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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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Polycomb group proteins are involved in the maintenance of latency of KSHV.(A) Overexpression of the wild-type HA-tagged H3K27me3 histone methyltransferases (HMTs) UTX and JMJD3 triggers the lytic reactivation of KSHV in Vero-rKSHV.219 as shown with the expression of RFP. In contrast, the H3K9me3 HMT JMJD2A and the enzymatically inactive UTXm showed little or no effect on KSHV lytic reactivation. (B) Quantification of RFP positive cells. (C and D) JSC-1 cells were treated with 5 uM DZNep for 1, 2 and 3 days and the cells were harvested for immunoblot analysis with the indicated specific antibodies against cellular proteins and histone modifications (C) or viral proteins (D). “Dpt” indicates days post-treatment. Whole cell lysate of NaB-treated JSC-1 cells was used as a control for Zta immunoblot. (E) JSC-1 cells were treated with DZNep as described in (C) and total RNAs were isolated for RT-qPCR analysis of some selected KSHV, EBV and cellular mRNAs. (F and G) BCBL-1 cells were infected by lentivirus expressing the indicated shRNAs and were then subject to immunoblotting analysis with the indicated antibodies (F) or RT-qPCR analysis was performed for the indicated viral transcripts (G).
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ppat-1001013-g005: Polycomb group proteins are involved in the maintenance of latency of KSHV.(A) Overexpression of the wild-type HA-tagged H3K27me3 histone methyltransferases (HMTs) UTX and JMJD3 triggers the lytic reactivation of KSHV in Vero-rKSHV.219 as shown with the expression of RFP. In contrast, the H3K9me3 HMT JMJD2A and the enzymatically inactive UTXm showed little or no effect on KSHV lytic reactivation. (B) Quantification of RFP positive cells. (C and D) JSC-1 cells were treated with 5 uM DZNep for 1, 2 and 3 days and the cells were harvested for immunoblot analysis with the indicated specific antibodies against cellular proteins and histone modifications (C) or viral proteins (D). “Dpt” indicates days post-treatment. Whole cell lysate of NaB-treated JSC-1 cells was used as a control for Zta immunoblot. (E) JSC-1 cells were treated with DZNep as described in (C) and total RNAs were isolated for RT-qPCR analysis of some selected KSHV, EBV and cellular mRNAs. (F and G) BCBL-1 cells were infected by lentivirus expressing the indicated shRNAs and were then subject to immunoblotting analysis with the indicated antibodies (F) or RT-qPCR analysis was performed for the indicated viral transcripts (G).

Mentions: The dynamic association of EZH2 with KSHV genome suggests that PcG-mediated H3K27me3 histone modification is involved in the repression of lytic gene expression during latency. To address this issue, HA-tagged JMJD2A, JMJD3 and UTX histone demethylases were expressed in Vero-rKSHV.219 cells to test if the elimination of histone methylations can trigger KSHV reactivation (Figure 5A). JMJD2A is an H3K9me3-specific histone demethylase [50], JMJD3 and UTX are H3K27me3-specific histone demethylases [21], [51], and UTXmut is an enzymatically inactive form of UTX that contains a single point mutation (H1146A) in the Fe2++ ion binding site [51]. JMJD3 and UTX have been shown to eradicate the H3K27me3 repressive mark, resulting in the upregulation of PcG-targeted gene expression [21], [52]. JMJD2A and JMJD3 or UTX expression detectably suppressed the steady-state levels of H3K9me3 or H3K27me3, respectively, in transfected Vero cells (Figure S12B, C). Since Vero-rKSHV.219 cells express red fluorescent protein (RFP) from the KSHV lytic PAN promoter and green fluorescent protein (GFP) from the EF-1α promoter, RFP expression has been extensively used as a marker of KSHV lytic reactivation [53]. Immunofluorescence analysis revealed that JMJD3 and UTX efficiently triggered KSHV reactivation, while JMJD2A and UTXmut did not (Figure 5A, B). Furthermore, coexpression of JMJD2A and JMJD3 showed no significant synergistic effect on KSHV reactivation (Figure 5A, B). Finally, JMJD2A, JMJD3, UTX and UTXmut were expressed at comparable levels (Figure S12A). These results bespeak the importance of the H3K27me3 histone modification in the maintenance of KSHV latency.


