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PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type.

ABSTRACT: 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.

SUBMITTER: Lupey-Green LN

PROVIDER: S-EPMC6146685 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type.

Lupey-Green Lena N LN Caruso Lisa B LB Madzo Jozef J Martin Kayla A KA Tan Yinfei Y Hulse Michael M Tempera Italo I

Journal of virology 20180829 18

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 transf ...[more]

PMID: 29976663

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Project description: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.

Project description:Epstein-Barr virus (EBV) is a human herpesvirus associated with various tumors. Rather than going through the lytic cycle, EBV maintains latency by limiting the expression of viral genes in tumors. Viral microRNAs (miRNAs) of some herpesviruses have been reported to directly target immediate early genes and suppress lytic induction. In this study, we investigated whether BamHI-A rightward transcript (BART) miRNAs targeted two EBV immediate early genes, BZLF1 and BRLF1. Bioinformatic analysis predicted that 12 different BART miRNAs would target BRLF1. Of these, the results of a luciferase reporter assay indicated that only one interacted with the 3' untranslated region (UTR) of BRLF1: miR-BART20-5p. miR-BART20-5p's effect on gene expression involved two putative seed match sites in the BRLF1 3' UTR, but a mutant version of the miRNA, miR-BART20-5pm, had no effect on expression. As expected from the fact that the entire 3' UTR of BZLF1 resides within the 3' UTR of BRLF1, miR-BART20-5p interacted with the 3' UTR of BZLF1 as well. BZLF1 and BRLF1 mRNA and protein expression was suppressed in cells of an AGS cell line infected with the recombinant Akata strain of EBV (AGS-EBV) transfected with a miR-BART20-5p mimic. The expression of various EBV early proteins was also suppressed by the miR-BART20-5p mimic. In contrast, BZLF1 and BRLF1 expression in AGS-EBV cells transfected with a miR-BART20-5p inhibitor was enhanced. Furthermore, progeny virus production was suppressed by the miR-BART20-5p mimic and enhanced by the miR-BART20-5p inhibitor in AGS-EBV cells induced for the lytic cycle. Our data suggest that miR-BART20-5p plays a key role in latency maintenance in EBV-associated tumors by directly targeting immediate early genes.Herpesviruses maintain latency using various mechanisms and establish lifelong infection in the host. From time to time, herpesviruses are reactivated and express immediate early genes which trigger a lytic cascade, leading to the production of progeny viruses. Recently, some herpesviruses have been shown to use their own microRNAs (miRNAs) to downregulate immediate early genes to inhibit the lytic cycle. This study presents evidence that EBV also downregulates two immediate early genes by miR-BART20-5p to suppress the lytic cycle and progeny virus production. Overall, this is the first study to report the direct regulation of EBV immediate early genes by an EBV miRNA, implying its likely importance in latency maintenance in EBV-associated tumors.

Project description:Epstein-Barr virus (EBV) establishes life-long latency in human B-cells by maintaining its chromatinized episomes within the nucleus. These circularized mini-chromosomes do not integrate into the host genome. Therefore, it is essential for EBV to organize its chromatin in a manner suitable for genomic stability, DNA replication, and efficient gene expression. Poly [ADP-ribose] polymerase 1 (PARP1) activity is significantly higher in B-cells infected with EBV than those without, and considerably higher in the transcriptionally active type III latency compared to the immunoevasive type I. In addition to its role in DNA damage response, PARP1 has been implicated in transcriptional regulation and structural maintenance of both the human and EBV genome at specific regions. To better understand PARP1's role in the regulation of the EBV episome, we have functionally characterized the effect of PARP enzymatic inhibition on total episomal structure through in situ Hi-C mapping, generating the first complete 3D structure of the EBV genome. We have also mapped intragenomic contact changes after PARP inhibition to global binding of the chromatin looping factors CTCF and cohesin across the EBV genome. Additionally, PARP inhibition was shown to alter gene expression at the regions where chromatin looping was most effected. Finally, we have identified a novel function of PARP, which regulates cohesin complex chromatin binding. In conclusion, PARP1 inhibition does not alter the location of cohesin binding but does increase its frequency of binding at these regions. Despite this, there are fewer overall unique intragenomic interactions after PARP inhibition, while some areas have new chromatin loops not seen in the untreated EBV episome, leading us to conclude that PARP does have an essential role in the regulation of global EBV chromatin structure. The altered expression profile after the structural rearrangement induced by PARP inhibition also supports the idea that PARP1 helps maintain EBV latency programs.

Dataset Information

PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type.

Publications

PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type.

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