Chi-Ju Chen

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

ABSTRACT 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.

Telomeric Proteins Regulate Episomal Maintenance of Epstein-Barr Virus Origin of Plasmid Replication

Episomal maintenance and DNA replication of EBV origin of plasmid replication (OriP) plasmid maintenance is mediated by the viral encoded origin binding protein, EBNA1, and unknown cellular factors. We found that telomeric repeat binding factor 2 (TRF2), TRF2-interacting protein hRap1, and the telomere-associated poly(ADP-ribose) polymerase (Tankyrase) bound to the dyad symmetry (DS) element of OriP in an EBNA1-dependent manner. TRF2 bound cooperatively with EBNA1 to the three nonamer sites (TTAGGGTTA), which resemble telomeric repeats. Mutagenesis of the nonamers reduced plasmid maintenance function and increased plasmid sensitivity to genotoxic stress. DS affinity-purified proteins possessed poly(ADP-ribose) polymerase (PARP) activity, and EBNA1 was subject to NAD-dependent posttranslational modification in vitro. OriP plasmid maintenance was sensitive to changes in cellular PARP/Tankyrase activity. These findings imply that telomere-associated proteins regulate OriP plasmid maintenance by PAR-dependent modifications.

Stimulation of CREB Binding Protein Nucleosomal Histone Acetyltransferase Activity by a Class of Transcriptional Activators

ABSTRACT The transcriptional coactivator CREB binding protein (CBP) possesses intrinsic histone acetyltransferase (HAT) activity that is important for gene regulation. CBP binds to and cooperates with numerous nuclear factors to stimulate transcription, but it is unclear if these factors modulate CBP HAT activity. Our previous work showed that CBP interacts with the Epstein-Barr virus-encoded basic region zipper (b-zip) protein, Zta, and augments its transcriptional activity. Here we report that Zta strongly enhances CBP-mediated acetylation of nucleosomal histones. Zta stimulated the HAT activity of CBP that had been partially purified or immunoprecipitated from mammalian cells as well as from affinity-purified, baculovirus expressed CBP. Stimulation of nucleosome acetylation required the CBP HAT domain, the Zta DNA binding and transcription activation domain, and nucleosomal DNA. In addition to Zta, we found that two other b-zip proteins, NF-E2 and C/EBPα, strongly stimulated nucleosomal HAT activity. In contrast, several CBP-binding proteins, including phospho-CREB, JUN/FOS, GATA-1, Pit-1, and EKLF, failed to stimulate HAT activity. These results demonstrate that a subset of transcriptional activators enhance the nucleosome-directed HAT activity of CBP and suggest that nuclear factors may regulate transcription by altering substrate recognition and/or the enzymatic activity of chromatin modifying coactivators.

HCMV IE2-mediated inhibition of HAT activity downregulates p53 function

Targeting of cellular histone acetyltransferases (HATs) by viral proteins is important in the development of virus‐associated diseases. The immediate‐early 2 protein (IE2) of human cytomegalovirus (HCMV) binds to the tumor suppressor, p53, and inactivates its functions by unknown mechanisms. Here, we show that IE2 binds to the HAT domain of the p53 coactivators, p300 and CREB‐binding protein (CBP), and blocks their acetyltransferase activity on both histones and p53. The minimal HAT inactivation region on IE2 involves the N‐terminal 98 amino acids. The in vivo DNA binding of p53 and local histone acetylation on p53‐dependent promoters are all reduced by IE2, but not by mutant IE2 proteins that lack the HAT inhibition region. Furthermore, the p53 acetylation site mutant, K320/373/382R, retains both DNA binding and promoter transactivation activity in vivo and these effects are repressed by IE2 as well. Together with the finding that only wild‐type IE2 exerts an antiapoptotic effect, our results suggest that HCMV IE2 downregulates p53‐dependent gene activation by inhibiting p300/CBP‐mediated local histone acetylation and that IE2 may have oncogenic activity.

EBV Zta protein induces the expression of interleukin-13, promoting the proliferation of EBV-infected B cells and lymphoblastoid cell lines

Epstein-Barr virus (EBV) infection can modify the cytokine expression profiles of host cells and determine the fate of those cells. Of note, expression of interleukin-13 (IL-13) may be detected in EBV-associated Hodgkin lymphoma and the natural killer (NK) cells of chronic active EBV-infected patients, but its biologic role and regulatory mechanisms are not understood. Using cytokine antibody arrays, we found that IL-13 production is induced in B cells early during EBV infection. Furthermore, the EBV lytic protein, Zta (also known as the BZLF-1 product), which is a transcriptional activator, was found to induce IL-13 expression following transfection. Mechanistically, induction of IL-13 expression by Zta is mediated directly through its binding to the IL-13 promoter, via a consensus AP-1 binding site. Blockade of IL-13 by antibody neutralization showed that IL-13 is required at an early stage of EBV-induced proliferation and for long-term maintenance of the growth of EBV immortalized lymphoblastoid cell lines (LCLs). Thus, Zta-induced IL-13 production facilitates B-cell proliferation and may contribute to the pathogenesis of EBV-associated lymphoproliferative disorders, such as posttransplantation lymphoproliferative disease (PTLD) and Hodgkin lymphoma.

