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Coordination of KSHV latent and lytic gene control by CTCF-cohesin mediated chromosome conformation.

ABSTRACT: Herpesvirus persistence requires a dynamic balance between latent and lytic cycle gene expression, but how this balance is maintained remains enigmatic. We have previously shown that the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) major latency transcripts encoding LANA, vCyclin, vFLIP, v-miRNAs, and Kaposin are regulated, in part, by a chromatin organizing element that binds CTCF and cohesins. Using viral genome-wide chromatin conformation capture (3C) methods, we now show that KSHV latency control region is physically linked to the promoter regulatory region for ORF50, which encodes the KSHV immediate early protein RTA. Other linkages were also observed, including an interaction between the 5' and 3' end of the latency transcription cluster. Mutation of the CTCF-cohesin binding site reduced or eliminated the chromatin conformation linkages, and deregulated viral transcription and genome copy number control. siRNA depletion of CTCF or cohesin subunits also disrupted chromosomal linkages and deregulated viral latent and lytic gene transcription. Furthermore, the linkage between the latent and lytic control region was subject to cell cycle fluctuation and disrupted during lytic cycle reactivation, suggesting that these interactions are dynamic and regulatory. Our findings indicate that KSHV genomes are organized into chromatin loops mediated by CTCF and cohesin interactions, and that these inter-chromosomal linkages coordinate latent and lytic gene control.

SUBMITTER: Kang H

PROVIDER: S-EPMC3158054 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Coordination of KSHV latent and lytic gene control by CTCF-cohesin mediated chromosome conformation.

Kang Hyojeung H Wiedmer Andreas A Yuan Yan Y Robertson Erle E Lieberman Paul M PM

PLoS pathogens 20110818 8

Herpesvirus persistence requires a dynamic balance between latent and lytic cycle gene expression, but how this balance is maintained remains enigmatic. We have previously shown that the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) major latency transcripts encoding LANA, vCyclin, vFLIP, v-miRNAs, and Kaposin are regulated, in part, by a chromatin organizing element that binds CTCF and cohesins. Using viral genome-wide chromatin conformation capture (3C) methods, we now show that KSHV latency ...[more]

PMID: 21876668

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Project description:Epigenetic modifications of the herpesviral genome play a key role in the transcriptional control of latent and lytic genes during a productive viral lifecycle. In this study, we describe for the first time a comprehensive genome-wide ChIP-on-Chip analysis of the chromatin associated with the Kaposi's sarcoma-associated herpesvirus (KSHV) genome during latency and lytic reactivation. Depending on the gene expression class, different combinations of activating [acetylated H3 (AcH3) and H3K4me3] and repressive [H3K9me3 and H3K27me3] histone modifications are associated with the viral latent genome, which changes upon reactivation in a manner that is correlated with their expression. Specifically, both the activating marks co-localize on the KSHV latent genome, as do the repressive marks. However, the activating and repressive histone modifications are mutually exclusive of each other on the bulk of the latent KSHV genome. The genomic region encoding the IE genes ORF50 and ORF48 possesses the features of a bivalent chromatin structure characterized by the concomitant presence of the activating H3K4me3 and the repressive H3K27me3 marks during latency, which rapidly changes upon reactivation with increasing AcH3 and H3K4me3 marks and decreasing H3K27me3. Furthermore, EZH2, the H3K27me3 histone methyltransferase of the Polycomb group proteins (PcG), colocalizes with the H3K27me3 mark on the entire KSHV genome during latency, whereas RTA-mediated reactivation induces EZH2 dissociation from the genomic regions encoding IE and E genes concurrent with decreasing H3K27me3 level and increasing IE/E lytic gene expression. Moreover, either the inhibition of EZH2 expression by a small molecule inhibitor DZNep and RNAi knockdown, or the expression of H3K27me3-specific histone demethylases apparently induced the KSHV lytic gene expression cascade. These data indicate that histone modifications associated with the KSHV latent genome are involved in the regulation of latency and ultimately in the control of the temporal and sequential expression of the lytic gene cascade. In addition, the PcG proteins play a critical role in the control of KSHV latency by maintaining a reversible heterochromatin on the KSHV lytic genes. Thus, the regulation of the spatial and temporal association of the PcG proteins with the KSHV genome may be crucial for propagating the KSHV lifecycle.

Project description:CTCF and the cohesin complex modify chromatin by binding to DNA and interacting with each other and with other cellular proteins. Both proteins regulate transcription by a variety of local effects on transcription and by long-range topological effects. CTCF and cohesin also bind to herpesvirus genomes at specific sites and regulate viral transcription during latent and lytic cycles of replication. Kaposi's sarcoma-associated herpesvirus (KSHV) transcription is regulated by CTCF and cohesin, with both proteins previously reported to act as restrictive factors for lytic cycle transcription and virion production. In this study, we examined the interdependence of CTCF and cohesin binding to the KSHV genome. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that cohesin binding to the KSHV genome is highly CTCF dependent, whereas CTCF binding does not require cohesin. Furthermore, depletion of CTCF leads to the almost complete dissociation of cohesin from sites at which they colocalize. Thus, previous studies that examined the effects of CTCF depletion actually represent the concomitant depletion of both CTCF and cohesin components. Analysis of the effects of single and combined depletion indicates that CTCF primarily activates KSHV lytic transcription, whereas cohesin has primarily inhibitory effects. Furthermore, CTCF or cohesin depletion was found to have regulatory effects on cellular gene expression relevant for the control of viral infection, with both proteins potentially facilitating the expression of multiple genes important in the innate immune response to viruses. Thus, CTCF and cohesin have both positive and negative effects on KSHV lytic replication as well as effects on the host cell that enhance antiviral defenses.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to Kaposi's sarcoma and several lymphoproliferative diseases. KSHV, like other herpesviruses, intermittently reactivates from latency and enters a lytic cycle in which numerous lytic mRNAs and proteins are produced, culminating in infectious virion production. These lytic proteins may also contribute to tumorigenesis. Reactivation from latency is controlled by processes that restrict or activate the transcription of KSHV lytic genes. KSHV gene expression is modulated by binding of the host cell proteins CTCF and cohesin complex to the KSHV genome. These proteins bind to and modulate the conformation of chromatin, thereby regulating transcription. We have analyzed the interdependence of binding of CTCF and cohesin and demonstrate that while CTCF is required for cohesin binding to KSHV, they have very distinct effects, with cohesin primarily restricting KSHV lytic transcription. Furthermore, we show that cohesin and CTCF also exert effects on the host cell that promote antiviral defenses.

Dataset Information

Coordination of KSHV latent and lytic gene control by CTCF-cohesin mediated chromosome conformation.

Publications

Coordination of KSHV latent and lytic gene control by CTCF-cohesin mediated chromosome conformation.

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