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Histone demethylase JMJD2A regulates Kaposi's sarcoma-associated herpesvirus replication and is targeted by a viral transcriptional factor.

ABSTRACT: The switch between the latency and lytic cycles of Kaposi's sarcoma-associated herpesvirus (KSHV) is accompanied by specific alterations of histone codes. Recently, comprehensive analysis of histone modifications of KSHV showed the deposition of H3K27me3 across the KSHV genome with two specific regions occupied by the heterochromatin marker H3K9me3. Here, we show that knockdown of JMJD2A, an H3K9me3 demethylase, attenuates viral titers, whereas its overexpression increases KSHV reactivation. JMJD2A is localized in regions of latent viral chromosomes that are deficient in the H3K9me3 mark, indicating that JMJD2A may be responsible for the low level of this mark on viral chromatin. The presence of JMJD2A on the latent genome maintains H3K9 in unmethylated form and signals the readiness of specific sets of viral genes to be reactivated. The demethylase activity of JMJD2A is important for KSHV reactivation, because a demethylase-deficient mutant cannot restore the JMJD2A knockdown phenotype. Interestingly, we found that the KSHV encoded K-bZIP associated with JMJD2A, resulting in the inhibition of demethylase activity of JMJD2A both in vivo and in vitro. Inhibition of JMJD2A by K-bZIP is likely due to a physical interaction which blocks substrate accessibility. A consequence of such an inhibition is increasing global levels of H3K9me3 and gene silencing. Consistently, K-bZIP overexpression resulted in a repression of ∼80% of the ≥2-fold differentially regulated genes compared to results for the uninduced control cells. The consequences of K-bZIP targeting JMJD2A during viral replication will be discussed. To our knowledge, this is the first description of a viral product shown to be a potent inhibitor of a host cellular histone demethylase.

SUBMITTER: Chang PC

PROVIDER: S-EPMC3067885 | biostudies-other | 2011 Apr

REPOSITORIES: biostudies-other

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Histone demethylase JMJD2A regulates Kaposi's sarcoma-associated herpesvirus replication and is targeted by a viral transcriptional factor.

Chang Pei-Ching PC Fitzgerald Latricia D LD Hsia Datsun A DA Izumiya Yoshihiro Y Wu Chun-Yi CY Hsieh Wen-Ping WP Lin Su-Fang SF Campbell Mel M Lam Kit S KS Luciw Paul A PA Tepper Clifford G CG Kung Hsing-Jien HJ

Journal of virology 20110112 7

The switch between the latency and lytic cycles of Kaposi's sarcoma-associated herpesvirus (KSHV) is accompanied by specific alterations of histone codes. Recently, comprehensive analysis of histone modifications of KSHV showed the deposition of H3K27me3 across the KSHV genome with two specific regions occupied by the heterochromatin marker H3K9me3. Here, we show that knockdown of JMJD2A, an H3K9me3 demethylase, attenuates viral titers, whereas its overexpression increases KSHV reactivation. JMJ ...[more]

PMID: 21228229

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Project description:The Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, open reading frame (ORF) K9 encodes a viral interferon regulatory factor (vIRF) that functions as a repressor for interferon-mediated signal transduction. Consequently, this gene is thought to play an important role in the tumorigenicity of KSHV. To understand the molecular mechanisms underlying vIRF expression, we studied the transcriptional regulation of this gene. Experiments using 5' rapid amplification of cDNA ends and primer extension revealed that vIRF had different transcriptional patterns during the latent and lytic phases. The promoter region of the minor transcript, which was mainly expressed in uninduced BCBL-1 cells, did not contain a canonical TATA box, but a cap-like element and an initiator element flanked the transcription start site. The promoter of the major transcript, which was mainly expressed in tetradecanoyl phorbol acetate-induced BCBL-1 cells, contained a canonical TATA box. A luciferase reporter assay using a deletion mutant of the vIRF promoter and a mutation in the TATA box showed that the TATA box was critical for the lytic activity of vIRF. The promoter activity in the latent phase was eight times stronger than that of the empty vector but was less than 10% of the activity in the lytic phase. Therefore, KSHV may use different functional promoter elements to regulate the expression of vIRF and to antagonize the cell's interferon-mediated antiviral activity. We have also identified a functional domain in the ORF 50 protein, an immediate-early gene product that is mainly encoded by ORF 50. The ORF 50 protein transactivated the vIRF and DNA polymerase promoters in BCBL-1, 293T, and CV-1 cells. Deleting one of its two putative nuclear localization signals (NLSs) resulted in failure of the ORF 50 protein to localize to the nucleus and consequently abrogated its transactivating activity. We further confirmed that the N-terminal region of the ORF 50 protein included an NLS domain. We found that this domain was sufficient to translocate beta-galactosidase to the nucleus. Analysis of deletions within the vIRF promoter suggested that two sequence domains were important for its transactivation by the ORF 50 protein, both of which included putative SP-1 and AP-1 binding sites. Competition gel shift assays demonstrated that SP-1 bound to these two domains, suggesting that the SP-1 binding sites in the vIRF promoter are involved in its transactivation by ORF 50.

Project description:Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional "factories," which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an "all-in-one" factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression.

Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. The virus-encoded bZIP family protein K8 plays an important role in viral DNA replication in both viral reactivation and de novo infection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here, we report that K8 is an RNA binding protein that also associates with many other proteins, including other RNA binding proteins. Many protein-protein interactions involving K8 are mediated by RNA. Using a UV cross-linking and immunoprecipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, including both viral (PAN, T1.4, T0.7, etc.) and cellular (MALAT-1, MRP, 7SK, etc.) RNAs. An RNA binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 to bind to many noncoding RNAs, as well as viral DNA replication during de novo infection, suggesting that the K8 functions in viral replication are carried out through RNA association. The functions of K8 and associated T1.4 RNA were investigated in detail, and the results showed that T1.4 mediates the binding of K8 to ori-Lyt DNA. The T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble a replication complex. Depletion of T1.4 abolished DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication during de novo infection.IMPORTANCE Genomewide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding regions is actually transcribed and gives rise to stable RNAs. The emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes, e.g., ribosomes or spliceosomes, are not ancient relics of the last ribo-organism but would be well adapted to a regulatory role in biology. K8 has been puzzling because of its unique characteristics, such as multiple regulatory roles in gene expression and DNA replication without DNA binding capability. This study reveals the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiates DNA replication in coordination with a noncoding RNA. It is suggested that many K8 functions, if not all, are carried out through its associated RNAs.

Dataset Information

Histone demethylase JMJD2A regulates Kaposi's sarcoma-associated herpesvirus replication and is targeted by a viral transcriptional factor.

Publications

Histone demethylase JMJD2A regulates Kaposi's sarcoma-associated herpesvirus replication and is targeted by a viral transcriptional factor.

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