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Paul M Lieberman

Publications and source records attributed to Paul M Lieberman.

At least 19 recordsLinked to original sources
PubMed2025

USP7 Inhibitors Destabilize EBNA1 and Suppress Epstein-Barr Virus Tumorigenesis.

Epstein-Barr virus (EBV) is a ubiquitous human ɣ-herpesvirus implicated in various malignancies, including Burkitt's lymphoma and gastric carcinomas. In most EBV-associated cancers, the viral genome is maintained as an extrachromosomal episome by the EBV nuclear antigen-1 (EBNA1). EBNA1 is considered to be a highly stable protein that interacts with the ubiquitin-specific protease 7 (USP7). Here, we show that pharmacological inhibitors and small interfering RNA (siRNA) targeting USP7 reduce EBNA1 protein levels in a proteosome-dependent manner. Proteomic analysis revealed that USP7 inhibitor GNE6776 altered the EBNA1 protein interactome, including disrupting USP7 association with EBNA1. GNE6776 also inhibited EBNA1 binding to EBV oriP DNA and reduced viral episome copy number. Transcriptomic studies revealed that USP7 inhibition affected chromosome segregation and mitotic cell division pathways in EBV+ cells. Finally, we show that GNE6776 selectively inhibited EBV+ gastric and lymphoid cell proliferation in cell culture and slowed EBV+ tumor growth in mouse xenograft models. These findings suggest that USP7 inhibitors perturb EBNA1 stability and function and may be exploited to treat EBV latent infection and tumorigenesis.

Ubiquitin-Specific Peptidase 7↗
PubMed2006

Multivalent sequence recognition by Epstein-Barr virus Zta requires cysteine 171 and an extension of the canonical B-ZIP domain.

Epstein-Barr virus (EBV) immediate-early protein Zta is a member of the basic-leucine zipper (B-ZIP) family of DNA binding proteins that has an unusual capacity to recognize multiple DNA recognition sites, including AP-1 and C/EBP binding sites. To better understand the structure and function of Zta, we have mutagenized cysteine residues within or adjacent to the B-ZIP domain. We found that serine substitution for cysteine 171 (C171S), which lies outside and amino terminal to the B-ZIP basic region, completely abrogates Zta capacity to initiate lytic cycle replication. C171S disrupted Zta transcription activation function of several EBV lytic cycle promoters, including the BMRF1 gene (EA-D) and the other lytic activator, Rta. Overexpression of Rta could not rescue the C171S defect for transcription reactivation or viral DNA replication. Zta C171S was defective for binding to these promoters in vivo, as measured by chromatin immunoprecipitation assay. Purified Zta C171S bound AP-1 sites similar to wild-type Zta, but it was incapable of binding several degenerate Zta sites, including a consensus C/EBP site. Zta truncation mutations reveal that residues N terminal to the B-ZIP (amino acids 156 to 178) confer C/EBP binding capacity to the otherwise AP-1-restricted DNA recognition function. Comparison among viral orthologues of Zta suggest that a conserved N-terminal extension of the consensus B-ZIP domain is required for this multivalent DNA recognition capacity of Zta and is essential for viral reactivation.

Amino Acid Substitution↗
PubMed2006

ORC binding to TRF2 stimulates OriP replication.

In higher eukaryotes, the origin recognition complex (ORC) lacks sequence-specific DNA binding, and it remains unclear what other factors specify an origin of DNA replication. The Epstein-Barr virus origin of plasmid replication (OriP) recruits ORC, but the precise mechanism of ORC recruitment and origin activation is not clear. We now show that ORC is recruited selectively to the dyad symmetry (DS) region of OriP as a consequence of direct interactions with telomere repeat factor 2 (TRF2) and ORC1. TRF-binding sites within DS stimulate replication initiation and facilitate ORC recruitment in vitro and in vivo. TRF2, but not TRF1 or hRap1, recruits ORC from nuclear extracts. The amino-terminal domain of TRF2 associated with a specific region of ORC1 and was necessary for stimulation of DNA replication. These results support a model in which TRF2 stimulates OriP replication activity by direct binding with ORC subunits.