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)

Polycomb group proteins are involved in the maintenance of latency of KSHV.(A) Overexpression of the wild-type HA-tagged H3K27me3 histone methyltransferases (HMTs) UTX and JMJD3 triggers the lytic reactivation of KSHV in Vero-rKSHV.219 as shown with the expression of RFP. In contrast, the H3K9me3 HMT JMJD2A and the enzymatically inactive UTXm showed little or no effect on KSHV lytic reactivation. (B) Quantification of RFP positive cells. (C and D) JSC-1 cells were treated with 5 uM DZNep for 1, 2 and 3 days and the cells were harvested for immunoblot analysis with the indicated specific antibodies against cellular proteins and histone modifications (C) or viral proteins (D). “Dpt” indicates days post-treatment. Whole cell lysate of NaB-treated JSC-1 cells was used as a control for Zta immunoblot. (E) JSC-1 cells were treated with DZNep as described in (C) and total RNAs were isolated for RT-qPCR analysis of some selected KSHV, EBV and cellular mRNAs. (F and G) BCBL-1 cells were infected by lentivirus expressing the indicated shRNAs and were then subject to immunoblotting analysis with the indicated antibodies (F) or RT-qPCR analysis was performed for the indicated viral transcripts (G).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001013-g005: Polycomb group proteins are involved in the maintenance of latency of KSHV.(A) Overexpression of the wild-type HA-tagged H3K27me3 histone methyltransferases (HMTs) UTX and JMJD3 triggers the lytic reactivation of KSHV in Vero-rKSHV.219 as shown with the expression of RFP. In contrast, the H3K9me3 HMT JMJD2A and the enzymatically inactive UTXm showed little or no effect on KSHV lytic reactivation. (B) Quantification of RFP positive cells. (C and D) JSC-1 cells were treated with 5 uM DZNep for 1, 2 and 3 days and the cells were harvested for immunoblot analysis with the indicated specific antibodies against cellular proteins and histone modifications (C) or viral proteins (D). “Dpt” indicates days post-treatment. Whole cell lysate of NaB-treated JSC-1 cells was used as a control for Zta immunoblot. (E) JSC-1 cells were treated with DZNep as described in (C) and total RNAs were isolated for RT-qPCR analysis of some selected KSHV, EBV and cellular mRNAs. (F and G) BCBL-1 cells were infected by lentivirus expressing the indicated shRNAs and were then subject to immunoblotting analysis with the indicated antibodies (F) or RT-qPCR analysis was performed for the indicated viral transcripts (G).
Mentions: The dynamic association of EZH2 with KSHV genome suggests that PcG-mediated H3K27me3 histone modification is involved in the repression of lytic gene expression during latency. To address this issue, HA-tagged JMJD2A, JMJD3 and UTX histone demethylases were expressed in Vero-rKSHV.219 cells to test if the elimination of histone methylations can trigger KSHV reactivation (Figure 5A). JMJD2A is an H3K9me3-specific histone demethylase [50], JMJD3 and UTX are H3K27me3-specific histone demethylases [21], [51], and UTXmut is an enzymatically inactive form of UTX that contains a single point mutation (H1146A) in the Fe2++ ion binding site [51]. JMJD3 and UTX have been shown to eradicate the H3K27me3 repressive mark, resulting in the upregulation of PcG-targeted gene expression [21], [52]. JMJD2A and JMJD3 or UTX expression detectably suppressed the steady-state levels of H3K9me3 or H3K27me3, respectively, in transfected Vero cells (Figure S12B, C). Since Vero-rKSHV.219 cells express red fluorescent protein (RFP) from the KSHV lytic PAN promoter and green fluorescent protein (GFP) from the EF-1α promoter, RFP expression has been extensively used as a marker of KSHV lytic reactivation [53]. Immunofluorescence analysis revealed that JMJD3 and UTX efficiently triggered KSHV reactivation, while JMJD2A and UTXmut did not (Figure 5A, B). Furthermore, coexpression of JMJD2A and JMJD3 showed no significant synergistic effect on KSHV reactivation (Figure 5A, B). Finally, JMJD2A, JMJD3, UTX and UTXmut were expressed at comparable levels (Figure S12A). These results bespeak the importance of the H3K27me3 histone modification in the maintenance of KSHV latency.

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