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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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Genome-wide binding of EZH2 to the KSHV genome correlates with the repression of lytic genes.(A) ChIP-on-chip was performed for EZH2 and its genome-wide binding was compared with the distribution of H3K27me3 on the KSHV genome during both latency and reactivation. Labels are the same as in Figure 1. The H3K27me3 graph was taken from Figure 1. (B) EZH2 and SUZ12 binding to the H3K27me3-rich lytic promoters are shown by independent ChIP assays. EZH2-interacting PcG protein SUZ12 is enriched only where EZH2 is present. (C) Recruitment of transcription activators to the activated lytic promoters during KSHV reactivation. An anti-RNA polymerase II antibody (H-224) that recognizes the RNAPII independently from its phosphorylation state (total RNAPII) was used for ChIP of total RNAPII, while the anti-RNA polymerase II antibody CTD4H8 specifically immunoprecipitates RNAPII phosphorylated at the 5th serine of its C-terminal domain (RNAPII Ser5).
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ppat-1001013-g004: Genome-wide binding of EZH2 to the KSHV genome correlates with the repression of lytic genes.(A) ChIP-on-chip was performed for EZH2 and its genome-wide binding was compared with the distribution of H3K27me3 on the KSHV genome during both latency and reactivation. Labels are the same as in Figure 1. The H3K27me3 graph was taken from Figure 1. (B) EZH2 and SUZ12 binding to the H3K27me3-rich lytic promoters are shown by independent ChIP assays. EZH2-interacting PcG protein SUZ12 is enriched only where EZH2 is present. (C) Recruitment of transcription activators to the activated lytic promoters during KSHV reactivation. An anti-RNA polymerase II antibody (H-224) that recognizes the RNAPII independently from its phosphorylation state (total RNAPII) was used for ChIP of total RNAPII, while the anti-RNA polymerase II antibody CTD4H8 specifically immunoprecipitates RNAPII phosphorylated at the 5th serine of its C-terminal domain (RNAPII Ser5).

Mentions: Because of the genome-wide distribution of H3K27me3 on the KSHV genome, we performed a ChIP-on-chip assay to determine whether EZH2, the H3K27me3 histone methyltransferase of the PcG proteins, associates with the KSHV genome. (Figure 4A). ChIP-on-chip assays showed that EZH2 almost completely colocalized with H3K27me3 throughout the entire KSHV genome during both latency and reactivation (Figure 4A, S7, S10). Additional ChIP assays showed that EZH2 and SUZ12 (another subunit of the PcG complex PRC2) were found on the RTA and ORF25 promoters enriched with H3K27me3, but not on the LANA promoter (Figure 4B).


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)

Genome-wide binding of EZH2 to the KSHV genome correlates with the repression of lytic genes.(A) ChIP-on-chip was performed for EZH2 and its genome-wide binding was compared with the distribution of H3K27me3 on the KSHV genome during both latency and reactivation. Labels are the same as in Figure 1. The H3K27me3 graph was taken from Figure 1. (B) EZH2 and SUZ12 binding to the H3K27me3-rich lytic promoters are shown by independent ChIP assays. EZH2-interacting PcG protein SUZ12 is enriched only where EZH2 is present. (C) Recruitment of transcription activators to the activated lytic promoters during KSHV reactivation. An anti-RNA polymerase II antibody (H-224) that recognizes the RNAPII independently from its phosphorylation state (total RNAPII) was used for ChIP of total RNAPII, while the anti-RNA polymerase II antibody CTD4H8 specifically immunoprecipitates RNAPII phosphorylated at the 5th serine of its C-terminal domain (RNAPII Ser5).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908616&req=5

ppat-1001013-g004: Genome-wide binding of EZH2 to the KSHV genome correlates with the repression of lytic genes.(A) ChIP-on-chip was performed for EZH2 and its genome-wide binding was compared with the distribution of H3K27me3 on the KSHV genome during both latency and reactivation. Labels are the same as in Figure 1. The H3K27me3 graph was taken from Figure 1. (B) EZH2 and SUZ12 binding to the H3K27me3-rich lytic promoters are shown by independent ChIP assays. EZH2-interacting PcG protein SUZ12 is enriched only where EZH2 is present. (C) Recruitment of transcription activators to the activated lytic promoters during KSHV reactivation. An anti-RNA polymerase II antibody (H-224) that recognizes the RNAPII independently from its phosphorylation state (total RNAPII) was used for ChIP of total RNAPII, while the anti-RNA polymerase II antibody CTD4H8 specifically immunoprecipitates RNAPII phosphorylated at the 5th serine of its C-terminal domain (RNAPII Ser5).
Mentions: Because of the genome-wide distribution of H3K27me3 on the KSHV genome, we performed a ChIP-on-chip assay to determine whether EZH2, the H3K27me3 histone methyltransferase of the PcG proteins, associates with the KSHV genome. (Figure 4A). ChIP-on-chip assays showed that EZH2 almost completely colocalized with H3K27me3 throughout the entire KSHV genome during both latency and reactivation (Figure 4A, S7, S10). Additional ChIP assays showed that EZH2 and SUZ12 (another subunit of the PcG complex PRC2) were found on the RTA and ORF25 promoters enriched with H3K27me3, but not on the LANA promoter (Figure 4B).

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