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Dynamic chromatin boundaries delineate a latency control region of Epstein-Barr virus.

ABSTRACT: The oncogenic potential of latent Epstein-Barr virus (EBV) can be regulated by epigenetic factors controlling LMP1 and EBNA2 gene transcription. The EBV latency control region (LCR) constitutes approximately 12 kb of viral sequence spanning the divergent promoters of LMP1 and EBNA2 and encompasses the EBV latent replication origin OriP and RNA polymerase III-transcribed EBV-encoded RNA genes. We have used the chromatin immunoprecipitation assay to examine the chromatin architecture of the LCR in different types of EBV latency programs. We have found that histone H3 K4 methylation (H3mK4) was enriched throughout a large domain that extended from internal repeat 1 (IR1) to the terminal repeat in type III latency where EBNA2 and LMP1 genes are expressed. In type I latency where EBNA2 and LMP1 genes are transcriptionally silent, the H3mK4 domain contracts and does not enter the EBNA2 or LMP1 promoters. In contrast, histone H3 K9 methylation (H3mK9), associated with silent heterochromatin, was enriched in the EBNA2 and LMP1 upstream control regions in type I but not type III cells. MTA [5'-deoxy-5'(methylthio)adenosine], a pharmacological inhibitor of protein methylation, globally reduced histone H3mK4 and inhibited EBNA2 transcription in type III cells. 5'-Azacytidine, an inhibitor of DNA methylation that derepresses EBNA2 transcription in type I latency, caused H3mK4 expansion and a corresponding loss of H3mK9 at IR1. The chromatin boundary protein and transcription repressor CCCTC-binding factor was enriched at the EBNA2 transcription control region in type I but not type III cells. We also present evidence that OriP binding factors EBNA1 and ORC2 can interact with sequences outside of OriP including a region within IR1 that may influence EBNA2 transcription status. These results indicate that types I and III latency programs have distinct histone methylation patterns in the LCR and suggest that chromatin architecture coordinates gene expression of LMP1 and EBNA2.

SUBMITTER: Chau CM

PROVIDER: S-EPMC525066 | biostudies-literature | 2004 Nov

REPOSITORIES: biostudies-literature

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Dynamic chromatin boundaries delineate a latency control region of Epstein-Barr virus.

Chau Charles M CM Lieberman Paul M PM

Journal of virology 20041101 22

The oncogenic potential of latent Epstein-Barr virus (EBV) can be regulated by epigenetic factors controlling LMP1 and EBNA2 gene transcription. The EBV latency control region (LCR) constitutes approximately 12 kb of viral sequence spanning the divergent promoters of LMP1 and EBNA2 and encompasses the EBV latent replication origin OriP and RNA polymerase III-transcribed EBV-encoded RNA genes. We have used the chromatin immunoprecipitation assay to examine the chromatin architecture of the LCR in ...[more]

PMID: 15507618

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Project description:Epstein Barr virus (EBV) is a potentially oncogenic gammaherpesvirus that establishes a chronic, latent infection in memory B cells. The EBV genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type. CTCF is posttranslationally modified by the host enzyme PARP1. PARP1, or poly(ADP-ribose) polymerase 1, catalyzes the transfer of a poly(ADP-ribose) (PAR) moiety from NAD+ onto acceptor proteins, including itself, histone proteins, and CTCF. PARylation of CTCF by PARP1 can affect CTCF's insulator activity, DNA binding capacity, and ability to form chromatin loops. Both PARP1 and CTCF have been implicated in the regulation of EBV latency and lytic reactivation. Thus, we predicted that pharmacological inhibition with PARP1 inhibitors would affect EBV latency type through a chromatin-specific mechanism. Here, we show that PARP1 and CTCF colocalize at specific sites throughout the EBV genome and provide evidence to suggest that PARP1 acts to stabilize CTCF binding and maintain the open chromatin landscape at the active Cp promoter during type III latency. Further, PARP1 activity is important in maintaining latency type-specific viral gene expression. The data presented here provide a rationale for the use of PARP inhibitors in the treatment of EBV-associated cancers exhibiting type III latency and ultimately could contribute to an EBV-specific treatment strategy for AIDS-related or posttransplant lymphomas.IMPORTANCE EBV is a human gammaherpesvirus that infects more than 95% of individuals worldwide. Upon infection, EBV circularizes as an episome and establishes a chronic, latent infection in B cells. In doing so, the virus utilizes host cell machinery to regulate and maintain the viral genome. In otherwise healthy individuals, EBV infection is typically nonpathological; however, latent infection is potentially oncogenic and is responsible for 1% of human cancers. During latent infection, EBV expresses specific sets of proteins according to the given latency type, each of which is associated with specific types of cancers. For example, type III latency, in which the virus expresses its full repertoire of latent proteins, is characteristic of AIDS-associated and posttransplant lymphomas associated with EBV infection. Understanding how viral latency type is regulated at the chromatin level may reveal potential targets for EBV-specific pharmacological intervention in EBV-associated cancers.

Project description:Deciphering the molecular pathogenesis of virally induced cancers is challenging due, in part, to the heterogeneity of both viral gene expression and host gene expression. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus prevalent in B-cell lymphomas of immune-suppressed individuals. EBV infection of primary human B cells leads to their immortalization into lymphoblastoid cell lines (LCLs), serving as a model of these lymphomas. In previous studies, reports from our laboratory have described a temporal model for immortalization with an initial phase characterized by expression of Epstein-Barr nuclear antigens (EBNAs), high levels of c-Myc activity, and hyperproliferation in the absence of the latent membrane proteins (LMPs), called latency IIb. This is followed by the long-term outgrowth of LCLs expressing the EBNAs along with the LMPs, particularly NFκB-activating LMP1, defining latency III. However, LCLs express a broad distribution of LMP1 such that a subset of these cells express LMP1 at levels similar to those seen in latency IIb, making it difficult to distinguish these two latency states. In this study, we performed mRNA sequencing (mRNA-Seq) on early EBV-infected latency IIb cells and latency III LCLs sorted by NFκB activity. We found that latency IIb transcriptomes clustered independently from latency III independently of NFκB. We identified and validated mRNAs defining these latency states. Indeed, we were able to distinguish latency IIb cells from LCLs expressing low levels of LMP1 using multiplex RNA-fluorescence in situ hybridization (RNA-FISH) targeting EBV EBNA2 or LMP1 and human CCR7 or MGST1 This report defines latency IIb as a bona fide latency state independent from latency III and identifies biomarkers for understanding EBV-associated tumor heterogeneity.IMPORTANCE EBV is a ubiquitous pathogen, with >95% of adults harboring a life-long latent infection in memory B cells. In immunocompromised individuals, latent EBV infection can result in lymphoma. The established expression profile of these lymphomas is latency III, which includes expression of all latency genes. However, single-cell analysis of EBV latent gene expression in these lymphomas suggests heterogeneity where most cells express the transcription factor, EBNA2, and only a fraction of the cells express membrane protein LMP1. Our work describes an early phase after infection where the EBNAs are expressed without LMP1, called latency IIb. However, LMP1 levels within latency III vary widely, making these states hard to discriminate. This may have important implications for therapeutic responses. It is crucial to distinguish these states to understand the molecular pathogenesis of these lymphomas. Ultimately, better tools to understand the heterogeneity of these cancers will support more-efficacious therapies in the future.

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Dynamic chromatin boundaries delineate a latency control region of Epstein-Barr virus.

Publications

Dynamic chromatin boundaries delineate a latency control region of Epstein-Barr virus.

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