene Enhancer with KSHV mini-chromatin

Abstract

Kaposis sarcoma-associated herpesvirus (KSHV) genome contains a terminal repeats (TR) sequence. Previous studies demonstrated that KSHV TR functions as a gene enhancer for inducible lytic gene promoters. Gene enhancers anchor bromodomain-containing protein 4 (BRD4) at specific genomic region, where BRD4 interacts flexibly with transcription-related proteins through its intrinsically disordered domain and exerts transcription regulatory function. Here, we generated recombinant KSHV with reduced TR copy numbers and studied BRD4 recruitment and its contributions to the inducible promoter activation. Reducing the TR copy numbers from 21 (TR21) to 5 (TR5) strongly attenuated viral gene expression during de novo infection and impaired reactivation. The EF1 promoter encoded in the KSHV BAC backbone also showed reduced promoter activity, suggesting a global attenuation of transcription activity within TR5 latent episomes. Isolation of reactivating cells confirmed that the reduced inducible gene transcription from TR-shortened DNA template and is mediated by decreased efficacies of BRD4 recruitment to viral gene promoters. Separating the reactivating iSLK cell population from non-responders showed that reactivatable iSLK cells harbored larger LANA nuclear bodies (NBs) compared to non-responders. The cells with larger LANA NBs, either due to prior transcription activation or TR copy number, supported KSHV reactivation more efficiently than those with smaller LANA NBs. With auxin-inducible LANA degradation, we confirmed that LANA is responsible for BRD4 occupancies on latent chromatin. Finally, with purified fluorescence-tagged proteins, we demonstrated that BRD4 is required for LANA to form liquid-liquid phase-separated dots. The inclusion of TR DNA fragments further facilitated the formation of larger BRD4-containing LLPS in the presence of LANA, similar to the "cellular enhancer dot" formed by transcription factor-DNA bindings. These results suggest that LANA binding to TR establishes an enhancer domain for infected KSHV episomes. The strength of this enhancer, regulated by TR length or transcription memory, determines the outcome of KSHV replication. ImportanceGene enhancers are genomic domains that regulate frequency and duration of transcription burst at gene promoters, with BRD4 playing a critical role in their enhancer functions. KSHV latent mini-chromosome also contains an enhancer domain made with multiple copies of 801 bp identical repeat DNA fragments, terminal repeats. Here, we utilized manipulable mini-scale chromatins with convenient inducible KSHV reactivation to systematically examine the association between enhancer strength and the outcome of inducible promoter activation. This study illustrated the amount of BRD4 recruitment at the enhancer associated with frequencies of BRD4 distribution to the inducible promoters during KSHV reactivation and, therefore, KSHV lytic replication. Recruitment of BRD4 to the TR is specifically regulated by KSHV latent protein, LANA. KSHV evolves clever enhancer elements designed to be regulated by the KSHV own latent protein, LANA.