A comparative epigenome analysis of gammaherpesviruses suggests cis-acting sequence features as critical mediators of rapid polycomb recruitment

Abstract

Latent Kaposi sarcoma-associated herpesvirus (KSHV) genomes rapidly acquire distinct patterns of the activating histone modification H3K4-me3 as well as repressive H3K27-me3 marks, a modification linked to transcriptional silencing by polycomb repressive complexes (PRC). Interestingly, PRCs have recently been reported to restrict viral gene expression in a number of other viral systems, suggesting they may play a broader role in controlling viral chromatin. If so, it is an intriguing possibility that latency establishment may result from viral subversion of polycomb-mediated host responses to exogenous DNA.To investigate such scenarios we sought to establish whether rapid repression by PRC constitutes a general hallmark of herpesvirus latency. For this purpose, we performed a comparative epigenome analysis of KSHV and the related murine gammaherpesvirus 68 (MHV-68). We demonstrate that, while latently replicating MHV-68 genomes readily acquire distinct patterns of activation-associated histone modifications upon de novo infection, they fundamentally differ in their ability to efficiently attract H3K27-me3 marks. Statistical analyses of ChIP-seq data from in vitro infected cells as well as in vivo latency reservoirs furthermore suggest that, whereas KSHV rapidly attracts PRCs in a genome-wide manner, H3K27-me3 acquisition by MHV-68 genomes may require spreading from initial seed sites to which PRC are recruited as the result of an inefficient or stochastic recruitment, and that immune pressure may be needed to select for latency pools harboring PRC-silenced episomes in vivo.Using co-infection experiments and recombinant viruses, we also show that KSHV’s ability to rapidly and efficiently acquire H3K27-me3 marks does not depend on the host cell environment or unique properties of the KSHV-encoded LANA protein, but rather requires specific cis-acting sequence features. We show that the non-canonical PRC1.1 component KDM2B, a factor which binds to unmethylated CpG motifs, is efficiently recruited to KSHV genomes, indicating that CpG island characteristics may constitute these features. In accord with the fact that, compared to MHV-68, KSHV genomes exhibit a fundamentally higher density of CpG motifs, we furthermore demonstrate efficient acquisition of H2AK119-ub by KSHV and H3K36-me2 by MHV-68 (but not vice versa), furthermore supporting the notion that KSHV genomes rapidly attract PRC1.1 complexes in a genome-wide fashion. Collectively, our results suggest that rapid PRC silencing is not a universal feature of viral latency, but that some viruses may rather have adopted distinct genomic features to specifically exploit default host pathways that repress epigenetically naive, CpG-rich DNA.

Epigenetic control in Kaposi sarcoma-associated herpesvirus infection and associated disease

“…This is of interest since KSHV genomes are very CpG rich molecules that enter the nucleus in a completely unmethylated state. Indeed, the entire KSHV genome effectively resembles one contiguous CpG island [98]. In accordance with this, we could recently demonstrate that KDM2B, a PRC component that directly binds to non-methylated CpGs [110,111,113], associates with KSHV genomes very early during infection.…”

“…In accordance with this, we could recently demonstrate that KDM2B, a PRC component that directly binds to non-methylated CpGs [110,111,113], associates with KSHV genomes very early during infection. In contrast, a related herpesvirus with a substantially lower CpG density did not show evidence of KDM2B binding and consequently did also not acquire repressive H3K27-me3 marks [98]. In addition to these cis-acting sequence features, there is also evidence that latent KSHV gene products favor PRC recruitment.…”

“…When considering the pathogenesis of KSHV-associated tumors (see Fig. 1a for a model of KS), epigenetic control is of interest in the context of viral as well as cellular gene regulation: Firstly, epigenetic mechanisms have been shown to control the viral latency program [94][95][96][97][98]. How the latent viral epigenome itself is established and regulated and to what extend alterations in its profile are directly responsible for the adoption of alternate latency programs are areas of active research.…”

“…Instead, viral episomes rapidly and globally acquire H3K27-me3 marks early in infection [95]. The enrichment of H3K27-me3 is highly significant, and overall patterns are very similar when compared between PEL cells and various in vitro infected cell lines or primary cells [94][95][96][97][98]. Only a few loci (including the major latency promoter upstream of the LANA-coding region) escape PRC-mediated silencing and instead maintain distinct peaks of activating histone marks such as H3K4-me3 [95,96].…”

“…How are PRCs attracted to KSHV episomes and to what extend is this process controlled by the virus? Our own recent data suggest that the composition of the viral genome sequence itself favors rapid silencing by PRCs [98]. There are two major forms of polycomb repressive complexes, PRC1 and PRC2.…”