Cell Line↗
PubMed2006

Chromatin regulation of virus infection.

Cellular chromatin forms a dynamic structure that maintains the stability and accessibility of the host DNA genome. Viruses that enter and persist in the nucleus must, therefore, contend with the forces that drive chromatin formation and regulate chromatin structure. In some cases, cellular chromatin inhibits viral gene expression and replication by suppressing DNA accessibility. In other cases, cellular chromatin provides essential structure and organization to the viral genome and is necessary for successful completion of the viral life cycle. Consequently, viruses have acquired numerous mechanisms to manipulate cellular chromatin to ensure viral genome survival and propagation.

Chromatin↗
PubMed2006

Regulation of Epstein-Barr virus latency type by the chromatin boundary factor CTCF.

Epstein Barr virus (EBV) can establish distinct latency types with different growth-transforming properties. Type I latency and type III latency can be distinguished by the expression of EBNA2, which has been shown to be regulated, in part, by the EBNA1-dependent enhancer activity of the origin of replication (OriP). Here, we report that CTCF, a chromatin boundary factor with well-established enhancer-blocking activity, binds to EBV sequences between the OriP and the RBP-Jkappa response elements of the C promoter (Cp) and regulates transcription levels of EBNA2 mRNA. Using DNA affinity, electrophoretic mobility shift assay, DNase I footprinting, and chromatin immunoprecipitation (ChIP), we found that CTCF binds both in vitro and in vivo to the EBV genome between OriP and Cp, with an approximately 50-bp footprint at EBV coordinates 10515 to 10560. Deletion of this CTCF binding site in a recombinant EBV bacterial artificial chromosome (BAC) increased EBNA2 transcription by 3.5-fold compared to a wild-type EBV BAC. DNA affinity and ChIP showed more CTCF binding at this site in type I latency cell lines (MutuI and KemI) than in type III latency cell lines (LCL3456 and Raji). CTCF protein and mRNA expression levels were higher in type I than type III cell lines. Short interfering RNA depletion of CTCF in type I MutuI cells stimulated EBNA2 mRNA levels, while overexpression of CTCF in type III Raji cells inhibited EBNA2 mRNA levels. These results indicate that increased CTCF can repress EBNA2 transcription. We also show that c-MYC, as well as EBNA2, can stimulate CTCF mRNA levels, suggesting that CTCF levels may contribute to B-cell differentiation as well as EBV latency type determination.

Binding Sites↗
PubMed2006

Acetylation of the latency-associated nuclear antigen regulates repression of Kaposi's sarcoma-associated herpesvirus lytic transcription.

Reactivation of the Kaposi's sarcoma-associated herpesvirus (KSHV) lytic cycle can be initiated by transcription activation of the ORF50 immediate early gene (Rta). We show that ORF50 transcription is actively repressed by the KSHV latency-associated nuclear antigen (LANA) during latency. Depletion of LANA by small interfering RNA derepressed ORF50 transcription in the latently infected BCBL1 pleural effusion lymphoma-derived cell line. In contrast, overexpression of LANA suppressed ORF50 mRNA levels in BCBL1 cells. ORF50 transcription was significantly elevated during primary infection with recombinant virus lacking LANA, further indicating that LANA plays a role in lytic gene silencing during the establishment of latency. Chromatin immunoprecipitation assays indicated that LANA interacts with the ORF50 promoter region in latently infected cells. Histone deacetylase inhibitors, including sodium butyrate (NaB) and trichostatin A, caused the rapid dissociation of LANA from the ORF50 promoter. NaB treatment of latently infected BCBL1 cells disrupted a stable interaction between LANA and the cellular proteins Sp1 and histone H2B. We also found immunological and radiochemical evidence that LANA is subject to lysine acetylation after NaB treatment. These findings support the role of LANA as a transcriptional repressor of lytic reactivation and provide evidence that lysine acetylation regulates LANA interactions with chromatin, Sp1, and ORF50 promoter DNA.

