Date Published: July 17, 2014
Publisher: Public Library of Science
Author(s): Jianhong Hu, Yajie Yang, Peter C. Turner, Vaibhav Jain, Lauren M. McIntyre, Rolf Renne, Dirk P. Dittmer.
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus associated with KS and two lymphoproliferative diseases. Recent studies characterized epigenetic modification of KSHV episomes during latency and determined that latency-associated genes are associated with H3K4me3 while most lytic genes are associated with the silencing mark H3K27me3. Since the latency-associated nuclear antigen (LANA) (i) is expressed very early after de novo infection, (ii) interacts with transcriptional regulators and chromatin remodelers, and (iii) regulates the LANA and RTA promoters, we hypothesized that LANA may contribute to the establishment of latency through epigenetic control. We performed a detailed ChIP-seq analysis in cells of lymphoid and endothelial origin and compared H3K4me3, H3K27me3, polII, and LANA occupancy. On viral episomes LANA binding was detected at numerous lytic and latent promoters, which were transactivated by LANA using reporter assays. LANA binding was highly enriched at H3K4me3 peaks and this co-occupancy was also detected on many host gene promoters. Bioinformatic analysis of enriched LANA binding sites in combination with biochemical binding studies revealed three distinct binding patterns. A small subset of LANA binding sites showed sequence homology to the characterized LBS1/2 sequence in the viral terminal repeat. A large number of sites contained a novel LANA binding motif (TCCAT)3 which was confirmed by gel shift analysis. Third, some viral and cellular promoters did not contain LANA binding sites and are likely enriched through protein/protein interaction. LANA was associated with H3K4me3 marks and in PEL cells 86% of all LANA bound promoters were transcriptionally active, leading to the hypothesis that LANA interacts with the machinery that methylates H3K4. Co-immunoprecipitation demonstrated LANA association with endogenous hSET1 complexes in both lymphoid and endothelial cells suggesting that LANA may contribute to the epigenetic profile of KSHV episomes.
Eukaryotic DNA is packaged into chromatin which plays a central role in the regulation of all DNA processes including replication, transcription, and repair. Chromatin contains nucleosomes with DNA wrapped around the core histones H2A, H2B, H3, and H4. Nucleosomes carry epigenetic information in the form of post-translational histone modifications. N-terminal histone modifications including acetylation, methylation, phosphorylation, and sumoylation are important in partitioning chromatin into transcriptionally active or repressive domains (reviewed in ). In mammalian cells, genome-wide ChIP-seq assays revealed that histone acetylation at H3K9 and H3K4 trimethylation (H3K4me3) correlate with active transcription, while H3K27 trimethylation (H3K27me3) is detected in promoters of repressed genes . The apparently opposite modifications H3K4me3 and H3K27me3 co-localize at some promoters (“bivalent marks”), poising these genes to be transcribed upon signaling. Histone modifications are also detected in regions outside promoters. All three states of H3K4 methylation are highly enriched at insulator sites, while only H3K4me and H3K4me3 are associated with enhancers , .
Previous reports by several independent groups demonstrated the importance of H3K27me3 deposition for both the establishment and maintenance of KSHV latency , , . Decreases of H3K27me3 by either overexpression of JMJD3, the H3K27 demethylase, or by blocking with small molecule inhibitors the H3K27 methyltransferase EZH2, disrupt latency and induce KSHV reactivation. Very recently, Toth et al. demonstrated that the deposition of H3K27me3 follows after an initial phase of lytic gene expression associated with H3K4me3 deposition . Hence, KSHV latency and reactivation seem to be largely controlled by a balance between H3K27me3 and H3K4me3 deposition to specific genomic regions. Recently, LANA was demonstrated to interact with and recruit KDM3A, which demethylates H3K9me1/2, a mark associated with heterochromatin. While inhibiting KDM3A affected the extent of lytic replication after induction, overexpression of KDM3A, unlike JMJD3, did not induce reactivation . However, very little is known about the potential mechanisms by which viral proteins prevent PRC2 complexes from silencing KSHV after de novo infection or during long-term latency.