The Amino-Terminal C/H1 Domain of CREB Binding Protein Mediates Zta Transcriptional Activation of Latent Epstein-Barr Virus

ABSTRACT Latent Epstein-Barr virus (EBV) is maintained as a nucleosome-covered episome that can be transcriptionally activated by overexpression of the viral immediate-early protein, Zta. We show here that reactivation of latent EBV by Zta can be significantly enhanced by coexpression of the cellular coactivators CREB binding protein (CBP) and p300. A stable complex containing both Zta and CBP could be isolated from lytically stimulated, but not latently infected RAJI nuclear extracts. Zta-mediated viral reactivation and transcriptional activation were both significantly inhibited by coexpression of the E1A 12S protein but not by an N-terminal deletion mutation of E1A (E1AΔ2-36), which fails to bind CBP. Zta bound directly to two related cysteine- and histidine-rich domains of CBP, referred to as C/H1 and C/H3. These domains both interacted specifically with the transcriptional activation domain of Zta in an electrophoretic mobility shift assay. Interestingly, we found that the C/H3 domain was a potent dominant negative inhibitor of Zta transcriptional activation function. In contrast, an amino-terminal fragment containing the C/H1 domain was sufficient for coactivation of Zta transcription and viral reactivation function. Thus, CBP can stimulate the transcription of latent EBV in a histone acetyltransferase-independent manner mediated by the CBP amino-terminal C/H1-containing domain. We propose that CBP may regulate aspects of EBV latency and reactivation by integrating cellular signals mediated by competitive interactions between C/H1, C/H3, and the Zta activation domain.

The CBP Bromodomain and Nucleosome Targeting Are Required for Zta-Directed Nucleosome Acetylation and Transcription Activation

ABSTRACT The Epstein-Barr virus (EBV)-encoded lytic activator Zta is a bZIP protein that can stimulate nucleosomal histone acetyltransferase (HAT) activity of the CREB binding protein (CBP) in vitro. We now show that deletion of the CBP bromo- and C/H3 domains eliminates stimulation of nucleosomal HAT activity in vitro and transcriptional coactivation by Zta in transfected cells. In contrast, acetylation of free histones was not affected by the addition of Zta or by deletions in the bromo or C/H3 domain of CBP. Zta stimulated acetylation of oligonucleosomes assembled on supercoiled DNA and dinucleosomes assembled on linear DNA, but Zta-stimulated acetylation was significantly reduced for mononucleosomes. Western blotting and amino-terminal protein sequencing indicated that all lysine residues in the H3 and H4 amino-terminal tails were acetylated by CBP and enhanced by the addition of Zta. Histone acetylation was also dependent upon the Zta basic DNA binding domain, which could not be substituted with the homologous basic region of c-Fos, indicating specificity in the bZIP domain nucleosome binding function. Finally, we show that Zta and CBP colocalize to viral immediate-early promoters in vivo and that overexpression of Zta leads to a robust increase in H3 and H4 acetylation at various regions of the EBV genome in vivo. Furthermore, deletion of the CBP bromodomain reduced stable CBP-Zta complex formation and histone acetylation at Zta-responsive viral promoters in vivo. These results suggest that activator- and bromodomain-dependent targeting to oligonucleosomal chromatin is required for stable promoter-bound complex formation and transcription activity.

Regulation of Epstein-Barr Virus OriP Replication by Poly(ADP-Ribose) Polymerase 1