Antigens, Viral↗
PubMed2005

Epigenetic control of replication origins.

Efficient duplication of the eukaryotic genome requires the spatial and temporal coordination of numerous replication origins on each chromosome. Epigenetic factors, like chromatin environment, can have profound effects on origin site selection, utilization frequency, and cell cycle firing time. Precisely how chromatin contributes to origin site selection and timing is not completely understood. Recently, we reported on the cell cycle changes in chromatin structure at the plasmid replication origins of Epstein-Barr Virus (EBV) and Kaposi's Sarcoma-Associated Herpes virus (KSHV). These studies and others suggest that cell cycle changes in histone modification and nucleosome remodeling regulate prereplication factor assembly and initiation of DNA replication at origins. We discuss how these studies of viral origins may provide important insights into epigenetic control of cellular chromosome origins.

Epigenesis, Genetic↗
PubMed2005

Cell cycle regulation of chromatin at an origin of DNA replication.

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.

Adenosine Triphosphatases↗
PubMed2005

Kaposi's sarcoma-associated herpesvirus virion-induced transcription activation of the ORF50 immediate-early promoter.

Lytic cycle reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) can be initiated by transcription activation of the ORF50 immediate-early (IE) gene promoter (ORF50p). We provide evidence that KSHV virions stimulate transcription of ORF50p. Virion activation was resistant to UV inactivation and cycloheximide treatment. The virion-responsive element was mapped to core promoter region -150 to + 1 relative to the ORF50 initiation codon. Electrophoretic mobility shift assays and chromatin immunoprecipitation suggest that KSHV virions indirectly alter the protein composition and chromatin modifications at ORF50p. These data suggest that KSHV virions possess an IE trans-inducing function similar to that observed in alpha- and betaherpesviruses.

Cell Line↗
PubMed2005

A redox-sensitive cysteine in Zta is required for Epstein-Barr virus lytic cycle DNA replication.

Epstein-Barr virus (EBV) reactivation from latency is known to be sensitive to redox regulation. The immediate-early protein Zta is a member of the basic-leucine zipper (bZIP) family of DNA binding proteins that stimulates viral and cellular transcription and nucleates a replication complex at the viral lytic origin. Zta shares with several members of the bZIP family a conserved cysteine residue (C189) that confers redox regulation of DNA binding. In this work, we show that replacement of C189 with serine (C189S) eliminated lytic cycle DNA replication function of Zta. The mechanistic basis for this replication defect was investigated. We show that C189S was not significantly altered for DNA binding activity in vitro or in vivo. We also show that C189S was not defective for transcription activation of EBV early gene promoters. C189S was deficient for transcription activation of several viral late genes that depend on lytic replication and therefore was consistent with a primary defect of C189S in activating lytic replication. C189S was not defective in binding methylated DNA binding sites and was capable of activating Rta from endogenous latent viral genomes, in contrast to the previously characterized S186A mutation. C189S was slightly impaired for its ability to form a stable complex with Rta, although this did not prevent Rta recruitment to OriLyt. C189S did provide some resistance to oxidation and nitrosylation, which potently inhibit Zta DNA binding activity in vitro. Interestingly, this redox sensitivity was not strictly dependent on C189S but involved additional cysteine residues in Zta. These results provide evidence that the conserved cysteine in the bZIP domain of Zta plays a primary role in EBV lytic cycle DNA replication.

Amino Acid Sequence↗
PubMed2005

Inhibition of Epstein-Barr virus OriP function by tankyrase, a telomere-associated poly-ADP ribose polymerase that binds and modifies EBNA1.

