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Epigenetic analysis of KSHV latent and lytic genomes.

Toth Z, Maglinte DT, Lee SH, Lee HR, Wong LY, Brulois KF, Lee S, Buckley JD, Laird PW, Marquez VE, Jung JU - PLoS Pathog. (2010)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
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.

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Dynamic association of histone modifications with viral genes during latency and reactivation.(A) Time-course ChIP analysis of histone modifications on the RTA promoter during latency and reactivation. Cellular controls can be seen in panel D. (B) Colocalization of H3K4me3 and H3K27me3 on the RTA promoter is confirmed by sequential ChIP assays. The first ChIP was performed with either H3K4me3-specific or H3K27me3-specific antibody, followed by the elution of the immunoprecipitated DNAs and a second ChIP with either H3K27me3 or H3K4me3 antibody. LANA and ORF25 promoters were used as controls. (C) Time-course ChIP analysis of histone modifications on the selected latent (LANA), early (K2, ORF56) and late (ORFs 8, 25, 64) genes. Cellular controls can be found in panel D. Pr: promoter, in: within gene body. (D) ChIP assays of histone modifications on cellular promoters. The promoters of the repressed cellular MYT1 and HTF6 genes as well as the active promoter of the actin (ACT) gene were also tested using the same ChIP samples that had been used in panels A and C. ND: not detectable.
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ppat-1001013-g003: Dynamic association of histone modifications with viral genes during latency and reactivation.(A) Time-course ChIP analysis of histone modifications on the RTA promoter during latency and reactivation. Cellular controls can be seen in panel D. (B) Colocalization of H3K4me3 and H3K27me3 on the RTA promoter is confirmed by sequential ChIP assays. The first ChIP was performed with either H3K4me3-specific or H3K27me3-specific antibody, followed by the elution of the immunoprecipitated DNAs and a second ChIP with either H3K27me3 or H3K4me3 antibody. LANA and ORF25 promoters were used as controls. (C) Time-course ChIP analysis of histone modifications on the selected latent (LANA), early (K2, ORF56) and late (ORFs 8, 25, 64) genes. Cellular controls can be found in panel D. Pr: promoter, in: within gene body. (D) ChIP assays of histone modifications on cellular promoters. The promoters of the repressed cellular MYT1 and HTF6 genes as well as the active promoter of the actin (ACT) gene were also tested using the same ChIP samples that had been used in panels A and C. ND: not detectable.

Mentions: By definition, IE genes are the first set of expressed viral genes whose induction does not require de novo expression of any viral proteins and can be rapidly induced upon reactivation of KSHV from latency. We found that although the IE genes (K4.2, K8, ORF45, ORF48, ORF50/RTA) are silenced during latency, their 2-kb regulatory regions are enriched in the activating histone modifications H3K4me3 and AcH3, and this is further increased upon lytic reactivation at ORFs 48, 50 and K8 (Figure 2, S8, S9). Since RTA is responsible for the switch between latency and lytic replication, not only is its promoter tightly repressed during latency, but its silencing should also be rapidly reversible upon reactivation. We found that the RTA promoter is enriched in both H3K4me3 and H3K27me3 during latency, suggesting that it possesses a bivalent chromatin that maintains the repression of the RTA promoter while keeping it poised for rapid activation (Figure 2, S8). An extensive ChIP analysis further confirmed the enrichment of AcH3, H3K4me3 and H3K27me3 and the depletion of H3K9me3 on the 2.5-kb promoter region of RTA during latency (Figure 3A). Upon lytic reactivation, however, the repressive H3K27me3 gradually decreased, while the activating histone modifications H3K4me3 and AcH3 increased on the RTA promoter (Figure 3A). These changes in the histone modification pattern are in concert with the induction of RTA expression.


Epigenetic analysis of KSHV latent and lytic genomes.

Toth Z, Maglinte DT, Lee SH, Lee HR, Wong LY, Brulois KF, Lee S, Buckley JD, Laird PW, Marquez VE, Jung JU - PLoS Pathog. (2010)

Dynamic association of histone modifications with viral genes during latency and reactivation.(A) Time-course ChIP analysis of histone modifications on the RTA promoter during latency and reactivation. Cellular controls can be seen in panel D. (B) Colocalization of H3K4me3 and H3K27me3 on the RTA promoter is confirmed by sequential ChIP assays. The first ChIP was performed with either H3K4me3-specific or H3K27me3-specific antibody, followed by the elution of the immunoprecipitated DNAs and a second ChIP with either H3K27me3 or H3K4me3 antibody. LANA and ORF25 promoters were used as controls. (C) Time-course ChIP analysis of histone modifications on the selected latent (LANA), early (K2, ORF56) and late (ORFs 8, 25, 64) genes. Cellular controls can be found in panel D. Pr: promoter, in: within gene body. (D) ChIP assays of histone modifications on cellular promoters. The promoters of the repressed cellular MYT1 and HTF6 genes as well as the active promoter of the actin (ACT) gene were also tested using the same ChIP samples that had been used in panels A and C. ND: not detectable.
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Related In: Results  -  Collection

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ppat-1001013-g003: Dynamic association of histone modifications with viral genes during latency and reactivation.(A) Time-course ChIP analysis of histone modifications on the RTA promoter during latency and reactivation. Cellular controls can be seen in panel D. (B) Colocalization of H3K4me3 and H3K27me3 on the RTA promoter is confirmed by sequential ChIP assays. The first ChIP was performed with either H3K4me3-specific or H3K27me3-specific antibody, followed by the elution of the immunoprecipitated DNAs and a second ChIP with either H3K27me3 or H3K4me3 antibody. LANA and ORF25 promoters were used as controls. (C) Time-course ChIP analysis of histone modifications on the selected latent (LANA), early (K2, ORF56) and late (ORFs 8, 25, 64) genes. Cellular controls can be found in panel D. Pr: promoter, in: within gene body. (D) ChIP assays of histone modifications on cellular promoters. The promoters of the repressed cellular MYT1 and HTF6 genes as well as the active promoter of the actin (ACT) gene were also tested using the same ChIP samples that had been used in panels A and C. ND: not detectable.
Mentions: By definition, IE genes are the first set of expressed viral genes whose induction does not require de novo expression of any viral proteins and can be rapidly induced upon reactivation of KSHV from latency. We found that although the IE genes (K4.2, K8, ORF45, ORF48, ORF50/RTA) are silenced during latency, their 2-kb regulatory regions are enriched in the activating histone modifications H3K4me3 and AcH3, and this is further increased upon lytic reactivation at ORFs 48, 50 and K8 (Figure 2, S8, S9). Since RTA is responsible for the switch between latency and lytic replication, not only is its promoter tightly repressed during latency, but its silencing should also be rapidly reversible upon reactivation. We found that the RTA promoter is enriched in both H3K4me3 and H3K27me3 during latency, suggesting that it possesses a bivalent chromatin that maintains the repression of the RTA promoter while keeping it poised for rapid activation (Figure 2, S8). An extensive ChIP analysis further confirmed the enrichment of AcH3, H3K4me3 and H3K27me3 and the depletion of H3K9me3 on the 2.5-kb promoter region of RTA during latency (Figure 3A). Upon lytic reactivation, however, the repressive H3K27me3 gradually decreased, while the activating histone modifications H3K4me3 and AcH3 increased on the RTA promoter (Figure 3A). These changes in the histone modification pattern are in concert with the induction of RTA expression.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus