Research Article: BET-Inhibitors Disrupt Rad21-Dependent Conformational Control of KSHV Latency

Date Published: January 20, 2017

Publisher: Public Library of Science

Author(s): Horng-Shen Chen, Alessandra De Leo, Zhuo Wang, Andrew Kerekovic, Robert Hills, Paul M. Lieberman, Dirk P. Dittmer.

http://doi.org/10.1371/journal.ppat.1006100

Abstract

Kaposi’s Sarcoma-associated Herpesvirus (KSHV) establishes stable latent infection in B-lymphocytes and pleural effusion lymphomas (PELs). During latency, the viral genome persists as an epigenetically constrained episome with restricted gene expression programs. To identify epigenetic regulators of KSHV latency, we screened a focused small molecule library containing known inhibitors of epigenetic factors. We identified JQ1, a Bromodomain and Extended Terminal (BET) protein inhibitor, as a potent activator of KSHV lytic reactivation from B-cells carrying episomal KSHV. We validated that JQ1 and other BET inhibitors efficiently stimulated reactivation of KSHV from latently infected PEL cells. We found that BET proteins BRD2 and BRD4 localize to several regions of the viral genome, including the LANA binding sites within the terminal repeats (TR), as well as at CTCF-cohesin sites in the latent and lytic control regions. JQ1 did not disrupt the interaction of BRD4 or BRD2 with LANA, but did reduce the binding of LANA with KSHV TR. We have previously demonstrated a cohesin-dependent DNA-loop interaction between the latent and lytic control regions that restrict expression of ORF50/RTA and ORF45 immediate early gene transcripts. JQ1 reduced binding of cohesin subunit Rad21 with the CTCF binding sites in the latency and lytic control regions. JQ1 also reduced DNA-loop interaction between latent and lytic control regions. These findings implicate BET proteins BRD2 and BRD4 in the maintenance of KSHV chromatin architecture during latency and reveal BET inhibitors as potent activators of KSHV reactivation from latency.

Partial Text

Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is a human gammaherpesvirus responsible for all forms of Kaposi’s Sarcoma (KS) and strongly associated with pleural effusion lymphomas (PELs) and Castleman’s Disease[1]. KSHV can establish long-term latent infection in B-lymphocytes where it persists as a stable, chromatin-associated circular minichromosome, commonly referred to as an episome [2, 3]. During latent infection, the viral genome expresses only a few viral genes required for maintaining the latent state and host-cell survival [4, 5]. The major latency transcripts include the multi-cistronic RNAs encoding LANA (ORF73), vCyclin (ORF72), vFLIP (ORF71), K1, and 21 miRNAs. The major immediate early genes are also regulated as a cluster of RNAs that can be initiated during the early stage of the reactivation process. These include the immediate early transcriptional activator RTA (ORF50), KbZip (ORF51), and a series of transcripts that are made in the opposite orientation that include ORF45-49. Lytic transcription is repressed during latency, while latency transcription occurs efficiently. How these regions are differentially regulated and how they communicate with each other remains an area of active interest.

Pharmacogenomics is a valuable tool for understanding biological process and pathways affected by small molecules and candidate pharmacological agents. Here, we have screened a focus library of small molecules with known inhibitory activities directed towards cellular epigenetic regulators and assayed these for their ability to stimulate KSHV lytic cycle gene expression in latently infected B-lymphoma cells. We found that bromodomain inhibitors, including JQ1, were among the more potent activators of KSHV lytic cycle gene expression. JQ1 was found to induce KSHV lytic cycle transcription, as well as DNA replication, in several different PEL cell lines. We investigated the mechanism of action of JQ1, focusing on the well-characterized JQ1 target proteins BRD2 and BRD4. BRD2 and BRD4 were found to interact with KSHV episomes at latency control regions, including the LANA binding sites in TR, and CTCF-cohesin sites at the latency and lytic control regions. Depletion of BRD2 or BRD4 partially phenocopied JQ1 activation of KSHV lytic transcription. JQ1 reduced binding of LANA to the TR and latency control region, but did not destabilize the interaction of BRD4 or BRD2 with LANA protein. JQ1 reduced RAD21 binding and disrupted a DNA loop interaction between the latent and lytic control regions. Taken together, these findings suggest that BRD2 and BRD4 contribute to maintaining the KSHV latent state, including a RAD21-dependent chromosome conformation important for KSHV latency control (Fig 9).

 

Source:

http://doi.org/10.1371/journal.ppat.1006100

 

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