Tankyrase (TNKS) is a telomere-associated poly-ADP ribose polymerase (PARP) that has been implicated along with several telomere repeat binding factors in the regulation of Epstein-Barr virus origin of plasmid replication (OriP). We now show that TNKS1 can bind to the family of repeats (FR) and dyad symmetry regions of OriP by using a chromatin immunoprecipitation assay and DNA affinity purification. TNKS1 and TNKS2 bound to EBNA1 in coimmunoprecipitation experiments with transfected cell lysates and with purified recombinant proteins in vitro. Two RXXPDG-like TNKS-interacting motifs in the EBNA1 amino-terminal domain mediated binding with the ankyrin repeat domain of TNKS. Mutations of both motifs at EBNA1 G81 and G425 abrogated TNKS binding and enhanced EBNA1-dependent replication of OriP. Small hairpin RNA targeted knock-down of TNKS1 enhanced OriP-dependent DNA replication. Overexpression of TNKS1 or TNKS2 inhibited OriP-dependent DNA replication, while a PARP-inactive form of TNKS2 (M1045V) was compromised for this inhibition. We show that EBNA1 is subject to PAR modification in vivo and to TNKS1-mediated PAR modification in vitro. These results indicate that TNKS proteins can interact directly with the EBNA1 protein, associate with the FR region of OriP in vivo, and inhibit OriP replication in a PARP-dependent manner.

Amino Acid Sequence↗
PubMed2005

DNA affinity purification of Epstein-Barr virus OriP-binding proteins.

DNA affinity purification has been used to identify cellular and viral proteins associated with the Epstein-Barr virus origin of plasmid DNA replication. This approach allows for a one- or two-step purification scheme of high-affinity DNA binding proteins from crude nuclear extracts. Additionally, this approach may be useful for isolation of proteins that are found in the insoluble fractions of the nuclear matrix or scaffold.

Blotting, Western↗
PubMed2004

Identification of protein tyrosine kinases required for B-cell- receptor-mediated activation of an Epstein-Barr Virus immediate-early gene promoter.

Epstein-Barr Virus (EBV) is a potentially oncogenic herpesvirus that infects >90% of the world's population. EBV exists predominantly as a latent infection in B lymphocytes, with periodic lytic-cycle reactivation essential for cellular and host transmission. Viral reactivation can be stimulated by ligand-induced activation of B-cell-receptor (BCR)-coupled signaling pathways. The critical first step in the transition from latency to the lytic cycle is the expression of the viral immediate-early gene BZLF1 through the transcription activation of its promoter, Zp. However, the BCR-coupled signal transduction cascade(s) leading to the induction of Zp and the expression of the BZLF1 gene product, Zta, is currently unclear. A major obstacle to delineating the relevant signal transduction events has been the lack of a model of EBV infection that is amenable to genetic manipulation. The use of the avian B-cell line DT40 has proven to be a powerful tool for delineating BCR-mediated signal transduction pathways that appear to be highly conserved between avian and mammalian systems. We demonstrate that the DT40 cell line is a robust and genetically tractable system for the study of BCR-mediated signaling pathways leading to transcriptional activation of BZLF1. Using this system, we demonstrate that activation of Zp requires the BCR-coupled protein tyrosine kinases Syk and Btk and that it is positively regulated by Lyn. Thus, the use of DT40 cells has allowed us to delineate the early signaling components required for BCR-dependent reactivation of latent EBV, and this system is likely to prove useful for further dissection of the downstream signaling cascades involved.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2004

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

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.

Cell Line↗
PubMed2004

ORC, MCM, and histone hyperacetylation at the Kaposi's sarcoma-associated herpesvirus latent replication origin.