ABSTRACT Poly(ADP-ribose) polymerase (PARP) is an abundant, chromatin-associated, NAD-dependent enzyme that functions in multiple chromosomal processes, including DNA replication and chromatin remodeling. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is a dynamic genetic element that confers stable episome maintenance, DNA replication initiation, and chromatin organization functions. OriP function depends on the EBV-encoded origin binding protein EBNA1. We have previously shown that EBNA1 is subject to negative regulation by poly(ADP-ribosyl)ation (PARylation). We now show that PARP1 physically associates with OriP in latently EBV-infected B cells. Short hairpin RNA depletion of PARP1 enhances OriP replication activity and increases EBNA1, origin recognition complex 2 (ORC2), and minichromosome maintenance complex (MCM) association with OriP. Pharmacological inhibitors of PARP1 enhance OriP plasmid maintenance and increase EBNA1, ORC2, and MCM3 occupancy at OriP. PARylation in vitro inhibits ORC2 recruitment and remodels telomere repeat factor (TRF) binding at the dyad symmetry (DS) element of OriP. Purified PARP1 can ribosylate EBNA1 at multiple sites throughout its amino terminus but not in the carboxy-terminal DNA binding domain. We also show that EBNA1 linking regions (LR1 and LR2) can bind directly to oligomers of PAR. We propose that PARP1-dependent PARylation of EBNA1 and adjacently bound TRF2 induces structural changes at the DS element that reduce EBNA1 DNA binding affinity and functional recruitment of ORC.

Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates.

Abstract The recombinant nucleocapsid (rN) protein of the coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS) was cloned and expressed in Escherichia coli, extracted from cell lysates containing 6M urea, then purified by Ni2+-affinity chromatography. In animal immunogenicity studies, we found that most anti-rN protein antibodies were IgG2a in BALB/c mice vaccinated with rN emulsified in Montanide ISA-51 containing the synthetic oligodeoxynucleotide, CpG. In contrast, anti-rN protein antibodies of mice immunized with rN protein in PBS were found to mainly be IgG1. These results indicated that ISA-51/CpG-formulated rN protein was dramatically biased toward a Th1 immune response. To identify the B-cell immunodominant epitopes of the rN protein in the mouse and monkey, the reactivities of antisera raised against purified rN proteins formulated in ISA-51/CpG were tested with a panel of overlapping synthetic peptides covering the entire N protein sequence. Three immunodominant linear B-cell epitope regions were mapped to residues 166–180, 356–375, and 396–410 of the rN protein. When the reactivities of these peptides were screened with human sera from five SARS patients, peptides corresponding to residues 156–175 reacted strongly with sera from two of the SARS patients. These results indicated that the region around residues 156–175 of the N protein is immunogenic in the mouse, monkey, and human. We found that peptides corresponding to residues 1–30, 86–100, 306–320, and 351–365 contained murine immunodominant T-cell epitopes. To identify functional CTL epitopes of the N protein, BALB/c mice were immunized with peptides containing the H-2Kd CTL motif emulsified in adjuvant ISA-51/CpG. Using an IFN-γ secretion cell assay and analysis by flow cytometry, peptides containing residues 81–95 were found to be capable of stimulating both CD4+ and CD8+ cell proliferation in vitro. We also only observed that peptides corresponding to residues 336–350 were capable of stimulating IFN-γ production in T-cell cultures derived from peripheral blood mononuclear cells (PBMCs) of macaques immunized with the rN protein emulsified in ISA/CpG adjuvant. Our current results together with those of others suggest that some immunodominant B-cell and T-cell epitopes are conserved in the mouse, monkey, and human. This information is very important for the development SARS diagnostic kits and a vaccine.

Decoy receptor 3, upregulated by Epstein-Barr virus latent membrane protein 1, enhances nasopharyngeal carcinoma cell migration and invasion

Decoy receptor 3 (DcR3), a member of tumor necrosis factor receptor superfamily, has been implicated in tumorigenesis through its abilities to modulate immune responses and induce angiogenesis. Epstein-Barr virus (EBV), a ubiquitous gamma-herpesvirus, is associated with malignancies including nasopharyngeal carcinoma (NPC). Previous studies show that DcR3 is overexpressed in EBV-positive lymphomas and Rta, an EBV transcription activator, can upregulate DcR3 in Burkitt lymphoma cell lines. However, DcR3 expression has not been demonstrated in EBV-associated NPC nor have there been any EBV latent genes linked to DcR3 upregulation. Here, we showed DcR3 was overexpressed in NPC. Higher DcR3 expression score and DcR3-positive rate were found in metastatic NPC than in primary NPC tissues, suggesting DcR3 may enhance cell metastatic potential. This hypothesis is supported by our observation that NPC HONE-1 cells overexpressing DcR3 exhibited significant higher migration and invasion abilities in vitro. We found besides Rta, EBV latent membrane protein (LMP) 1 can upregulate DcR3 via nuclear factor-kappaB and phosphatidylinositol 3-kinase-signaling events. Approximate 75% of LMP1-positive NPC tissues overexpressed DcR3, suggesting LMP1 may enhance DcR3 expression in vivo. Data herein suggested that increasing DcR3 expression by LMP1 not only helps EBV-associated cancer cells gain survival advantage by preventing host immune detection but also increases the chance of cancer metastasis by enhancing cell migration and invasion. All these DcR3-mediated events facilitate normal cells to gain cancer hallmarks.