THE THREE-DIMENSIONAL STRUCTURE OF THE EPSTEIN-BARR VIRUS GENOME VARIES BY LATENCY TYPE AND IS REGULATED BY PARP1 ENZYMATIC ACTIVITY

Abstract

Epstein-Barr virus (EBV) establishes life-long latency in human B-cells by maintaining its chromatinized episomes within the nucleus. These circularized mini-chromosomes do not integrate into the host genome. Therefore, it is essential for EBV to organize its chromatin in a manner suitable for genomic stability, DNA replication, and efficient gene expression. Poly [ADP-ribose] polymerase 1 (PARP1) activity is significantly higher in B-cells infected with EBV than those without, and considerably higher in the transcriptionally active type III latency compared to the immunoevasive type I. In addition to its role in DNA damage response, PARP1 has been implicated in transcriptional regulation and structural maintenance of both the human and EBV genome at specific regions. To better understand PARP1's role in the regulation of the EBV episome, we have functionally characterized the effect of PARP enzymatic inhibition on total episomal structure through in situ Hi-C mapping, generating the first complete 3D structure of the EBV genome. We have also mapped intragenomic contact changes after PARP inhibition to global binding of the chromatin looping factors CTCF and cohesin across the EBV genome. In doing so, we found that PARP inhibition stabilizes cohesin-chromatin binding while decreasing CTCF binding. PARP inhibition resulted in widespread alterations to chromatin structure, with fewer overall unique intragenomic interactions. Despite this, some areas have new chromatin loops not seen in the untreated EBV episome. Additionally, PARP inhibition was shown to alter gene expression at the regions where chromatin looping was most affected. The altered expression profile after the structural rearrangement induced by PARP inhibition also supports the idea that PARP1 helps maintain EBV latency programs, as many of the significantly upregulated genes were indicative of lytic reactivation. The data presented herein allows us to conclude that PARP1 does have an essential role in the regulation of global EBV chromatin structure. And furthermore, it provides the first study showing that PARP enzymatic activity alone can regulate 3D chromatin structure on a global scale.