The viral genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists as an extrachromosomal plasmid in latently infected cells. The KSHV latency-associated nuclear antigen (LANA) stimulates plasmid maintenance and DNA replication by binding to an approximately 150-bp region within the viral terminal repeats (TR). We have used chromatin immunoprecipitation assays to demonstrate that LANA binds specifically to the replication origin sequence within the KSHV TR in latently infected cells. The latent replication origin within the TR was also bound by LANA-associated proteins CBP, double-bromodomain-containing protein 2 (BRD2), and the origin recognition complex 2 protein (ORC2) and was enriched in hyperacetylated histones H3 and H4 relative to other regions of the latent genome. Cell cycle analysis indicated that the minichromosome maintenance complex protein, MCM3, bound TR in late-G(1)/S-arrested cells, which coincided with the loss of histone H3 K4 methylation. Micrococcal nuclease studies revealed that TRs are embedded in a highly ordered nucleosome array that becomes disorganized in late G(1)/S phase. ORC binding to TR was LANA dependent when reconstituted in transfected plasmids. DNA affinity purification confirmed that LANA, CBP, BRD2, and ORC2 bound TR specifically and identified the histone acetyltransferase HBO1 (histone acetyltransferase binding to ORC1) as a potential TR binding protein. Disruption of ORC2, MCM5, and HBO1 expression by small interfering RNA reduced LANA-dependent DNA replication of TR-containing plasmids. These findings are the first demonstration that cellular replication and origin licensing factors are required for KSHV latent cycle replication. These results also suggest that the KSHV latent origin of replication is a unique chromatin environment containing histone H3 hyperacetylation within heterochromatic tandem repeats.

Acetylation↗
PubMed2004

The c-MYC oncoprotein is a substrate of the acetyltransferases hGCN5/PCAF and TIP60.

The c-MYC oncoprotein functions as a sequence-specific transcription factor. The ability of c-MYC to activate transcription relies in part on the recruitment of cofactor complexes containing the histone acetyltransferases mammalian GCN5 (mGCN5)/PCAF and TIP60. In addition to acetylating histones, these enzymes have been shown to acetylate other proteins involved in transcription, including sequence-specific transcription factors. This study was initiated in order to determine whether c-MYC is a direct substrate of mGCN5 and TIP60. We report here that mGCN5/PCAF and TIP60 acetylate c-MYC in vivo. By using nanoelectrospray tandem mass spectrometry to examine c-MYC purified from human cells, the major mGCN5-induced acetylation sites have been mapped. Acetylation of c-MYC by either mGCN5/PCAF or TIP60 results in a dramatic increase in protein stability. The data reported here suggest a conserved mechanism by which acetyltransferases regulate c-MYC function by altering its rate of degradation.

Acetylation↗
PubMed2003

Chromatin remodeling of the Kaposi's sarcoma-associated herpesvirus ORF50 promoter correlates with reactivation from latency.

The switch from latent to lytic infection of Kaposi's sarcoma-associated herpesvirus is initiated by the immediate early transcriptional activator protein Rta/open reading frame 50 (ORF50). We examined the transcriptional regulation of the ORF50 core promoter in response to lytic cycle stimulation. We show that the ORF50 promoter is highly responsive to sodium butyrate (NaB) and trichostatin A (TSA), two chemicals known to inhibit histone deacetylases. The NaB and TSA responsive element was mapped to a 70-bp minimal promoter containing an essential GC box that binds Sp1/Sp3 in vitro and in vivo. Micrococcal nuclease mapping studies revealed that a nucleosome is positioned over the transcriptional initiation and the Sp1/3 binding sites. Stimulation with NaB or TSA increased histone acetylation and restriction enzyme accessibility of the ORF50 promoter transcription initiation site. Chromatin immunoprecipitation assay was used to demonstrate that the ORF50 promoter is associated with several different histone deacetylase proteins (including HDAC1, 5, and 7) in latently infected cells. NaB treatment led to the rapid association of Ini1/Snf5, a component of the Swi/Snf family of chromatin remodeling proteins, with the ORF50 promoter. Ectopic expression of the CREB-binding protein (CBP) histone acetyltransferase (HAT) stimulated plasmid-based ORF50 transcription in a HAT-dependent manner, suggesting that CBP recruitment to the ORF50 promoter can be an initiating event for transcription and viral reactivation. Together, these results suggest that remodeling of a stably positioned nucleosome at the transcriptional initiation site of ORF50 is a regulatory step in the transition from latent to lytic infection.

Base Sequence↗